US 2017/0137801 A1Application

TISSUE-SPECIFIC GENOME ENGINEERING USING CRISPR-CAS9

Publication Date:2017-05-18

•29 Claims

•19 Drawing Sheets

Abstract

The present application provides compounds, compositions, uses thereof for the treatment of diseases, conditions and/or disorders, and uses thereof as asialoglycoprotein receptor (ASGPR) targeting agents.

Metadata

Assignees

Pfizer Inc.
The Regents of the University of California

Inventors

Spiros Liras
Vincent Mascitti
Benjamin Aaron Thuma
Jennifer A. Doudna
Romain Rouet

Application Information

Application Number:US 15/347,339

Filing Date:2016-11-09

Priority Date:2015-11-12

Classifications

IPC:

C12N9/96C12N15/90C12N9/22

Patent Drawings (19 sheets)

Description

RELATED APPLICATIONS

[0001] This application claims the benefit of and priority from U.S. Provisional Patent Applications 62/254,652, filed Nov. 12, 2015, and 62/384,660, filed Sep. 7, 2016. Each of the foregoing applications is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 7, 2016, is named PCFC-989-101_SL.txt and is 8,027,538 bytes in size.

FIELD OF THE INVENTION

[0003] The present invention relates to compounds, compositions and uses thereof as gene targeting agents.

BACKGROUND

[0004] Considerable attention has been devoted to developing reagents and methods for delivering bioactive agents to particular tissues, cells, and/or subcellular locations. For example, delivery of large molecules, such as an antisense or RNAi molecules or proteins, is difficult as such compounds are generally not able to penetrate cell membranes; when they can, they can often lack of selectivity for the tissue of interest and therefore increase risk of off-target pharmacology and present serious safety concerns. Furthermore, selective drug delivery to targeted delivery sites is often a challenge because molecules that are cell permeable are often not selective. One solution to cell diffusion and targeted delivery is drug conjugation to targeting agents.

[0005] In the context of gene therapy, most gene editing agents have often been delivered via plasmid DNA encapsulated in viral-derived vectors such as adeno viruses and adeno-associated viruses. Unfortunately, this type of approach has been plagued with serious issues for the patients mainly i) increased risk of insertional mutagenesis, ii) increased risks of hepatotoxicity upon interaction of viral vector with Kupffer cells and iii) only transient pharmacological benefit for the patient triggered by immunogenic response against the treated cells. As a result, there remains a need to find more effective and safer ways to deliver gene editing agents. Attempts thus far have been elusive and in this context, delivering gene editing agents in the form of the protein, and not its DNA or RNA, represent an immense therapeutic opportunity.

[0006] Targeting agents often enhance pharmaceutical attributes including pharmacokinetics and pharmacodynamics. Targeting agents allow the drug payload attached to the targeting agent to be efficiently distributed to and up-taken by specific cells. Certain sugars, such as galactose, N-acetyl galactosamine, and other galactose derivatives including those described by M. G. Finn and V. Mascitti et al. in the Journal of the American Chemical Society, 134, 1978 (2012) have been used as targeting agents for hepatocytes due to the binding to asialoglycoprotein receptors (ASGPR) that are present on the surface of hepatocytes.

[0007] As a further issue for drug delivery, in receptor mediated endocytosis mechanisms, endosomal uptake pathway is known to be a rate-limiting barrier for delivering bioactive agents to the target subcellular locations. Bioactive molecules often get trapped in the endosomal vesicles and degraded in the lysosomal compartment. Various reagents, such as chloroquine, polyethyleneimine [PEI], certain highly charged cationic compounds, fusogenic peptides, and inactivated adenoviruses, have been developed that are intended to quickly disrupt the endosome in order to minimize the amount of time that a delivered bioactive agent spends in the endosome-like environment. However, these agents often lack of generality and present suboptimal ability to promote endosomal escape of the cargo and are associated with various disadvantages, including toxicity problems.

[0008] Consequently, a versatile and biocompatible cell delivery system would be important for the clinical success of gene targeting and the delivery of therapeutic agents.

SUMMARY OF THE INVENTION

[0009] One aspect of the present invention provides compounds of Formula (A-1), (A-2), or (A-3):

or a pharmaceutically acceptable salt thereof,
wherein:

[0010] R^xxis —H, -alkyl, -cycloalkyl, -alkenyl, -alkynyl, -aryl, -heteroaryl, —OR⁵, —N(R⁴)—R⁵, —SR⁵, wherein a —CH₂— group of said R^xxmay each be independently replaced with a heteroatom group selected from —O—, —S—, —N(R⁴)— and wherein a —CH₃of said R^xxmay be replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms, and wherein the alkyl, cycloalkyl, alkenyl and alkynyl may each be independently substituted with one or more halo atoms;
[0011] R^yyis —CN, —CH₂—CN, —C≡CH, —CH₂—N₃, —CH₂—NH₂, —CH₂—N(R⁴)—S(O)₂—R⁵, —CH₂—CO₂H, —CO₂H, —CH₂—OH, —CH₂—SH, —CH═CH—R⁵, —CH₂—R⁵, —CH₂—S—R⁵, —CH₂—N(R⁴)—R⁵, —CH₂—N(R⁴)—C(O)—R⁵, —CH₂—N(R⁴)—C(O)—O—R⁵, —CH₂—N(R⁴)—C(O)—N(R⁴)—R⁵, —CH₂—O—R⁵, —CH₂—O—C(O)—R⁵, —CH₂—O—C(O)—N(R⁴)—R⁵, —CH₂—O—C(O)—O—R⁵, —CH₂—S(O)—R⁵, —CH₂—S(O)₂—R⁵, —CH₂—S(O)₂—N(R⁴)—R⁵, —C(O)—NH₂, —C(O)—O—R⁵, —C(O)—N(R⁴)—R⁵, or aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with R⁵;
[0012] each R¹is independently —CN, —CH₂—CN, —C≡CH, —CH₂—N₃, —CH₂—NH₂, —CH₂—N(R⁴)—S(O)₂—R⁵, —CH₂—CO₂H, —CO₂H, —CH₂—OH, —CH₂—SH, —CH═CH—R⁵, —CH₂—R⁵, —CH₂—S—R⁵, —CH₂—N(R⁴)—R⁵, —CH₂—N(R⁴)—C(O)—R⁵, —CH₂—N(R⁴)—C(O)—O—R⁵, —CH₂—N(R⁴)—C(O)—N(R⁴)—R⁵, —CH₂—O—R⁵, —CH₂—O—C(O)—R⁵, —CH₂—O—C(O)—N(R⁴)—R⁵, —CH₂—O—C(O)—O—R⁵, —CH₂—S(O)—R⁵, —CH₂—S(O)₂—R⁵, —CH₂—S(O)₂—N(R⁴)—R⁵, —C(O)—NH₂, —C(O)—O—R⁵, —C(O)—N(R⁴)—R⁵, or aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with R⁵,
[0013] or R¹is —Z—X—Y, —Z—Y, —X—Y, —Z—X⁺Y⁻, —Z—X⁻Y⁺, —X⁻Y⁺, or —Y;
[0014] X is a linker;
[0015] X⁺ is a positively charged linker;
[0016] X⁻ is a negatively charged linker;
[0017] Y is a ribonucleoprotein or endonuclease comprising a site-directed modifying polypeptide, or Y is a site-directed modifying polypeptide, or Y is Cas9 ribonucleoprotein, or Y is a Cas9 protein, or Y is a single guide RNA sequence (sgRNA) or Y is a dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA);
[0018] Y⁺ is a positively charged ribonucleoprotein or endonuclease comprising a site-directed modifying polypeptide, or Y⁺ a positively charged site-directed modifying polypeptide, or Y⁺ is a positively charged Cas9 protein;
[0019] Y⁻ is a negatively charged ribonucleoprotein or endonuclease comprising a site-directed modifying polypeptide, or Y⁻ is a negatively charged site-directed modifying polypeptide, or Y⁻ is a negatively charged Cas9 ribonucleoprotein, or Y⁻ is a negatively charged sgRNA, or Y⁻ is a negatively charged dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA);
[0020] Z is absent or is —C≡C—, —CH═CH—, —CH₂—, —CH₂—O—, —C(O)—N(R⁴)—, —CH₂—S(O)—, —CH₂—S(O)₂—, —CH₂—S(O)₂—N(R⁴)—, —C(O)—O—, —CH₂—N(R⁴)—, —CH₂—N(R⁴)—C(O)—, —CH₂—N(R⁴)—S(O)₂—, —CH₂—N(R⁴)—C(O)—O—, —CH₂—N(R⁴)—C(O)—N(R⁴)—, —CH₂—O—C(O)—, —CH₂—O—C(O)—N(R⁴)—, —CH₂—O—C(O)—O—, or aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with R⁵;
[0021] R²is —OH, —N₃, —N(R³)₂, —N(R³)—C(O)—R³, —N(R³)—C(O)—N(R³)₂, —N(R³)—C(O)—OR³, —N(R³)—S(O)₂—R³, tetrazole, or triazole, wherein the tetrazole and triazole are optionally substituted with R³;
[0022] each R³is independently —H, halo-substituted (C₁-C₅)alkyl, halo substituted (C₃-C₆)cycloalkyl, (C₃-C₆)cycloalkyl, —(C₁-C₅)alkenyl, —(C₁-C₅)alkynyl, halo substituted —(C₁-C₅)alkenyl, or halo substituted —(C₁-C₅)alkynyl, wherein a —CH₂— group of the alkyl or cycloalkyl may each be independently replaced with a heteroatom group selected from —O—, —S—, and —N(R⁴)— and —CH₃of the alkyl may each be independently replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms;
[0023] each R⁴is independently —H, —(C₁-C₂₀)alkyl, —(C₁-C₂₀)alkenyl, —(C₁-C₂₀)alkynyl, or (C₃-C₆)cycloalkyl wherein one to six —CH₂— groups of the alkyl or cycloalkyl separated by at least two carbon atoms may each be independently replaced with a heteroatom independently selected from —O—, —S—, or —N(R⁴)—, and —CH₃of the alkyl may each be independently replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms; and wherein the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with halo atoms;
[0024] each R⁵is independently —H, (C₃-C₂₀)cycloalkyl, —(C₁-C₆₀)alkenyl, —(C₁-C₆₀)alkynyl, or (C₁-C₆₀)alkyl wherein one to six —CH₂— groups of the cycloalkyl or one to 20 —CH₂— groups of the alkyl may each be independently replaced with heteroatoms independently selected from —O—, —S—, and —N(R⁴)— wherein the heteroatoms are separated by at least two carbon atoms, and —CH₃of the alkyl may each be independently replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms; and wherein the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with halo atoms.

[0025] Another aspect of the present invention provides a compound of Formula (B):

or a pharmaceutically acceptable salt thereof,
wherein:

[0026] R¹is —Z—X—Y, —Z—Y, —X—Y, —Z—X⁺Y⁻, —X⁺Y⁻, —Z—X⁻Y⁺, —X⁻Y⁺, or —Y;
[0027] X is a linker;
[0028] X⁺ is a positively charged linker;
[0029] X⁻ is a negatively charged linker;
Y is a ribonucleoprotein or endonuclease comprising a site-directed modifying polypeptide, or Y is a site-directed modifying polypeptide, or Y is Cas9 ribonucleoprotein, or Y is a Cas9 protein, or Y is a single guide RNA sequence (sgRNA) or Y is a dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA);
[0030] Y⁺ is a positively charged ribonucleoprotein or endonuclease comprising a site-directed modifying polypeptide, or Y⁺ a positively charged site-directed modifying polypeptide, or Y⁺ is a positively charged Cas9 protein;
[0031] Y⁻ is a negatively charged ribonucleoprotein or endonuclease comprising a site-directed modifying polypeptide, or Y⁻ is a negatively charged site-directed modifying polypeptide, or Y⁻ is a negatively charged Cas9 ribonucleoprotein, or Y⁻ is a negatively charged sgRNA, or Y⁻ is a negatively charged dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA);
[0032] Z is absent or is —C≡C—, —C═CH—, —CH₂—, —CH₂—O—, —C(O)—N(R⁴)—, —CH₂—S—, —CH₂—S(O)—, —CH₂—S(O)₂—, —CH₂—S(O)₂—N(R⁴)—, —C(O)—O—, —CH₂—N(R⁴)—, —CH₂—N(R⁴)—C(O)—, —CH₂—N(R⁴)—S(O)₂—, —CH₂—N(R⁴)—C(O)—O—, —CH₂—N(R⁴)—C(O)—N(R⁴)—, —CH₂—O—C(O)—, —CH₂—O—C(O)—N(R⁴)—, —CH₂—O—C(O)—O—, or aryl or heteroaryl, wherein the aryl or heteroaryl is optionally substituted with R⁵;
[0033] R²is —OH, —N₃, —N(R³)₂, —N(R³)—C(O)—R³, —N(R³)—C(O)—N(R³)₂, —N(R³)—C(O)—OR³, —N(R³)—S(O)₂—R³, tetrazole, or triazole, wherein the tetrazole and triazole are optionally substituted with R³;
[0034] each R³is independently —H, halo-substituted (C₁-C₅)alkyl, halo substituted (C₃-C₆)cycloalkyl, —(C₁-C₅)alkenyl, —(C₁-C₅)alkynyl, halo substituted —(C₁-C₅)alkenyl, halo substituted —(C₁-C₅)alkynyl, or (C₃-C₆)cycloalkyl, wherein a —CH₂— group of the alkyl or cycloalkyl may each be independently replaced with a heteroatom group selected from —O—, —S—, and —N(R⁴)— and —CH₃of the alkyl may each be independently replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms;
[0035] each R⁴is independently —H, —(C₁-C₂₀)alkyl, —(C₁-C₂₀)alkenyl, —(C₁-C₂₀)alkynyl, or (C₃-C₆)cycloalkyl wherein one to six —CH₂— groups of the alkyl or cycloalkyl separated by at least two carbon atoms may each be independently replaced with a heteroatom independently selected from —O—, —S—, or —N(R⁴)—, and —CH₃of the alkyl may each be independently replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms; and wherein the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with halo atoms;
[0036] each R⁵is independently —H, (C₃-C₂₀)cycloalkyl, —(C₁-C₆₀)alkenyl, —(C₁-C₆₀)alkynyl, or (C₁-C₆₀)alkyl wherein one to six —CH₂— groups of the cycloalkyl or one to 20 —CH₂— groups of the alkyl may each be independently replaced with heteroatoms independently selected from —O—, —S—, and —N(R⁴)— wherein the heteroatoms are separated by at least two carbon atoms, and —CH₃of the alkyl may each be independently replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms; and wherein the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with halo atoms.

[0037] Another aspect of the present invention includes a method for treating a liver disease or condition or a liver modulated disease or condition in a subject, including, but not limited to, hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, Alpha-1-antitrypsin deficiency, Bile acid synthesis and metabolism defects, Biliary Atresia, Cystic Fibrosis liver disease, Idiopathic neonatal hepatitis, Mitochondrial hepatopathies, Progressive familial intrahepatic cholestasis, Primary sclerosing cholangitis, Transthyretin amyloidosis, Hemophilia, Homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia like Type II diabetes mellitus, and diseases involving abnormally high hepatic glucose production similar to Type II diabetes mellitus, comprising the administration of an effective amount of a compound or composition described herein. In some embodiments, the disease or condition is hyperlipidemia, non-alcoholic steatohepatitis (NASH), or non-alcoholic fatty liver disease (NAFLD).

[0038] Another aspect of the present invention provides a method for selectively modulating transcription of a target DNA in a liver cell of a subject, said DNA being associated with a liver disease or condition or a liver modulated disease or condition in a subject, such as, but are not limited to hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, Alpha-1-antitrypsin deficiency, Bile acid synthesis and metabolism defects, Biliary Atresia, Cystic Fibrosis liver disease, Idiopathic neonatal hepatitis, Mitochondrial hepatopathies, Progressive familial intrahepatic cholestasis, Primary sclerosing cholangitis, Transthyretin amyloidosis, Hemophilia, Homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia like Type II diabetes mellitus, and diseases involving abnormally high hepatic glucose production similar to Type II diabetes mellitus, comprising the administration of an effective amount of a compound or composition described herein.

[0039] Another aspect of the present invention includes a method for editing a nucleic acid molecule encoding a protein associated with a liver disease or condition or a liver modulated disease or condition in a subject, such as, but are not limited to hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, Alpha-1-antitrypsin deficiency, Bile acid synthesis and metabolism defects, Biliary Atresia, Cystic Fibrosis liver disease, Idiopathic neonatal hepatitis, Mitochondrial hepatopathies, Progressive familial intrahepatic cholestasis, Primary sclerosing cholangitis, Transthyretin amyloidosis, Hemophilia, Homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia like Type II diabetes mellitus, and diseases involving abnormally high hepatic glucose production similar to Type II diabetes mellitus, comprising the administration of an effective amount of a compound or composition described herein.

[0040] Another aspect of the present invention includes a method for modulating the expression of level of at least one gene product associated with a liver disease or condition or a liver modulated disease or condition, such as, but are not limited to hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, Alpha-1-antitrypsin deficiency, Bile acid synthesis and metabolism defects, Biliary Atresia, Cystic Fibrosis liver disease, Idiopathic neonatal hepatitis, Mitochondrial hepatopathies, Progressive familial intrahepatic cholestasis, Primary sclerosing cholangitis, Transthyretin amyloidosis, Hemophilia, Homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia like Type II diabetes mellitus, and diseases involving abnormally high hepatic glucose production similar to Type II diabetes mellitus, comprising the administration of an effective amount of a compound or composition described herein.

[0041] Another aspect of the present invention provides a composition comprising a ribonucleoprotein described herein (e.g., a RNP comprising a site-directed modifying polypeptide described herein, such as a Cas9 RNP or a Cpf1 RNP) and an endosomal escape agent. In some embodiments, the ribonucleoprotein (e.g., a RNP comprising a site-directed modifying polypeptide described herein, such as a Cas9 RNP or a Cpf1 RNP) or the endosomal escape agent is conjugated to an antibody. In some embodiments, the ribonucleoprotein (e.g., a RNP comprising a site-directed modifying polypeptide described herein, such as a Cas9 RNP or a Cpf1 RNP) is modified to include glycosylation sites. In some embodiments, the ribonucleoprotein (e.g., a RNP comprising a site-directed modifying polypeptide described herein, such as a Cas9 RNP or a Cpf1 RNP) is modified to include transduction or translocation domains.

[0042] Another aspect of the present invention provides a composition comprising a Cas9 ribonucleoprotein and an endosomal escape agent. In some embodiments, the Cas9 ribonucleoprotein or the endosomal escape agent is conjugated to an antibody. In some embodiments, the Cas9 ribonucleoprotein is modified to include glycosylation sites. In some embodiments, the Cas9 ribonucleoprotein is modified to include transduction or translocation domains.

[0043] Another aspect of the present invention includes pharmaceutical compositions comprising (i) a composition comprising a Cas9 ribonucleoprotein and an endosomal escape agent, as described herein; and (ii) a pharmaceutically acceptable excipient, diluent, or carrier.

[0044] Another aspect of the present invention provides a composition comprising a Cpf1 ribonucleoprotein and an endosomal escape agent. In some embodiments, the Cpf1 ribonucleoprotein or the endosomal escape agent is conjugated to an antibody. In some embodiments, the Cpf1 ribonucleoprotein is modified to include glycosylation sites. In some embodiments, the Cpf1 ribonucleoprotein is modified to include transduction or translocation domains.

[0045] Another aspect of the present invention includes pharmaceutical compositions comprising (i) a composition comprising a Cpf1 ribonucleoprotein and an endosomal escape agent, as described herein; and (ii) a pharmaceutically acceptable excipient, diluent, or carrier.

[0046] Another aspect of the present invention includes a method for treating a disease or condition selected from, but not limited to, blood disorders, cell dysregulation or oncology diseases and disorders, inflammation and immune related diseases, metabolic, liver, kidney and protein diseases and disorders, muscular or skeletal diseases, neurological and neuronal diseases and disorders, and ocular diseases and disorders, comprising the administration of a composition comprising a ribonucleoprotein described herein (e.g., a RNP comprising a site-directed modifying polypeptide described herein, such as a Cas9 RNP or a Cpf1 RNP) and an endosomal escape agent described herein.

[0047] Another aspect of the present invention includes a method for selectively modulating transcription of a target DNA in a subject, said DNA being associated with a disease or condition selected from, but not limited to, blood disorders, cell dysregulation or oncology diseases and disorders, inflammation and immune related diseases, metabolic, liver, kidney and protein diseases and disorders, muscular or skeletal diseases, neurological and neuronal diseases and disorders, and ocular diseases and disorders, comprising the administration of a composition comprising a ribonucleoprotein described herein (e.g., a RNP comprising a site-directed modifying polypeptide described herein, such as a Cas9 RNP or a Cpf1 RNP) and an endosomal escape agent described herein.

[0048] Another aspect of the present invention includes a method for editing a nucleic acid molecule encoding a protein associated with a disease or condition selected from, but not limited to, blood disorders, cell dysregulation or oncology diseases and disorders, inflammation and immune related diseases, metabolic, liver, kidney and protein diseases and disorders, muscular or skeletal diseases, neurological and neuronal diseases and disorders, and ocular diseases and disorders, comprising the administration of a composition comprising a ribonucleoprotein described herein (e.g., a RNP comprising a site-directed modifying polypeptide described herein, such as a Cas9 RNP or a Cpf1 RNP) and an endosomal escape agent described herein.

[0049] Another aspect of the present invention includes a method for modulating the expression level of at least one gene product associated with a disease or condition selected from, but not limited to, blood disorders, cell dysregulation or oncology diseases and disorders, inflammation and immune related diseases, metabolic, liver, kidney and protein diseases and disorders, muscular or skeletal diseases, neurological and neuronal diseases and disorders, and ocular diseases and disorders, comprising the administration of a composition comprising a ribonucleoprotein described herein (e.g., a RNP comprising a site-directed modifying polypeptide described herein, such as a Cas9 RNP or a Cpf1 RNP) and an endosomal escape agent described herein

[0050] Another aspect of the present invention provides a method for site-specific endonucleolytic cleavage of a single-stranded RNA (ssRNA), comprising the administration of a compound, a RNP, or a composition described herein, in the presence of a protospacer adjacent motif (PAM)-presenting oligonucleotides (PAMmer).

[0051] Another aspect of the present invention provides a method for site-specific endonucleolytic cleavage of a single-stranded RNA (ssRNA), comprising the administration of a compound or RNP, which comprises a CRISPR/Cas Type III-B Cmr complex, or a composition thereof, as described herein.

[0052] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 represents the refined crystal structure for the compound 23 which was plotted using the SHELXTL plotting package with ellipsoids drawn at 50% confidence level.

[0054] FIG. 2 represents the results of microscopic imaging studies of RNP constructs.

Panels A-C: RNP construct Y53aASGPRL-RNP-EMX1 (A) uptake in HepG2 cells is evident within 60 minutes as indicated by intracellular puncta with some surface binding still evident. Lysosomes are labeled with Dextran-647 (B) and nuclei are labeled with Hoechst (C).
Panels D-F: RNP construct Y1C80S-3N-m-RNP-EMX1 (D) shows only cell surface staining at 60 minutes with no evidence of internalization. Lysosomes are labeled with Dextran-647 (E) and nuclei are labeled with Hoechst (F).
Panels G-I: RNP construct Y53aASGPRL-RNP-EMX1 (G) is completely internalized in HepG2 cells by 20 hours as indicated by intracellular puncta. Lysosomes are labeled with Dextran-647 (H) and nuclei are labeled with Hoechst (I).
Panels J-L: RNP construct Y53aASGPRL-RNP-EMX1 (J) accumulates on the surface of SK-Hep cells within 60 minutes and shows no evidence of internalization. Lysosomes are labeled with Dextran-647 (K) and nuclei are labeled with Hoechst (L).

[0055] FIG. 3 represents mass spectrometry of Cas9 constructs. Panel A represents a mass spectrometry of Cas9 construct Y1C80S-3N-m. Panel B represents a mass spectrometry of Cas9 construct Y53Aasgprl.

[0056] FIG. 4 represents EMX1 editing results in HepG2 and SkHep cells, which were treated with 50 pmol of RNP construct Y53aASGPRL-RNP-EMX1 or Y1C80S-3N-m-RNP-EMX1 and in the presence of (co-incubation) increasing concentrations of ppTG21 endosomolytic peptide (62.5, 250, 1000 and 2000 nM). RNP only treatment and lipofection treatment of the RNP constructs were included as controls.

[0057] FIG. 5 represents PCSK9 exons 4 and 5 editing results in HepG2 and SkHep cells, which were treated with 50 pmol of RNP construct Y53aASGPRL-RNP-PCS4 and in the presence of (co-incubation) increasing concentrations of ppTG21 endosomolytic peptide (250 and 1000 nM).

[0058] FIG. 6 provides exemplary Cas9, Cas9 construct, RNP, RNP construct and Cpf1 sequences.

DETAILED DESCRIPTION

Definitions

[0059] The present invention may be understood even more readily by reference to the following detailed description of exemplary embodiments of the invention and the examples included therein.

[0060] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, chemistry, cell and tissue culture, molecular biology, cell and cancer biology, neurobiology, neurochemistry, virology, immunology, microbiology, pharmacology, genetics and protein and nucleic acid chemistry, described herein, are those well known and commonly used in the art.

[0061] The methods and techniques of the present invention are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout this specification. See, e.g. “Principles of Neural Science,” McGraw-Hill Medical, New York, N.Y. (2000); Motulsky, “Intuitive Biostatistics,” Oxford University Press, Inc. (1995); Lodish et al., “Molecular Cell Biology, 4th ed.,” W. H. Freeman & Co., New York (2000); Griffiths et al., “Introduction to Genetic Analysis, 7th ed.,” W. H. Freeman & Co., N.Y. (1999); and Gilbert et al., “Developmental Biology, 6th ed.,” Sinauer Associates, Inc., Sunderland, Mass. (2000).

[0062] Chemistry terms used herein are used according to conventional usage in the art, as exemplified by “The McGraw-Hill Dictionary of Chemical Terms,” Parker S., Ed., McGraw-Hill, San Francisco, Calif. (1985).

[0063] All of the publications, patents and published patent applications referred to in this application are specifically incorporated by reference herein. In case of conflict, the present specification, including its specific definitions, will control.

[0064] Throughout this specification, the word “comprise” or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated integer (or components) or group of integers (or components), but not the exclusion of any other integer (or components) or group of integers (or components).

[0065] The singular forms “a,” “an,” and “the” include the plurals unless the context clearly dictates otherwise.

[0066] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0067] Each embodiment of the invention described herein may be taken alone or in combination with one or more other embodiments of the invention.

[0068] The term “agent” is used herein to denote a chemical compound (such as an organic or inorganic compound (including, such as, a compound of the present invention), a mixture of chemical compounds), a biological macromolecule (such as a nucleic acid, an antibody, including parts thereof as well as humanized, chimeric and human antibodies and monoclonal antibodies, a protein or portion thereof, e.g., a peptide, a lipid, a carbohydrate), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. Agents include, for example, agents which are known with respect to structure, and those which are not known with respect to structure.

[0069] A “patient,” “subject,” or “individual” are used interchangeably and refer to either a human or a non-human animal. These terms include mammals, such as humans, primates, livestock animals (including bovine, porcine, etc.), companion animals (e.g., canine, feline, etc.) and rodents (e.g., mice and rats).

[0070] Before the present compounds, compositions and methods are disclosed and described, it is to be understood that this invention is not limited to specific synthetic methods of making that may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The plural and singular should be treated as interchangeable, other than the indication of number:

[0071] As used herein, the term “alkyl” refers to a hydrocarbon radical of the general formula C_nH_2n+1. The alkane radical may be straight or branched. For example, the term “(C₁-C₆)alkyl” refers to a monovalent, straight, or branched aliphatic group containing 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, 3,3-dimethylpropyl, hexyl, 2-methylpentyl, and the like). Similarly, the alkyl portion (i.e., alkyl moiety) of an alkoxy, acyl (e.g., alkanoyl), alkylamino, dialkylamino, alkylsulfonyl, and alkylthio group have the same definition as above. When indicated as being “optionally substituted”, the alkane radical or alkyl moiety may be unsubstituted or substituted with one or more substituents (generally, one to three substituents except in the case of halogen substituents such as perchloro or perfluoroalkyls) independently selected from the group of substituents listed below in the definition for “substituted.” “Halo-substituted alkyl” refers to an alkyl group substituted with one or more halogen atoms (e.g., fluoromethyl, difluoromethyl, trifluoromethyl, perfluoroethyl, 1,1-difluoroethyl and the like).

[0072] Similarly, “alkylene” refers to a divalent hydrocarbon radical of the general formula C_nH_2nwhich may be straight or branched.

[0073] The term “alkenyl” refers to a univalent unsaturated hydrocarbon radical having one or more carbon-carbon double bonds. The alkenyl moiety may be straight or branched. Exemplary alkenyl groups include ethylenyl. “Alkenylene” as used herein refers to a divalent unsaturated hydrocarbon radical having one or more carbon-carbon double bonds and which may be straight or branched.

[0074] The term “alkynyl” refers to a univalent unsaturated hydrocarbon radical having one or more carbon-carbon triple bonds. The alkynyl moiety may be straight or branched.

[0075] “Alkynylene” as used herein refers to a divalent unsaturated hydrocarbon radical having one or more carbon-carbon triple bonds which may be straight or branched.

[0076] The term “aryl” means a carbocyclic aromatic system containing one, two or three rings wherein such rings may be fused. If the rings are fused, one of the rings must be fully unsaturated and the fused ring(s) may be fully saturated, partially unsaturated or fully unsaturated. The term “fused” means that a second ring is present (i.e., attached or formed) by having two adjacent atoms in common (i.e., shared) with the first ring. The term “fused” is equivalent to the term “condensed”. The term “aryl” embraces aromatic radicals such as benzyl, phenyl, naphthyl, tetrahydronaphthyl, indanyl, biphenyl, benzo[b][1,4]oxazin-3(4H)-onyl, 2,3-dihydro-1H indenyl, and 1,2,3,4-tetrahydronaphthalenyl.

[0077] The term “cycloalkyl” refers to nonaromatic rings that are fully hydrogenated and may exist as a single ring, bicyclic ring or a spiro ring. Unless specified otherwise, the carbocyclic ring is generally a 3- to 20-membered ring. For example, cycloalkyl include groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, and the like.

[0078] The term “heteroaryl” means an aromatic carbocyclic system containing one, two, three or four heteroatoms selected independently from oxygen, nitrogen and sulfur and having one, two or three rings wherein such rings may be fused, wherein fused is defined above. The term “heteroaryl” includes but is not limited to furyl, thienyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, pyridinyl, pyridiazinyl, pyrimidinyl, pyrazinyl, pyridin-2(1H)-onyl, pyridazin-2(1H)-onyl, pyrimidin-2(1H)-onyl, pyrazin-2(1H)-onyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, 5,6,7,8-tetrahydroisoquinolinyl, 5,6,7,8-tetrahydroquinolinyl, 6,7-dihydro-5H-cyclopenta[b]pyridinyl, 6,7-dihydro-5H-cyclopenta[c]pyridinyl, 1,4,5,6-tetrahydrocyclopenta[c]pyrazolyl, 2,4,5,6-tetrahydrocyclopenta[c]pyrazolyl, 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazolyl, 6,7-dihydro-5H-pyrrolo[1,2-b][1,2,4]triazolyl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridinyl, 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridinyl, 4,5,6,7-tetrahydro-1H-indazolyl and 4,5,6,7-tetrahydro-2H-indazolyl.

[0079] The term “drug delivery system” refers to a means of delivering a therapeutically effective amount of a ligand and includes, but is not limited to polymers such as PEG (Poly(ethylene glycol) methyl ether), PEG-PLA (Poly(ethylene glycol) methyl ether-poly(D,L lactide)), PEG-PLGA (Poly(ethylene glycol) methyl ether-poly(lactide-co-glycolide)), and PEG-PCL (Poly(ethylene glycol)-poly(ε-caprolactone) methyl ether), Quantum Dots (Q dots), liposomes, immuno-liposomes, micelles, nanoparticles, and nanogels. Exemplary drug delivery systems are described in Tiwari, G., “Drug Delivery Systems: an Updated Review”, Int J Pharm Investig 2 (1) p. 2-11 (January 2012), which is incorporated herein by reference for all purposes.

[0080] The term “small molecule” means an organic compound having a molecular weight between 100 and 2,000 daltons, including but not limited to synthetic compounds and natural products.

[0081] An “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (such as Fab, Fab′, F(ab′)2, Fv), single chain (ScFv) and domain antibodies), and fusion proteins comprising an antibody portion, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or sub-class thereof), and the antibody need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0082] As used herein, “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256:495, or may be made by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature 348:552-554, for example.

[0083] A “variable region” of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. As known in the art, the variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) that contain hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al, 1997, J. Molec. Biol. 273:927-948). As used herein, a CDR may refer to CDRs defined by either approach or by a combination of both approaches.

[0084] As known in the art a “constant region” of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[0085] The phrase “therapeutically effective amount” means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

[0086] The term “animal” refers to humans (male or female), companion animals (e.g., dogs, cats and horses), food-source animals, zoo animals, marine animals, birds and other similar animal species. “Edible animals” refers to food-source animals such as cows, pigs, sheep and poultry.

[0087] The phrase “pharmaceutically acceptable” indicates that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients comprising a formulation, and/or the mammal being treated therewith.

[0088] The terms “treating”, “treat”, or “treatment” embrace both preventative, i.e., prophylactic, and palliative treatment.

[0089] As used herein, “pharmaceutically acceptable carrier” or “pharmaceutical acceptable excipient” includes any material which, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the subject's immune system.

[0090] Examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline (PBS) or normal (0.9%) saline. Compositions comprising such carriers are formulated by well known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990; and Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing, 2000).

[0091] The term “compounds of the present invention” (unless specifically identified otherwise) refer to compounds of any of the Formulas described herein, and all enantiomers, tautomers and isotopically labeled compounds thereof. Hydrates and solvates of the compounds of the present invention are considered compositions of the present invention, wherein the compound is in association with water or solvent, respectively. The compounds may also exist in one or more crystalline states, i.e. as co-crystals, polymorphs, or they may exist as amorphous solids. All such forms are encompassed by the claims.

[0092] The term “linker” is a chemical group that connects one or more other chemical groups via at least one covalent bond. The linker may include one or more spacing groups including, but not limited to alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl and the like. The linker may be charge neutral, charge positive or charge negative. In addition, the linker may be cleavable such that the linker's covalent bond that connects the linker to another chemical group within the linker or that bonds to the ligand may be broken or cleaved under certain conditions (see for example H. Bruyere, et al., “Tuning the pH Sensitivities of Orthoester based compounds for Drug Delivery Applications by Simple Chemical Modification”, Bioorganic and Medicinal Chemistry Letters, 20, 2200 (2010) and A. A. Kislukhin et al., “Degradable Conjugates from Oxanorbornadiene Reagents”, Journal of the American Chemical Society, 134, 6491 (2012). These conditions include pH, temperature, salt concentration, a catalyst, or an enzyme. (G. M. Dubowchik et al., “Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates: Model Studies of Enzymatic Drug Release and Antigen-Specific In Vitro Anticancer Activity”, Bioconjugate Chemistry, 13, 855 (2002), G. Leriche et al., “Cleavable Linkers in Chemical Biology”, Bioorganic and Medicinal Chemistry, 20, 571 (2012), C. P. R. Hackenberger et al., “Chemoselective Ligation and Modification Strategies for Peptides and Proteins”, Angewandte Chemie International Edition, 47, 10030 (2008); D. M. Patterson et al., “Finding the Right (Bioorthogonal) Chemistry”, ACS Chemical Biology, 9, 592 (2014); C. A. Blencowe et al., “Self-immolative Linkers in Polymeric Delivery Systems”, Polymer Chemistry, 2, 773 (2011). The disclosures of the above publications are incorporated herein by reference in their entireties for all purposes.

[0093] In some embodiments, the linker is cleavable under intracellular conditions, such that the cleavage of the linker releases the ligand unit from the compound of Formulae A and B in the intracellular environment. In some embodiments, the linker is cleavable by a cleaving agent that is present in the intracellular environment (e.g. within a lysosome or endosome or caveolus). One example of a cleavable linker is an enzymatically cleaved linker i.e., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to, a lysosomal or endosomal protease. In some embodiments, the peptidyl linker is at least two amino acids long or at least three amino acids long. Enzymatic cleaving agents include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drug inside the target cells (see Dubowchik, Gene M. et al., Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin, Bioconjugate Chem. 2002, 13, 855-869). Such linkers include peptides and dipeptides including those described in the above publications which are incorporated herein by reference in their entireties for all purposes.

[0094] Other cleavable linkers may be cleaved by nucleophilic/basic reagents, reducing reagents, photo-irradiation, and electrophilic/acidic reagents. See, e.g., Leriche, Geoffray, et al., Cleavable Linkers in Chemical Biology, Bioorganic & Medicinal Chemistry 20 (2012) 571-582.

[0095] In yet another embodiment, the linker unit is not cleavable and the drug is released by the compound of Formulae A and B by degradation. This process is often referred to as self-immolative elimination, which works by cyclization or electronic cascade reactions driven by entropy and thermodynamics. One example of a noncleavable linker is a polysubstituted, electron-rich aromatic species with an amino or hydroxyl group or other electron-donating group that is conjugated to a leaving group at a benzylic position (see Blencowe, Christopher A. et al., Self-immolative Linkers in Polymeric Delivery Systems, Polymer Chemistry, 2011, 2, 773-790). Self-immolative elimination linkers include, but are not limited to, aniline based linkers, N-hydroxyaniline based linkers, phenol based linkers, 1,8 elimination based linkers, cyclization based linkers (i.e., hydroxyl based linkers, amino based linkers and thiol based linkers), polymer-dendron conjugates, and polymer conjugates (i.e., N-(2-hydroxypropyl)-methacrylamide (HPMA) polymer conjugates, poly-ethylene glycol (PEG) polymer conjugates) (see I. Tranoy-Opalinski, et al., Design of Self-Immolative Linkers for Tumour-Activated Prodrug Therapy, Anti-Cancer Agents in Medicinal Chemistry, 2008, 8, 618-637; Blencowe, Christopher A. et al., Self-immolative Linkers in Polymeric Delivery Systems, Polymer Chemistry, 2011, 2, 773-790).

[0096] Typically, the linker is not substantially cleaved in the extracellular environment. As used herein, “not substantially cleaved in the extracellular environment” in the context of a linker means that no more than 20%, typically no more than about 15%, more typically no more than about 10%, and even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers in a sample of compound of Formulae A and B which includes the X—Y group, are cleaved when the compound is present in an extracellular environment (e.g., plasma). Whether a linker is not substantially cleaved in the extracellular environment can be determined for example by incubating the compound with plasma for a predetermined time period up to 24 hours (e.g. 2, 4, 8, 16 or 24 hours) and then quantitating the amount of free ligand present in the plasma.

[0097] The linker may be a monovalent, bivalent or trivalent branched linker. In one embodiment, the linker is a disulfide bridge. In another embodiment, the linker is any of structures L1-L10, which show the linkage to Y and Z (wherein Y and Z represent groups as presented in the summary):

wherein each T is independently absent or is alkylene, alkenylene, or alkynylene, wherein one or more —CH₂— groups of the alkylene, alkenylene, or alkynylene may each independently be replaced with a heteroatom group independently selected from —O—, —S—, and —N(R⁴)— wherein the heteroatom groups are separated by at least 2 carbon atoms, wherein said alkylene, alkenylene, and alkynylene may each be independently substituted with one or more halo atoms;
each Q is independently absent or is C(O), C(O)—NR⁴, NR⁴—C(O), O—C(O)—NR⁴, NR⁴—C(O)—O, —CH₂—, a heteroaryl, or a heteroatom group selected from O, S, S—S, S(O), S(O)₂, and NR⁴, wherein at least two carbon atoms separate the heteroatom groups O, S, S—S, S(O), S(O)₂and NR⁴from any other heteroatom group;
each R⁴is independently —H, —(C₁-C₂₀)alkyl, or (C₃-C₆)cycloalkyl wherein one to six —CH₂— groups of the alkyl or cycloalkyl separated by at least two carbon atoms may be replaced with —O—, —S—, or —N(R⁴)—, and —CH₃of the alkyl may be replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms; and wherein the alkyl and cycloalkyl may be substituted with halo atoms; and each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. Each T and each Q of each (T-Q-T-Q) is independently selected.

[0098] In one embodiment, Q is a heteroaryl selected from 1H-1,2,3-triazolyl, pyridinyl, and 1,2,3,4-tetrazolyl.

[0099] The linker length could be adjusted by the value of n to optimize accessibility to the target molecule. In some cases, the optimal length of the linker could be designed by analyzing the drug-target interaction site or the space needed to adequately cleave the compound of Formulas (A) and (B).

[0100] By “genetically derived material” is meant to include proteins, endonucleases, and CRISPR/Cas from class I, II, or III (e.g., a Cas9 protein), plasmids (e.g., a plasmid that encodes the Cas9 protein or a cas9 protein and a guide sequence), RNA sequences such as mRNA, siRNA sequences and Cas9 ribonucleoproteins.

[0101] A “site-directed modifying polypeptide” as used herein refers to a CRISPR/Cas endonuclease or a derivative thereof (from class I, II, or III). In some embodiments, the site-directed modifying polypeptide is a Class 2 CRISPR/Cas endonuclease or a derivative thereof. In some embodiments, the site-directed modifying polypeptide is a type II CRISPR/Cas endonuclease, such as Cas9, or a derivative thereof. In some embodiments, the site-directed modifying polypeptide is a type V CRISPR/Cas endonuclease, such as Cpf1, or a derivative thereof. In some embodiments, the site-directed modifying polypeptide is a type III CRISPR/Cas endonuclease. In some embodiments, the site-directed modifying polypeptide is a Type III-B Cmr complex, e.g., a Type III-B Cmr complex derived from Pyrococcus furiosus, Sulfolobus solfataricus, or Therms thermophilus. See, e.g., Hale, C. R. et al. Genes & Development, 2014, 28:2432-2443, and Makarova K. S. et al. Nature Reviews Microbiology, 2015, 13, 1-15.

[0102] A “derivative” refers to any polypeptide having a substantially identical amino acid sequence to the naturally occurring polypeptide, in which one or more amino acids have been modified at side groups of the amino acids. The term “derivative” shall also include any polypeptide which has one or more amino acids deleted from, added to, or substituted from the natural polypeptide sequence, but which retains a substantial amino acid sequence homology to the natural sequence. A substantial sequence homology is any homology greater than 50 percent. “Derivative” shall also include fusion Cas9-fluorescent polypeptide fusion proteins such as Cas9/mcherry, Cas9/transient domains, Cas9/endosomal escape agent and Cas9/NLS. “Derivative” shall also include fusion Cpf1-fluorescent polypeptide fusion proteins such as Cpf1/mcherry, Cpf1/transient domains, Cpf1/endosomal escape agent and Cpf1/NLS. Moreover, “derivative” shall also include mutations such as glycosylation site mutations on the polypeptide.

[0103] A “ribonucleoprotein” or “RNP” or “RNP construct” refers to an association that combines ribonucleic acid (RNA) and protein. In some embodiments, a ribonucleoprotein comprises a site-directed modifying polypeptide as described herein. In some embodiments, the ribonucleoprotein comprises a Cas9 protein. In some embodiments, the ribonucleoprotein comprises a Cpf1 protein.

[0104] A “Cas9 ribonucleoprotein” or “Cas9 RNP” or “Cas9 RNP construct” may comprise two linked or associated elements: (1) a first element comprising a recognition element comprising either a dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or a single guide RNA sequence (sgRNA), wherein when expressed, the guide sequence directs sequence-specific binding of the Cas9 ribonucleoprotein to a target sequence, and the first element optionally comprises one or more endosomal escape agents and (2) a second element comprising a Cas9 construct wherein the Cas9 construct comprises a Cas9 protein and optionally one or more nuclear localization sequences (NLSs) and optionally one or more fluorescent proteins, and one or more endosomal escape agents; wherein said first element is associated with said second element. Wherein when the present compounds of formula A-1, A-2, A-3 or B are present, said compounds are conjugated to either the first or second element of the RNP construct.

[0105] A “Cpf1 ribonucleoprotein” comprising: (1) a first element comprising a recognition element comprising a guide sequence, wherein when expressed, the guide sequence directs sequence-specific binding of the Cpf1 ribonucleoprotein to a target sequence, and the first element optionally comprises one or more endosomal escape agents, and (2) a second element comprising a Cpf1 construct wherein the Cpf1 construct comprises a Cpf1 protein and optionally one or more nuclear localization sequences (NLSs) and optionally one or more fluorescent proteins, and one or more endosomal escape agents; wherein said first element is associated with said second element. Wherein when the present compounds of formula A-1, A-2, A-3 or B are present, said compounds are conjugated to either the first or second element of the RNP construct.

[0106] In some embodiments of a ribonucleoprotein, the guide sequence directs sequence-specific binding of the ribonucleoprotein to a target sequence in a cell. The cell may be eukaryotic or prokaryotic. In some embodiments, the second element comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NLSs at or near the amino-terminus, the carboxy-terminus, or a combination of these (e.g. one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies.

[0107] A “guide RNA sequence” is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a ribonucleoprotein described herein to the target sequence. In some embodiments, a “guide RNA sequence” is any polynucleotide sequence having (i) a portion with sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and (ii) a portion that binds to and directs a site-directed modifying protein (e.g., Cas9, cpf1, etc., as described herein) to the target sequence. See, e.g., Jinek et al., Science 2012; Briner et al., Mol. Cell 2014. Exemplary Cas9 proteins, Cas9 guide RNA sequences, plasmids, and ribonucleoproteins are described in US20140068797, published on Mar. 6, 2014; US2015031134, published on Jan. 29, 2015; and US2015079681, published on Mar. 19, 2015, all of which are incorporated herein in their entireties for all purposes.

[0108] In some embodiments, the guide sequence comprises at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 nucleotides where the degree of complementarity between the guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0109] Cas9 protein may be derived from S. aureus, S. pneumoniae, S. pyogenes, S. thermophilus, N. meningitidis or A. ebreus. In some embodiments, the Cas9 protein exhibits conserved architecture having an HNH homing endonuclease domain and a split RuvC/RNaseH endonuclease domain whereby each Cas9 protein shares 4 primary motifs: motifs 1, 2, and 4, which are RuvC like motifs and motif 3, which is an HNH motif. For Streptococcus pyogenes (SEQ ID No:8), motifs 1 is SEQ ID NO:260, motif 2 is SEQ ID NO:261, motif 3 is SEQ ID NO:262, and motif 4 is SEQ ID NO:263. Therefore, by “Cas9 protein sequence” or “Cas9 protein” is meant a polypeptide which comprises an amino acid sequence having at least 4 motifs within the sequence which have at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent amino acid sequence identity to the motifs 1, 2, 3, and 4 of the Cas9 amino acid sequence of any of SEQ ID NOs:260-263, or to the corresponding portions in any of the amino acid sequences set forth in SEQ ID NOs:1-829. In another embodiment, the Cas9 amino acid sequence is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent amino acid sequence identity to the amino acids at positions 7 to 166 or 731 to 1003 of SEQ ID NO:8 or the corresponding amino acids of those set forth in SEQ ID NOs:1-7, 9-829.

[0110] In some embodiments, the Cas9 protein is selected from: S. pyogenes Cas9 (wild type) (SEQ ID NO:848), S. pyogenes Cas9-mutation M1C (SEQ ID NO:849), S. pyogenes Cas9-mutation M1C & C80S (SEQ ID NOs:850), S. pyogenes Cas9 nickase-mutation D10A (SEQ ID NO:851), S. pyogenes Cas9 nickase-mutation H840A (SEQ ID NO:852), S. pyogenes Cas9 nickase-mutations E923P & T924P (SEQ ID NO:853), Acidovorax ebreus Cas9 (SEQ ID NO:854), Acid mine drainage bacteria Ga0052161_JGI Cas9 (SEQ ID NO:855), S. pyogenes Cas9 null-mutation D10A& H840A (SEQ ID NO:1027), and Uranium mine bacteria FW106_JGI Cas9 (SEQ ID NO:856).

[0111] Cpf1 protein may be derived from A. sp., L. bacterium, P. macacae, and P. disiens. In some embodiments, the Cpf1 protein is selected from, but is not limited to, A. sp Cpf1 (SEQ ID NO:857), L. bacterium Cpf1 (SEQ ID NO:858), P. macacae Cpf1 (SEQ ID NO:859), P. disiens Cpf1 (SEQ ID NO:860). In some embodiments, A Cpf1 protein can comprise an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to any one of SEQ ID NOs:857-860.

[0112] “Nuclear localization sequence” (NLS) refers to an amino acid sequence which assists the ribonucleoprotein of the present invention, e.g., a Cas-9 ribonucleoprotein to enter the nucleus of a eukaryotic cell. Consequently, an NLS typically comprises one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Exemplary NLSs include, but are not limited to, an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 830); the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 831); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 832) or RQRRNELKRSP (SEQ ID NO: 833); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 834); the sequence RMRIXFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (wherein X is any amino acid) (SEQ ID NO: 835) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 836) and PPKKARED (SEQ ID NO: 837) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 838) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 839) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 840) and PKQKKRK (SEQ ID NO: 841) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 842) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 843) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 844) of the human poly(ADP-ribose) polymerase; the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 845) of the steroid hormone receptors (human) glucocorticoid; the sequence MAPKKKRKVGIHRGVP (SEQ ID NO:1035); and the sequence PKKKRKVEDPKKKRKVD (SEQ ID NO:1036).

[0113] “Cas9 construct Y1C80S-3N-m” or “Y1C80S-3N-m” as used herein refers to S. pyogenes Cas9-mutation M1C & C80S (amino acid sequence)—3NLS and mCherry (SEQ ID NO:1015).

[0114] “Cas9 construct Y1C80S-2N” or “Y1C80S-2N” as used herein refers to S. pyogenes Cas9-mutation M1C & C80S (amino acid sequence)—2NLS (SEQ ID NO:1013).

[0115] “Cas9 construct Y53aASGPRL” or “Y53aASGPRL” as used herein refers to S. pyogenes Cas9-mutation M1C & C80S (amino acid sequence)—3NLS and mCherry (SEQ ID NO:1015) labeled with two copies of fragment 53a (using compound 53 as reactant, see experimental part for further details), at cysteines position 1 and 574 (addition of 2×2165 Da) via formation of disulfide bond with S atom of cysteine:

[0116] “RNP construct Y53aASGPRL-RNP-EMX1” or “Y53aASGPRL-RNP-EMX1” as used herein refers to a RNP construct prepared by co-incubation of (1) Cas9 construct Y53aASGPRL, and (2) EMX1 single guide sgRNA sequence (SEQ ID NO: 907), at a molar ratio of 1:1.2.

[0117] “RNP construct Y1C80S-3N-m-RNP-EMX1” or “Y1C80S-3N-m-RNP-EMX1” as used herein refers to a RNP construct prepared by co-incubation of (1) Cas9 construct Y1C80S-3N-m, and (2) EMX1 single guide sgRNA sequence (SEQ ID NO: 907), at a molar ratio of 1:1.2.

[0118] “RNP construct Y53aASGPRL-RNP-PCS4” or “Y53aASGPRL-RNP-PCS4” as used herein refers to a RNP construct prepared by co-incubation of (1) Cas9 construct Y53aASGPRL, and (2) sgRNA targeting the PCSK9 exons 4&5 locus (SEQ ID NO:906), at a molar ratio of 1:1.2.

[0119] “RNP construct Y53aASGPRL-RNP-PCS1” or “Y53aASGPRL-RNP-PCS1” as used herein refers to a RNP construct prepared by co-incubation of (1) Cas9 construct Y53aASGPRL, and (2) sgRNA targeting the PCSK9 gene (SEQ ID NO:896), at a molar ratio of 1:1.2.

[0120] An “endosomal escape agent” as used herein refers to an agent that facilitates an agent, such as compounds, DNA, siRNA, polypeptides, or ribonucleoproteins, to escape the cell endosome. Endosomal escape agents for use in the present invention include, but are not limited to, a lysosomotropic agent, a cell penetrating peptide, a fusogenic peptide, an endosomolytic peptide, a pore forming agent, and a proton sponge agent. Lysosomotropic agents are weak bases that can penetrate in lysosome as protonated form and increase the intracellular pH. Cell penetrating peptides (CPPs) are a class of diverse peptides, typically with 5-30 amino acids, that can cross the cellular membrane. Fusogenic peptides are short peptides that destabilize the phospholipid membrane. Pore-forming agents are agents, such as peptides, that induce pore formation through the membrane thereby disrupting endosome. Proton sponge agents are agents having multiple proton acceptor sites that disrupt the endosome by osmolytic action. An endosomolytic peptide may be a polyanionic peptide or peptidomimetic, or a peptide having a neutral or near neutral charge at physiological pH which shows pH-dependent membrane lytic activity and promotes endosomal lysis or leakage. In certain embodiments, the endosomolytic peptide assumes its active conformation at endosomal pH. The “active” conformation is that conformation in which the endosomolytic component promotes lysis of the endosome and/or transport of the modular composition of the invention, or its components, from the endosome to the cytoplasm of the cell.

[0121] In some embodiments, the endosomal escape agent is associated with the RNP construct. In other embodiments, the endosomal escape agent is conjugated to the first or second element of the RNP construct. In further embodiments, the endosomal escape agent is conjugated to the compound of formula A-1, A-2, A-3 or B.

Compounds

[0122] One aspect of the present invention includes compounds of Formula (A-1), (A-2), or (A-3):

or a pharmaceutically acceptable salt thereof, as described in the summary.

[0123] In some embodiments of a compound of Formula (A-1), (A-2), or (A-3), the site-directed modifying polypeptide is a Cas9 protein or a Cpf1 protein. In some embodiments of a compound of Formula (A-1), (A-2), or (A-3), Y is a Cas9 ribonucleoprotein, a Cas9 protein, a single guide RNA sequence (sgRNA) or a dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA); Y⁺ is a positively charged Cas9 protein; and Y⁻ is a negatively charged Cas9 ribonucleoprotein, a negatively charged sgRNA, or a negatively charged dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA).

[0124] Another aspect of the present invention provides a compound of Formula (B):

or a pharmaceutically acceptable salt thereof, as described in the summary.

[0125] In some embodiments of a compound of Formula (B), the site-directed modifying polypeptide is a Cas9 protein or a Cpf1 protein. In some embodiments of a compound of Formula (B), Y is a Cas9 ribonucleoprotein, a Cas9 protein, a single guide RNA sequence (sgRNA) or a dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA); Y⁺ is a positively charged Cas9 protein; and Y⁻ is a negatively charged Cas9 ribonucleoprotein, a negatively charged sgRNA, or a negatively charged dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA).

[0126] In some embodiments of a compound of Formula (A-1), (A-2), (A-3) or (B), R²is —NH—C(O)—CH₃.

[0127] In some embodiments of a compound of Formula (A-1), (A-2), (A-3) or (B), R¹is —Z—X⁺Y⁻. In some embodiments, R¹is —Z—X⁻Y⁺. In some embodiments, R¹is —Z—X—Y. In some embodiments, R¹is —Z—X—Y that is selected from the group consisting of L1-L10:

wherein each T is independently absent or is (C₁-C₁₀) alkylene, (C₂-C₁₀) alkenylene, or (C₂-C₁₀) alkynylene, wherein one or more carbon groups of said T may each independently be replaced with a heteroatom group independently selected from —O—, —S—, and —N(R⁴)— wherein the heteroatom groups are separated by at least 2 carbon atoms, wherein said alkylene, alkenylene, and alkynylene may each be independently substituted with one or more halo atoms;
each Q is independently absent or is C(O), C(O)—NR⁴, NR⁴—C(O), O—C(O)—NR⁴, NR⁴—C(O)—O, —CH₂—, a heteroaryl, or a heteroatom group selected from O, S, S—S, S(O), S(O)₂, and NR⁴, wherein at least two carbon atoms separate the heteroatom groups O, S, S—S, S(O), S(O)₂and NR⁴from any other heteroatom group;
each R⁴is independently —H, —(C₁-C₂₀)alkyl, —(C₁-C₂₀)alkenyl, —(C₁-C₂₀)alkynyl, or (C₃-C₆)cycloalkyl wherein one to six —CH₂— groups of the alkyl or cycloalkyl separated by at least two carbon atoms may be replaced with —O—, —S—, or —N(R⁴)—, and —CH₃of the alkyl may be replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms; and wherein the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with halo atoms; and
each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40. In some embodiments, Q is independently a heteroaryl selected from 1H-1,2,3-triazolyl, pyridinyl, and 1,2,3,4-tetrazolyl.

[0128] In some embodiments of a compound of Formula (A-1), (A-2), (A-3) or (B), X, X⁺, or X⁻ comprises a disulfide bond.

[0129] Another aspect of the present invention provides a compound of Formula (C-1), (C-2), (C-3) or (C4):

or a pharmaceutical acceptable salt thereof,
wherein:
n is 1, 2, or 3;
W is absent or a peptide;
L is -(T-Q-T-Q)_m-, wherein:
each T is independently absent or is (C₁-C₁₀) alkylene, (C₂-C₁₀) alkenylene, or (C₂-C₁₀) alkynylene, wherein one or more carbon groups of said T may each independently be replaced with a heteroatom group independently selected from —O—, —S—, and —N(R⁴)— wherein the heteroatom groups are separated by at least 2 carbon atoms, wherein said alkylene, alkenylene, and alkynylene may each be independently substituted with one or more halo atoms;
each Q is independently absent or is C(O), C(O)—NR⁴, NR⁴—C(O), O—C(O)—NR⁴, NR⁴—C(O)—O, —CH₂—, a heteroaryl, or a heteroatom group selected from O, S, S—S, S(O), S(O)₂, and NR⁴, wherein at least two carbon atoms separate the heteroatom groups O, S, S—S, S(O), S(O)₂and NR⁴from any other heteroatom group;
each R⁴is independently —H, —(C₁-C₂₀)alkyl, —(C₁-C₂₀)alkenyl, —(C₁-C₂₀)alkynyl, or (C₃-C₆)cycloalkyl wherein one to six —CH₂— groups of the alkyl or cycloalkyl separated by at least two carbon atoms may be replaced with —O—, —S—, or —N(R⁴)—, and —CH₃of the alkyl may be replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms; and wherein the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with halo atoms;
each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; and
the other moieties are as defined in Formula (A-1), (A-2) or (A-3).

[0130] Another aspect of the present invention provides a compound of Formula (D-1) or (D-2):

or a pharmaceutically acceptable salt thereof,
wherein Y is as defined in Formula (A-1), (A-2) or (A-3).

[0131] Another aspect of the present invention provides a compound of Formula (E):

or a pharmaceutically acceptable salt thereof,
wherein:
n is 1, 2 or 3;
W is absent or is a peptide;
L is -(T-Q-T-Q)_m-, wherein:
each T is independently absent or is (C₁-C₁₀) alkylene, (C₂-C₁₀) alkenylene, or (C₂-C₁₀) alkynylene, wherein one or more carbon groups of said T may each independently be replaced with a heteroatom group independently selected from —O—, —S—, and —N(R⁴)— wherein the heteroatom groups are separated by at least 2 carbon atoms, wherein said alkylene, alkenylene, alkynylene, may each independently be substituted by one or more halo atoms;
each Q is independently absent or is C(O), C(O)—NR⁴, NR⁴—C(O), O—C(O)—NR⁴, NR⁴—C(O)—O, —CH₂—, a heteroaryl, or a heteroatom group selected from O, S, S—S, S(O), S(O)₂, and NR⁴, wherein at least two carbon atoms separate the heteroatom groups O, S, S—S, S(O), S(O)₂and NR⁴from any other heteroatom group;
each R⁴is independently —H, —(C₁-C₂₀)alkyl, —(C₁-C₂₀)alkenyl, —(C₁-C₂₀)alkynyl, or (C₃-C₆)cycloalkyl wherein one to six —CH₂— groups of the alkyl or cycloalkyl separated by at least two carbon atoms may be replaced with —O—, —S—, or —N(R⁴)—, and —CH₃of the alkyl may be replaced with a heteroatom group selected from —N(R⁴)₂, —OR⁴, and —S(R⁴) wherein the heteroatom groups are separated by at least 2 carbon atoms; and wherein the alkyl, alkenyl, alkynyl, and cycloalkyl may be substituted with halo atoms;
each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40; and
the other moieties are as defined in Formula (B).

[0132] Another aspect of the present invention provides a compound of Formula (F-1) or (F-2):

or a pharmaceutically acceptable salt thereof, and
Y is as defined in Formula (B).

[0133] In some embodiments of a compound of Formula (C-1), (C-2), (C-3), (C4), (D-1), (D-2), (E), (F-1), or (F-2), n is 1. In some embodiments, n is 2. In some embodiments, n is 3.

[0134] In some embodiments of a compound of Formula (E), the compound is:

or a pharmaceutically acceptable salt thereof.

[0135] In some embodiments of a compound of the present invention, W is absent. In some embodiments, W is a peptide that comprises an endosomolytic peptide or a nuclear localization peptide. In some embodiments, the endosomolytic peptide may be a polyanionic, peptidomimetic or a peptide having a neutral or near-neutral charge at physiological pH, which shows pH-dependent membrane lytic activity and leads to endosome lysis or leakage. In certain embodiments, the endosomolytic peptide assumes its active conformation at endosomal pH. The “active” conformation is that conformation in which the endosomolytic component promotes lysis of the endosome and/or transport of the modular composition of the invention, or its components, from the endosome to the cytoplasm of the cell. See, e.g., Martin M. E. et al. Peptide-guided gene delivery, the AAPS Journal, 2007, 9(1), article 3.

[0136] In some embodiments, W is a peptide that comprises an endosomolytic histidine-rich peptide selected from, but not limited to, CHK₆HC (SEQ ID NO: 861); H5WYG (SEQ ID NO: 1029), GLFHAIAHFIHGGWHGLIHGWYG (SEQ ID NO: 862); and derivatives thereof.

[0137] In some embodiments, W is a peptide that comprises a fusogenic peptide selected from, but not limited to, an influenza HA-2 peptide, ppTG21 (SEQ ID NO: 1012); Aurein 1.2, GLFDIIKKIAESF (SEQ ID NO: 1023); GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 863); melittin, GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 864); tat (48-60), GRKKRRQRRRPPQ (SEQ ID NO: 865); penetratin, RQIKIWFQNRRMKWKK (SEQ ID NO: 866); transportan, GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 867); GALA peptide, WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 868); KALA peptide, WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 869); JST-1, GLFEALLELLESLWELLLEA (SEQ ID NO: 870); ppTG1, GLFKALLKLLKSLWKLLLKA (SEQ ID NO: 871); ppTG20, GLFRALLRLLRSLWRLLLRA (SEQ ID NO: 872); and derivatives thereof.

[0138] In some embodiments, W is a peptide that comprises a proteasome peptide with Gly-Ala repeat, such as CWK₁₈(GA)₄(SEQ ID NO: 873).

[0139] In some embodiments, W is a peptide that comprises a monopartite NLS including, but not limited to, the SV40 T antigen, PKKKRKV (SEQ ID NO: 830); the SV40 Vp3, KKKRK (SEQ ID NO: 874); the Adenovirus Ela, KRPRP (SEQ ID NO: 875); the human c-myc, PAAKRVKLD (SEQ ID NO: 832), RQRRNELKRSP (SEQ ID NO: 833); and derivatives thereof. In some embodiments, the peptide comprises a bipartite NLS including, but not limited to, nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 831); Xenopus N1, VRKKRKTEEESPLKDKDAKKSKQE (SEQ ID NO: 876); mouse FGF3, RLRRDAGGRGGVYEHLGGAPRRRK (SEQ ID NO: 877); PARP, KRKGDEVDGVDECAKKSKK (SEQ ID NO: 878), and derivatives thereof. In some embodiments, the peptide comprises a nonclassical NLS such as M9 peptide, NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 834).

[0140] In some embodiments, W is a peptide that comprises a cellular targeting peptide selected from, but not limited to, RGD peptide, ICRRARGDNPDDRCT (SEQ ID NO: 1037); integrin binding peptide, PLAEIDGIELTY (SEQ ID NO: 1038); secretin, HSDGTFTSELSRLRDSARLQRLLQGLV (SEQ ID NO: 879); GE7 (from EGF), NPVVGYIGERPQYRDL (SEQ ID NO: 880); neurotensin, ELYENKPRRPYIL (SEQ ID NO: 881); LOX-1 binding peptide, LSIPPKA (SEQ ID NO: 882), FQTPPQL (SEQ ID NO: 883), LTPATAI (SEQ ID NO: 884); or derivatives thereof.

[0141] In some embodiments of a compound of the present invention, Y is a Cas9 ribonucleoprotein. In some embodiments, Y is a Cas9 ribonucleoprotein comprising: (1) a first element comprising a recognition element comprising either a dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or a single guide RNA sequence (sgRNA), wherein when expressed, the guide sequence directs sequence-specific binding of the Cas9 ribonucleoprotein to a target sequence, and (2) a second element comprising a Cas9 protein and optionally one or more nuclear localization sequences (NLSs); wherein said first element is associated with said second element. In some embodiments, the target sequence is a eukaryotic cell target sequence. In other embodiments, the target sequence is a prokaryotic cell target sequence.

[0142] In some embodiments, the tracrRNA, the crRNA or the sgRNA used herein each may be optionally independently chemically modified. Suitable chemical modifications include modifications to the backbone, modifications to the base, and modifications to the sugar. Exemplary chemical modifications include, but are not limited to, use of phosphorothioate linkages (e.g., replacing one non-bridging oxygen atom on the backbone phosphate between two ribonucleotides with a sulphur atom creates a phosphorothioate (PS) linkage); use of boranophosphate linkages (e.g., the introduction of a boron atom in place of one of the nonbridging oxygen atoms to create a boron-phosphorous linkage); use of locked nucleic acid (LNA) nucleotides which contain a methylene bridge between the 2′ and 4′ carbons of the ribose ring; chemical modifications at the 2′-position of the ribose sugar (e.g., use of RNA analogs include 2′-O-methyl RNA, 2′-O-methoxyethyl (2′-MOE) RNA, and 2′-fluoro RNA); modification at the 4′-thio position by introducing a sulphur atom in place of oxygen attached to the 4′ carbon of the ribose ring; introduction of ribo-difluorotoluyl (rF) nucleotides; and introduction of PNA or morpholino monomers.

[0143] In some embodiments, the first element of the Cas9 ribonucleoprotein comprises a dual guide RNA sequence comprising a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), wherein the remainder of the compound is linked to said Cas9 ribonucleoprotein via one or more interactions each independently to the tracrRNA or to the crRNA, where the tracrRNA or the crRNA may optionally be chemically modified. In some embodiments, the tracrRNA and the crRNA are assembled to form a dual-RNA guide. In some embodiments, the tracrRNA-crRNA dual-RNA guide has the degree of complementarity between the dual RNA-guide and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is at least 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0144] In some embodiments of a Cas9 ribonucleoprotein, the tracrRNA comprises a sequence that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent nucleotide sequence identity to the sequence of: CAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCUUUU (SEQ ID NO:1028), wherein said sequence optionally contains 2′-O-methyl or 2′-F modification at any one or more of the first 3 bases, wherein said tracrRNA may be optionally further modified. In some embodiments of a Cas9 ribonucleoprotein, the tracrRNA comprises a sequence that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent nucleotide sequence identity to the sequence of: CAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACC GAGUCGGUGCUUUU (SEQ ID NO:1028), wherein said sequence optionally contains 2′-O-methyl or 2′-F modifications at the first 3 bases, wherein said tracrRNA may be optionally further modified.

[0145] In some embodiments of a Cas9 ribonucleoprotein, the crRNA comprises a sequence that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent nucleotide sequence identity to a sequence selected from:

PCSK9 crRNA sequence 1:

(SEQ ID NO: 885)

GGUGCUAGCCUUGCGUUCCGGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modification at any one or more of the first 3 of

the last 4 bases (GCU);

PCSK9 crRNA sequence 2:

(SEQ ID NO: 886)

CGUGCUCGGGUGCUUCGGCCGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modification at any one or more of the first 3 of

the last 4 bases (GCU);

PCSK9 crRNA sequence 3:

(SEQ ID NO: 887)

GCCGUCCUCCUCGGAACGCAGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modification at any one or more of the first 3 of

the last 4 bases (GCU);

PCSK9 crRNA sequence 4:

(SEQ ID NO: 888)

GGACGAGGACGGCGACUACGGUUUUAGAGCUAUGCUG, optionally

containing 2′-O-methyl or 2′-F modification at any

one or more of the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 5:

(SEQ ID NO: 889)

ACCACCGGGAAAUCGAGGGCGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modification at any one or more of the first 3 of

the last 4 bases (GCU);

PCSK9 crRNA sequence 6:

(SEQ ID NO: 890)

CGACUUCGAGAAUGUGCCCGGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modification at any one or more of the first 3 of

the last 4 bases (GCU);

PCSK9 crRNA sequence 7:

(SEQ ID NO: 891)

GAGUGACCACCGGGAAAUCGGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modification at any one or more of the first 3 of

the last 4 bases (GCU);

PCSK9 crRNA sequence 8:

(SEQ ID NO: 892)

CUCGGGCACAUUCUCGAAGUGUUUUAGAGCUAUGCUG, optionally

containing 2′-O-methyl or 2′-F modification at any

one or more of the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 9:

(SEQ ID NO: 893)

GGAAGCCAGGAAGAAGGCCAGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modification at any one or more of the first 3 of

the last 4 bases (GCU);

PCSK9 crRNA sequence 10:

(SEQ ID NO: 894)

UCUUUGCCCAGAGCAUCCCGGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modification at any one or more of the first 3 of

the last 4 bases (GCU);

and

PCSK9 crRNA sequence 11:

(SEQ ID NO: 895)

CUAGGAGAUACACCUCCACCGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modification at any one or more of the first 3 of

the last 4 bases (GCU);

wherein said crRNA may optionally be further chemically modified.

[0146] In some embodiments of a Cas9 ribonucleoprotein, the crRNA comprises a sequence that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent nucleotide sequence identity to a sequence selected from:

PCSK9 crRNA sequence 1:

(SEQ ID NO: 885)

GGUGCUAGCCUUGCGUUCCGGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modifications at the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 2:

(SEQ ID NO: 886)

CGUGCUCGGGUGCUUCGGCCGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modifications at the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 3:

(SEQ ID NO: 887)

GCCGUCCUCCUCGGAACGCAGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modifications at the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 4:

(SEQ ID NO: 888)

GGACGAGGACGGCGACUACGGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modifications at the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 5:

(SEQ ID NO: 889)

ACCACCGGGAAAUCGAGGGCGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modifications at the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 6:

(SEQ ID NO: 890)

CGACUUCGAGAAUGUGCCCGGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modifications at the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 7:

(SEQ ID NO: 891)

GAGUGACCACCGGGAAAUCGGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-

modifications a the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 8:

(SEQ ID NO: 892)

CUCGGGCACAUUCUCGAAGUGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modifications at the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 9:

(SEQ ID NO: 893)

GGAAGCCAGGAAGAAGGCCAGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modifications at the first 3 of the last 4 bases

(GCU);

PCSK9 crRNA sequence 10:

(SEQ ID NO: 894)

UCUUUGCCCAGAGCAUCCCGGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modifications at the first 3 of the last 4 bases

(GCU);

and

PCSK9 crRNA sequence 11:

(SEQ ID NO: 895)

CUAGGAGAUACACCUCCACCGUUUUAGAGCUAUGCUG,

optionally containing 2′-O-methyl or 2′-F

modifications at the first 3 of the last 4 bases

(GCU);

wherein said crRNA may optionally be further chemically modified.

[0147] In some embodiments, the first element of the Cas9 ribonucleoprotein comprises a single guide RNA sequence (sgRNA), wherein the remainder of the compound is linked to said Cas9 ribonucleoprotein via one or more interactions to the sgRNA. In some embodiments, the sgRNA comprises 20 or more nucleotides. In some embodiments, the sgRNA comprises at least 8 nucleotides.

[0148] In some embodiments of a Cas9 ribonucleoprotein, the degree of complementarity between the sgRNA and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is at least 50%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0149] In some embodiments of a Cas9 ribonucleoprotein, said sgRNA has at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent nucleotide sequence identity to the sequence selected from the group consisting of:

PCSK9 single guide RNA sequence 1:

(SEQ ID NO: 896)

GGUGCUAGCCUUGCGUUCCGGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

PCSK9 single guide RNA sequence 2:

(SEQ ID NO: 897)

CGUGCUCGGGUGCUUCGGCCGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

PCSK9 single guide RNA sequence 3:

(SEQ ID NO: 898)

GCCGUCCUCCUCGGAACGCAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

PCSK9 guide RNA sequence 4:

(SEQ ID NO: 899)

GGACGAGGACGGCGACUACGGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

PCSK9 single guide RNA sequence 5:

(SEQ ID NO: 900)

ACCACCGGGAAAUCGAGGGCGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

PCSK9 single guide RNA sequence 6:

(SEQ ID NO: 901)

CGACUUCGAGAAUGUGCCCGGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

PCSK9 single guide RNA sequence 7:

(SEQ ID NO: 902)

GAGUGACCACCGGGAAAUCGGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

PCSK9 single guide RNA sequence 8:

(SEQ ID NO: 903)

CUCGGGCACAUUCUCGAAGUGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

PCSK9 single guide RNA sequence 9:

(SEQ ID NO: 904)

GGAAGCCAGGAAGAAGGCCAGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

PCSK9 single guide RNA sequence 10:

(SEQ ID NO: 905)

UCUUUGCCCAGAGCAUCCCGGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

PCSK9 single guide RNA sequence 11:

(SEQ ID NO: 906)

CUAGGAGAUACACCUCCACCGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

EMX1 single guide RNA sequence:

(SEQ ID NO: 907)

GUCACCUCCAAUGACUAGGGGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

ROSA26 single guide RNA sequence:

(SEQ ID NO: 908)

CGAACCCUACACAUUCAACGGUUUAAGAGCUAUGCUGGAAACAGCAUAGC

AAGUUUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUUUUU;

wherein said sequence is optionally chemically modified.

[0150] In some embodiments of a compound of the present invention, Y is a Cpf1 ribonucleoprotein. In some embodiments, Y is a Cpf1 ribonucleoprotein comprising: (1) a first element comprising a recognition element comprising a guide sequence, wherein when expressed, the guide sequence directs sequence-specific binding of the Cpf1 ribonucleoprotein to a target sequence, and (2) a second element comprising a Cpf1 protein and optionally one or more nuclear localization sequences (NLSs); wherein said first element is associated with said second element. In some embodiments, the target sequence is a eukaryotic cell target sequence. In some embodiments, the target sequence is a prokaryotic cell target sequence.

[0151] In some embodiments, a Cpf1 ribonucleoprotein comprises a crRNA, said crRNA comprising a sequence that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent nucleotide sequence identity to a sequence selected from:

	EMX1 crRNA sequence (Asp Cpf1):
(SEQ ID NO: 909)
	UAAUUUCUACUCUUGUAGAUUCAUCUGUGCCCCUCCCUCCCUG;

	EMX1 crRNA sequence (Lba Cpf1):
(SEQ ID NO: 910)
	UAAUUUCUACUCUAAGUAGAUUCAUCUGUGCCCCUCCCUCCCUG;

	PCSK9 crRNA sequence 12 (Asp Cpf1):
(SEQ ID NO: 911)
	UAAUUUCUACUCUUGUAGAUCCCAGAGCAUCCCGUGGAACCUG;

	PCSK9 crRNA sequence 12 (Lba Cpf1):
(SEQ ID NO: 912)
	UAAUUUCUACUCUAAGUAGAUCCCAGAGCAUCCCGUGGAACCUG;

	PCSK9 crRNA sequence 13 (Asp Cpf1):
(SEQ ID NO: 913)
	UAAUUUCUACUCUUGUAGAUCCGGUGGUCACUCUGUAUGCUGG;
	and

	PCSK9 crRNA sequence 13 (Lba Cpf1):
(SEQ ID NO: 914)
	UAAUUUCUACUCUAAGUAGAUCCGGUGGUCACUCUGUAUGCUGG,

wherein said sequence is optionally chemically modified.

[0152] In some embodiments of a compound described herein, Y is a Type III-B Cmr complex, e.g., a Type III-B Cmr complex derived from Pyrococcus furiosus, Sulfolobus solfataricus, and Therms thermophilus. In some embodiments, the Cmr proteins suitable for use herein include but are not limited to those described in Hale, C. R. et al. Genes & Development, 2014, 28:2432-2443, and Makarova K. S. et al. Nature Reviews Microbiology, 2015, 13, 1-15.

[0153] In some embodiments of a compound described herein, Y further comprises a fluorescent probe. In some embodiments, the fluorescent probe comprises the mCherry sequence (SEQ ID NO:915). Other suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilised EGFP (dEGFP), destabilised ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, mPlum (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, is suitable for use.

[0154] In some embodiments, Y comprises one or more NLSs. In some embodiments, Y comprises one or more NLSs, each being independently selected from, but are not limited to, an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 830); the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 831); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 832) or RQRRNELKRSP (SEQ ID NO: 833); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 834); the sequence RMRIXFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV, wherein X is any amino acid (SEQ ID NO: 835) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 836) and PPKKARED (SEQ ID NO: 837) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 838) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 839) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 840) and PKQKKRK (SEQ ID NO: 841) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 842) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 843) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 844) of the human poly(ADP-ribose) polymerase; the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 845) of the steroid hormone receptors (human) glucocorticoid; the sequence MAPKKKRKVGIHRGVP (SEQ ID NO:1035); and the sequence PKKKRKVEDPKKKRKVD (SEQ ID NO:1036). In the embodiments where Y comprises two or more NLSs, there may optionally be an intervening amino acid sequence between each two NLSs. Such intervening sequences may comprise one or more amino acid residues. In some embodiments, Y comprises two NLSs, each comprising the amino acid sequence PKKKRKV (SEQ ID NO: 830). Y comprises three NLSs, each comprising the amino acid sequence PKKKRKV (SEQ ID NO: 830).

[0155] In some embodiments of a compound of the present invention, Y comprises a Cas9 protein that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent amino acid sequence identity to a Cas9 protein derived from S. aureus, S. pneumoniae, S. pyogenes, S. thermophilus, S. Aureus, N. meningitidis or A. ebreus. See, e.g., Hou et al, PNAS 2013. In some embodiments, Y comprises a Cas9 protein that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent amino acid sequence identity to a Type II Cas9 protein. In some embodiments, Y comprises a Cas9 protein that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent amino acid sequence identity to the amino acids at positions 7 to 166 or 731 to 1003 of SEQ ID NO:8 or the corresponding amino acids of those set forth in SEQ ID NOs:1-7, 9-829. In some embodiments, Y comprises a Cas9 protein that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent amino acid sequence identity to the amino acids at positions 7 to 166 or 731 to 1003 of SEQ ID NO:8. In some embodiments, Y comprises a Cas9 protein having at least 4 motifs within the sequence which have at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent amino acid sequence identity to the motifs 1, 2, 3, and 4 of the Cas9 amino acid sequence of any of SEQ ID NOs: 260-263.

[0156] In some embodiments, Y comprises a Type II-C Cas9 protein. In some embodiments, Y comprises a Cas9 protein that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent amino acid sequence identity to a sequence selected from: S. pyogenes Cas9 (wild type) (SEQ ID NO:848), S. pyogenes Cas9-mutation M1C (SEQ ID NO:849), S. pyogenes Cas9-mutation M1C & C80S (SEQ ID NO:850), S. pyogenes Cas9 nickase-mutation D10A (SEQ ID NO:851), S. pyogenes Cas9 nickase-mutation H840A (SEQ ID NO:852), S. pyogenes Cas9 nickase-mutations E923P & T924P (SEQ ID NO:853), Acidovorax ebreus Cas9 (SEQ ID NO:854), Acid mine drainage bacteria Ga0052161_JGI Cas9 (SEQ ID NO:855), S. pyogenes Cas9 null-mutation D10A& H840A (SEQ ID NO:1027), and Uranium mine bacteria FW106_JGI Cas9 (SEQ ID NO:856).

[0157] In some embodiments of a Cas9 protein suitable for use herein, the Cas9 protein sequence may be modified such as being codon-optimized for expression in a eukaryotic cell or to include modifications to the sequence to impact its function. In some embodiments, the Cas9 protein sequence directs cleavage of one or two strands of DNA at the location of the target sequence such as with a Cas9 nickase (i.e., Cas9-D10A) used in combination with guide sequence(s), e.g., two guide sequences, which target respectively sense and antisense strands of the DNA target thereby allowing both strands to be nicked and resulting in non-homologous end-joining. Cas9-D10A with a single guide RNA sequence can create indels. However, in other embodiments, the Cas9 protein sequence lacks DNA strand cleavage activity such as with selective use of catalytically inactive Cas (dCas) domains. In other embodiments, the Cas9 protein sequence is modified to allow covalent linkage to the Cas9 protein or Cas9 ribonucleoprotein, including lysine, glutamine, and cysteine residue modifications. In yet other embodiments, the Cas9 ribonucleoprotein is capable of directing the cleavage of RNA strands as described in O'Connell, Mitchell R., et al., Programmable RNA recognition and cleavage by CRISPR/Cas9, Nature, 2014, 516, p 263-266.

[0158] In another embodiment the Cas9 protein has mutated residues, such as solvent exposed lysines or arginines residues (not involved in either catalytic activity, binding of DNA, or binding of RNA) mutated to hystidines, Cysteins, or glutamines.

[0159] Modifications of the Cas9 protein sequence can include a D10A (aspartate to alanine at amino acid position 10 of SEQ ID NO: 8) mutation (or the corresponding mutation of any of the proteins set forth as SEQ ID NOs: 1-829) that can cleave the complementary strand of the target DNA but has reduced ability to cleave the non-complementary strand of the target DNA (thus resulting in a single strand break instead of a double strand break). Another modification is a H840A (histidine to alanine at amino acid position 840 of SEQ ID NO: 8) mutation (or the corresponding mutation of any of the proteins set forth as SEQ ID NOs:1-829) that can cleave the non-complementary strand of the target DNA but has reduced ability to cleave the complementary strand of the target DNA (thus resulting in a single strand break instead of a double strand break). The use of the D10A or H840A variant of Cas9 (or the corresponding mutations in any of the proteins set forth as SEQ ID NOs: 1-829) can alter the expected biological outcome because the non-homologous end joining is much more likely to occur when double strand breaks are present as opposed to single strand breaks.

[0160] Other residues can be mutated to also inactivate a particular nuclease from motif 1, 2, 3, or 4. As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and/or A987 (or the corresponding mutations of any of the proteins set forth as SEQ ID NOs: 1-829) can be modified. Mutations can include substitutions, additions, and deletions, or any combination thereof. In some instances, the mutation converts the mutated amino acid to another amino acid, such as alanine. Other modifications include a base modification, a backbone modification, and/or an internucleoside linkage modification.

[0161] In some embodiments of a compound of the present invention, Y is Y1C80S-3N-m or Y1C80S-2N. In some embodiments, Y is Y1C80S-3N-m-RNP-EMX1. In some embodiments, the compound is Y53aASGPRL. In some embodiments, the compound is Y53aASGPRL-RNP-EMX1, Y53aASGPRL-RNP-PCS4 or Y53aASGPRL-RNP-PCS1.

[0162] In some embodiments of a compound of the present invention, Y comprises a Cpf1 protein that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent amino acid sequence identity to a Cpf1 protein derived from A. sp., L. bacterium, P. macacae, and P. disiens. In some embodiments, Y comprises a Cpf1 protein that is at least about 75 percent, at least about 80 percent, at least about 85 percent, at least about 90 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, at least about 99 percent or 100 percent amino acid sequence identity to a Cpf1 protein selected from, but is not limited to, A. sp Cpf1 (SEQ ID NO:857), L. bacterium Cpf1 (SEQ ID NO:858), P. macacae Cpf1 (SEQ ID NO:859), P. disiens Cpf1 (SEQ ID NO:860).

[0163] In some embodiments, the compounds of the present invention further comprise an endosomal escape agent. In some embodiments of a compound of the present invention, the Y moiety of the compound further comprises an endosomal escape agent. In some embodiments, the endosomal escape agent is linked to the remainder of the compound via one or more covalent bonds. In some endosomal embodiments, the endosomal escape agent is associated with the remainder of the compound, e.g., via electrostatic interactions. In some embodiments, the endosomal escape agent is co-incubated with a compound of the present invention.

[0164] Suitable endosomal escape agents for use in the present invention include, but are not limited to, a lysosomotropic agent, a cell penetrating peptide, a fusogenic peptide, an endosomolytic peptide, a pore forming agent, and a proton sponge agent.

[0165] Lysosomotropic agents are weak bases that can penetrate in lysosome as protonated form and increase the intracellular pH. Suitable lysosomotropic agents for use in the present application include, but are not limited to chlorpromazine, amantadine, 4-aminoquinoline, amiodarone, amodiaquine, azithromycin, chloroquine, clindamycin, N-(3-[(2,4-dinitrophenyl)-amino]-propyl)-N-(3-aminopropyl-methylamine)dihydrochloride (DAMP), imipramine, methylamine, monensin, monodansylcadaverine, NH₄Cl, perhexilene, phenylalanine methyl ester, primaquine, quinacrine, suramin, thioridazine, tilorone, tributylamine, ketotifen fumarate, glycerol, and sucrose. In some embodiments, the lysosomotropic agent is chloroquine, glycerol or sucrose.

[0166] Cell penetrating peptides (CPPs) are a class of diverse peptides, typically with 5-30 amino acids, that can cross the cellular membrane. In some embodiments, suitable CPPs for use herein include cationic CPPs, amphipathic CPPs, and hydrophobic CPPs. Suitable CPPs for use herein include, but are not limited to, CPPs derived from heparin-, RNA-, and DNA-binding proteins such as those listed in Table 1, CPPs derived from signal peptides such as those listed in Table 2, CPPs derived from antimicrobial peptides such as those listed in Table 3, CPPs derived from viral proteins such as those listed in Table 4, CPPs derived from various natural proteins such as those listed in Table 5, and designed CPPs and CPPs derived from peptide libraries such as those listed in Table 6. See, e.g., Milletti, F. Drug Discovery Today, Volume 17, Numbers 15/16, 2012, 850-860.

TABLE 1

CPPs derived from heparan-, RNA- and DNA-
binding proteins

CPPs derived from heparan binding proteins
RKKRRRESRKKRRRES (SEQ ID NO: 916)	DPV3

GRPRESGKKRKRKRLKP (SEQ ID NO: 917)	DPV6

GKRKKKGKLGKKRDP (SEQ ID NO: 918)	DPV7

GKRKKKGKLGKKRPRSR (SEQ ID NO: 919)	DPV7b

RKKRRRESRRARRSPRHL (SEQ ID NO: 920)	DPV3/10

SRRARRSPRESGKKRKRKR (SEQ ID NO: 921)	DPV10/6

VKRGLKLRHVRPRVTRMDV (SEQ ID NO: 922)	DPV1047

SRRARRSPRHLGSG (SEQ ID NO: 923)	DPV10

LRRERQSRLRRERQSR (SEQ ID NO: 924)	DPV15

GAYDLRRRERQSRLRRRERQSR (SEQ ID NO:	DPV15b
925)

CPPs derived from RNA binding proteins
RKKRRQRRR (SEQ ID NO: 926)	HIV-1 Tat

RRRRNRTRRNRRRVR (SEQ ID NO: 927)	FHV coat

TRQARRNRRRRWRERQR (SEQ ID NO: 928)	HIV-1 Rev

TRRQRTRRARRNR (SEQ ID NO: 929)	HTLV-II Rex

KMTRAQRRAAARRNRWTAR (SEQ ID NO: 930)	BMV Gag

NAKTRRHERRRKLAIER (SEQ ID NO: 931)	P22 N

MDAQTRRRERRAEKQAQWKAAN (SEQ ID NO:	λN(1-22)
932)

TAKTRYKARRAELIAERR (SEQ ID NO: 933)	φ21N(12-29)

TRRNKRNRIQEQLNRK (SEQ ID NO: 934)	Yeast PrP6

CPPs derived from DNA binding proteins
PRRRRSSSRPVRRRRRPRVSRRRRRRGGRRRR	Protamine 1
(SEQ ID NO: 935)

Leucine zipper
RIKAERKRMRNRIAASKSRKRKLERIAR (SEQ	Human cJun
ID NO: 936)

KRRIRRERNKMAAAKSRNRRRELTDT (SEQ ID	Human cFos
NO: 937)

Transcription factors
KRARNTEAARRSRARKLQRMKQ (SEQ ID NO:	Yeast GCN4
938)

Homeoproteins
RQIKIWFQNRRMKWKK (SEQ ID NO: 866)	Penetratin

RVIRVWFQNKRCKDKK (SEQ ID NO: 939)	Islet-1

SKRTRQTYTRYQTLELEKEFHFNRYITRRRRIDI	Fushi-tarazu
ANALSLSERQIKIWFQNRRMKSKKDR (SEQ ID
NO: 940)

SQIKIWFQNKRAKIKK (SEQ ID NO: 941)	Engrailed-2

RQVTIWFQNRRVKEKK (SEQ ID NO: 942)	HoxA-13

KQINNWFINQRKRHWK (SEQ ID NO: 943)	Knotted-1

RHIKIWFQNRRMKWKK (SEQ ID NO: 1039)	PDX-1

TABLE 2

CPPs derived from signal peptides.

Amphipathic (I): signal peptide + NLS
	MGLGLHLLVLAAALQGAKKKRKV (SEQ ID	Ig(v)
	NO: 944)

	MVKSKIGSWILVLFVAMWSDVGLCKKRPKP	BPrPp(1-30)
	(SEQ ID NO: 945)

	MANLGYWLLALFVTMWTDVGLCKKRPKP	MPrPp(1-28)
	(SEQ ID NO: 946)

Hydrophobic: signal peptide alone
	AAVLLPVLLAAP (SEQ ID NO: 947)	K-FGF

TABLE 3

CPPs derived from antimicrobial peptides.

Pro-rich
RRIRPRPPRLPRPRPRPLPFPRPG	Bac7
(SEQ ID NO: 948)

VDKGSYLPRPTPPRPIYNRN (SEQ ID NO: 949)	Pyrrhocoricin

Amphipathic
KCFQWQRNMRKVRGPPVSCIKR	Human
(SEQ ID NO: 950)	lactoferrin
	(19-40)

TRSSRAGLQWPVGRVHRLLRK	Buforin 2
(SEQ ID NO: 951)

GIGAVLKVLTTGLPALISWIKRKRQQ	Melittin
(SEQ ID NO: 864)

GIGKWLHSAKKFGKAFVGEIMNS	Magainin 2
(SEQ ID NO: 952)

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTE	LL-37
SC (SEQ ID NO: 953)

RGGRLSYSRRRFSTSTGR (SEQ ID NO: 954)	SynB1

YKQCHKKGGKKGSG (SEQ ID NO: 955)	Crotamine

ALWKTLLKKVLKAPKKKRKV (SEQ ID NO: 956)	S413-PVrev

HARIKPTFRRLKWKYKGKFW (SEQ ID NO: 957)	L-2

TABLE 4

CPPs derived from viral proteins.


TKRRITPKDVIDVRSVTTEINT (SEQ ID NO: 958)	Inv3

Amphipathic
RQGAARVTSWLGRQLRIAGKRLEGRSK (SEQ ID	E^ms
NO: 959)

NAATATRGRSAASRPTQRPRAPARSASRPRRPVQ	VP22
(SEQ ID NO: 960)

RHSRIGIIQQRRTRNG (SEQ ID NO: 961)	HIV-1 VPR
	77-92

KLIKGRTPIKFGKADCDRPPKHSQNGMGK (SEQ	Ribotoxin2
ID NO: 962)	L3 loop

PLSSIFSRIGDP (SEQ ID NO: 963)	PreS2-TLM

Amphipathic (β-sheet)
DPKGDPKGVTVTVTVTVTGKGDPKPD (SEQ ID	VT5
NO: 964)

TABLE 5

CPPs derived from various natural proteins.

Cationic
RRIPNRRPRR (SEQ ID NO: 965)	HRSV

RLRWR (SEQ ID NO: 966)	AIP6

Amphipathic (I)
MVRRFLVTLRIRRACGPPRVRV (SEQ ID	ARF(1-22)
NO: 967)

MVTVLFRRLRIRRACGPPRVRV (SEQ ID	M918
NO: 968)

LLIILRRRIRKQAHAHSK (SEQ ID NO: 969)	pVEC

Amphipathic (helical)
LSTAADMQGVVTDGMASG (SEQ ID NO:	Azurin p18
970)

LSTAADMQGVVTDGMASGLDKDYLKPDD	Azurin p28

(SEQ ID NO: 971)
KFHTFPQTAIGVGAP (SEQ ID NO: 972)	hCT18-32

Hydrophobic
VPTLK (SEQ ID NO: 973); PMLKE (SEQ ID	Bip
NO: 1030), VPALR (SEQ ID NO: 1031),
VSALK (SEQ ID NO: 1032), IPALK (SEQ ID
NO: 1033)

PFVYLI (SEQ ID NO: 974)	C105Y

PIEVCMYREP (SEQ ID NO: 975)	FGF12

TABLE 6

Designed CPPs and CPPs derived from peptide
libraries.
Designed

Cationic
R8 (SEQ ID NO: 1040), R9(SEQ ID NO:	Polyarginine
1041), R10 (SEQ ID NO: 1042),
R12 (SEQ ID NO: 976)

Amphipathic (cationic)
KETWWETWWTEWSQPKKRKV (SEQ ID NO: 977)	Pep-1

GLAFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID	MPG
NO: 978)

Amphipathic (cationic)
GWTLNSAGYLLGKINLKALAALAKKIL	Transportan
(SEQ ID NO: 867)

AGYLLGHINLHHLAHLAibHHIL	TH
(SEQ ID NO: 979)

KLALKALKALKAALKLA (SEQ ID NO: 980)	MAP

RRWWRRWRR (SEQ ID NO: 981)	W/R

GLWRALWRLLRSLWRLLWRA (SEQ ID NO: 982)	CADY

LIRLWSHLIHIWFQNRRLKWKKK	EB-1
(SEQ ID NO: 983)

Amphipathic (cationic)
WEAALAEALAEALAEHLAEALAEALEALAA (SEQ	GALA
ID NO: 868)

WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ	KALA
ID NO: 869)

LKTLTETLKELTKTLTEL (SEQ ID NO: 984)	MAP12

Amphipathic (zero-charge)
QLALQLALQALQAALQLA (SEQ ID NO: 1034)	MAP17

Amphipathic (Proline-rich)
(PPR)3 (SEQ ID NO: 1043), (PPR)4	(PPR)n
(SEQ ID NO: 1044), (PPR)5 (SEQ ID NO:
1045), (PPR)6 (SEQ ID NO: 985)

(PRR)3 (SEQ ID NO: 1046), (PRR)4	(PPR)n
(SEQ ID NO: 1047), (PRR)5 (SEQ ID NO:
1048), (PRR)6 (SEQ ID NO: 986)

GPSQPTYPGDDAPVRDLIRFYRDLQRYLNVVTRHRY	aPP4R1
(SEQ ID NO: 987)

GPSQPTYPGDDAPVRDLIRFYRDLRRYLNVVTRHRY	aPP5R1
(SEQ ID NO: 988)

GPSQPTYPGDDAPVRDLRRFYRDLRRYLNVVTRHRY	aPP6R1
(SEQ ID NO: 989)

G(P_LXX)_nP_L, where P_Lis a proline-	PoliProline-
based mimic of leucine, and X is a	based
proline-based mimic of
lysine or arginine (SEQ ID NO: 990)

VRLPPPVRLPPPVRLPPP (SEQ ID NO: 991)	SAP

VELPPPVELPPPVELPPP (SEQ ID NO: 992)	SAP(E)

Peptide libraries
Support-vector machine model
FKIYDKKVRTRVVKH (SEQ ID NO: 993)	SVM1

RASKRDGSWVKKLHRILE (SEQ ID NO: 994)	SVM2

KGTYKKKLMRIPLKGT (SEQ ID NO: 995)	SVM3

LYKKGPAKKGRPPLRGWFH (SEQ ID NO: 996)	SVM4

HSPIIPLGTRFVCHGVT (SEQ ID NO: 997)	SVM5

YTAIAWVKAFIRKLRK (SEQ ID NO: 998)	YTA2

IAWVKAFIRKLRKGPLG (SEQ ID NO: 999)	YTA4

Plasmid display
Amphipathic
RLSGMNEVLSFRWL (SEQ ID NO: 1000)	SG3

Phage display
Hydrophobic
SDLWEMMMVSLACQY (SEQ ID NO: 1001)	Pep-7

VTWTPQAWFQWV (SEQ ID NO: 1002)

GSPWGLQHHPPRT (SEQ ID NO: 1003)	439a

GPFHFYQFLFPPV (SEQ ID NO: 1004)	435b

TSPLNIHNGQKL (SEQ ID NO: 1005)	HN-1

Other
CAYHRLRRC (SEQ ID NO: 1006)

Phylomer library
Cationic
RCGRASRCRVRWMRRRRI (SEQ ID NO: 1007)	BEN_1079

Other
PYSRPHVQLWYPNRESCRSLIRSLGP	BEN_0805
(SEQ ID NO: 1008)

Peptide arrays
Hydrophobic
PLILLRLLRGQF (SEQ ID NO: 1009)	Pept1

PLIYLRLLRGQF (SEQ ID NO: 1010)	Pept2

KLWMRWYSPTTRRYG (SEQ ID NO: 1011)	IVV-14

[0167] Fusogenic peptides are short peptides that destabilize the phospholipid membrane. In some embodiments, a fusogenic peptide comprises 20-30 amino acids. They primarily elicit endosomal escape by two main mechanisms: membrane fusion and pore formation. Suitable fusogenic peptides for use herein include, but are not limited to, an influenza HA-2 peptide, GLFGAIAGFIENGWEGMIDGWYG (SEQ ID NO: 863); melittin, GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 864); tat (48-60), GRKKRRQRRRPPQ (SEQ ID NO: 865); penetratin, RQIKIWFQNRRMKWKK (SEQ ID NO: 866); transportan, GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 867); GALA peptide, WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO: 868); KALA peptide, WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 869); JST-1, GLFEALLELLESLWELLLEA (SEQ ID NO: 870); ppTG1, GLFKALLKLLKSLWKLLLKA (SEQ ID NO: 871); ppTG20, GLFRALLRLLRSLWRLLLRA (SEQ ID NO: 872); and derivatives thereof. In some embodiments, the endosomal escape agent comprises a KALA peptide, WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 869).

[0168] In some embodiments, the endosomal escape agent an endosomolytic histidine-rich peptide selected from, but not limited to, CHK₆HC (SEQ ID NO:861); HSWYG (SEQ ID NO: 1029), GLFHAIAHFIHGGWHGLIHGWYG (SEQ ID NO:862); and derivatives thereof. In some embodiments, the endosomal escape agent comprises a HSWYG (SEQ ID NO: 1029), or GLFHAIAHFIHGGWHGLIHGWYG (SEQ ID NO:862) peptide.

[0169] In some embodiments, the endosomal escape agent is a synthetic peptide. In some embodiments, the endosomal escape agent is peptide ppTG21: GLFHALLHLLHSLWHLLLHA (SEQ ID NO: 1012), having the structure:

wherein the hydrogen atoms are omitted from the peptide structure for clarity of view.

See Rittner, Karola, et al., New Basic Membrane-Destabilizing Peptides for Plasmid-Based Gene Delivery in Vitro and in Vivo, MOLECULAR THERAPY 5(2) 104-114 (2002).

[0170] Pore-forming agents are agents, such as peptides, that induce pore formation through the membrane thereby disrupting endosome. Suitable pore-forming agents for use herein include, but are not limited to, cecropin (insects), magainin, CPF 1, PGLa, Bombinin BLP-1 (all three from amphibians), melittin (bees), seminalplasmin (bovine), indolicidin, bactenecin (both from bovine neutrophils), tachyplesin 1 (crabs), protegrin (porcine leukocytes), and defensins (from human, rabbit, bovine, fungi, and plants), Gramicidin A and gramicidin S (bacillus brevis), the lantibiotics such as nisin (lactococcus lactis), androctonin (scorpion), cardiotoxin I (cobra), caerin (frog litoria splendida), dermaseptin (frog). Viral peptides have also been shown to have pore-forming activity, examples include hemagglutinin subunit HA-2 (influenza virus), E1 (Semliki forest virus), F1 (Sendai and measles viruses), gp41 (HIV), gp32 (SIV), and vp1 (Rhino, polio, and coxsackie viruses). In some embodiments, the endosomal escape agent is a vp1 peptide derived from rhino virus. In some embodiments, the endosomal escape agent is the mellitin peptide derived from bee venom, or a masked analog thereof.

[0171] Proton sponge agents are agents having multiple proton acceptor sites that disrupt the endosome by osmolytic action. In some embodiments, the proton sponge agent suitable for use herein comprises a plurality of proton acceptor sites having pKa values between physiological and lysosomal pH. In some embodiments, the proton sponge agent suitable for use herein comprises a plurality of proton acceptor sites having pKa values within the range of 4 to 7, which endosomal lysing component is polycationic at pH 4. In some embodiments, the endosomal escape agent is a proton sponge agent selected from imidazole-containing compounds such as compounds or peptides comprising one or more histidine, histamine, vinylimidazole, or combinations thereof. In some embodiments, the suitable proton sponge agents for use herein include polymers have one or more secondary or tertiary amines and exhibit pKa values between physiological and lysosomal pH. In some embodiments, the proton sponge agents suitable for use herein include, but are not limited to polymers such as plyethylenimines, plyamidoamine dendrimers, PEG-oligo(glutamic acid)-PEI; poly(L-histidines); chloroquine, methylamine, ammonium chloride.

[0172] In another embodiment, a compound describe is capable of binding to a receptor present on a hepatocyte. In another embodiment, the receptor present on a hepatocyte is an asialoglycoprotein receptor.

Preparation Methods

[0173] Compounds of the present invention may be synthesized by synthetic routes that include processes analogous to those well-known in the chemical arts, particularly in light of the description contained herein. The starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, Wis.) or are readily prepared using methods well known to those skilled in the art (e.g., prepared by methods generally described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley, New York (1967-1999 ed.), or Beilsteins Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin, including supplements (also available via the Beilstein online database)).

[0174] For illustrative purposes, the reaction schemes depicted below provide potential routes for synthesizing the compounds of the present invention as well as key synthetic intermediates. For a more detailed description of the individual reaction steps, see the Examples section below. Those skilled in the art will appreciate that other synthetic routes may be used to synthesize the inventive compounds. Although specific starting materials and reagents are depicted in the schemes and discussed below, other starting materials and reagents can be easily substituted to provide a variety of derivatives and/or reaction conditions. In addition, many of the compounds prepared by the methods described below can be further modified in light of this disclosure using conventional chemistry well known to those skilled in the art.

[0175] In the preparation of compounds of the present invention, protection of remote functionality (e.g., primary or secondary amine) of intermediates may be necessary. The need for such protection will vary depending on the nature of the remote functionality and the conditions of the preparation methods. Suitable amino-protecting groups (NH-Pg or NPg) include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBz), 9-fluorenylmethyleneoxycarbonyl (Fmoc), and phthalimide (Pht). A “hydroxy-protecting group” refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable hydroxyl-protecting groups (O-Pg) include for example, allyl, acetyl (Ac), silyl (like trimethylsily (TMS) or tert-butyldimethylsilyl (TBS)), benzyl (Bn), para-methoxybenzyl (PMB), trityl (Tr), para-bromobenzoyl, para-nitrobenzoyl and the like (benzylidene, cyclic ketals, orthoesters, orthoamides for protection of 1,2- or 1,3-diols). The need for such protection is readily determined by one skilled in the art. For a general description of protecting groups and their use, see T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

[0176] Scheme 1 outlines the general procedures one could use to provide compounds of the present invention. In step 1 of Scheme 1, synthetic intermediate (I-a), that can be prepared by procedures described by H. Paulsen and M. Paal in Carbohydrate Research, 135, 53 (1984), is persylilated under classical conditions [using trimethylsilyl chloride and pyridine at room temperature (about 23° C.)] followed by selective cleavage of the trimethyl silyl group protecting the primary alcohol (by treatment under basic conditions such as potassium carbonate in an alcoholic solvent like methanol at a temperature ranging from about −10 degrees Celsius to room temperature) to reveal primary alcohol intermediate (I-b). In step 2 of Scheme 1, the additional hydroxymethylene group found in intermediate (I-c) can be introduced onto the glycoside by means of a Parikh-Doering oxidation described by J. R. Parikh and William v. E. Doering in Journal of the American Chemical Society, 89, 5505-5507 (1967) followed by treatment with a formaldehyde source (e.g., solution of formaldehyde in water, solid paraformaldehyde) in the presence of an alkali metal hydroxide (e.g., sodium hydroxide, sodium alkoxide) in water or in an alcoholic solvent at a temperature ranging from about room temperature to about 60 degrees Celsius. This is referred to as an aldol-Cannizzaro reaction. Modifications of this process known to those of skill in the art may also be used. For example, other oxidants, like stabilized 2-iodoxybenzoic acid described by Ozanne, A. et al. in Organic Letters, 5, 2903 (2003), the Swern oxidation described in Kanji Omura and Daniel Swern in Tetrahedron, 34, 1651 (1978), as well as other oxidants known by those skilled in the art can also be used. The aldol Cannizzaro sequence has been described by Robert Schaffer in the Journal of The American Chemical Society, 81, 5452 (1959) and Amigues, E. J., et al., in Tetrahedron, 63, 10042 (2007). Experimental conditions of step 2 in Scheme 1 also promote cleavage of the trimethylsilyl groups protecting the secondary alcohols. In step 3 of Scheme 1, intermediate (I-c) is treated with an organic or inorganic acid (e.g., sulfuric acid) or an acidic resin in a solvent like water at a temperature ranging from about room temperature to about 100 degrees Celsius to produce compound (1). In step 4 of Scheme 1, compound (1) can be treated with a reducing agent known to reduce azido groups to the corresponding amine (e.g., transition-metal mediated catalytic hydrogenation, use of triphenylphosphine in water under classical experimental conditions well known by those skilled in the art). Subsequent treatment in presence of an acylating agent (e.g., acetic anhydride or acetyl chloride in presence of pyridine or triethylamine in a solvent such as dichloromethane or tetrahydrofuran at a temperature ranging from 0 to 80 degrees Celsius) provides compound (2). In step 5 of Scheme 1, treatment of compound (2) in presence of an alkoxide (e.g., sodium methoxide) in a solvent, or mixture of solvents, such as an alcoholic solvent or tetrahydrofuran at a temperature ranging from about 0 to room temperature provides compound (3). Furthermore, the compounds thus obtained can then be easily functionalized to other claimed compounds from the present invention using well known protective and functional groups manipulation sequences known by those skilled in the art. Thus, in steps 6 and 7 of Scheme 1, secondary hydroxyl groups in compounds (1) and (3) respectively can be further protected by a suitable protecting group (e.g., as a cyclic ketal upon treatment with 2,2-dimethoxypropane under acidic conditions in a solvent such as N,N-dimethylformamide at a temperature ranging from about room temperature to about 90 degrees Celsius) to access intermediates such as (I-d) and (I-e). In turn, using synthetic transformations and functional and protecting groups manipulations well known by those skilled in the art, (I-d) and (I-e) are primed for further functionalization and derivatization of the primary hydroxyl group to link the desired linker X and ligand Y of interest to produce the XY-containing compounds claimed in the present invention. Removal of the protecting groups (e.g., Pg), using reagents and conditions well known to those skilled in the art (e.g., in the case where the two Pg form a cyclic ketal such as an acetonide, it can be removed under acidic conditions using an acid such as acetic acid in a solvent or mixture of solvents such as acetic acid, an alcoholic solvent, water, tetrahydrofuran at a temperature ranging from room temperature to about 80 degrees Celsius), to reveal the secondary hydroxyl groups leads to XY-containing compounds claimed in the present invention. For example, alkylation of the primary hydroxyl group in (I-e) can lead to, after protecting group manipulation and removal, the corresponding ether-linked XY-containing compounds claimed in this invention. Ester-linked, carbonate-linked and carbamate-linked XY-containing compounds claimed in the present invention can also be conveniently accessed from (3) or intermediate (I-e) using the appropriate reactants and reagents well known by those skilled in the art. Conversion of the primary hydroxyl group in (I-e) to the corresponding triflate (III-e-1) followed by nucleophilic displacement with the appropriate nucleophile can lead to, after protecting group manipulation and removal, the corresponding ether- and thioether-linked XY-containing compounds claimed in this invention. Oxidation of the thioether intermediate can also lead to the corresponding sulfoxide- and sulfone-linked XY-containing compounds claimed in this invention. In addition, displacement of the primary triflate in (III-e-1) by potassium thioacetate followed by thioester hydrolysis can provide the corresponding thiol (III-e-2) which provides compound (IV-e-1) after protecting group manipulation and removal; further alkylation of the thiol (III-e-2) and protecting group manipulation and removal can also produce thioether-linked XY-containing compounds claimed in this invention. (III-e-2) can also be converted to the corresponding sulfonyl chloride and treated with the appropriate amine to produce, after protecting group manipulation and removal, sulfonamide linked XY-containing compounds of the present invention. Displacement of the primary triflate (III-e-1) with sodium azide can also produce the corresponding azide-containing compound (III-e-3) which after protecting group manipulation and removal provides compound (IV-e-2). Reduction of compound (III-e-3) can produce the corresponding primary amine (III-e-4) primed for further functionalization (e.g., amide bond formation, reductive amination, sulfonamide formation, urea formation, carbamate formation, etc.) to link the XY substituent and produce compounds claimed in this invention after protecting group manipulation and removal. (III-e-4) can also produce compound (IV-e-3) of the present invention after protecting group manipulation and removal. Treatment of the above azide intermediate (III-e-3) with an alkyne or nitrile containing reagent or synthetic intermediate followed by protecting group manipulation and removal under conditions well known by those skilled in the art can also produce a triazole- or tetrazole-linked XY-containing compounds claimed in this invention, respectively. Oxidation of the primary hydroxyl group in (I-e) to the corresponding aldehyde (III-e-5) followed by reductive amination, under classical conditions known to those skilled in the art, with the appropriate amine, or olefination (such as Wittig-, Horner-Wadsworth-Emmons-, Petterson, Julia-type and modification thereof) followed by reduction of the olefin formed (using for instance a metal mediated catalytic hydrogenation or a diimide mediated reduction well known by those skilled in the art), can lead to the desired nitrogen- or carbon-linked X-Y-containing compounds claimed in this invention after functional group manipulation and protecting group manipulation and removal, respectively. Conversion of the aldehyde (III-e-5) to the corresponding alkyne (III-e-6) (using a Corey-Fuchs type reaction or using a Seyferth-Gilbert type reagent) followed by protecting group manipulation and removal can lead to compound (IV-e-4). In turn, treatment of alkyne (III-e-6) or (IV-e-4) with an azide containing reagent or synthetic intermediate followed by protecting group manipulation and removal under conditions well known by those skilled in the art can also produce a triazole-linked XY-containing compounds claimed in this invention. Alkyne (III-e-6) can also serve as a useful synthetic intermediate to access other compounds claimed in this invention upon treatment with the appropriate reagents under metal mediated cross couplings known to those skilled in the art (such as a Sonogashira-type reaction). Oxidation of the primary hydroxyl group in (I-e) to the corresponding acid (III-e-7) provides, using synthetic transformations well known by those skilled in the art, access to ester- and amide-linked XY-containing compound claimed in this invention, after protecting group removal. Protecting group manipulation and removal in (III-e-7) also provides readily access to compound (IV-e-5). Conversion of (IV-e-5) or (III-e-7) to the corresponding primary amide under conditions well known by those skilled in the art directly provides compound (IV-e-6) or compound (III-e-8) which after protecting group manipulation and removal gives (IV-e-6). In addition, dehydration of the amide functionality in (III-e-8) can provide the corresponding nitrile (III-e-9) which after functional group manipulation and removal provides compound (IV-e-7). Displacement of the primary triflate (III-e-1) with a cyanide anion can also produce the corresponding nitrile-containing compound (III-e-10) which after protecting group manipulation and removal provides compound (IV-e-8). In turn, hydrolysis of the nitrile in (IV-e-8) or (III-e-10) can provide directly access to acid (IV-e-9) or to (III-e-11) which after protecting group manipulation and removal provides (IV-e-9). Alkyne containing compounds such as (III-e-6)/(IV-e-4), primary amide containing compounds such as (III-e-8)/(IV-e-6), nitrile containing compounds such as (III-e-9)/(IV-e-7)/(III-e-10)/(IV-e-8), acid containing compound such as (III-e-7)/(IV-e-5)/(III-e-11)/(IV-e-9), aldehyde containing compounds such as (III-e-5) can also be further functionalized and reacted with the appropriate reagent and synthetic intermediate under conditions well known by those skilled in the art (and summarized in J. A. Joule and K. Mills, Heterocyclic Chemistry, 5^thedition, Wiley Ed., (2010); J. J. Li, Name Reactions in heterocyclic chemistry, Wiley, (2005); M. R. Grimmett Advances in Heterocyclic Chemistry, 27, 241, (1981); I. G. Turchi et al., Chemical Reviews, 75, 389, (1975); K. T. Potts, Chemical Reviews, 61, 87, (1961); R. H. Wiley, Organic Reactions, 6, 367, (1951); L. B. Clapp, Advances in Heterocyclic Chemistry, 20, 65, (1976); A. Hetzheim et al., Advances in Heterocyclic Chemistry, 7, 183, (1967); J. Sandstrom, Advances in Heterocyclic Chemistry, 9, 165, (1968); S. J. Wittenberger, Organic Preparations and Procedures International, 26, 499, (1994); M. G. Finn et al., Angewandte Chemie International Edition, 48, 9879, (2009)) to produce additional 5 and 6 membered ring (such as isoxazole, isothiazole, pyrazole, oxazole, thiazole, imidazole, 1,2,4-oxadiazole, 1,2,4-thiadiazole, 1,2,4-triazole, 1,3,4-oxadiazole, 1,3,4-thiadiazole, tetrazole, 1,2,3-triazole) linked XY-containing compounds claimed in this invention. Aryl ring-linked XY-containing compounds claimed in this invention can also be accessed from alkynes such as (III-e-6) and (IV-e-4), or a heterosubstituted analogue of these alkynes (i.e., by replacing the alkyne hydrogen in (III-e-6)/(IV-e-4) by OR⁴, N(R⁴)₂, SR⁴; these compounds can be accessed using conditions and reagents known to those skilled in the art), via a benzannulation reaction known from those skilled in the art (such as a Danheiser-type or Dotz-type benzannulation).

[0177] Similar chemistry described above for compounds (3) and (I-e) can also be applied to compound (1) and intermediate (I-d) to provide additional compounds claimed in the present invention. See examples section for further details.

[0178] The use of trifluoroacetic anhydride in step 4 of Scheme 1 can also provide access to N-((1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octan-4-yl)-2,2,2-trifluoroacetamide, compound (24).

[0179] Particularly when R¹contains an aliphatic-, PEG-derived chain, PEG-derived oligo- or polymer, compounds of the present invention can be further functionalized, reacted, and formulated under conditions known to those skilled in the art to access additional compounds claimed in the present invention that can be used and incorporated in the formulation of hepatoselective drug delivery systems such as Biodegradable PLGA-b-PEG polymeric nanoparticles (see, Erica Locatelli et al., Journal of Nanoparticle Research, 14, 1316, (2012)) and lipid based platforms such as liposomes, lipid nanoparticles, stable nucleic acids lipid nanoparticles (see, Sara Falsini et al., Journal of Medicinal Chemistry, 57, 1138 (2014)).

[0180] Diastereomeric mixtures can be separated into their individual diastereoisomers on the basis of their physical chemical differences by methods well known to those skilled in the art, such as by chromatography and/or fractional crystallization, distillation, sublimation. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., chiral auxiliary such as a chiral alcohol or Mosher's acid chloride), separating the diastereoisomers and converting (e.g., hydrolyzing) the individual diastereoisomers to the corresponding pure enantiomers. Also, some of the compounds of the present invention may be atropisomers (e.g., substituted biaryls) and are considered as part of this invention. Enantiomers can also be separated by use of a chiral HPLC (high pressure liquid chromatography) column.

[0181] It is also possible that the intermediates and compounds of the present invention may exist in different tautomeric forms, and all such forms are embraced within the scope of the invention. The term “tautomer” or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. A specific example of a proton tautomer is the imidazole moiety where the proton may migrate between the two ring nitrogens. Valence tautomers include interconversions by reorganization of some of the bonding electrons. The equilibrium between closed and opened form of some intermediates (and/or mixtures of intermediates) is reminiscent of the process of mutarotation involving aldoses, known by those skilled in the art.

[0182] Scheme 3 provides exemplary Cas9 modifications in preparing the compounds of the present invention, wherein each occurrence of n, o, p, q, or r is an integer independently selected from 0 to 50.

[0183] Scheme 4 provides exemplary processes for linking a Cas9 protein or a Cas9 ribonucleoprotein to one or more other moieties via disulfide linkages, wherein each occurrence of n is an integer independently selected from 0 to 50. A person with ordinary skill in the art would appreciate that the disulfide linkages depicted herein may be replaced by other suitable linkages.

“ASGPRL” or “ASGPrL” refer to as a ligand, or dendrimer thereof, for the asialoglycoprotein receptor, such as compounds described in the present invention.

[0184] Scheme 5 provides exemplary processes for preparing Cas9 ribonucleoproteins where the RNA is associated with the Cas9 protein via electrostatic interactions, wherein each occurrence of n is an integer independently selected from 0 to 50.

[0185] Scheme 6 provides exemplary syntheses of the linker of type L1-L10 wherein Z is peptide ppTG21 and wherein the hydrogen atoms are omitted from the peptide structures for clarity of view. A person with ordinary skill in the art would readily appreciate that the peptide ppTG21 may be replaced with any suitable small molecule such as ASGPrL or an endoosmolytic agent, or a peptide as described herein.

[0186] The present invention also embraces isotopically-labeled compounds of the present invention which are identical to those recited herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ¹²³I, ¹²⁵I and ³⁶Cl, respectively.

[0187] Certain isotopically-labeled compounds of the present invention (e.g., those labeled with ³H and ¹⁴C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., ²H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Positron emitting isotopes such as ¹⁵O, ¹³N, ¹¹C, and ¹⁸F are useful for positron emission tomography (PET) studies to examine substrate occupancy. Isotopically labeled compounds of the present invention can generally be prepared by following procedures analogous to those disclosed in the Schemes and/or in the Examples herein below, by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

Endosomal Escape Agent-Containing Compositions

[0188] Another aspect of the present invention provides a composition comprising a compound described herein and an endosomal escape agent described herein. In some embodiments of such a composition, the compound is co-incubated with the endosomal escape agent to form the composition.

[0189] Another aspect of the present invention provides a composition comprising a ribonucleoprotein described herein (e.g., a RNP comprising a site-directed modifying polypeptide, such as a Cas9 RNP or a Cpf1 RNP) and an endosomal escape agent described herein. In some embodiments of such a composition, the ribonucleoprotein (e.g., a RNP comprising a site-directed modifying polypeptide, such as a Cas9 RNP or a Cpf1 RNP) is co-incubated with the endosomal escape agent to form the composition. In some embodiments, the ribonucleoprotein (e.g., a RNP comprising a site-directed modifying polypeptide, such as a Cas9 RNP or a Cpf1 RNP) or the endosomal escape agent is conjugated to an antibody or a fragment thereof. In some embodiments, the ribonucleoprotein (e.g., a RNP comprising a site-directed modifying polypeptide, such as a Cas9 RNP or a Cpf1 RNP) is modified to include glycosylation sites. In some embodiments, the ribonucleoprotein (e.g., a RNP comprising a site-directed modifying polypeptide, such as a Cas9 RNP or a Cpf1 RNP) is modified to include transduction or translocation domains.

Pharmaceutical Compositions and Modes of Administration

[0190] Compounds and compositions of the present invention are useful for treating diseases, conditions and/or disorders; therefore, another embodiment of the present invention is a pharmaceutical composition comprising a therapeutically effective amount of a compound, or an endosomal escape agent containing composition, of the present invention and a pharmaceutically acceptable excipient, diluent or carrier. The compounds of the present invention (including the compositions and processes used therein) may also be used in the manufacture of a medicament for the therapeutic applications described herein.

[0191] The pharmaceutical compositions of this invention may be in liquid solutions (e.g., injectable and infusible solutions). The preferred form depends on the intended mode of administration and therapeutic application. Typical pharmaceutical compositions are in the form of injectable or infusible solutions, such as pharmaceutical compositions similar to those used for passive immunization of humans. One mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intradermal, and intrasternally) or by infusion techniques, in the form of sterile injectable liquid or olagenous suspensions. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In a preferred embodiment, the compound or composition is administered by intravenous infusion or injection. In another preferred embodiment, the compound or composition is administered by intramuscular or subcutaneous injection.

[0192] Therapeutic pharmaceutical compositions typically are sterile and stable under the conditions of manufacture and storage.

[0193] The pharmaceutical composition can be formulated as a solution, microemulsion, dispersion, or liposome. Sterile injectable solutions can be prepared by incorporating the compound of the present invention in the required amount in an appropriate diluent with one or a combination of ingredients enumerated above, as required, followed by sterilization (e.g., filter sterilization). Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. Such suspensions may be formulated according to the known art using those suitable dispersing of wetting agents and suspending agents or other acceptable agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. In addition, n−3 polyunsaturated fatty acids may find use in the preparation of injectables.

[0194] In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

[0195] Prolonged absorption of injectable pharmaceutical compositions can be brought about by including in the pharmaceutical composition an agent that delays absorption, for example, monostearate salts and gelatin or by formulating the pharmaceutical composition into prolonged absorption forms such as, depots, liposomes, polymeric microspheres, polymeric gels, and implants.

[0196] Other methods for administration of the compound of the present invention described herein include dermal patches that release the medications directly into a subject's skin. Such patches can contain the compound of the present invention in an optionally buffered, liquid solution, dissolved and/or dispersed in an adhesive, or dispersed in a polymer.

[0197] The compound may be administered once, but may also be administered multiple times. For example, the compound may be administered from once daily to once every six months or longer. The administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months, once every six months, once a year, and once every two years.

[0198] The compound may also be administered continuously via a minipump. The compound may be administered at the site of the diseased body part or at a site distant from the site of the diseased body part. The compound may be administered once, at least twice or for at least the period of time until the disease is treated, palliated or cured. The compound generally may be administered for as long as the disease is present. The compound typically would be administered as part of a pharmaceutical composition as described supra.

[0199] The pharmaceutical compositions of the invention may include a therapeutically effective amount or a prophylactically effective amount of compound of the invention. In preparing the pharmaceutical composition, the therapeutically effective amount of the compound present in the pharmaceutical composition can be determined, for example, by taking into account the desired dose volumes and mode(s) of administration, the nature and severity of the condition to be treated, and the age and size of the subject.

[0200] Exemplary, non limiting dose ranges for administration of the pharmaceutical compositions of the present invention to a subject are from about 0.01 mg/kg to about 200 mg/kg (expressed in terms of milligrams (mg) of compound of Formula (A) or (B) administered per kilogram (kg) of subject weight), from about 0.1 mg/kg to about 100 mg/kg, from about 1.0 mg/kg to about 50 mg/kg, from about 5.0 mg/kg to about 20 mg/kg, or about 15 mg/kg. For purposes of the present invention, an average human subject weighs about 70 kg. Ranges intermediate to any of the dosages cited herein, e.g., about 0.02 mg/kg-199 mg/kg, are also intended to be part of this invention. For example, ranges of values using a combination of any of the recited values as upper and/or lower limits are intended to be included.

[0201] Dosage regimens can also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response) by administering several divided doses to a subject over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral pharmaceutical compositions in dosage unit form for ease of administration and uniformity of dosage.

[0202] Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the compound or portion and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an antibody for the treatment of sensitivity in individuals.

[0203] The liquid pharmaceutical compositions of the present invention can be prepared as unit dosage forms. For example, a unit dosage per vial may contain from 1 to 1000 milliliters (mls) of different concentrations of the compound of Formula (A) or (B). In other embodiments, a unit dosage per vial may contain about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml or 100 ml of different concentrations of the compound of Formula (A) or (B). If necessary, these preparations can be adjusted to a desired concentration by adding a sterile diluent to each vial. The liquid pharmaceutical compositions of the present invention can also be prepared as unit dosage forms in sterile bags or containers, which are suitable for connection to an intravenous administration line or catheter.

[0204] Another typical formulation is prepared by mixing a compound of the present invention and a carrier, diluent or excipient. Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like. The particular carrier, diluent or excipient used will depend upon the means and purpose for which the compound of the present invention is being applied. Solvents are generally selected based on solvents recognized by persons skilled in the art as safe (GRAS) to be administered to a mammal. In general, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG400, PEG300), etc. and mixtures thereof. The formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).

[0205] The formulations may be prepared using conventional dissolution and mixing procedures. For example, the bulk drug substance (i.e., compound of the present invention or stabilized form of the compound (e.g., complex with a cyclodextrin derivative or other known complexation agent)) is dissolved in a suitable solvent in the presence of one or more of the excipients described above. The compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable dosage of the drug and to give the patient an elegant and easily handleable product.

[0206] The pharmaceutical compositions also include solvates and hydrates of the compounds of Formula (A) or (B). The term “solvate” refers to a molecular complex of a compound represented by Formula (A) or (B) (including pharmaceutically acceptable salts thereof) with one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical art, which are known to be innocuous to the recipient, e.g., water, ethanol, ethylene glycol, and the like, The term “hydrate” refers to the complex where the solvent molecule is water. The solvates and/or hydrates preferably exist in crystalline form. Other solvents may be used as intermediate solvates in the preparation of more desirable solvates, such as methanol, methyl t-butyl ether, ethyl acetate, methyl acetate, (S)-propylene glycol, (R)-propylene glycol, 1,4-butyne-diol, and the like. The crystalline forms may also exist as complexes with other innocuous small molecules, such as L-phenylalanine, L-proline, L-pyroglutamic acid and the like, as co-crystals or solvates or hydrates of the co-crystalline material. The solvates, hydrates and co-crystalline compounds may be prepared using procedures described in PCT Publication No. WO 08/002824, incorporated herein by reference, or other procedures well-known to those of skill in the art.

[0207] The pharmaceutical composition (or formulation) for application may be packaged in a variety of ways depending upon the method used for administering the drug. Generally, an article for distribution includes a container having deposited therein the pharmaceutical formulation in an appropriate form. Suitable containers are well-known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include a tamper-proof assemblage to prevent indiscreet access to the contents of the package. In addition, the container has deposited thereon a label that describes the contents of the container. The label may also include appropriate warnings.

Methods

[0208] Compounds and compositions of the present application are useful in treating a variety of diseases or conditions. In some embodiments, the compounds and compositions described herein are useful in treating a liver disease or condition or a liver modulated disease or condition in a subject including, but not limited to, hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, Alpha-1-antitrypsin deficiency, Bile acid synthesis and metabolism defects, Biliary Atresia, Cystic Fibrosis liver disease, Idiopathic neonatal hepatitis, Mitochondrial hepatopathies, Progressive familial intrahepatic cholestasis, Primary sclerosing cholangitis, Transthyretin amyloidosis, Hemophilia, Homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia like Type II diabetes mellitus, and diseases involving abnormally high hepatic glucose production similar to Type II diabetes mellitus.

[0209] Compounds and compositions of the present application are useful in selectively modulating transcription of a target DNA in a liver cell of a subject, said DNA being associated with a liver disease or condition or a liver modulated disease or condition in a subject, such as, but are not limited to hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, Alpha-1-antitrypsin deficiency, Bile acid synthesis and metabolism defects, Biliary Atresia, Cystic Fibrosis liver disease, Idiopathic neonatal hepatitis, Mitochondrial hepatopathies, Progressive familial intrahepatic cholestasis, Primary sclerosing cholangitis, Transthyretin amyloidosis, Hemophilia, Homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia like Type II diabetes mellitus, and diseases involving abnormally high hepatic glucose production similar to Type II diabetes mellitus, comprising the administration of an effective amount of a compound or composition described herein. In some embodiments, target DNA is the PCSK9 gene.

[0210] Compounds and compositions of the present application are useful in editing a nucleic acid molecule encoding a protein associated with a liver disease or condition or a liver modulated disease or condition in a subject, such as, but are not limited to hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, Alpha-1-antitrypsin deficiency, Bile acid synthesis and metabolism defects, Biliary Atresia, Cystic Fibrosis liver disease, Idiopathic neonatal hepatitis, Mitochondrial hepatopathies, Progressive familial intrahepatic cholestasis, Primary sclerosing cholangitis, Transthyretin amyloidosis, Hemophilia, Homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia like Type II diabetes mellitus, and diseases involving abnormally high hepatic glucose production similar to Type II diabetes mellitus, comprising the administration of an effective amount of a compound or composition described herein.

[0211] Compounds and compositions of the present application are useful in modulating the expression of level of at least one gene product associated with a liver disease or condition or a liver modulated disease or condition in a subject, such as, but are not limited to hereditary angioedema, familial tyrosinemia type I, Alagille syndrome, Alpha-1-antitrypsin deficiency, Bile acid synthesis and metabolism defects, Biliary Atresia, Cystic Fibrosis liver disease, Idiopathic neonatal hepatitis, Mitochondrial hepatopathies, Progressive familial intrahepatic cholestasis, Primary sclerosing cholangitis, Transthyretin amyloidosis, Hemophilia, Homozygous familial hypercholesterolemia, familial chylomicronemia, hyperlipidemia, steatohepatitis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), hyperglycemia like Type II diabetes mellitus, and diseases involving abnormally high hepatic glucose production similar to Type II diabetes mellitus, comprising the administration of an effective amount of a compound or composition described herein. In some embodiments, said method modulates the level of low-density lipoproteins (LDLs). In some embodiments, said method modulates the level of cholesterol in the blood of said subject. In some embodiments, said method reduces the blood cholesterol level in said subject.

[0212] In some embodiments, the disease or condition targeted by the methods or use described herein is hyperlipidemia, non-alcoholic steatohepatitis (NASH), or non-alcoholic fatty liver disease (NAFLD). In some embodiments, said subject is human.

[0213] Compositions comprising a ribonucleoprotein described herein (e.g., a RNP comprising a site-directed modifying polypeptide described herein, such as a Cas9 RNP or a Cpf1 RNP) and an endosomal escape agent described herein are useful in treating a disorder or disease in a subject. In some embodiments, said subject is human. In some embodiments, the disorder or disease is selected from, but is not limited to, blood disorders, cell dysregulation or oncology diseases and disorders, inflammation and immune related diseases, metabolic, liver, kidney and protein diseases and disorders, muscular or skeletal diseases, neurological and neuronal diseases and disorders, and ocular diseases and disorders. In some embodiments, the blood disorder or disease is selected from, but is not limited to, anemia, bare lymphocyte syndrome, bleeding disorders, deficiency in Factor H and Factor H-like 1, V, VIII, VII, X, XI, XII, XIIIA, or XIIIB, Fanconi anemia, hemophagocytic lymphohistiocytosis disorders, hemophilia A or B, hemorrhagic disorders, leukocyde deficiencies and disorders, sickle cell anemia (HBB) and thalassemia. In some embodiments, the cell dysregulation and oncology disease or disorder is selected from, but is not limited to, B-cell non-Hodgkin lymphoma and Leukemia. In some embodiments, the inflammation and immune-related disease is selected from, but is not limited to AIDS, autoimmune lymphoproliferative syndrome, combined immunodeficiency, HIV susceptibility or infection, severe combined immunodeficiencies. In some embodiments, the metabolic, liver, kidney or protein disease is selected from, but is not limited to amyloid neuropathy, amyloidosism, cirrhosis, cystic fibrosis, glycogen storage diseases, hepatic adenoma, hepatic failure, hepatic lipase deficiency, hepatoblastoma, cancer and carcinomas, medullary cystic kidney disease, phenylketonuria, polycystic kidney and hepatic disease. In some embodiments, the muscular or skeletal disease is selected from, but is not limited to Becker muscular dystrophy, Duchenne Muscular Dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, muscular dystrophy, osteopetrosis, muscular atrophy. In some embodiments, the neurological and neuronal disease or disorder is selected from, but is not limited to ALS, Alzheimer disease, autism; Fragile X Syndrome, Huntington's disease and disease like disorders, Parkinson disease, Rett syndrome, Schizophrenia, secretase related disorders, and trinucleotide repeat disorders. In some embodiments, the ocular disease or disorder is selected from, but is not limited to, age-related macular degeneration, cataract, corneal clouding and dystrophy, cornea plana congenital, glaucoma, leber congenital amaurosis, and macular dystrophy.

[0214] In some embodiments, the disorder or disease is selected from, but is not limited to, neoplasia, age-related macular degeneration, CNS disorders (such as schizophrenia or bipolar, Alzheimer's Disease, Parkinson's Disease and autism), trinucleotide repeat disorders, Fragile X Syndrome, secretase related disorders, prion-related disorders, ALS, and substance addition.

[0215] Compositions comprising a ribonucleoprotein described herein (e.g., a RNP comprising a site-directed modifying polypeptide, such as a Cas9 RNP or a Cpf1 RNP) and an endosomal escape agent described herein are useful in modulating a cellular function in a subject. In some embodiments, the cellular signaling is selected, but is not limited to, PI3K/AKT Signaling, ERK/MAPK Signaling, Glucocorticoid Receptor Signaling, Axonal Guidance Signaling, Ephrin Receptor Signaling, Actin Cytoskeleton Signaling, Huntington's Disease Signaling, Apoptosis Signaling, B Cell Receptor Signaling, Leukocyte Extravasation Signaling, Integrin Signaling, Acute Phase Response Signaling, PTEN Signaling, p53 Signaling, Aryl Hydrocarbon Receptor Signaling, Xenobiotic Metabolism Signaling, SAPK/JNK Signaling, PPAr/RXR Signaling, NF-KB Signaling, Neuregulin Signaling, Wnt & beta catenin Signaling, Insulin Receptor Signaling, IL-6 Signaling, Hepatic Cholestasis, IGF-1 Signaling, NRF2-mediated Oxidative Stress Response, Hepatic Fibrosis/Hepatic Stellate Cell Activation, PPAR Signaling, Fc Epsilon RI Signaling, G-Protein Coupled Receptor Signaling, Inositol Phosphate Metabolism, PDGF Signaling, VEGF Signaling, Natural Killer Cell Signaling, Cell Cycle: G1/S Checkpoint Regulation, T Cell Receptor Signaling, Death Receptor Signaling, FGF Signaling, GM-CSF Signaling, Amyotrophic Lateral Sclerosis Signaling, JAK/Stat Signaling, Nicotinate and Nicotinamide Metabolism, Chemokine Signaling, IL-2 Signaling, Synaptic Long Term Depression, Estrogen Receptor Signaling, Protein Ubiquitination Pathway, IL-10 Signaling, VDR/RXR Activation, TGF-beta Signaling, Toll-like Receptor Signaling, p38 MAPK Signaling, Neurotrophin/TRK Signaling, FXR/RXR Activation, Synaptic Long Term Potentiation, Calcium Signaling, EGF Signaling, Hypoxia Signaling in the Cardiovascular System, LPS/IL-1 Mediated Inhibition of RXR Function, LXR/RXR Activation, Amyloid Processing, IL-4 Signaling, Cell Cycle: G2/M DNA Damage Checkpoint Regulation, Nitric Oxide Signaling in the Cardiovascular System, Purine Metabolism, cAMP-mediated Signaling, Mitochondrial Dysfunction, Notch Signaling, Endoplasmic Reticulum Stress Pathway, Pyrimidine Metabolism, Parkinson's Signaling, Cardiac & Beta Adrenergic Signaling, Glycolysis/Gluconeogenesis, Interferon Signaling, Sonic Hedgehog Signaling, Glycerophospholipid Metabolism, Phospholipid Degradation, Tryptophan Metabolism, Lysine Degradation, Nucleotide Excision Repair Pathway, Starch and Sucrose Metabolism, Aminosugars Metabolism, Arachidonic Acid Metabolism, Circadian Rhythm Signaling, Coagulation System, Dopamine Receptor Signaling, Glutathione Metabolism, Glycerolipid Metabolism, Linoleic Acid Metabolism, Methionine Metabolism, Pyruvate Metabolism, Arginine and Proline Metabolism, Eicosanoid Signaling, Fructose and Mannose Metabolism, Galactose Metabolism, Stilbene, Coumarine and Lignin Biosynthesis, Antigen Presentation Pathway, Biosynthesis of Steroids, Butanoate Metabolism, Citrate Cycle, Fatty Acid Metabolism, Glycerophospholipid Metabolism, Histidine Metabolism, Inositol Metabolism, Metabolism of Xenobiotics by Cytochrome p450, Methane Metabolism, Phenylalanine Metabolism, Propanoate Metabolism, Selenoamino Acid Metabolism, Sphingolipid Metabolism, Aminophosphonate Metabolism, Androgen and Estrogen Metabolism, Ascorbate and Aldarate Metabolism, Bile Acid Biosynthesis, Cysteine Metabolism, Fatty Acid Biosynthesis, Glutamate Receptor Signaling, NRF2-mediated Oxidative Stress Response, Pentose Phosphate Pathway, Pentose and Glucuronate Interconversions, Retinol Metabolism, Riboflavin Metabolism, Tyrosine Metabolism, Ubiquinone Biosynthesis, Valine, Leucine and Isoleucine Degradation, Glycine, Serine and Threonine Metabolism, Lysine Degradation, Pain/Taste, Pain, Mitochondrial Function, and Developmental Neurology.

[0216] Compositions comprising a ribonucleoprotein (e.g., a RNP comprising a site-directed modifying polypeptide, such as a Cas9 RNP or a Cpf1 RNP) and an endosomal escape agent, as described herein, are useful in selectively modulating transcription of a target DNA in a subject, said DNA being associated with a disease or disorder as described herein. In some embodiments, the target DNA is associated with neoplasia, said DNA being selected from PTEN; ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; AKT; AKT2; AKT3; HIF; HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1 (Wilms Tumor); FGF Receptor Family members (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB (retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR (Androgen Receptor); TSG101; IGF; IGF Receptor; Igf1 (4 variants); Igf2 (3 variants); Igf 1 Receptor; Igf 2 Receptor; Bax; Bcl2; caspases family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; Apc.

[0217] In some embodiments, the target DNA is associated with age-related macular degeneration, said DNA being selected from Abcr; Ccl2; Cc2; cp (ceruloplasmin); Timp3; cathepsinD; Vldlr; Ccr2.

[0218] In some embodiments, the target DNA is associated with a CNS disorder, said DNA being selected from Neuregulin1 (Nrg1); Erb4 (receptor for Neuregulin); Complexin1 (Cplx1); Tph1 Tryptophan hydroxylase; Tph2 Tryptophan hydroxylase 2; Neurexin 1; GSK3; GSK3a; GSK3b; 5-HTT (Slc6a4); COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1); Mecp2; BZRAP1; MDGA2; Sema5A; Neurexin 1; Fragile X (FMR2 (AFF2); FXR1; FXR2; Mglur5); E1; CHIP; UCH; UBB; Tau; LRP; PICALM; Clusterin; PS1; SORL1; CR1; Vldlr; Uba1; Uba3; CHIP28 (Aqp1, Aquaporin 1); Uchl1; Uchl3; APP; x-Synuclein; DJ-1; LRRK2; Parkin; PINK1.

[0219] In some embodiments, the target DNA is associated with trinucleotide repeat disorders, said DNA being selected from HTT (Huntington's Dx); SBMA/SMAX1/AR (Kennedy's Dx); FXN/X25 (Friedrich's Ataxia); ATX3 (Machado-Joseph's Dx); ATXN1 and ATXN2 (spinocerebellar ataxias); DMPK (myotonic dystrophy); Atrophin-1 and Atn1 (DRPLA Dx); CBP (Creb-BP—global instability); VLDLR (Alzheimer's); Atxn7; Atxn10.

[0220] In some embodiments, the target DNA is associated with Fragile X Syndrome, said DNA being selected from FMR2; FXR1; FXR2; mGLUR5.

[0221] In some embodiments, the target DNA is associated with secretase related disorders, said DNA being selected from APH-1 (alpha and beta); Presenilin (Psen1); nicastrin (Ncstn); PEN-2; Nos1; Parp1; Nat1; Nat2.

[0222] In some embodiments, the target DNA is associated with prion-related disorders, said DNA being Prp.

[0223] In some embodiments, the target DNA is associated with ALS, said DNA being selected from SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c).

[0224] In some embodiments, the target DNA is associated with addiction, said DNA being selected from Prkce (alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2; Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol).

[0225] In some embodiments, the target DNA is associated with inflammation, said DNA being selected from IL-10; IL-1 (IL-1a; IL-1b); IL-13; IL-17 (IL-17a (CTLA8); IL-17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1; ptpn22; TNFa; NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4; Cx3cl1.

[0226] In some embodiments, the target DNA is associated with blood and coagulation diseases and disorders, said DNA being selected from CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT, TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5, TBXA2R, P2RX1, P2X1, HF1, CFH, HUS, MCFD2, F7, F10, F11, F12, HAF, F13A1, F13A, F13B, FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, F1134064, FANCB, FANCC, FACC, BRCA2, FANCD1, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1, BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596, PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3, F8, F8C, HEMA, F9, HEMB, PI, ATT, F5, ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4, HBB, HBA2, HBB, HBD, LCRB, HBA1.

[0227] In some embodiments, the target DNA is associated with cell dysregulation and oncology diseases and disorders, said DNA being selected from BCL7A, BCL7, TAL1, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3, FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN.

[0228] In some embodiments, the target DNA is associated with inflammation and immune related diseases and disorders, said DNA being selected from KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1, TNFRSF6, APT1, FAS, CD95, ALPS1A, IL2RG, SCIDX1, SCIDX, IMD4, CCL5, SCYA5, D17S136E, TCP228, IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI, IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3cr1, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1, JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4).

[0229] In some embodiments, the target DNA is associated with metabolic, liver, kidney and protein diseases and disorders, said DNA being selected from TTR, PALB, APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PALB, KRT18, KRT8, CIRH1A, NAIC, TEX292, KIAA1988, CFTR, ABCC7, CF, MRP7, SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM, TCF1, HNF1A, MODY3, SCOD1, SCO1, LIPC, CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5, UMOD, HNFJ, FJHN, MCKD2, ADMCKD2, PAH, PKU1, QDPR, DHPR, PTS, FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63.

[0230] In some embodiments, the target DNA is associated with muscular/skeletal diseases and disorders, said DNA being selected from DMD, BMD, MYF6, DMD, BMD, LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A, FSHMD1A, FSHD1A, FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7, OC116, OPTB1, VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1.

[0231] In some embodiments, the target DNA is associated with neurological and neuronal diseases and disorders, said DNA being selected from SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c, APP, AAA, CVAP, AD1, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3, Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR2, FXR1, FXR2, mGLUR5, HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17, NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2, MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-1, Neuregulin1 (Nrg1), Erb4 (receptor for Neuregulin), Complexin1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1), APH-1 (alpha and beta), Presenilin (Psen1), nicastrin, (Ncstn), PEN-2, Nos1, Parp1, Nat1, Nat2, HTT (Huntington's Dx), SBMA/SMAX1/AR (Kennedy's Dx), FXN/X25 (Friedrich's Ataxia), ATX3 (Machado-Joseph's Dx), ATXN1 and ATXN2 (spinocerebellar ataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atn1 (DRPLA Dx), CBP (Creb-BP—global instability), VLDLR (Alzheimer's), Atxn7, Atxn10.

[0232] In some embodiments, the target DNA is associated with ocular diseases and disorders, said DNA being selected from Abcr, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsinD, Vldlr, Ccr2, CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1, APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD, KERA, CNA2, MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A, CRB1, RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3, ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2.

[0233] Compositions comprising a ribonucleoprotein (e.g., a RNP comprising a site-directed modifying polypeptide, such as a Cas9 RNP or a Cpf1 RNP) and an endosomal escape agent, as described herein, are useful in selectively modulating transcription of a target DNA in a subject, said DNA being associated with a cellular function as described herein.

[0234] In some embodiments, the target DNA is associated with PI3K/AKT Signaling, said DNA being selected from PRKCE; ITGAM; ITGA5; IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; PRKCZ; GRK6; MAPK1; TSC1; PLK1; AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8; BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1; MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB; DYRK1A; CDKN1A; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK; PDPK1; PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN; ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1; RPS6KB1.

[0235] In some embodiments, the target DNA is associated with ERK/MAPK Signaling, said DNA being selected from PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2; RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8; MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3; ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF; STAT1; SGK.

[0236] In some embodiments, the target DNA is associated with Glucocorticoid Receptor Signaling, said DNA being selected from RAC1; TAF4B; EP300; SMAD2; TRAF6; PCAF; ELK1; MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA; CREB1; FOS; HSPA5; NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8; BCL2L1; MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A; MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3; MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8; NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1; SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1.

[0237] In some embodiments, the target DNA is associated with Axonal Guidance Signaling, said DNA being selected from PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; IGF1; RAC1; RAP1A; E1F4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ; PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA.

[0238] In some embodiments, the target DNA is associated with Ephrin Receptor Signaling, said DNA being selected from PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; PRKAA2; EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1; AKT2; DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4, AKT1; JAK2; STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK; CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3; SGK.

[0239] In some embodiments, the target DNA is associated with Actin Cytoskeleton Signaling, said DNA being selected from ACTN4; PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; PRKAA2; EIF2AK2; RAC1; INS; ARHGEF7; GRK6; ROCK2; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; PTK2; CFL1; PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN; VIL2; RAF1; GSN; DYRK1A; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1; PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF; VAV3; SGK.

[0240] In some embodiments, the target DNA is associated with Huntington's Disease Signaling, said DNA being selected from PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2; MAPK1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2; PIK3CA; HDAC5; CREB1; PRKC1; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1; GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11; MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1; CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; AKT3; PRKCA; CLTC; SGK; HDAC6; CASP3.

[0241] In some embodiments, the target DNA is associated with Apoptosis Signaling, said DNA being selected from PRKCE; ROCK1; BID; IRAK1; PRKAA2; EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2L1; CAPN1; MAPK3; CASP8; KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG; RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA; CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1.

[0242] In some embodiments, the target DNA is associated with B Cell Receptor Signaling, said DNA being selected from RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; AKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3; MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9; EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN; GSK3B; ATF4; AKT3; VAV3; RPS6KB1.

[0243] In some embodiments, the target DNA is associated with Leukocyte Extravasation Signaling, said DNA being selected from ACTN4; CD44; PRKCE; ITGAM; ROCK1; CXCR4; CYBA; RAC1; RAP1A; PRKCZ; ROCK2; RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8; PRKD1; ABL1; MAPK10; CYBB; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A; BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1; CTNNB1; CLDN1; CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9.

[0244] In some embodiments, the target DNA is associated with Integrin Signaling, said DNA being selected from ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1; ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3; MAPK8; CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7; PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1; TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3.

[0245] In some embodiments, the target DNA is associated with Acute Phase Response Signaling, said DNA being selected from IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11; AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8; RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1; TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3; IL1R1; IL6.

[0246] In some embodiments, the target DNA is associated with PTEN Signaling, said DNA being selected from ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11; MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2; PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1; IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1; MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1; CASP3; RPS6KB1.

[0247] In some embodiments, the target DNA is associated with p53 Signaling, said DNA being selected from PTEN; EP300; BBC3; PCAF; FASN; BRCA1; GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3; MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1; HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1; RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2; GSK3B; BAX; AKT3.

[0248] In some embodiments, the target DNA is associated with Aryl Hydrocarbon Receptor Signaling, said DNA being selected from HSPB1; EP300; FASN; TGM2; RXRA; MAPK1; NQO1; NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; SMARCA4; NFKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73; GSTP1; RB1; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2; APAF1; NFKB1; CCND1; ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; IL6; CYP1B1; HSP90AA1.

[0249] In some embodiments, the target DNA is associated with Xenobiotic Metabolism Signaling, said DNA being selected from PRKCE; EP300; PRKCZ; RXRA; MAPK1; NQO1; NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB; PPP2R1A; PIK3C3; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13; PRKCD; GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A; PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1; NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1.

[0250] In some embodiments, the target DNA is associated with SAPK/JNK Signaling, said DNA being selected from PRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2; PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1; IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3; CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK.

[0251] In some embodiments, the target DNA is associated with PPAr/RXR Signaling, said DNA being selected from PRKAA2; EP300; INS; SMAD2; TRAF6; PPARA; FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB; NCOR2; ABCA1; GNAQ; NFKB2; MAP3K14; STAT5B; MAPK8; IRS1; MAPK3; KRAS; RELA; PRKAA1; PPARGC1A; NCOA3; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1; ADIPOQ.

[0252] In some embodiments, the target DNA is associated with NF-KB Signaling, said DNA being selected from IRAK1; EIF2AK2; EP300; INS; MYD88; PRKCZ: TRAF6; TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2; MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A; TRAF2; TLR4: PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1; PIK3R1; CHUK; PDGFRA; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1.

[0253] In some embodiments, the target DNA is associated with Neuregulin Signaling, said DNA being selected from ERBB4; PRKCE; ITGAM; ITGA5: PTEN; PRKCZ; ELK1; MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3; ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17; AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC; NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1.

[0254] In some embodiments, the target DNA is associated with Wnt & Beta catenin Signaling, said DNA being selected from CD44; EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; AKT2; PIN1; CDH1; BTRC; GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2: ILK; LEF1; SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1; TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2.

[0255] In some embodiments, the target DNA is associated with Insulin Receptor Signaling, said DNA being selected from PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; PTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3; TSC2; KRAS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B; AKT3; FOXO1; SGK; RPS6KB1.

[0256] In some embodiments, the target DNA is associated with IL-6 Signaling, said DNA being selected from HSPB1; TRAF6; MAPKAPK2; ELK1; MAPK1; PTPN11; IKBKB; FOS; NFKB2: MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1; CEBPB; JUN; IL1R1; SRF; IL6.

[0257] In some embodiments, the target DNA is associated with Hepatic Cholestasis, said DNA being selected from PRKCE; IRAK1; INS; MYD88; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8; PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN; IL1R1; PRKCA; IL6.

[0258] In some embodiments, the target DNA is associated with IGF-1 Signaling, said DNA being selected from IGF1; PRKCZ; ELK1; MAPK1; PTPN11; NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R; IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF; CTGF; RPS6KB1.

[0259] In some embodiments, the target DNA is associated with NRF2-mediated Oxidative Stress Response, said DNA being selected from PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1; NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A; MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN; KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1.

[0260] In some embodiments, the target DNA is associated with Hepatic Fibrosis/Hepatic Stellate Cell Activation, said DNA being selected from EDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; SMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4; PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1; CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9.

[0261] In some embodiments, the target DNA is associated with PPAR Signaling, said DNA being selected from EP300; INS; TRAF6; PPARA; RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3; NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF; INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1; NFKB1; JUN; IL1R1; HSP90AA1.

[0262] In some embodiments, the target DNA is associated with Fc Epsilon RI Signaling, said DNA being selected from PRKCE; RAC1; PRKCZ; LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK; MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; VAV3; PRKCA.

[0263] In some embodiments, the target DNA is associated with G-Protein Coupled Receptor Signaling, said DNA being selected from PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB; PIK3CA; CREB1; GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3; MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1; PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCA.

[0264] In some embodiments, the target DNA is associated with Inositol Phosphate Metabolism, said DNA being selected from PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; MAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1; MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK.

[0265] In some embodiments, the target DNA is associated with PDGF Signaling, said DNA being selected from EIF2AK2; ELK1; ABL2; MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3; KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA; STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2.

[0266] In some embodiments, the target DNA is associated with VEGF Signaling, said DNA being selected from ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA; ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA; AKT3; FOXO1; PRKCA.

[0267] In some embodiments, the target DNA is associated with Natural Killer Cell Signaling, said DNA being selected from PRKCE; RAC1; PRKCZ; MAPK1; RAC2; PTPN11; KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; PRKD1; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4; AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA.

[0268] In some embodiments, the target DNA is associated with Cell Cycle: G1/S Checkpoint Regulation, said DNA being selected from HDAC4; SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; ATR; ABL1; E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1; HDAC6.

[0269] In some embodiments, the target DNA is associated with T Cell Receptor Signaling, said DNA being selected from RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS; NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA, PIK3C2A; BTK; LCK; RAF1; IKBKG; RELB, FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK; BCL10; JUN; VAV3.

[0270] In some embodiments, the target DNA is associated with Death Receptor Signaling, said DNA being selected from CRADD; HSPB1; BID; BIRC4; TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX; TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1; CASP2; BIRC2; CASP3; BIRC3.

[0271] In some embodiments, the target DNA is associated with FGF Signaling, said DNA being selected from RAC1; FGFR1; MET; MAPKAPK2; MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3; MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1; FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF.

[0272] In some embodiments, the target DNA is associated with GM-CSF Signaling, said DNA being selected from LYN; ELK1; MAPK1; PTPN11; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1; MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1.

[0273] In some embodiments, the target DNA is associated with Amyotrophic Lateral Sclerosis Signaling, said DNA being selected from BID; IGF1; RAC1; BIRC4; PGF; CAPNS1; CAPN2; PIK3CA; BCL2; PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3.

[0274] In some embodiments, the target DNA is associated with JAK/Stat Signaling, said DNA being selected from PTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS; SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1.

[0275] In some embodiments, the target DNA is associated with Nicotinate and Nicotinamide Metabolism, said DNA being selected from PRKCE; IRAK1; PRKAA2; EIF2AK2; GRK6; MAPK1; PLK1; AKT2; CDK8; MAPK8; MAPK3; PRKCD; PRKAA1; PBEF1; MAPK9; CDK2; PIM1; DYRK1A; MAP2K2; MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK.

[0276] In some embodiments, the target DNA is associated with Chemokine Signaling, said DNA being selected from CXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8; MAPK3; KRAS; MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1; JUN; CCL2; PRKCA.

[0277] In some embodiments, the target DNA is associated with IL-2 Signaling, said DNA being selected from ELK1; MAPK1; PTPN11; AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3.

[0278] In some embodiments, the target DNA is associated with Synaptic Long Term Depression, said DNA being selected from PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1; GNAS; PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCA.

[0279] In some embodiments, the target DNA is associated with Estrogen Receptor Signaling, said DNA being selected from TAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2; SMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2.

[0280] In some embodiments, the target DNA is associated with Protein Ubiquitination Pathway, said DNA being selected from TRAF6; SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; CBL; UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1; VHL; HSP90AA1; BIRC3.

[0281] In some embodiments, the target DNA is associated with IL-10 Signaling, said DNA being selected from TRAF6; CCR1; ELK1; IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN; IL1R1; IL6.

[0282] In some embodiments, the target DNA is associated with VDR/RXR Activation, said DNA being selected from PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKC1; CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1; LRP5; CEBPB; FOXO1; PRKCA.

[0283] In some embodiments, the target DNA is associated with TGF-beta Signaling, said DNA being selected from EP300; SMAD2; SMURF1; MAPK1; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1; RAF1; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5.

[0284] In some embodiments, the target DNA is associated with Toll-like Receptor Signaling, said DNA being selected from IRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN.

[0285] In some embodiments, the target DNA is associated with p38 MAPK Signaling, said DNA being selected from HSPB1; IRAK1; TRAF6; MAPKAPK2; ELK1; FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF; STAT1.

[0286] In some embodiments, the target DNA is associated with Neurotrophin/TRK Signaling, said DNA being selected from NTRK2; MAPK1; PTPN11; PIK3CA; CREB1; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42; JUN; and ATF4.

[0287] In some embodiments, the target DNA is associated with FXR/RXR Activation, said DNA being selected from INS; PPARA; FASN; RXRA; AKT2; SDC1; MAPK8; APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; and FOXO1.

[0288] In some embodiments, the target DNA is associated with Synaptic Long Term Potentiation, said DNA being selected from PRKCE; RAP1A; EP300; PRKCZ; MAPK1; CREB1; PRKCI; GNAQ; CAMK2A; PRKD1; MAPK3; KRAS; PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4; and PRKCA.

[0289] In some embodiments, the target DNA is associated with Calcium Signaling, said DNA being selected from RAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1; CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4; and HDAC6.

[0290] In some embodiments, the target DNA is associated with EGF Signaling, said DNA being selected from ELK1; MAPK1; EGFR; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; and STAT1.

[0291] In some embodiments, the target DNA is associated with Hypoxia Signaling in the Cardiovascular System, said DNA being selected from EDN1; PTEN; EP300; NQO1; UBE2I; CREB1; ARNT; HIF1A; SLC2A4; NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN; ATF4; VHL; and HSP90AA1.

[0292] In some embodiments, the target DNA is associated with LPS/IL-1 Mediated Inhibition of RXR Function, said DNA being selected from IRAK1; MYD88; TRAF6; PPARA; RXRA; ABCA1, MAPK8; ALDH1A1; GSTP1; MAPK9; ABCB1; TRAF2; TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN; and IL1R1.

[0293] In some embodiments, the target DNA is associated with LXR/RXR Activation, said DNA being selected from FASN; RXRA; NCOR2; ABCA1; NFKB2; IRF3; RELA; NOS2A; TLR4; TNF; RELB; LDLR; NR1H2; NFKB1; SREBF1; IL1R1; CCL2; IL6; and MMP9.

[0294] In some embodiments, the target DNA is associated with Amyloid Processing, said DNA being selected from PRKCE; CSNK1E; MAPK1; CAPNS1; AKT2; CAPN2; CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1; GSK3B; AKT3; and APP.

[0295] In some embodiments, the target DNA is associated with IL-4 Signaling, said DNA being selected from AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1; KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1; AKT3; and RPS6KB1.

[0296] In some embodiments, the target DNA is associated with Cell Cycle: G2/M DNA Damage Checkpoint Regulation, said DNA being selected from EP300; PCAF; BRCA1; GADD45A; PLK1; BTRC; CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A; PRKDC; ATM; SFN; and CDKN2A.

[0297] In some embodiments, the target DNA is associated with Nitric Oxide Signaling in the Cardiovascular System, said DNA being selected from KDR; FLT1; PGF; AKT2; PIK3CA; PIK3CB; PIK3C3; CAV1; PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1; VEGFA; AKT3; and HSP90AA1.

[0298] In some embodiments, the target DNA is associated with Purine Metabolism, said DNA being selected from NME2; SMARCA4; MYH9; RRM2; ADAR; EIF2AK4; PKM2; ENTPD1; RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; and NME1.

[0299] In some embodiments, the target DNA is associated with cAMP-mediated Signaling, said DNA being selected from RAP1A; MAPK1; GNAS; CREB1; CAMK2A; MAPK3; SRC; RAF1; MAP2K2; STAT3; MAP2K1; BRAF; and ATF4.

[0300] In some embodiments, the target DNA is associated with Mitochondrial Dysfunction, said DNA being selected from SOD2; MAPK8; CASP8; MAPK10; MAPK9; CASP9; PARK7; PSEN1; PARK2; APP; and CASP3.

[0301] In some embodiments, the target DNA is associated with Notch Signaling, said DNA being selected from HES1; JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3; NOTCH1; and DLL4.

[0302] In some embodiments, the target DNA is associated with Endoplasmic Reticulum Stress Pathway, said DNA being selected from HSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4; EIF2AK3; and CASP3.

[0303] In some embodiments, the target DNA is associated with Pyrimidine Metabolism, said DNA being selected from NME2; AICDA; RRM2; EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; and NME1.

[0304] In some embodiments, the target DNA is associated with Parkinson's Signaling, said DNA being selected from UCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; and CASP3.

[0305] In some embodiments, the target DNA is associated with Cardiac & Beta Adrenergic Signaling, said DNA being selected from GNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; and PPP2R5C.

[0306] In some embodiments, the target DNA is associated with Glycolysis/Gluconeogenesis, said DNA being selected from HK2; GCK; GPI; ALDH1A1; PKM2; LDHA; and HK1.

[0307] In some embodiments, the target DNA is associated with Interferon Signaling, said DNA being selected from IRF1; SOCS1; JAK1; JAK2; IFITM1; STAT1; and IFIT3.

[0308] In some embodiments, the target DNA is associated with Sonic Hedgehog Signaling, said DNA being selected from ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B; and DYRKIB.

[0309] In some embodiments, the target DNA is associated with Glycerophospholipid Metabolism, said DNA being selected from PLD1; GRN; GPAM; YWHAZ; SPHK1; and SPHK2.

[0310] In some embodiments, the target DNA is associated with Phospholipid Degradation, said DNA being selected from PRDX6; PLD1; GRN; YWHAZ; SPHK1; and SPHK2.

[0311] In some embodiments, the target DNA is associated with Tryptophan Metabolism, said DNA being selected from SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1; and SIAH1.

[0312] In some embodiments, the target DNA is associated with Lysine Degradation, said DNA being selected from SUV39H1; EHMT2; NSD1; SETD7; and PPP2R5C.

[0313] In some embodiments, the target DNA is associated with Nucleotide Excision Repair Pathway, said DNA being selected from ERCC5; ERCC4; XPA; XPC; and ERCC1.

[0314] In some embodiments, the target DNA is associated with Starch and Sucrose Metabolism, said DNA being selected from UCHL1; HK2; GCK; GPI; and HK1.

[0315] In some embodiments, the target DNA is associated with Aminosugars Metabolism, said DNA being selected from NQO1; HK2; GCK; and HK1.

[0316] In some embodiments, the target DNA is associated with Arachidonic Acid Metabolism, said DNA being selected from PRDX6; GRN; YWHAZ; and CYP1B1.

[0317] In some embodiments, the target DNA is associated with Circadian Rhythm Signaling, said DNA being selected from CSNK1E; CREB1; ATF4; and NR1D1.

[0318] In some embodiments, the target DNA is associated with Coagulation System, said DNA being selected from BDKRB1; F2R; SERPINE1; and F3.

[0319] In some embodiments, the target DNA is associated with Dopamine Receptor Signaling, said DNA being selected from PPP2R1A; PPP2CA; PPP1CC; and PPP2R5C.

[0320] In some embodiments, the target DNA is associated with Glutathione Metabolism, said DNA being selected from IDH2; GSTP1; ANPEP; and IDH1.

[0321] In some embodiments, the target DNA is associated with Glycerolipid Metabolism, said DNA being selected from ALDH1A1; GPAM; SPHK1; and SPHK2.

[0322] In some embodiments, the target DNA is associated with Linoleic Acid Metabolism, said DNA being selected from PRDX6; GRN; YWHAZ; and CYP1B1.

[0323] In some embodiments, the target DNA is associated with Methionine Metabolism, said DNA being selected from DNMT1; DNMT3B; AHCY; and DNMT3A.

[0324] In some embodiments, the target DNA is associated with Pyruvate Metabolism, said DNA being selected from GLO1; ALDH1A1; PKM2; and LDHA.

[0325] In some embodiments, the target DNA is associated with Arginine and Proline Metabolism, said DNA being selected from ALDH1A1; NOS3; and NOS2A.

[0326] In some embodiments, the target DNA is associated with Eicosanoid Signaling, said DNA being selected from PRDX6; GRN; and YWHAZ.

[0327] In some embodiments, the target DNA is associated with Fructose and Mannose Metabolism, said DNA being selected from HK2; GCK; and HK1.

[0328] In some embodiments, the target DNA is associated with Galactose Metabolism, said DNA being selected from HK2; GCK; and HK1.

[0329] In some embodiments, the target DNA is associated with Stilbene, Coumarine and Lignin Biosynthesis, said DNA being selected from PRDX6; PRDX1; and TYR.

[0330] In some embodiments, the target DNA is associated with Antigen Presentation Pathway, said DNA being selected from CALR; and B2M.

[0331] In some embodiments, the target DNA is associated with Biosynthesis of Steroids, said DNA being selected from NQO1; and DHCR7.

[0332] In some embodiments, the target DNA is associated with Butanoate Metabolism, said DNA being selected from ALDH1A1; and NLGN1.

[0333] In some embodiments, the target DNA is associated with Citrate Cycle, said DNA being selected from IDH2; and IDH1.

[0334] In some embodiments, the target DNA is associated with Fatty Acid Metabolism, said DNA being selected from ALDH1A1; and CYP1B1.

[0335] In some embodiments, the target DNA is associated with Glycerophospholipid Metabolism, said DNA being selected from PRDX6; and CHKA.

[0336] In some embodiments, the target DNA is associated with Histidine Metabolism, said DNA being selected from PRMT5; and ALDH1A1.

[0337] In some embodiments, the target DNA is associated with Inositol Metabolism, said DNA being selected from ERO1L; and APEX1.

[0338] In some embodiments, the target DNA is associated with Metabolism of Xenobiotics by Cytochrome p450, said DNA being selected from GSTP1; and CYP1B1.

[0339] In some embodiments, the target DNA is associated with Methane Metabolism, said DNA being selected from PRDX6; and PRDX1.

[0340] In some embodiments, the target DNA is associated with Phenylalanine Metabolism, said DNA being selected from PRDX6; and PRDX1.

[0341] In some embodiments, the target DNA is associated with Propanoate Metabolism, said DNA being selected from ALDH1A1; and LDHA.

[0342] In some embodiments, the target DNA is associated with Selenoamino Acid Metabolism, said DNA being selected from PRMT5; and AHCY.

[0343] In some embodiments, the target DNA is associated with Sphingolipid Metabolism, said DNA being selected from SPHK1; and SPHK2.

[0344] In some embodiments, the target DNA is associated with Aminophosphonate Metabolism, said DNA being selected from PRMT5.

[0345] In some embodiments, the target DNA is associated with Androgen and Estrogen Metabolism, said DNA being PRMT5.

[0346] In some embodiments, the target DNA is associated with Ascorbate and Aldarate Metabolism, said DNA being ALDH1A1.

[0347] In some embodiments, the target DNA is associated with Bile Acid Biosynthesis, said DNA being ALDH1A1.

[0348] In some embodiments, the target DNA is associated with Cysteine Metabolism, said DNA being LDHA.

[0349] In some embodiments, the target DNA is associated with Fatty Acid Biosynthesis, said DNA being FASN.

[0350] In some embodiments, the target DNA is associated with Glutamate Receptor Signaling, said DNA being GNB2L1.

[0351] In some embodiments, the target DNA is associated with NRF2-mediated Oxidative Stress Response, said DNA being PRDX1.

[0352] In some embodiments, the target DNA is associated with Pentose Phosphate Pathway, said DNA being GPI.

[0353] In some embodiments, the target DNA is associated with Pentose and Glucuronate Interconversions, said DNA being UCHL1.

[0354] In some embodiments, the target DNA is associated with Retinol Metabolism, said DNA being ALDH1A1.

[0355] In some embodiments, the target DNA is associated with Riboflavin Metabolism, said DNA being TYR.

[0356] In some embodiments, the target DNA is associated with Tyrosine Metabolism, said DNA being selected from PRMT5, and TYR.

[0357] In some embodiments, the target DNA is associated with Ubiquinone Biosynthesis, said DNA being PRMT5.

[0358] In some embodiments, the target DNA is associated with Valine, Leucine and Isoleucine Degradation, said DNA being ALDH1A1.

[0359] In some embodiments, the target DNA is associated with Lysine Degradation, said DNA being ALDH1A1.

[0360] In some embodiments, the target DNA is associated with Glycine, Serine and Threonine Metabolism, said DNA being CHKA.

[0361] In some embodiments, the target DNA is associated with Pain or Taste, said DNA being selected from TRPM5; TRPA1; TRPM7; TRPC5; TRPC6; TRPC1; Cnr1; cnr2; Grk2; Trpa1; Pomc; Cgrp; Crf; Pka; Era; Nr2b; TRPM5; Prkaca; Prkacb; Prkar1a; and Prkar2a.

[0362] In some embodiments, the target DNA is associated with Mitochondrial Function, said DNA being selected from AIF; CytC; SMAC (Diablo); Aifm-1; and Aifm-2.

[0363] In some embodiments, the target DNA is associated with Developmental Neurology, said DNA being selected from BMP-4; Chordin (Chrd); Noggin (Nog); WNT (Wnt2; Wnt2b; Wnt3a; Wnt4; Wnt5a; Wnt6; Wnt7b; Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b; Wnt16); beta-catenin; Dkk-1; Frizzled related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dabl; unc-86 (Pou4fl or Brn3a); Numb; Reln.

[0364] Another aspect of the present invention provides a method for site-specific endonucleolytic cleavage of a single-stranded RNA (ssRNA), comprising the administration of a compound, a RNP, or a composition described herein, in the presence of a protospacer adjacent motif (PAM)-presenting oligonucleotides (PAMmer). Suitable PAMmers for use in the present invention include but are not limited to those described in O'Connell, M. R. et al. Nature, 2014, 516 (7530): 263-6.

[0365] Another aspect of the present invention provides a method for site-specific endonucleolytic cleavage of a single-stranded RNA (ssRNA), comprising the administration of a compound or RNP, which comprises a CRISPR/Cas Type III-B Cmr complex, or a composition thereof, as described herein. In some embodiments, the Type III-B Cmr complex may be derived from Pyrococcus furiosus, Sulfolobus solfataricus, or Thermus thermophilus. In some embodiments, the Cmr proteins suitable for use herein include but are not limited to those described in Hale, C. R. et al. Genes & Development, 2014, 28:2432-2443, and Makarova K. S. et al. Nature Reviews Microbiology, 2015, 13, 1-15.

[0366] Embodiments of the present invention are illustrated by the following Examples. It is to be understood, however, that the embodiments of the invention are not limited to the specific details of these Examples, as other variations thereof will be known, or apparent in light of the instant disclosure, to one of ordinary skill in the art.

EXAMPLES

[0367] Unless specified otherwise, starting materials are generally available from commercial sources such as Aldrich Chemicals Co. (Milwaukee, Wis.), Lancaster Synthesis, Inc. (Windham, N.H.), Acros Organics (Fairlawn, N.J.), Maybridge Chemical Company, Ltd. (Cornwall, England), Tyger Scientific (Princeton, N.J.), AstraZeneca Pharmaceuticals (London, England), and Accela ChemBio (San Diego, Calif.).

General Experimental Procedures

[0368] NMR spectra were recorded on a Varian Unity™ 400 (available from Varian Inc., Palo Alto, Calif.) at room temperature at 400 MHz for proton. Chemical shifts are expressed in parts per million (delta) relative to residual solvent as an internal reference. The peak shapes are denoted as follows: s, singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quartet; m, multiplet; bs or br.s., broad singlet; 2s, two singlets; br.d., broad doublet. In some cases only representative ¹H NMR peaks are given. Column chromatography was performed with either Baker™ silica gel (40 micron; J. T. Baker, Phillipsburg, N.J.) or Silica Gel 50 (EM Sciences™, Gibbstown, N.J.) in glass columns or in Flash 40 Biotage™ columns (ISC, Inc., Shelton, Conn.). MPLC (medium pressure liquid chromatography) was performed using a Biotage™ SP purification system or a Combiflash® Companion® from Teledyne™ Isco™; Biotage™ SNAP cartridge KPsil or Redisep Rf silica (from Teledyne™ Isco™) under low nitrogen pressure were used. Except where otherwise noted, all reactions were run under an inert atmosphere of nitrogen gas using anhydrous solvents. Also, except where otherwise noted, all reactions were run at room temperature (˜23° C.). When doing TLC (thin layer chromatography), R_fis defined as the ratio of the distance traveled by the compound divided by the distance traveled by the eluent. R_t(retention time). H-Cube® Continuous-flow Hydrogenation Reactor: A bench-top standalone hydrogenation reactor, combining continuous-flow microchemistry with endogenous on-demand hydrogen generation and a disposable catalyst cartridge system.

[0369] LC/MS TOF (ESI):

[0370] All data were gathered on an Agilent 1100 LC with MSD TOF (Agilent model G1969A) mass spec detectors running with electrospray spray ionization source. The LC instrument includes a binary pump (Agilent model G1312A) with upper pressure limit of 400 bar attached to autosampler (Agilent model G1313A) which uses external try for sample submission. The column compartment (Agilent model G1316A) which is attached to diode array (Agilent model G1315A). The instrument acquisition and data handling was done with Agilent MassHunter TOF/Q-TOF B.02 (B11285) Patches 1.2.3. Elution Conditions: Column: No column was used. Flow Injection: Injection Volume: 1.0 microL; Flow Rate: 0.5 mL/min. Run Time: 1.0 min; Solvent: Methanol (0.1% formic acid and 0.05% ammonium formate). TOF Conditions: Ionization Source: Electrospray spray ionization source in Positive Mode; Gas Temp: 325 C; Drying Gas: 6 L/min; Nebulizer: 50 psg; VCap: 3500V; Mass Range 110-100 m/z; Acquisition Rate: 0.99 spectra/s: Acquisition Time; 1012.8 ms/spectrum. All solvents were of HPLC Chromasolv grade, from Sigma Aldrich (St. Louis, Mo.). A majority of the chemicals and buffers were purchased from Sigma Aldrich, all 97% in purity or higher.

[0371] Method C 1.5 minute run LRMS (low resolution mass spectroscopy): Waters Acquity HSS T3, 2.1 mm×50 mm, C18, 1.7 μm; Mobile Phase: A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Flow-1.25 ml/minute; Initial conditions: A—95%:B—5%; hold at initial from 0.0-0.1 minute; Linear Ramp to A—5%:B—95% over 0.1-1.0 minute; hold at A—5%:B—95% from 1.0-1.1 minute; return to initial conditions 1.1-1.5 minute.

[0372] Method C 3.0 minute run LRMS (low resolution mass spectroscopy): Waters Acqity HSS T3, 2.1 mm×50 mm, C18, 1.7 μm; Mobile Phase: A: 0.1% formic acid in water (v/v); Mobile phase B: 0.1% formic acid in acetonitrile (v/v); Flow-1.25 ml/minute; Initial conditions: A—95%:B—5%; hold at initial from 0.0-0.1 minute; Linear Ramp to A—5%:B—95% over 0.1-2.6 minute; hold at A—5%:B—95% from 2.6-2.95 minute; return to initial conditions 2.95-3.0 minute.

Procedures

((2R,3S,4R,5R,6R)-5-azido-6-methoxy-3,4-bis((trimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)methanol (I-b)

[0373]

[0374] (2R,3R,4R,5R,6R)-5-azido-2-(hydroxymethyl)-6-methoxytetrahydro-2H-pyran-3,4-diol (I-a) (5 g, 23 mmol) was dissolved in anhydrous pyridine (100 mL) and trimethylsilyl chloride (17.5 mL, 139 mmol) was added. The reaction mixture was stirred for 12 hours at room temperature and then pyridine was evaporated. The residue was taken up in ethyl acetate/water. The aqueous phase was extracted once with ethyl acetate and the combined organic layers were washed with water, a saturated aqueous solution of sodium chloride, dried over magnesium sulfate, filtered and concentrated to give 9.9 g (98% yield) of the corresponding per-silylated compound as a yellow oil. The material was used in the next step without any further purification. To a solution of the above per-silylated compound (9.71 g, 22.3 mmol) in anhydrous methanol (45 mL) cooled to 0 degrees Celsius was added 9.06 mL of a solution of potassium carbonate in methanol (0.032M). The reaction mixture was stirred at 0° C. for 1 hour and then neutralized by the addition of 17 microL of acetic acid. The solvent was evaporated and the residue was dissolved in ethyl acetate. Water was added and the aqueous phase was extracted twice with ethyl acetate. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated. The crude material was purified by flash chromatography (30% ethyl acetate/hexanes) over silica gel to afford 6.77 g (84%) of (I-b) as an oil. [α]_D7 (c 1, chloroform); ¹H NMR (400 MHz, CHLOROFORM-d) delta ppm 0.14 (s, 9H), 0.20 (s, 9H), 1.80 (br. s., 1H), 3.36-3.42 (m, 1H), 3.45 (dd, J=7.3, 4.6 Hz, 1H), 3.54 (dd, J=10.0, 8.0 Hz, 1H), 3.59 (s, 3H), 3.65 (dd, J=11.3, 4.7 Hz, 1H), 3.77 (d, J=2.7 Hz, 1H), 3.87 (dd, J=11.2, 7.3 Hz, 1H), 4.14 (d, J=8.0 Hz, 1H); ¹³C NMR (100 MHz, CHLOROFORM-d) delta ppm 0.27 (3C), 0.6 (3C), 57.3, 62.6, 64.0, 71.1, 73.7, 75.2, 103.4; HRMS (ESI) calcd for C₁₃H₂₉N₃O₅Si₂(m/z) [M+Na]⁺386.1538, found 386.1539.

(3R,4R,5R,6R)-5-azido-2,2-bis(hydroxymethyl)-6-methoxytetrahydro-2H-pyran-3,4-diol (I-c)

[0375]

[0376] (I-b) (7.73 g, 21.3 mmol) was dissolved in dichloromethane (70 mL). Dimethyl sulfoxide (10.6 mL, 150 mmol) and triethylamine (9 mL, 60 mmol) were added and the reaction mixture was cooled to 0 degrees Celsius. Sulfur trioxide pyridine complex (10.2 g, 64 mmol) was added and the mixture was stirred at 0 degrees Celsius for 1 hour and then warmed up to room temperature over 30 minutes. The reaction was quenched with a saturated solution of sodium chloride and diluted with dichloromethane. The aqueous phase was extracted 3 times with dichloromethane and the combined organic layers were washed with a saturated aqueous solution of sodium chloride, dried over magnesium sulfate, filtered and concentrated to afford the corresponding aldehyde. The aldehyde was dissolved in anhydrous ethanol (106 mL) and paraformaldehyde powder (40.3 g, 425 mmol) followed by sodium ethoxide 21% wt solution in ethanol (16 mL, 42.5 mmol) were added. The reaction mixture was stirred at room temperature for 12 hours and then ethanol was evaporated. To the crude mixture was added methanol and the solid was filtered and thoroughly rinsed with methanol. To the filtrate containing the desired product was added silica gel and methanol was evaporated. The resulting dry load was dried under high vacuum and loaded on a column. The crude material was purified by flash chromatography (10% methanol/dichloromethane) over silica gel to give 3.03 g of (I-c) as a colorless oil (57% over 2 steps). [α]_D−20 (c 1.25, methanol); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 3.46 (dd, J=10.2, 8.1 Hz, 1H), 3.51 (s, 3H), 3.64-3.80 (m, 5H), 3.80-3.83 (m, 1H), 4.54 (d, J=8.0 Hz, 1H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 57.2, 61.2, 63.6, 65.9, 69.9, 71.2, 80.9, 101.2; HRMS (ESI) calcd for C₈H₁₅N₃O₆(m/z) [M+Na]⁺272.0853, found 272.0856.

N-((3aR,4S,7S,8R,8aR)-4-(hydroxymethyl)-2,2-dimethylhexahydro-4,7-epoxy[1,3]dioxolo[4,5-d]oxepin-8-yl)acetamide (I-e-1)

[0377]

[0378] To a solution of compound (3) (230 mg, 0.986 mmol) in 6.6 mL of dimethylformamide was added 2,2-dimethoxypropane (0.8 mL, 6 mmol) followed by (+/−)-camphor-10-sulphonic acid (101 mg, 0.435 mmol). The reaction mixture was stirred at 70 degrees Celsius for 24 hours, cooled down to room temperature and then methanol was added (1.2 mL). The reaction mixture was stirred at room temperature for 30 minutes and then neutralized with triethylamine (56 microL). The solvent was evaporated and the residue was coevaporated 3 times with toluene. The crude material was purified by flash chromatography (15/1 ethyl acetate/methanol) over silica gel to afford compound (I-e-1) as a white solid (246 mg, 91% yield). m.p.: 164.7-166.0° C.; [α]_D147 (c 1, methanol); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.34 (s, 3H), 1.48 (s, 3H), 1.98 (s, 3H), 3.77 (d, J=7.8 Hz, 1H), 3.83 (d, J=7.8 Hz, 1H), 3.86 (d, J=11.6 Hz, 1H), 3.90 (d, J=11.3 Hz, 1H), 3.91-3.94 (m, 1H), 4.14-4.19 (m, 1H), 4.29 (d, J=6.0 Hz, 1H), 5.23 (d, J=2.0 Hz, 1H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 22.7, 26.9, 28.5, 56.8, 61.9, 70.2, 76.1, 76.6, 83.0, 102.6, 112.5, 173.6; HRMS (ESI) calcd for C₁₂H₁₉NO₆(m/z) [M+H]⁺274.1285, found 274.1274.

(1S,2R,3R,4R,5S)-4-azido-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3-diol (1)

[0379]

[0380] The tetra-ol (I-c) (3 g, 12 mmol) was dissolved in water (40 mL) and concentrated sulfuric acid (6.7 mL) was added. The reaction mixture was stirred at 100 degrees Celsius for 40 hours, cooled to room temperature, and then neutralized by the addition of concentrated ammonium hydroxide. Water was evaporated and methanol was added to the resulting mixture. The solid was filtered and thoroughly rinsed with methanol. To the filtrate containing the desired product was added silica gel and methanol was evaporated. The resulting dry load was dried under high vacuum and loaded on a column. The crude material was purified by flash chromatography (10% methanol/dichloromethane) over silica gel to give 2.2 g (84%) of (1) as a colorless oil. [α]_D160 (c 1.1, methanol); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 3.35 (dd, J=9.2, 1.6 Hz, 1H), 3.70 (d, J=8.2 Hz, 1H), 3.76 (d, J=8.0 Hz, 1H), 3.80 (d, J=11.3 Hz, 1H), 3.83-3.89 (m, 2H), 3.90 (d, J=11.5 Hz, 1H), 5.32 (d, J=1.4 Hz, 1H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 61.9, 66.1, 69.5, 69.6, 71.0, 85.3, 102.7; HRMS (ESI) calcd for C₇H₁₁N₃O₅(m/z) [M+Na]⁺ 240.0591, found 240.0596.

(1R,2R,3R,4R,5S)-4-acetamido-1-(acetoxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3-diyl diacetate (2)

[0381]

[0382] In a round bottom flask, compound (1) (1.93 g, 8.9 mmol) was dissolved in ethanol (45 mL) and the system was flushed with nitrogen. Lindlar catalyst (1.89 g, 0.9 mmol) was added and the system was flushed with nitrogen and then with hydrogen. The reaction mixture was stirred at room temperature under an atmosphere of hydrogen (using a balloon) for 24 hours. The palladium was filtered using a nylon membrane and thoroughly rinsed with methanol and then water. Solvent was evaporated and the residue was dissolved in water and lyophilized. The resulting crude material was then dissolved in pyridine (40 mL) and acetic anhydride was added (9 mL, 100 mmol). The reaction mixture was stirred at room temperature for 48 hours and the pyridine was evaporated. The residue was dissolved in ethyl acetate and washed with a saturated solution of sodium bicarbonate. The aqueous phase was extracted twice with ethyl acetate and then the combined organic layers were washed with a saturated aqueous solution of sodium chloride, dried over magnesium sulfate, filtered and evaporated. The crude material was purified by flash chromatography (3% methanol/dichloromethane) over silica gel to give (2) (3.19 g, quant.). [α]_D75 (c 1, chloroform); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.95 (s, 3H), 1.95 (s, 3H), 2.04 (s, 3H), 2.15 (s, 3H), 3.75 (d, J=8.6 Hz, 1H), 4.06 (d, J=8.6 Hz, 1H), 4.13 (d, J=11.6 Hz, 1H), 4.20 (d, J=10.6 Hz, 1H), 4.46 (d, J=11.3 Hz, 1H), 5.13 (dd, J=10.4, 4.4 Hz, 1H), 5.35 (d, J=1.0 Hz, 1H), 5.38 (d, J=4.3 Hz, 1H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 20.6, 20.7 (2C), 22.6, 53.3, 63.0, 68.9, 69.1, 70.3, 82.6, 103.0, 171.8, 171.9, 172.1, 173.8; HRMS (ESI) calcd for C₁₅H₂₁NO₉(m/z) [M+H]⁺ 360.1289, found 360.1290.

N-((1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octan-4-yl)acetamide (3)

[0383]

[0384] Compound (2) (3.19 g, 8.88 mmol) was dissolved in tetrahydrofuran (50 mL) and sodium methoxide 0.5M in methanol (100 mL, 50 mmol) was added. The reaction mixture was stirred at room temperature for 12 hours and then neutralized by the addition of H⁺ Amberlyte™ IR-120 resin. The resin was filtered and solvent was evaporated to give 1.71 g of (3) as a white solid (83%). m.p.: 175.7-176.1° C.; [α]_D164 (c 1, methanol); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.99 (s, 3H), 3.68 (d, J=8.1 Hz, 1H), 3.70-3.73 (m, 1H), 3.75 (d, J=7.8 Hz, 1H), 3.81 (d, J=11.3 Hz, 1H), 3.87 (d, J=4.3 Hz, 1H), 3.92 (d, J=11.3 Hz, 1H), 3.95 (dd, J=9.9, 1.1 Hz, 1H), 5.22 (d, J=1.3 Hz, 1H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 22.7, 56.4, 62.1, 69.2, 69.3, 70.6, 85.1, 102.8, 174.1; HRMS (ESI) calcd for C₉H₁₅NO₆(m/z) [M+H]⁺ 234.0972, found 234.0974.

benzyl (4-((2-((1-(1-((1S,2R,3R,4R,5S)-4-acetamido-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octan-1-yl)-2,5,8,11-tetraoxatridecan-13-yl)-1H-1,2,3-triazol-4-yl)methoxy)ethyl)amino)-4-oxobutyl)carbamate (4), benzyl (4-((1,3-bis((1-(1-((1S,2R,3R,4R,5S)-4-acetamido-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octan-1-yl)-2,5,8,11-tetraoxatridecan-13-yl)-1H-1,2,3-triazol-4-yl)methoxy)propan-2-yl)amino)-4-oxobutyl)carbamate (5), benzyl (4-((1,3-bis((1-(1-((1S,2R,3R,4R,5S)-4-acetamido-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octan-1-yl)-2,5,8,11-tetraoxatridecan-13-yl)-1H-1,2,3-triazol-4-yl)methoxy)-2-(((1-(1-((1S,2R,3R,4R,5S)-4-acetamido-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octan-1-yl)-2,5,8,11-tetraoxatridecan-13-yl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)propan-2-yl)amino)-4-oxobutyl)carbamate (6)

[0385]

[0386] In a microwave vial was dissolved compound (I-e-1) (50 mg, 0.18 mmol) in 1 mL of dichloromethane. 12.5M aqueous sodium hydroxide (0.5 mL) was added followed by 15-crown-5-ether (5 microL, 0.02 mmol) and 1-azido-2-(2-(2-(2-iodoethoxy)ethoxy)ethoxy)ethane (described in J. Am. Chem. Soc. 132, 1523 (2010)) (301 mg, 0.915 mmol). The reaction mixture was vigorously stirred at 55° C. for 24 hours. The organic phase was removed and dried over magnesium sulfate, filtered and concentrated. The crude material was purified by flash chromatography (5% methanol/ethyl acetate) over silica gel to afford compound (I-e-2) as an oil (52 mg, 60% yield). [α]_D74 (c 1, chloroform); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.34 (s, 3H), 1.49 (s, 3H), 1.98 (s, 3H), 3.37 (t, J=4.9 Hz, 2H), 3.62-3.71 (m, 14H), 3.75-3.80 (m, 2H), 3.86 (d, J=8.1 Hz, 1H) 3.90-3.97 (m, 2H), 4.12-4.19 (m, 1H), 4.31 (d, J=5.8 Hz, 1H), 5.23 (d, J=2.0 Hz, 1H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 22.7, 26.9, 28.5, 51.9, 56.7, 70.9, 71.1, 71.3, 71.6, 71.7, 71.8, 71.81, 71.82, 72.7, 76.1, 76.5, 82.1, 102.4, 112.4, 173.6; HRMS (ESI) calcd for C₂₀H₃₄N₄O₉(m/z) [M+H]⁺ 475.2399, found 475.2386.

Intermediate (I-f-1) is known and is described in WO06120545.

[0387] Intermediate (I-f-2) can be synthesized as follow: To a solution of Boc-serinol (1000 mg, 5.1 mmol) in tetrahydrofuran (21 mL) was added at room temperature tetrabutylammonium iodide (287 mg, 0.76 mmol), sodium iodide (153 mg, 1.02 mmol) and propargyl bromide (1.8 mL, 16 mmol, 80% in toluene). Potassium hydroxide (569 mg, 10.1 mmol) was added portion wise over 30 minutes and then the mixture was stirred at room temperature for 16 hours. The reaction mixture was diluted with ethyl acetate and water. The aqueous phase was extracted once with ethyl acetate. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The crude material was purified by flash chromatography (30% ethyl acetate/hexanes) over silica gel to afford compound (I-f-2) as an oil (530 mg, 39% yield). ¹H NMR (400 MHz, CHLOROFORM-d/TMS) delta ppm 1.44 (s, 9H), 2.44 (t, J=2.4 Hz, 2H), 3.53-3.67 (m, 4H), 3.92 (br. s., 1H), 4.16 (d, J=2.5 Hz, 4H), 4.90 (br. s., 1H); ¹³C NMR (100 MHz, CHLOROFORM-d/TMS) delta ppm 28.4 (3C), 49.5, 58.5 (2C), 68.6 (2C), 74.6 (2C), 77.2, 79.5 (2C), 155.4; HRMS (ESI) calcd for C₁₄H₂₁NO₄(m/z) [M+H]⁺268.1543, found 268.1536.

Intermediate (I-f-3) is known and is described in R. Roy et al. J. Org. Chem. 73, 5602 (2008).

[0388] Compound (I-f-1), (I-f-2), or (I-f-3) (1 equiv.) was dissolved in dichloromethane (0.2M) and hydrogen chloride 4M in dioxane (5 to 10 equiv.) was added. The reaction mixture was stirred at room temperature for 2-3 hours and then the solvent was evaporated. The residue was dried under high vacuum for 1 hour. The resulting intermediate was used in the next step without any further purification. The above resulting intermediate (1 equiv.) and 4-(((benzyloxy)carbonyl)amino)butanoic acid (1 equiv.) were dissolved in a mixture of dioxane and dimethylformamide (0.09M, 3:1). (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (1.2 equiv.) was added followed by N,N-diisopropylethylamine (5 equiv.). The reaction mixture was stirred at room temperature for 16 hours. Dichloromethane and water were added and the aqueous phase was extracted twice with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The crude material was dissolved in a minimum amount of toluene, loaded on a column and purified by flash chromatography over silica gel.

[0389] Intermediate (I-g-1): Purification conditions: 100% ethyl acetate, quantitative, oil. ¹H NMR (400 MHz, CHLOROFORM-d/TMS) delta ppm 1.80-1.91 (m, 2H), 2.24 (t, J=7.1 Hz, 2H), 2.46 (t, J=2.3 Hz, 1H), 3.22-3.31 (m, 2H), 3.43-3.51 (m, 2H), 3.56-3.64 (m, 2H), 4.16 (d, J=2.3 Hz, 2H), 5.07 (br. s., 1H), 5.10 (s, 2H), 6.09 (br. s., 1H), 7.28-7.42 (m, 5H); ¹³C NMR (100 MHz, CHLOROFORM-d/TMS) delta ppm 25.9, 33.7, 39.1, 40.5, 58.3, 66.7, 68.7, 74.8, 79.4, 128.1, 128.5 (4C), 136.6, 156.7, 172.5; HRMS (ESI) calcd for C₁₇H₂₂N₂O₄(m/z) [M+H]⁺ 319.1652, found 319.1646.

[0390] Intermediate (I-g-2): Purification conditions: 70% ethyl acetate/hexanes, 65 mg, oil (76% yield), oil. ¹H NMR (400 MHz, CHLOROFORM-d/TMS) delta ppm 1.79-1.91 (m, 2H), 2.24 (t, J=7.1 Hz, 2H), 2.44 (t, J=2.4 Hz, 2H), 3.20-3.29 (m, 2H), 3.54-3.69 (m, 4H), 4.16 (d, J=1.5 Hz, 4H), 4.22-4.33 (m, 1H), 5.10 (br. s, 3H), 6.04 (d, J=7.8 Hz, 1H), 7.28-7.42 (m, 5H); ¹³C NMR (100 MHz, CHLOROFORM-d/TMS) delta ppm 25.8, 33.7, 40.4, 48.2, 58.4 (2C), 66.6, 68.3 (2C), 74.7 (2C), 79.4 (2C), 128.1, 128.5 (4C), 136.6, 156.6, 172.2; HRMS (ESI) calcd for C₂₁H₂₆N₂O₅(m/z) [M+H]⁺ 387.1914, found 387.1904.

[0391] Intermediate (I-g-3): Purification conditions: 70% ethyl acetate/hexanes, 42 mg, oil (60% yield). ¹H NMR (400 MHz, CHLOROFORM-0 delta ppm 1.76-1.88 (m, 2H), 2.21 (t, J=7.1 Hz, 2H), 2.44 (t, J=2.3 Hz, 3H), 3.18-3.30 (m, 2H), 3.84 (s, 6H), 4.14 (d, J=2.3 Hz, 6H), 5.10 (s, 2H), 5.12 (br. s., 1H), 5.89 (br. s., 1H), 7.28-7.40 (m, 5H); ¹³C NMR (100 MHz, CHLOROFORM-d/TMS) delta ppm 25.7, 34.3, 40.3, 58.6 (3C), 59.2, 66.6, 68.5 (3C), 74.6 (3C), 79.5 (3C), 128.1, 128.5 (4C), 136.6, 156.6, 172.6; HRMS (ESI) calcd for C₂₅H₃₀N₂O₆(m/z) [M+H]⁺ 455.2177, found 455.2167.

Intermediate (I-h-1):

[0392] tris(3-hydroxypropyltriazolylmethyl)amine (THPTA; see M. G. Finn et al. in Angewandte Chemie International Edition 48, 9879 (2009)) (2 mg, 0.005 mmol) and copper sulfate (1 mg, 0.004 mmol) were dissolved in water (50 microL) and then added to a solution of (I-e-2) (42 mg, 0.089 mmol) and alkyne (I-g-1) (40 mg, 0.125 mmol) in methanol (0.9 mL). Then sodium ascorbate (1.8 mg, 0.009 mmol), dissolved in water (30 microL), was added and the reaction mixture was stirred at room temperature for 24 hours. Solvent was evaporated and the crude material was purified by flash chromatography (5%-10% methanol in dichloromethane) over silica gel to afford the desired compound (I-h-1) as an oil (54 mg, 76% yield); [α]_D48.2 (c 0.54, methanol); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.33 (s, 3H), 1.48 (s, 3H), 1.70-1.83 (m, 2H), 1.98 (s, 3H), 2.21 (t, J=7.4 Hz, 2H), 3.13 (t, J=6.9 Hz, 2H), 3.37 (t, J=5.4 Hz, 2H), 3.51-3.70 (m, 14H), 3.71-3.95 (m, 7H), 4.15 (t, J=6.5 Hz, 1H), 4.29 (d, J=5.8 Hz, 1H), 4.56 (t, J=5.0 Hz, 2H), 4.60 (s, 2H), 5.06 (s, 2H), 5.22 (d, J=1.8 Hz, 1H), 7.25-7.38 (m, 5H), 8.01 (s, 1H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 22.7, 27.0, 27.4, 28.5, 34.4, 40.5, 41.4, 51.6, 56.7, 62.4, 64.9, 67.5, 70.0, 70.5, 70.8, 71.1, 71.5, 71.6, 71.65, 71.7, 71.73, 72.6, 73.8, 76.2, 76.5, 82.1, 102.4, 112.4, 126.1, 129.0, 129.1, 129.6, 138.6, 146.1, 159.0, 173.6, 175.7; HRMS (ESI) calcd for C₃₇H₅₆N₆O₁₃(m/z) [M+H]⁺ 793.3978, found 793.3959.

Intermediate (I-h-2)

[0393] THPTA (22 mg, 0.051 mmol) and copper sulfate (2.5 mg, 0.01 mmol) were dissolved in water (70 microL) and then added to a solution of (I-e-2) (48 mg, 0.1 mmol) and alkyne (I-g-2) (20 mg, 0.051 mmol) in methanol (1 mL). Then sodium ascorbate (4 mg, 0.02 mmol), dissolved in water (30 microL), was added and the reaction mixture was stirred at room temperature for 72 hours. Solvent was evaporated and the residue was taken up in dichloromethane and a saturated aqueous solution of ammonium chloride. The aqueous phase was extracted three times with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The crude material was used in the next step without any further purification.

Intermediate (I-h-3)

[0394] THPTA (34 mg, 0.079 mmol) and copper sulfate (4 mg, 0.016 mmol) were dissolved in water (200 microL) and then added to a solution of (I-e-2) (50 mg, 0.1 mmol) and alkyne (I-g-3) (24 mg, 0.053 mmol) in methanol (1 mL). Then sodium ascorbate (6.5 mg, 0.032 mmol), dissolved in water (30 microL), was added and the reaction mixture was stirred at room temperature for 72 hours. Solvent was evaporated and the residue was taken up in dichloromethane and a saturated aqueous solution of ammonium chloride. The aqueous phase was extracted three times with dichloromethane. The combined organic layers were dried over magnesium sulfate, filtered and concentrated. The crude material was used in the next step without any further purification.

General Procedure for Acetonide Removal:

[0395] Compound (I-h-1), (I-h-2), or (I-h-3) (0.030-0.068 mmol) was dissolved in a mixture of acetic acid, methanol and water (1.6-1.8 mL, 0.5 mL, 0.5 mL respectively) and stirred at 70° C. for 24 hours. Solvent was evaporated and the residue was co-evaporated twice with toluene. The crude material was purified by flash chromatography over silica gel.

(4):

[0396] Purification conditions: 10% methanol in dichloromethane, 43.3 mg, oil (85% yield). [α]_D45 (c 1, methanol); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.72-1.83 (m, 2H), 1.99 (s, 3H), 2.22 (t, J=7.4 Hz, 2H), 3.13 (t, J=6.8 Hz, 2H), 3.37 (t, J=5.4 Hz, 2H), 3.52-3.79 (m, 20H), 3.85-4.00 (m, 3H), 4.57 (t, J=5.0 Hz, 2H), 4.61 (s, 2H), 5.07 (s, 2H), 5.21 (s, 1H), 7.24-7.41 (m, 5H), 8.02 (s, 1H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 22.8, 27.4, 34.4, 40.4, 41.4, 51.6, 56.4, 64.9, 67.5, 69.0, 70.0, 70.1, 70.4, 70.5, 71.4, 71.5 (2C), 71.6, 71.65, 71.7, 72.5, 84.3, 102.6, 126.0, 129.0 (2C), 129.1, 129.6 (2C), 138.6, 145.8, 159.0, 174.0, 175.8; HRMS (ESI) calcd for C₃₄H₅₂N₆O₁₃(m/z) [M+H]⁺ 753.3665, found 753.3679.

(5):

[0397] Purification conditions: 20% methanol in dichloromethane, 25 mg, oil (20% yield over 2 steps). [α]_D56 (c 1.25, methanol); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.72-1.81 (m, 2H), 1.99 (s, 6H), 2.23 (t, J=7.5 Hz, 2H), 3.13 (t, J=6.9 Hz, 2H), 3.50-3.80 (m, 36H), 3.85-3.91 (m, 6H), 3.92-4.00 (m, 4H), 4.13-4.25 (m, 1H), 4.52-4.63 (m, 8H), 5.07 (s, 2H), 5.21 (d, J=1.3 Hz, 2H), 7.23-7.40 (m, 5H), 8.01 (s, 2H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 22.6 (2C), 27.2, 34.2, 41.2, 50.2, 51.4 (2C), 56.2 (2C), 65.0 (2C), 67.3, 68.8 (2C), 69.9 (2C), 70.0 (2C), 70.2 (2C), 70.3 (2C), 71.2 (2C), 71.3 (4C), 71.4 (2C), 71.5 (2C), 71.52 (2C), 72.3 (2C), 84.1 (2C), 102.4 (2C), 125.8 (2C), 128.8 (2C), 128.9, 129.4 (2C), 138.4, 145.6 (2C), 158.8, 173.8 (2C), 175.3; HRMS (ESI) calcd for C₅₅H₈₆N₁₀O₂₃(m/z) [M+H]⁺1255.5940, found 1255.5925.

(6):

[0398] Purification conditions: 20% methanol in dichloromethane, 31 mg, oil (18% yield over 2 steps). [α]_D53 (c 1, methanol); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.65-1.78 (m, 2H), 1.98 (s, 9H), 2.19 (t, J=7.3 Hz, 2H), 3.11 (t, J=6.8 Hz, 2H), 3.51-3.80 (m, 54H), 3.86-3.91 (m, 9H), 3.91-3.99 (m, 6H), 4.51-4.63 (m, 12H), 5.06 (s, 2H), 5.21 (d, J=1.3 Hz, 3H), 7.24-7.40 (m, 5H), 7.98 (s, 3H); HRMS (ESI) calcd for C₇₆H₁₂₀N₁₄O₃₃(m/z) [M+2H]⁺/2 879.4144, found 879.4148.

N-(2-((1-(1-((1S,2R,3R,4R,5S)-4-acetamido-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]octan-1-yl)-2,5,8,11-tetraoxatridecan-13-yl)-1H-1,2,3-triazol-4-yl)methoxy)ethyl)-4-aminobutanamide (7), 4-amino-N-{1,3-bis[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]propan-2-yl}butanamide (8), 4-amino-N-(1,3-bis[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}propan-2-yl)butanamide (9)

[0399]

[0400] In a round bottom flask, compound (4), (5), or (6) (1 equiv.) was dissolved in methanol (0.01M) and the flask was flushed with nitrogen. Palladium on carbon (10%, 0.7 equiv.) was added and the flask was flushed with nitrogen and then with hydrogen. The reaction mixture was stirred at room temperature for 12-24 hours under an atmosphere of hydrogen (a balloon filled with hydrogen was used). The palladium was filtered using a 0.45 microm PTFE Acrodisc Cr and rinsed once with methanol. Solvent was evaporated.

(7):

[0401] 25.5 mg, oil, 76% yield; [α]_D57.6 (c 1.25, methanol); ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.70-1.81 (m, 2H), 1.99 (s, 3H), 2.24 (t, J=7.4 Hz, 2H), 2.67 (t, J=6.8 Hz, 2H), 3.36-3.41 (m, 2H), 3.51-3.80 (m, 19H), 3.84-4.01 (m, 4H), 4.59 (t, J=5.2 Hz, 2H), 4.61 (s, 2H), 5.21 (s, 1H), 8.03 (s, 1H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 22.8, 29.6, 34.5, 40.4, 42.0, 51.6, 56.4, 64.9, 69.0, 70.0, 70.1, 70.5, 70.6, 71.4, 71.5 (2C), 71.6, 71.66, 71.7, 72.5, 84.3, 102.6, 126.0, 145.8, 174.1, 175.9; HRMS (ESI) calcd for C₂₆H₄₆N₆O₁₁(m/z) [M+H]⁺ 619.3297, found 619.3278.

(8):

[0402] The crude material was dissolved in 0.5 mL methanol/water (50:50) and injected on a HPLC column. Preparative high-performance liquid chromatography (HPLC) was performed using a Waters XBridge BEH C18 OBD Prep Column, 130 Å, 5 microm, 19 mm×100 mm (Waters, part number 186002978), eluting with a linear slope gradient at 17 mL/min flow rate. Solvent gradient: acetonitrile/water/trifluoroacetic acid (2:98:0.1) to (22:58:0.1) in 40 min. Collected fractions were analyzed by analytical LCMS, and the fractions at 25.7-27.3 minutes judged as having adequate purity were pooled and evaporated to afford 10.7 mg of (8) as an oil, 49% yield; [α]_D56 (c 1, methanol); ¹H NMR (500 MHz, METHANOL-d₄) delta ppm 1.86-1.95 (m, 2H), 1.99 (s, 6H), 2.37 (t, J=7.0 Hz, 2H), 2.96 (t, J=7.4 Hz, 2H), 3.50-3.80 (m, 36H), 3.84-4.00 (m, 10H), 4.17-4.26 (m, 1H), 4.57-4.62 (m, 8H), 5.21 (s, 2H), 8.03 (s, 2H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 22.8 (2C), 29.2, 34.5, 41.8, 50.4, 51.6 (2C), 56.4 (2C), 65.1 (2C), 69.0 (2C), 70.1 (2C), 70.3 (2C), 70.5 (2C), 70.6 (2C), 71.4 (2C), 71.5 (4C), 71.6 (2C), 7.67, (2C), 71.7 (2C), 72.5 (2C), 84.3 (2C), 102.6 (2C), 126.1 (2C), 145.8 (2C), 174.1 (2C), 175.6; HRMS (ESI) calcd for C₄₇H₈₀N₁₀O₂₁(m/z) [M+H]⁺1121.5572, found 1121.5558.

(9):

[0403] The crude material was dissolved in 0.5 mL methanol/water (50:50), and injected on HPLC column. Preparative high-performance liquid chromatography (HPLC) was performed using a Waters XBridge BEH C18 OBD Prep Column, 130 Å, 5 microm, 19 mm×100 mm (Waters, part number 186002978), eluting with a linear slope gradient at 17 mL/min flow rate. Solvent gradient: acetonitrile/water/trifluoroacetic acid (2:98:0.1) to (22:58:0.1) in 40 min. Collected fractions were analyzed by analytical LCMS, and the fractions at 30.3-32.0 minutes judged as having adequate purity were pooled and evaporated to afford 15 mg of (9) as an oil, 63% yield; [α]_D59.1 (c 1.1, methanol); ¹H NMR (500 MHz, METHANOL-d₄) delta ppm 1.84-1.92 (m, 2H), 2.00 (s, 9H), 2.31-2.38 (m, 2H), 2.97 (t, J=7.3 Hz, 2H), 3.54-3.80 (m, 54H), 3.86-3.93 (m, 9H), 3.93-4.00 (m, 6H), 4.57 (s, 6H), 4.60 (t, J=4.9 Hz, 6H), 5.22 (s, 3H), 8.02 (s, 3H); HRMS (ESI) calcd for C₆₈H₁₁₄N₁₄O₃₁(m/z) [M+H]⁺1623.7847, found 1623.7803.

(10), (11), and (12); Alexa Fluor® 647 Conjugates

[0404]

[0405] Alexa Fluor® 647 carboxylic acid succinimidyl ester was from Invitrogen (Catalog NumberA-20106). The molecular weight was reported by Invitrogen to be ˜1250. The Alexa647 labeled compound molecular weight was estimated based on the found [M+H]⁺ of 955.07 of Alexa Fluor 647 carboxylic acid succinimidyl ester from LCMS. Extinction coefficient for λ_max650 is ˜270000±20000, which varies from batch to batch.

General Procedure for HPLC Purification:

[0406] Preparative high-performance liquid chromatography (HPLC) was performed using a Waters XBridge BEH C18 OBD Prep Column, 130 Å, 5 microm, 19 mm×100 mm (Waters, part number 186002978), eluting with a linear slope gradient at 17 mL/min flow rate. Solvent gradient: acetonitrile/water/trifluoroacetic acid (2:98:0.1) to (22:78:0.1) in 40 min. Collected fractions were analyzed by analytical LCMS, and those judged as having adequate purity were pooled and evaporated.

(10):

[0407] To a solution of compound (7) (3.0 mg, 4.8 micromol) in dimethyl sulfoxide (200 microL) were added Alexa Fluor® 647 carboxylic acid succinimidyl ester (5.0 mg, 4 micromol) and N,N-diisopropylethylamine (10 microL, 10 equiv.). The reaction mixture was shaken at room temperature for 1 hour and then directly purified by preparative HPLC. Collected fractions were analyzed by analytical LCMS, and those judged as having adequate purity were pooled (R_t=22.7-24 minutes). 3.2 mg of (10) was obtained (55% yield). The solution was aliquoted and evaporated in a vacuum centrifuge, and the product was stored at 4° C. MS (ESI) calcd (m/z) for [M+H]⁺ ˜1456, found 1456.82.

(11):

[0408] To a solution of compound (8) (6.0 mg, 5 micromol) in dimethyl sulfoxide (200 microL) were added Alexa Fluor® 647 carboxylic acid succinimidyl ester (5.0 mg, 4 micromol) and N,N-diisopropylethylamine (10 microL, 10 equiv.). The reaction mixture was shaken at room temperature for 1 hour and then directly purified by preparative HPLC. Collected fractions were analyzed by analytical LCMS, and those judged as having adequate purity were pooled (R_t=25.3-26.7 minutes). 4.8 mg of (11) was obtained (62% yield). The solution was aliquoted and evaporated in a vacuum centrifuge, and the product was stored at 4° C. MS (ESI) calcd (m/z) for [M+H]⁺ ˜1958, found 1958.74.

(12):

[0409] To a solution of compound (9) (9.8 mg, 6 micromol) in dimethyl sulfoxide (200 microL) were added Alexa Fluor® 647 carboxylic acid succinimidyl ester (5.0 mg, 4.8 micromol) and N,N-diisopropylethylamine (10 microL, 10 equiv.). The reaction mixture was shaken at room temperature for 1 hour and then directly purified by preparative HPLC. Collected fractions were analyzed by analytical LCMS, and those judged as having adequate purity were pooled (R_t=27.7 minutes). 5.2 mg of (12) was obtained (52% yield). The solution was aliquoted and evaporated in a vacuum centrifuge, and the product was stored at 4° C. MS (ESI) calcd (m/z) for [M+H]⁺ ˜2460, found 2461.18.

4-amino-N-[1,31-bis(1-{[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]methyl}-1H-1,2,3-triazol-4-yl)-2,6,10,14,18,22,26,30-octaoxahentriacontan-16-yl]butanamide (13) and 4-amino-N-{1,31-bis(1-{[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]methyl}-1H-1,2,3-triazol-4-yl)-16-[15-(1-{[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]methyl}-1H-1,2,3-triazol-4-yl)-2,6,10,14-tetraoxapentadec-1-yl]-2,6,10,14,18,22,26,30-octaoxahentriacontan-16-yl}butanamide (14)

[0410]

[0411] Compound (I-e-1) (247 mg, 0.904 mmol) was dissolved in dichloromethane (15 mL) and pyridine was added (1.46 mL, 18.1 mmol). The reaction mixture was cooled at −20° C. and trifluoromethanesulfonic anhydride (0.23 mL, 1.4 mmol) in dichloromethane (0.6 mL) was added dropwise and the mixture was stirred while allowing warming to 0 degrees C. over 50 minutes. The reaction mixture was diluted with dichloromethane and washed with an aqueous solution of 1M hydrogen chloride, a saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium chloride. The organic phase was dried over magnesium sulfate, filtered and concentrated. The crude material was used in the next step without any further purification. Sodium azide (270 mg, 4.1 mmol) was added to a solution of the above triflate in dimethylformamide (4.1 mL). The reaction mixture was stirred at 60° C. for 16 hours. Solvent was evaporated and the crude material was purified by flash chromatography (15/1 ethyl acetate/methanol) over silica gel to afford the desired compound (I-e-3) as a yellow oil (227 mg, 92% yield). [α]_D127 (c 1, methanol); ¹H NMR (500 MHz, CHLOROFORM-d) delta ppm 1.34 (s, 3H), 1.53 (s, 3H), 2.00 (s, 3H), 3.67 (d, J=12.7 Hz, 1H), 3.72 (d, J=7.8 Hz, 1H), 3.74 (d, J=7.8 Hz, 1H), 3.75 (d, J=12.7 Hz, 1H), 4.02-4.10 (m, 2H), 4.11 (d, J=5.9 Hz, 1H), 5.35 (d, J=2.4 Hz, 1H), 5.95 (d, J=8.8 Hz, 1H); ¹³C NMR (125 MHz, CHLOROFORM-d) delta ppm 23.2, 26.2, 27.7, 51.0, 54.2, 69.3, 74.8, 76.1, 80.6, 101.2, 111.6, 170.1; HRMS (ESI) calcd for C₁₂H₁₈N₄O₅(m/z) [M+H]⁺299.1350, found 299.1344.

[0412] Compound (I-j-2) and (I-j-3) could be made starting from propargyl bromide, (I-i-2) (commercially available from Dalton Pharma; DC-001760) and (I-i-3) (B. Ernst et al. in Bioorganic & Medicinal Chemistry, 16, 5216 (2008)) respectively, following the same procedure described for the formation of compound (I-f-2).

[0413] Compound (I-j-2): Purification conditions: 20% ethyl acetate/hexanes, 85 mg, oil (8% yield); ¹H NMR (400 MHz, CHLOROFORM-d/TMS) delta ppm 1.77-1.90 (m, 14H), 2.23 (t, J=7.1 Hz, 2H), 2.43 (t, J=2.3 Hz, 2H), 3.20-3.29 (m, 2H), 3.39-3.55 (m, 24H), 3.60 (t, J=6.3 Hz, 4H), 4.13 (d, J=2.5 Hz, 4H), 4.15-4.24 (m, 1H), 5.09 (s, 2H), 5.16 (br. s., 1H), 6.05 (d, J=8.1 Hz, 1H), 7.28-7.41 (m, 5H); ¹³C NMR (100 MHz, CHLOROFORM-d/TMS) delta ppm 25.8, 29.8 (2C), 29.9 (2C), 30.0 (2C), 33.7, 40.4, 48.5, 58.1 (2C), 66.6, 67.2 (2C), 67.6 (2C), 67.7 (2C), 67.8 (2C), 67.9 (2C), 68.3 (2C), 69.0 (2C), 74.2 (2C), 79.9 (2C), 128.1, 128.5 (4C), 136.6, 156.6, 172.1; HRMS (ESI) calcd for C₃₉H₆₂N₂O₁₁(m/z) [M+H]⁺ 735.4426, found 735.4424.

[0414] Compound (I-j-3): Purification conditions: 85% ethyl acetate/hexanes, 32.6 mg, oil, (71% yield); ¹H NMR (400 MHz, CHLOROFORM-d/TMS) delta ppm 1.75-1.90 (m, 20H), 2.18 (t, J=6.9 Hz, 2H), 2.43 (t, J=2.4 Hz, 3H), 3.23 (q, J=6.3 Hz, 2H), 3.40-3.53 (m, 30H), 3.59 (t, J=6.3 Hz, 6H), 3.67 (s, 6H), 4.13 (d, J=2.3 Hz, 6H), 5.08 (s, 2H), 5.27 (br. s., 1H), 5.85 (s, 1H), 7.27-7.40 (m, 5H); ¹³C NMR (100 MHz, CHLOROFORM-d/TMS) delta ppm 25.7, 29.7 (3C), 29.9 (3C), 30.0 (3C), 34.4, 40.4, 58.1 (3C), 59.8, 66.5, 67.1 (3C), 67.6 (3C), 67.7 (3C), 67.8 (3C), 67.82 (3C), 68.4 (3C), 69.1 (3C), 74.2 (3C), 79.9 (3C), 128.0, 128.4 (4C), 136.6, 156.6, 172.3; HRMS (ESI) calcd for C₅₂H₈₄N₂O₁₅(m/z) [M+H]⁺ 977.5944, found 977.5943.

Compound (I-k-2):

[0415] THPTA (22.6 mg, 0.052 mmol) and copper sulfate (2.5 mg, 0.01 mmol) were dissolved in water (200 microL) and then added to a solution of (I-e-3) (45 mg, 0.152 mmol) and (I-j-2) (51 mg, 0.069 mmol) in methanol (1.1 mL). Then sodium ascorbate (4.2 mg, 0.021 mmol), dissolved in water (100 microL), was added and the reaction mixture was stirred at 50 degrees Celsius for 24 hours. Solvent was evaporated and the crude material was purified by flash chromatography (5% methanol in dichloromethane) over silica gel to afford the desired compound (I-k-2) as an oil (72 mg, 78% yield). ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.34 (s, 6H), 1.52 (s, 6H), 1.73-1.86 (m, 12H), 1.97 (s, 6H), 2.24 (t, J=7.4 Hz, 2H), 3.15 (t, J=6.8 Hz, 2H), 3.43-3.54 (m, 22H), 3.58 (t, J=6.3 Hz, 4H), 3.86 (d, J=8.1 Hz, 2H), 3.97 (dd, J=6.2, 1.9 Hz, 2H), 4.11-4.23 (m, 5H), 4.58 (s, 4H), 4.78 (s, 6H), 4.91 (d, J=14.1 Hz, 2H), 4.98 (d, J=14.4 Hz, 2H), 5.07 (s, 2H), 5.24 (d, J=1.8 Hz, 2H), 7.25-7.40 (m, 5H), 7.99 (s, 2H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 22.7 (2C), 26.8 (2C), 27.5, 28.4 (2C), 31.1 (4C), 31.2 (2C), 34.5, 41.4, 50.6, 51.0 (2C), 56.3 (2C), 64.8 (2C), 67.5 (2C), 68.7 (2C), 68.8 (2C), 68.9 (3C), 69.0 (2C), 69.4 (2C), 69.7 (2C), 70.9 (2C), 76.3 (2C), 76.6 (2C), 81.5 (2C), 102.5 (2C), 112.8 (2C), 127.2 (2C), 129.0 (2C), 129.1, 129.6 (2C), 138.6, 146.3 (2C), 159.0, 173.5 (2C), 175.5; HRMS (ESI) calcd for C₆₃H₉₈N₁₀O₂₁(m/z) [M+H]⁺ 1331.6981, found 1331.6971.

Compound (I-k-3):

[0416] THPTA (16 mg, 0.037 mmol) and copper sulfate (1.7 mg, 0.007 mmol) were dissolved in water (100 microL) and then added to a solution of (I-e-3) (32.5 mg, 0.109 mmol) and (I-j-3) (32 mg, 0.033 mmol) in methanol (1.1 mL). Then sodium ascorbate (3 mg, 0.015 mmol), dissolved in water (100 microL), was added and the reaction mixture was stirred at 50 degrees Celsius for 24 hours. Solvent was evaporated and the crude material was purified by flash chromatography (10% methanol in dichloromethane) over silica gel to afford the desired compound (I-k-3) as an oil (43.5 mg, 70% yield). ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 1.33 (s, 9H), 1.52 (s, 9H), 1.71-1.87 (m, 18H), 1.97 (s, 9H), 2.20 (t, J=7.3 Hz, 2H), 3.15 (t, J=6.8 Hz, 2H), 3.43-3.52 (m, 28H), 3.58 (t, J=6.3 Hz, 6H), 3.67 (s, 6H), 3.86 (d, J=8.3 Hz, 3H), 3.97 (dd, J=6.0, 1.8 Hz, 3H), 4.14-4.22 (m, 5H), 4.58 (s, 6H), 4.78 (s, 8H), 4.91 (d, J=14.6 Hz, 3H), 4.97 (d, J=14.6 Hz, 3H), 5.07 (s, 2H), 5.24 (d, J=2.0 Hz, 3H), 7.26-7.38 (m, 5H), 7.98 (s, 3H); ¹³C NMR (100 MHz, METHANOL-d₄) delta ppm 22.7 (3C), 26.9 (3C), 27.7, 28.4 (3C), 31.1 (3C), 31.2 (3C), 31.3 (3C), 35.2, 41.3, 51.0 (3C), 56.3 (3C), 61.8, 64.9 (3C), 67.5 (3C), 68.7 (3C), 68.8 (3C), 68.9 (3C), 69.0 (4C), 69.6 (3C), 69.7 (3C), 70.0 (3C), 76.3 (3C), 76.6 (3C), 81.5 (3C), 102.5 (3C), 112.8 (3C), 127.2 (3C), 129.0 (2C), 129.1, 129.7 (2C), 138.6, 146.4 (3C), 159.0, 173.5 (3C), 175.6; HRMS (ESI) calcd for C₈₈H₁₃₈N₄O₃₀(m/z) [M+H]⁺ 1871.9776, found 1871.9713.

[0417] Compound (I-k-2) or (I-k-3) (0.068 mmol) was dissolved in a mixture of acetic acid, methanol and water (2.5-3 mL, 0.6-0.9 mL, 0.6-0.9 mL respectively) and stirred at 70 degrees Celsius for 24 hours. Solvent was evaporated and the residue was co-evaporated twice with toluene. The crude material obtained was used in the next step without any further purification.

[0418] In a round bottom flask, compound (I-l-2) or (I-l-3) (1 equiv.) was dissolved in methanol (0.01M) and the flask was flushed with nitrogen. Palladium on carbon (10%, 0.7 equiv.) was added and the flask was flushed with nitrogen and then with hydrogen. The reaction mixture was stirred at room temperature for 24 hours under an atmosphere of hydrogen (a balloon filled with hydrogen was used). The palladium was filtered using a 0.45 microm PTFE Acrodisc Cr and rinsed once with methanol. Solvent was evaporated.

(13):

[0419] The crude material was dissolved in 0.5 mL methanol/water (50:50) and injected on a HPLC column. Preparative high-performance liquid chromatography (HPLC) was performed using a Waters XBridge BEH C18 OBD Prep Column, 130 Å, 5 microm, 19 mm×100 mm (Waters, part number 186002978), eluting with a linear slope gradient at 17 mL/min flow rate. Solvent gradient: acetonitrile/water/trifluoroacetic acid (2:98:0.1) to (22:58:0.1) in 40 min. Collected fractions were analyzed by analytical LCMS, and the fractions at 34.7-35.6 minutes judged as having adequate purity were pooled and evaporated to afford 12.8 mg of (13) as an oil, (17% yield over 2 steps); ¹H NMR (500 MHz, METHANOL-d₄) delta ppm 1.73-1.87 (m, 12H), 1.88-1.96 (m, 2H), 1.98 (s, 6H), 2.39 (t, J=7.0 Hz, 2H), 2.98 (t, J=7.4 Hz, 2H), 3.42 (d, J=8.5 Hz, 2H), 3.45-3.55 (m, 24H), 3.59 (t, J=6.3 Hz, 4H), 3.71-3.75 (m, 4H), 3.77 (d, J=8.3 Hz, 2H), 3.96-4.02 (m, 2H), 4.13-4.20 (m, 1H), 4.58 (s, 4H), 4.91-4.95 (m, 4H), 5.20 (d, J=1.5 Hz, 2H), 7.98 (s, 2H); HRMS (ESI) calcd for C₄₉H₈₄N₁₀O₁₉(m/z) [M+H]⁺ 1117.5987, found 1117.5977.

(14):

[0420] The crude material was dissolved in 0.5 mL methanol/water (50:50) and injected on a HPLC column. Preparative high-performance liquid chromatography (HPLC) was performed using a Waters XBridge BEH C18 OBD Prep Column, 130 Å, 5 microm, 19 mm×100 mm (Waters, part number 186002978), eluting with a linear slope gradient at 17 mL/min flow rate. Solvent gradient: acetonitrile/water/trifluoroacetic acid (2:98:0.1) to (42:58:0.1) in 40 min. Collected fractions were analyzed by analytical LCMS, and the fractions at 24.7-25.6 minutes judged as having adequate purity were pooled and evaporated to afford 5.5 mg of (14) as an oil (10% yield over 2 steps); ¹H NMR (500 MHz, METHANOL-d₄) delta ppm 1.75-1.86 (m, 18H), 1.87-1.94 (m, 2H), 1.98 (s, 9H), 2.37 (t, J=6.8 Hz, 2H), 2.98 (t, J=7.4 Hz, 2H), 3.43 (d, J=8.5 Hz, 3H), 3.46-3.53 (m, 31H), 3.59 (t, J=6.3 Hz, 6H), 3.68 (s, 6H), 3.72-3.76 (m, 5H), 3.77 (d, J=8.3 Hz, 3H), 3.97-4.02 (m, 3H), 4.59 (s, 6H), 4.90-4.96 (m, 6H), 5.20 (d, J=1.2 Hz, 3H) 7.98 (s, 3H); HRMS (ESI) calcd for C₇₁H₁₂₀N₁₄O₂₈(m/z) [M+H]⁺ 1617.8469, found 1617.8415.

(15) and (16); Alexa Fluor® 647 (AF647) Conjugates

[0421]

[0422] Alexa Fluor® 647 carboxylic acid succinimidyl ester was from Invitrogen (Catalog NumberA-20106). The molecular weight was reported by Invitrogen to be ˜1250. The Alexa647 labeled compound molecular weight was estimated based on the found [M+H]⁺ of 955.07 of Alexa Fluor 647 carboxylic acid succinimidyl ester from LCMS. Extinction coefficient for λ_max650 is ˜270000±20000, which varies from batch to batch.

General Procedure for HPLC Purification:

[0423] Preparative high-performance liquid chromatography (HPLC) was performed using a Waters XBridge BEH C18 OBD Prep Column, 130 Å, 5 microm, 19 mm×100 mm (Waters, part number 186002978), eluting with a linear slope gradient at 17 mL/min flow rate. Solvent gradient: acetonitrile/water/trifluoroacetic acid (2:98:0.1) to (22:78:0.1) in 40 min. Collected fractions were analyzed by analytical LCMS, and those judged as having adequate purity were pooled and evaporated.

(15):

[0424] To a solution of compound (13) (4.5 mg, 4 micromol) in dimethyl sulfoxide (200 microL) were added Alexa Fluor® 647 carboxylic acid succinimidyl ester (5.0 mg, 4 micromol) and N,N-diisopropylethylamine (10 microL, 10 equiv.). The reaction mixture was shaken at room temperature for 1 hour and then directly purified by preparative HPLC. Collected fractions were analyzed by analytical LCMS, and those judged as having adequate purity were pooled (R_t=37.3-39 minutes). 4.0 mg of (15) was obtained (50% yield). The solution was aliquoted and evaporated in a vacuum centrifuge, and the product was stored at 4 degrees Celsius. MS (ESI) calcd (m/z) for [M+H]⁺˜1955, found 1955.32.

(16):

[0425] To a solution of compound (14) (5.2 mg, 3.2 micromol) in dimethyl sulfoxide (200 microL) were added Alexa Fluor® 647 carboxylic acid succinimidyl ester (5.0 mg, 4 micromol) and N,N-diisopropylethylamine (10 microL, 10 equiv.). The reaction mixture was shaken at room temperature for 1 hour and then directly purified by preparative HPLC. Collected fractions were analyzed by analytical LCMS, and those judged as having adequate purity were pooled (R_t=24.3-25.3 minutes). 4.0 mg of (16) was obtained (51% yield). The solution was aliquoted and evaporated in a vacuum centrifuge, and the product was stored at 4° C. MS (ESI) calcd (m/z) for [M+H]⁺˜2455, found 2456.90.

General Alkylation/Deprotection Conditions to Access (17)-(21):

[0426] To a solution of (I-e-1) in dichloromethane was added the desired iodoalkyl, tetrabutylammonium hydrogen sulfate, and 12.5M sodium hydroxide aqueous solution. The reaction mixture was allowed to stir overnight at room temperature, was diluted with water and dichloromethane and the aqueous phase was extracted two additional times with dichloromethane. The combined organic layers were washed with an aqueous solution of 1M hydrochloric acid, water, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting material can be either carried on crude to the next reaction or purified using flash chromatography over silica gel. The resulting material was dissolved in a mixture of acetic acid/methanol/water (3:1:1 v/v) and the solution was heated to 60-70 degrees Celsius overnight. The reaction mixture was concentrated under reduced pressure, co-evaporated two times with toluene and the crude material was purified by flash chromatography over silica gel or reverse phase chromatography.

N-[(1S,2R,3R,4R,5S)-1-(ethoxymethyl)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (17)

[0427]

[0428] (17) was synthesized as described in the general procedure above using iodoethane (20 equiv.). The crude product was dissolved in methanol and to which was added activated charcoal. The mixture was stirred for 15 minutes, filtered and concentrated under reduced pressure. The crude material was purified by flash chromatography over silica gel eluting with ethyl acetate/methanol (15:1). The fractions containing the desired product were collected and concentrated under reduced pressure. To the crude material was added ethyl acetate/methanol (15:1) which resulted in a precipitate that was filtered yielding 9.1 mg (32% yield) of the desired product as a solid. ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 5.23 (d, J=1.5 Hz, 1H), 3.97 (dd, J=9.7, 1.4 Hz, 1H), 3.94 (d, J=9.3 Hz, 1H), 3.88 (d, J=4.3 Hz, 1H), 3.79 (d, J=8.1 Hz, 1H), 3.73 (dd, J=9.8, 4.3 Hz, 1H), 3.66 (d, J=8.1 Hz, 1H), 3.60 (d, J=9.6 Hz, 1H), 3.59 (dq, J=9.6, 7.1 Hz, 1H), 3.55 (dq, J=9.6, 7.1 Hz, 1H), 2.00 (s, 3H), 1.19 (t, J=6.9 Hz, 3H). LCMS (APCI) m/z: 262.1 [M+H] (100%).

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(propoxymethyl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (18)

[0429]

[0430] (18) was synthesized as described in the general procedure above using iodopropane (20 equiv.). The crude product was purified using flash chromatography over silica gel eluting with ethyl acetate/methanol (15:2) yielding 13.9 mg (80% yield) of the desired product as an oil. ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 5.23 (d, J=1.5 Hz, 1H), 3.97 (dd, J=9.7, 1.4 Hz, 1H), 3.95 (d, J=9.6 Hz, 1H), 3.89 (d, J=4.3 Hz, 1H), 3.79 (d, J=7.8 Hz, 1H), 3.73 (dd, J=9.8, 4.3 Hz, 1H), 3.66 (d, J=8.1 Hz, 1H), 3.59 (d, J=9.3 Hz, 1H), 3.49 (dt, 6.5 Hz, 1H), 3.46 (dt, 6.5 Hz, 1H), 2.01 (s, 3H), 1.60 (qt, J=7.4, 6.5 Hz, 2H), 0.94 (t, J=7.4 Hz, 3H). LCMS (APCI) m/z: 276.2 [M+H] (100%).

N-[(1S,2R,3R,4R,5S)-1-(butoxymethyl)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (19)

[0431]

[0432] (19) was synthesized as described in the general procedure above using iodobutane (20 equiv.). The desired crude product was purified using flash chromatography over silica gel eluting with ethyl acetate/methanol (15:1) yielding 18 mg (100% yield) of the desired product as an oil. ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 5.23 (d, J=1.3 Hz, 1H), 3.97 (dd, J=9.6, 1.3 Hz, 1H), 3.94 (d, J=9.6 Hz, 1H), 3.88 (d, J=4.3 Hz, 1H), 3.79 (d, J=7.8 Hz, 1H), 3.73 (dd, J=9.8, 4.3 Hz, 1H), 3.66 (d, J=7.8 Hz, 1H), 3.59 (d, J=9.3 Hz, 1H), 3.54 (dt, 6.5 Hz, 1H), 3.50 (dt, 6.5 Hz, 1H), 2.00 (s, 3H), 1.52-1.61 (m, 2H), 1.34-1.45 (m, 2H), 0.95 (t, J=7.3 Hz, 3H). LCMS (APCI) m/z: 290.2 [M+H] (100%).

N-{(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-[(pentyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}acetamide (20)

[0433]

[0434] (20) was synthesized as described in the general procedure above using iodopentane (20 equiv.). The desired crude product was purified using flash chromatography over silica gel eluting with ethyl acetate/methanol (15:1) yielding 17 mg (68% yield) of the desired product as an oil. ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 5.23 (d, J=1.5 Hz, 1H), 3.97 (dd, J=9.8, 1.3 Hz, 1H), 3.94 (d, J=9.6 Hz, 1H), 3.88 (d, J=4.3 Hz, 1H), 3.79 (d, J=8.1 Hz, 1H), 3.73 (dd, J=9.8, 4.3 Hz, 1 H), 3.66 (d, J=8.1 Hz, 1H), 3.59 (d, J=9.6 Hz, 1H), 3.53 (dt, 6.5 Hz, 1H), 3.49 (dt, 6.5 Hz, 1H), 2.01 (s, 3H), 1.53-1.63 (m, 2H), 1.29-1.41 (m, 4H), 0.89-0.97 (m, 3H). LCMS (APCI) m/z: 304.1 [M+H] (100%).

N-{(1S,2R,3R,4R,5S)-1-[(hexyloxy)methyl]-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-4-yl}acetamide (21)

[0435]

[0436] (21) was synthesized as described in the general procedure above using iodohexane (20 equiv.). The desired crude product was purified using flash chromatography over silica gel eluting with ethyl acetate/methanol (15:1) yielding 56 mg of product as an oil. This material was repurified using reverse phase chromatography yielding 7.1 mg (15% yield) of the desired product as a solid. ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 5.23 (d, J=1.5 Hz, 1H), 3.96 (dd, J=10.1, 1.3 Hz, 1H), 3.94 (d, J=9.6 Hz, 1H), 3.88 (d, J=4.3 Hz, 1H), 3.79 (d, J=7.8 Hz, 1H), 3.73 (dd, J=9.8, 4.3 Hz, 1H), 3.66 (d, J=8.1 Hz, 1H), 3.59 (d, J=9.6 Hz, 1H), 3.53 (dt, 6.5 Hz, 1H), 3.49 (dt, J=9.3, 6.5 Hz, 1H), 2.00 (s, 3H), 1.53-1.62 (m, 2H), 1.27-1.50 (m, 6H), 0.89-0.97 (m, 3H). LCMS (APCI) m/z: 318.1 [M+H] (100%).

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(2,5,8,11,14-pentaoxapentadec-1-yl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (22)

[0437]

[0438] To a solution of (I-e-1) in dichloromethane (3 mL) was added pyridine (0.3 mL, 4 mmol), the mixture was cooled to −20 degrees Celsius, and trifluoromethanesulfonic anhydride (0.047 mL, 0.28 mmol) in dichloromethane (0.6 mL) was added. The reaction mixture was allowed to warm to −10 degrees Celsius over 1 hour, was diluted with dichloromethane and successively washed with an aqueous solution of 1M hydrochloric acid, saturated sodium bicarbonate aqueous solution, brine, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to afford the desired crude material which was used in the next step without further purification. To a solution of 2,5,8,11-tetraoxatridecan-13-ol (207 mg, 0.994 mmol) in N,N-dimethylformamide cooled to 0 degrees Celsius was added sodium hydride (39.9 mg, 1.0 mmol) and the reaction mixture was stirred for 10 minutes. The above crude ((3aR,4R,7S,8R,8aR)-8-acetamido-2,2-dimethyltetrahydro-4,7-epoxy[1,3]dioxolo[4,5-d]oxepin-4(5H)-yl)methyl trifluoromethanesulfonate in N,N-dimethylformamide (0.5 mL) was added dropwise and the reaction mixture was allowed to stir for 25 minutes at 0 degrees Celsius. The reaction was quenched with methanol and the reaction mixture was allowed to stir for 5 minutes. The resulting mixture was then concentrated under reduced pressure and the residue obtained was dissolved in dichloromethane and washed with water. The aqueous layer was extracted two additional times with dichloromethane. The combined organic layers were washed with water and concentrated under reduced pressure. The crude material was purified by flash chromatography over silica gel eluting with ethyl acetate/methanol (15:2) yielding 85 mg (100%) of the desired product. A solution of N-((3aR,4S,7S,8R,8aR)-2,2-dimethyl-4-(2,5,8,11,14-pentaoxapentadecyl)hexahydro-4,7-epoxy[1,3]dioxolo[4,5-d]oxepin-8-yl)acetamide (85 mg, 0.18 mmol) in a mixture of acetic acid/methanol/water (3.9:1.3:1.3 v/v) was heated to 70 degrees Celsius overnight. The reaction mixture was concentrated under reduced pressure, the resulting crude material co-evaporated two times with toluene and purified by flash chromatography over silica gel eluting with 10% methanol/dichloromethane yielding 15 mg of the desired product (22) as an oil. ¹H NMR (400 MHz, METHANOL-d₄) delta ppm 5.22 (d, J=1.3 Hz, 1H), 3.96 (d, J=9.6 Hz, 1H), 3.95 (dd, J=9.9, 1.3 Hz, 1H), 3.89 (d, J=4.3 Hz, 1H), 3.78 (d, J=8.1 Hz, 1H), 3.59-3.74 (m, 17H), 3.53-3.56 (m, 2H), 3.36 (s, 3H), 1.99 (s, 3H). LCMS (APCI) m/z: 424.2 [M+H] (13%), 441.3 [M+NH₄] (100%).

(1R,2R,3R,4R,5S)-4-acetamido-1-(((4-bromobenzoyl)oxy)methyl)-6,8-dioxabicyclo[3.2.1]octane-2,3-diyl bis(4-bromobenzoate) (23)

[0439]

[0440] To a solution of (3) (9 mg) in anhydrous N,N-dimethylformamide (500 microL) cooled at room temperature were added N,N-diisopropylethylamine (34 microL) and 4-(dimethylamino)pyridine (4.3 mg) followed by p-bromobenzoyl chloride (44 mg), and the resulting mixture was stirred at room temperature for 4.5 h. Water was added, the resulting mixture extracted three times with ethyl acetate, and the combined organic phase was successively washed with 0.5 M aqueous hydrochloric acid solution and brine. The organic phase was dried over magnesium sulfate, filtered, and concentrated, and the crude material was purified by flash chromatography over silica gel, eluting with a gradient of 0-100% ethyl acetate in heptane, to afford 23 mg of product (23) (80% yield). 1H NMR (400 MHz, CDCl3): delta (ppm) 7.89-7.95 (m, 2H), 7.78-7.84 (m, 2H), 7.54-7.66 (m, 6H), 7.41-7.46 (m, 2H), 5.87 (d, J=8.8 Hz, 1H), 5.80 (d, J=4.3 Hz, 1H), 5.60 (d, J=1.1 Hz, 1H), 5.44 (dd, J=10.2, 4.5 Hz, 1H), 4.54-4.64 (m, 3H), 4.15 (d, J=8.6 Hz, 1H), 3.93 (d, J=8.6 Hz, 1H), 1.95 (s, 3H). ¹³C NMR (101 MHz, CDCl3) delta ppm 170.6, 165.6, 165.0, 164.9, 132.1, 131.9, 131.8, 131.4, 131.2, 131.2, 129.3, 129.0, 128.9, 127.7, 127.5, 127.4, 101.8, 81.6, 69.5, 68.8, 68.4, 62.5, 52.7, 23.2. Single crystals were obtained by vapor diffusion technique using methanol and heptane as solvents. Single crystal X-Ray analysis: Data collection was performed on a Bruker APEX diffractometer at room temperature. Data collection consisted of 3 omega scans and low angle and three at high angle; each with 0.5 step. In addition, 2 phi scans were collected to improve the quality of the absorption correction. Structure is a non-merohedrial twin; refined by ignoring the second domain. The structure was solved by direct methods using SHELX software suite (see SHELXTL, Version 5.1, Bruker AXS, 1997) in the space group P2(1). The structure was subsequently refined by the full-matrix least squares method. All non-hydrogen atoms were found and refined using anisotropic displacement parameters. The hydrogen atom located on nitrogen was placed in this position and constrained in reasonable position. The remaining hydrogen atoms were placed in calculated positions and were allowed to ride on their carrier atoms. The final refinement included isotropic displacement parameters for all hydrogen atoms. Analysis of the absolute structure using likelihood methods (R. W. W. Hooft et al. J. Appl. Cryst., 41, 96-103 (2008)) was performed using PLATON (A. L. Spek, J. Appl. Cryst., 36, 7-13 (2003)). The results indicate that the absolute structure has been correctly assigned. The method calculates that the probability that the structure is correct is 100.0. The Hooft parameter is reported as 0.036 with an esd of 0.013. Additionally, the Flack parameter is 0.03 with and esd of 0.04. The final R-index was 5.6%. A final difference Fourier revealed no missing or misplaced electron density. Pertinent crystal, data collection and refinement are summarized in table 1 and FIG. 1.

TABLE 1

Crystal data and structure refinement for (23).


Empirical formula	C15 H12 Br1.50 N0.50 O4.50
Formula weight	391.12
Temperature	296(2) K
Wavelength	1.54178 Å
Crystal system	Monoclinic
Space group	P2(1)
Unit cell dimensions	a = 12.5748(9) Å	α = 90°.
	b = 5.6465(4) Å	β = 97.453(4)°.
	c = 21.2806(16) Å	γ = 90°.
Volume	1498.23(19) Å3
Z	4
Density (calculated)	1.734 Mg/m3
Absorption coefficient	5.476 mm−1
F(000)	776
Crystal size	0.37 × 0.22 × 0.15 mm3
Theta range for data collection	2.09 to 68.30°.
Index ranges	−13 <= h <= 15, −5 <=
	k <= 6, −24 <= l <= 24
Reflections collected	8050
Independent reflections	4408 [R(int) = 0.0247]
Completeness to theta = 68.30°	93.9%
Absorption correction	Empirical
Refinement method	Full-matrix least-squares on F2
Data/restraints/parameters	4408/32/389
Goodness-of-fit on F2	1.031
Final R indices [I > 2sigma(I)]	R1 = 0.0563, wR2 = 0.1658
R indices (all data)	R1 = 0.0589, wR2 = 0.1701
Absolute structure parameter	0.04(3)
Largest diff. peak and hole	0.949 and −0.685 e.Å−3

(1S,2R,3R,4R,5S)-4-azido-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]octane (I-m-1)

[0441]

[0442] To a solution of (1S,2R,3R,4R,5S)-4-azido-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3-diol (1) (445 mg, 2.05 mmol) in N,N-dimethylformamide (5 mL) was added sodium hydride (60% dispersion in mineral oil, 410 mg, 10.2 mmol) at room temperature. The reaction became very thick and would not stir well. An additional 5 mL N,N-dimethylformamide was added and the reaction was stirred for 30 minutes at room temperature before the addition of benzylbromide (1.23 mL, 10.2 mmol) drop wise. The reaction was allowed to stir overnight at room temperature. The following morning the reaction was quenched with water and extracted with ethyl acetate three times. The combined organic layer was washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 40 g silica gel column) and eluting with a gradient of 0-30% ethyl acetate/heptane yielding the title compound (890.0 mg, 89.1%). Method C: 3 minute run LRMS [M+Na=510]. ¹H NMR (METHANOL-d₄) δ: 7.07-7.52 (m, 15H), 5.31 (s, 1H), 4.81 (d, J=5.1 Hz, 1H), 4.78 (d, J=5.5 Hz, 1H), 4.68-4.74 (m, 1H), 4.47-4.51 (m, 1H), 4.46 (d, J=6.6 Hz, 1H), 4.35-4.42 (m, 1H), 4.12 (d, J=3.9 Hz, 1H), 3.87-3.91 (m, 2H), 3.86 (d, J=8.2 Hz, 1H), 3.62 (d, J=8.2 Hz, 1H), 3.58 (d, J=9.4 Hz, 1H), 3.46 (d, J=8.6 Hz, 1H)

(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]octan-4-amine (1-n-1)

[0443]

[0444] A mixture of (1S,2R,3R,4R,5S)-4-azido-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]octane (I-m-1) (310 mg, 0.64 mmol), triphenylphosphine (334 mg, 1.27 mmol), water (92 mg, 5.1 mmol), and tetrahydrofuran (10 mL) was stirred at 65° C. for 16 hours. The reaction mixture was concentrated under reduced pressure and the residue was loaded on a silica gel column. Chromatography eluting with a gradient from 20% to 80% of ethyl acetate in heptane gave the title product as a colorless gum (210 mg, 72%). ¹H NMR (CHLOROFORM-d) δ: 7.20-7.37 (m, 15H), 5.29 (d, J=1.2 Hz, 1H), 4.90 (d, 11.5 Hz, 1H), 4.79 (d, J=11.5 Hz, 1H), 4.57 (d, J=11.7 Hz, 1H), 4.56 (d, J=12.1 Hz, 1H), 4.43 (d, J=12.1 Hz, 1H), 4.39 (d, J=11.7 Hz, 1H), 3.97 (d, J=3.9 Hz, 1H), 3.90 (d, J=9.0 Hz, 1H), 3.69 (d, J=8.2 Hz, 1H), 3.59 (d, J=8.2 Hz, 1H), 3.42 (d, J=8.6 Hz, 1H), 3.37 (dd, J=9.4, 3.5 Hz, 1H), 3.07 (dd, J=9.4, 1.2 Hz, 1H); ¹³C NMR (CHLOROFORM-d) δ: 131.8, 131.6, 131.5, 128.2, 128.2, 128.1, 128.1, 128.0, 127.7, 127.6, 127.6, 127.4, 103.5, 82.6, 80.3, 74.5, 73.3, 73.1, 72.2, 69.9, 69.3, 55.1; LCMS (ES+): 1.18 min, 484.2 (M+Na)⁺.

N-{(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}acetamide (I-n-2)

[0445]

[0446] To a stirred mixture of (1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]octan-4-amine (1-n-1) (25 mg, 0.054 mmol), pyridine (43 mg, 0.54 mmol), and 2-methyl-tetrahydrofuran (1 mL) was added acetic anhydride (46 mg, 0.43 mmol) in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate and water. The organic extract was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified on a silica gel column, eluting with a gradient from 20% to 60% of ethyl acetate in heptane to obtain the title product as a white solid (20 mg, 73%). ¹H NMR (CHLOROFORM-d) δ: 7.24-7.43 (m, 15H), 5.35 (d, J=1.2 Hz, 1H), 5.06 (d, J=8.6 Hz, 1H), 4.96 (d, J=11.3 Hz, 1H), 4.74 (d, J=12.5 Hz, 1H), 4.58 (d, J=11.3 Hz, 1H), 4.42 (d, J=12.5 Hz, 1H), 4.40 (s, 2H), 4.30-4.36 (m, 1H), 4.04 (d, J=3.9 Hz, 1H), 3.96 (d, J=8.6 Hz, 1H), 3.67-3.70 (m, 1H), 3.58-3.61 (m, 1H), 3.41-3.47 (m, 2H), 1.87 (s, 3H); ¹³C NMR (CHLOROFORM-d) δ: 170.0, 138.2, 137.8, 137.4, 128.7, 128.5, 128.4, 128.3, 128.3, 128.2, 128.1, 128.0, 127.8, 101.6, 82.8, 75.7, 75.0, 73.8, 73.2, 71.5, 70.1, 69.5, 53.5, 23.3; LCMS (ES+): 1.87 min, 526.3 (M+Na)⁺.

N-{(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}-2,2,2-trifluoroacetamide (1-n-3)

[0447]

[0448] To a stirred mixture of (1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-amine (1-n-1) (75 mg, 0.16 mmol), pyridine (129 mg, 1.62 mmol), and 2-methyl-tetrahydrofuran (1 mL) was added trifluoroacetic anhydride (102 mg, 0.49 mmol) in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate and water. The organic extract was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified on a silica gel column, eluting with a gradient from 10% to 40% of ethyl acetate in heptane to obtain the title product as a white solid (60 mg, 66%). ¹H NMR (CHLOROFORM-d) δ: 7.25-7.42 (m, 15H), 5.91 (d, J=8.6 Hz, 1H), 5.35 (d, J=1.2 Hz, 1H), 4.95 (d, J=11.3 Hz, 1H), 4.72 (d, J=12.5 Hz, 1H), 4.58 (d, J=11.3 Hz, 1H), 4.41 (s, 2H), 4.40 (d, J=12.5 Hz, 1H), 4.36 (t, J=9.8 Hz, 1H), 4.08 (d, J=3.9 Hz, 1H), 3.96 (d, J=9.0 Hz, 1H), 3.68-3.72 (m, 1H), 3.60-3.63 (m, 1H), 3.50 (dd, J=10.0, 3.7 Hz, 1H), 3.45 (d, J=8.6 Hz, 1H); ¹³C NMR (CHLOROFORM-d) δ: 137.9, 137.1, 136.1, 128.9, 128.5, 128.5, 128.4, 128.4, 128.1, 128.1, 128.1, 127.9, 100.6, 83.0, 75.0, 74.9, 73.8, 72.7, 71.4, 70.3, 69.2, 54.1; ¹⁹F NMR (CHLOROFORM-d) δ: −75.7 (s); LCMS (ES−): 2.11 min, 556.2 (M−H)⁻.

N-{(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}methanesulfonamide (1-n-4)

[0449]

[0450] To a stirred mixture of (1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-amine (1-n-1) (50 mg, 0.11 mmol), triethylamine (0.100 mL, 0.72 mmol), and 2-methyl-tetrahydrofuran (1 mL) was added methanesulfonyl chloride (0.025 mL, 0.33 mmol) drop wise at 0° C. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate and saturated aqueous sodium bicarbonate. The organic extract was washed with water, brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified on a silica gel column, eluting with a gradient from 10% to 50% of ethyl acetate in heptane to obtain the title product as a white solid (58 mg, 65%). ¹H NMR (CHLOROFORM-d) δ: 7.22-7.41 (m, 15H), 5.43 (d, J=1.2 Hz, 1H), 4.93 (d, J=11.3 Hz, 1H), 4.79 (d, J=11.7 Hz, 1H), 4.62 (br.s., 1H), 4.56 (d, J=11.7 Hz, 1H), 4.54 (d, J=11.3 Hz, 1H), 4.41 (d, J=12.1 Hz, 1H), 4.37 (d, J=12.1 Hz, 1H), 4.05 (d, J=3.9 Hz, 1H), 3.92 (d, J=8.6 Hz, 1H), 3.74 (d, J=8.6 Hz, 1H), 3.68-3.72 (m, 1H), 3.62 (d, J=8.6 Hz, 1H), 3.54 (dd, J=10.0, 3.7 Hz, 1H), 3.44 (d, J=9.0 Hz, 1H), 2.90 (s, 3H); ¹³C NMR (CHLOROFORM-d) δ: 138.0, 137.3, 137.2, 128.7(2), 128.5(2), 128.4(2), 128.3, 128.1(2), 128.1, 127.9(2), 127.8(2), 102.7, 82.9, 77.2, 77.1, 75.0, 73.7, 73.5, 72.8, 70.2, 69.3, 57.7, 41.2; LCMS (ES−): 1.97 min, 538.3 (M−H)⁻.

N-{(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}propanamide (1-n-5)

[0451]

[0452] To a stirred mixture of propionic acid (23 mg, 0.31 mmol) in tetrahydrofuran (1 mL) was added 1,1′-carbonyldiimidazole (33 mg, 0.20 mmol) in one portion at room temperature and the clear solution was stirred at room temperature for 3 hours. Triethylamine (0.028 mL, 0.20 mmol) and (1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-amine (1-n-1) (47 mg, 0.10 mmol) were added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate (3 mL), brine (2 mL), and water (2 mL). The organic extract was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified on a silica gel column, eluting with a gradient from 10% to 50% of ethyl acetate in heptane to obtain the title product as a white solid (43 mg, 82%). ¹H NMR (CHLOROFORM-d) δ: 7.03-7.58 (m, 15H), 5.35 (s, 1H), 5.09 (d, J=8.2 Hz, 1H), 4.95 (d, J=11.3 Hz, 1H), 4.72 (d, J=12.1 Hz, 1H), 4.57 (d, J=11.3 Hz, 1H), 4.43 (d, J=12.1 Hz, 1H), 4.39 (s, 2H), 4.31-4.38 (m, 1H), 4.04 (d, J=3.9 Hz, 1H), 3.95 (d, J=9.0 Hz, 1H), 3.68 (d, J=8.2 Hz, 1H), 3.59 (d, J=8.2 Hz, 1H), 3.44-3.49 (m, 1H), 3.44 (d, J=8.6 Hz, 1H), 2.08 (q, J=7.4 Hz, 2H), 1.11 (t, J=7.6 Hz, 3H); ¹³C NMR (CHLOROFORM-d) δ: 173.5, 138.1, 137.8, 137.4, 128.6, 128.5, 128.3, 128.3, 128.2, 128.1, 128.0, 128.0, 127.7, 101.6, 82.7, 75.7, 74.9, 73.7, 73.2, 71.5, 70.1, 69.4, 53.3, 29.6, 9.5; LCMS (ES−): 1.94 min, 516.4 (M−H)⁻; LCMS (AP+): 1.94 min, 518.5 (M+H)⁺.

N-{(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}-3,3,3-trifluoropropanamide (1-n-6)

[0453]

[0454] To a stirred mixture of 3,3,3-trifluoropropionic acid (39 mg, 0.31 mmol) in tetrahydrofuran (1 mL) was added 1,1′-carbonyldiimidazole (33 mg, 0.20 mmol) in one portion at room temperature and the clear solution was stirred at room temperature for 3 hours. Triethylamine (0.028 mL, 0.20 mmol) and (1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-amine (1-n-1) (47 mg, 0.10 mmol) were added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate (3 mL), brine (2 mL), and water (2 mL). The organic extract was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was subjected to chromatography on a silica gel column, eluting with a gradient from 10% to 50% of ethyl acetate in heptane to obtain the title product as a white solid (40 mg, 69%). ¹H NMR (CHLOROFORM-d) δ: 7.24-7.42 (m, 15H), 5.37 (d, J=8.6 Hz, 1H), 5.35 (d, J=1.6 Hz, 1H), 4.95 (d, J=11.3 Hz, 1H), 4.71 (d, J=12.1 Hz, 1H), 4.56 (d, J=11.3 Hz, 1H), 4.45 (d, J=12.1 Hz, 1H), 4.40 (s, 2H), 4.35-4.40 (m, 1H), 4.05 (d, J=3.5 Hz, 1H), 3.95 (d, J=8.6 Hz, 1H), 3.71 (d, J=8.2 Hz, 1H), 3.61 (d, J=8.2 Hz, 1H), 3.50 (dd, J=10.0, 3.7 Hz, 1H), 3.45 (d, J=8.6 Hz, 1H), 2.92 (q, J=10.5 Hz, 2H); ¹³C NMR (CHLOROFORM-d) δ: 162.3, 138.0, 137.6, 137.3, 128.7, 128.5, 128.4, 128.3, 128.3, 128.2, 128.0, 128.0, 127.8, 101.1, 82.8, 75.8, 75.0, 73.7, 73.2, 71.9, 70.2, 69.3, 54.0, 41.7; ¹⁹F NMR (CHLOROFORM-d) δ: −62.8 (s); LCMS (ES−): 2.05 min, 570.3 (M−H)⁻.

N-{(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}-2,2-difluoroacetamide (1-n-7)

[0455]

[0456] To a stirred mixture of difluoroacetic acid (29 mg, 0.31 mmol) in N,N-dimethylformamide (1 mL) was added 1,1′-carbonyldiimidazole (33 mg, 0.20 mmol) in one portion at room temperature and the clear solution was stirred at room temperature for 3 hours. Triethylamine (0.028 mL, 0.20 mmol) and (1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-amine (1-n-1) (47 mg, 0.10 mmol) were added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 h and partitioned between ethyl acetate (3 mL), brine (2 mL), and water (2 mL). The organic extract was washed with brine, dried over anhydrous magnesium sulfate, and concentrated. The residue was subjected to chromatography on a silica gel column, eluting with a gradient from 10% to 50% of ethyl acetate in heptane to obtain the title product as a white solid (32 mg, 58%). ¹H NMR (CHLOROFORM-d) δ: 7.14-7.47 (m, 15H), 6.02 (d, J=8.6 Hz, 1H), 5.83 (t, J=54.2 Hz, 1H), 5.35 (s, 1H), 4.96 (d, J=11.3 Hz, 1H), 4.73 (d, J=12.5 Hz, 1H), 4.58 (d, J=11.3 Hz, 1H), 4.45 (d, J=12.1 Hz, 1H), 4.41 (s, 2H), 4.34-4.40 (m, 1H), 4.08 (d, J=3.9 Hz, 1H), 3.96 (d, J=9.0 Hz, 1H), 3.67-3.75 (m, 1H), 3.59-3.65 (m, 1H), 3.53 (dd, J=9.8, 3.9 Hz, 1H), 3.46 (d, J=8.6 Hz, 1H); ¹³C NMR (CHLOROFORM-d) δ: 162.6, 138.0, 137.3, 128.8, 128.5, 128.4, 128.3, 128.1, 128.0, 127.8, 108.3 (t, J=253.1 Hz), 100.9, 82.9, 75.3, 75.0, 73.8, 72.9, 71.6, 70.2, 69.3, 53.5; ¹⁹F NMR (CHLOROFORM-d) δ: −126.1 (d, J=53.1 Hz); LCMS (ES−): 2.05 min, 538.2 (M−H)⁻.

tert-butyl {(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}carbamate (I-n-8)

[0457]

[0458] To a stirred mixture of (1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-amine (1-n-1) (120 mg, 0.26 mmol) and N,N-dimethylaminopyridine (DMAP) (6.4 mg, 0.052 mmol), and tetrahydrofuran (2 mL) was added di-tert-butyl dicarbonate (113 mg, 0.52 mmol) in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and concentrated under reduced pressure. The residue was purified on a silica gel column, eluting with a gradient from 10% to 40% of ethyl acetate in heptane to obtain the title product as a white solid (104 mg, 71%). ¹HNMR (CHLOROFORM-d) δ: 7.17-7.45 (m, 15H), 5.35 (s, 1H), 4.97 (d, J=11.3 Hz, 1H), 4.74 (d, J=12.1 Hz, 1H), 4.57 (d, J=11.3 Hz, 2H), 4.47 (d, J=7.0 Hz, 1H), 4.39 (s, 2H), 4.09 (br. s., 1H), 4.01 (d, J=3.9 Hz, 1H), 3.93 (d, J=9.0 Hz, 1H), 3.63-3.71 (m, 1H), 3.54-3.62 (m, 1H), 3.38-3.48 (m, 2H), 1.47 (s, 9H); ¹³C NMR (CHLOROFORM-d) δ: 155.2, 138.2, 137.8, 137.4, 128.5, 128.4, 128.3, 128.3, 128.0, 127.9, 127.8, 127.7, 102.2, 82.8, 79.4, 74.8, 73.7, 73.4, 72.0, 70.0, 69.4, 54.6, 31.9, 28.4; LCMS (AP+): 2.25 min, 462.2 (M-Boc+H)⁺.

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]propanamide (25)

[0459]

[0460] A mixture of N-((1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-yl)propionamide (1-n-5) (42 mg, 0.081 mmol), 1-methyl-1,4-cyclohexadiene (0.093 mL, 0.81 mmol), 10% Pd on activated carbon (20 mg), and 2-propanol (2.5 mL) was stirred at 80° C. for 3 hours. Water (0.2 ml) was added and the whole mixture was loaded on silica gel and dried on a rotary evaporator. The material was purified on a silica gel column eluting with a gradient from 4% to 15% of methanol in dichloromethane gave the title compound as a colorless gum (12 mg, 60%). ¹H NMR (METHANOL-d₄) δ: 5.21 (d, J=1.6 Hz, 1H), 3.95 (dd, J=10.1, 1.2 Hz, 1H), 3.92 (d, J=11.3 Hz, 1H), 3.87 (d, J=3.9 Hz, 1H), 3.81 (d, J=11.3 Hz, 1H), 3.75 (d, J=8.2 Hz, 1H), 3.71-3.76 (m, 2H), 3.68 (d, J=8.2 Hz, 1H), 3.35 (s, 1H), 2.26 (q, J=7.4 Hz, 2H), 1.13 (t, J=7.4 Hz, 3H); ¹³C NMR (METHANOL-d₄) δ: 177.7, 102.6, 84.9, 70.4, 69.1, 69.0, 61.9, 56.0, 30.0, 10.3; LCMS (AP+): 0.25 min, 248.2 (M+H)⁺.

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]-2,2,2-trifluoroacetamide (24)

[0461]

[0462] A mixture of N-((1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-yl)-2,2,2-trifluoroacetamide (1-n-3) (20 mg, 0.036 mmol), 1-methyl-1,4-cyclohexadiene (0.093 mL, 0.81 mmol), 10% Pd on activated carbon (20 mg), and 2-propanol (2.5 mL) was stirred at 80° C. for 3 hours. Water (0.2 ml) was added and the whole mixture was loaded on silica gel and dried on a rotary evaporator. The material was purified on a silica gel column, eluting with a gradient from 4% to 15% of methanol in dichloromethane gave the title compound as a colorless gum (7.2 mg, 69%). ¹H NMR (METHANOL-d₄) δ: 5.25 (d, J=1.2 Hz, 1H), 4.02 (d, J=8.6 Hz, 1H), 3.90 (d, J=7.0 Hz, 1H), 3.88-3.95 (m, 2H), 3.82 (d, J=11.7 Hz, 1H), 3.78 (d, J=7.8 Hz, 1H), 3.71 (d, J=7.8 Hz, 1H), 3.35 (s, 1H); ¹³C NMR (METHANOL-d₄) δ: 159.8, 102.3, 85.5, 70.8, 69.7, 68.4, 62.2, 57.4; ¹⁹F NMR (CHLOROFORM-d) δ: −77.0 (s); LCMS (AP+): 0.42 min, 288.2 (M+H)⁺.

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]methanesulfonamide (26)

[0463]

[0464] A mixture of N-((1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-yl)methanesulfonamide (1-n-4) (19 mg, 0.035 mmol), 1-methyl-1,4-cyclohexadiene (0.093 mL, 0.81 mmol), 10% Pd on activated carbon (20 mg), and 2-propanol (2.5 mL) was stirred at 80° C. for 3 hours. Water (0.2 ml) was added and the whole mixture was loaded on silica gel and dried on a rotary evaporator. The material was purified on a silica gel column, eluting with a gradient from 4% to 15% of methanol in dichloromethane gave the title compound as a colorless gum (6.4 mg, 68%). ¹H NMR (METHANOL-d₄) δ: 5.26 (d, J=1.6 Hz, 1H), 3.91 (d, J=11.3 Hz, 1H), 3.87 (d, J=4.3 Hz, 1H), 3.80 (d, J=11.3 Hz, 1H), 3.73 (d, J=7.8 Hz, 1H), 3.68 (d, J=7.8 Hz, 1H), 3.66-3.71 (m, 1H), 3.37 (dd, J=9.8, 1.6 Hz, 1H), 3.35 (s, 1H), 3.04 (s, 3H); ¹³C NMR (METHANOL-d₄) δ: 104.6, 85.1, 70.9, 69.8, 69.3, 62.0, 60.2, 41.7; LCMS (ES−): 0.15 min, 268.0 (M−H)⁻.

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]-2,2-difluoroacetamide (27)

[0465]

[0466] To a solution of N-{(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}-2,2-difluoroacetamide (1-n-7) (32.0 mg, 0.059 mmol) in 2-propanol (1.0 mL) and tetrahydrofuran (0.5 mL) in a 5 mL microwave vial was added 1-methyl-1,4-cyclohexadiene (0.2 mL, 2 mmol) followed by the addition of 10% Palladium on carbon (50% wet wt/wt, 20.0 mg, 0 mmol). The vial was sealed and heated to 80° C. for 4 hours. After 4 hours, the TLC (10% methanol/dichloromethane) showed that the reaction was not complete but there was formation of the desired product. An additional 1-methyl-1,4-cyclohexadiene (0.2 mL mg, 2 mmol) was added and the reaction was resealed and heated to 80° C. overnight (18 hours). After 22 total hours, the reaction was diluted with methanol and filtered through a Life Sciences Acrodisc 25 mm syringe filter. The filtrate was concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 4 g silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound as a solid (5.0 mg, SOLID, 31%). Method C: 3 minute run LRMS [M+Na=292]. ¹H NMR (METHANOL-d₄) δ: 6.06 (t, J=54.2 Hz, 1H), 5.25 (s, 1H), 4.02 (d, J=9.4 Hz, 1H), 3.92 (d, J=11.7 Hz, 1H), 3.84-3.90 (m, 2H), 3.81 (d, J=11.7 Hz, 1H), 3.78 (d, J=8.2 Hz, 1H), 3.70 (d, J=8.2 Hz, 1H)

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]-3,3,3-trifluoropropanamide (28)

[0467]

[0468] To a solution of N-{(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}-3,3,3-trifluoropropanamide (1-n-6) (40.0 mg, 0.070 mmol) in 2-propanol (1.0 mL) and tetrahydrofuran (0.5 mL) in a 5 mL microwave vial was added 1-methyl-1,4-cyclohexadiene (0.2 mL, 1.75 mmol) followed by the addition of 10% palladium on carbon (50% wet wt/wt., 20.0 mg, 0 mmol). The vial was sealed and heated to 80° C. for 4 hours. After 4 hours, the reaction was diluted with methanol and filtered through a Life Sciences Acrodisc 25 mm syringe filter. The filtrate was concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 4 g silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound as a solid (15.3 mg, 73%). LRMS [M+1=302]. ¹H NMR (METHANOL-d₄) δ: 5.23 (s, 1H), 3.99 (d, J=9.8 Hz, 1H), 3.92 (d, J=11.3 Hz, 1H), 3.87 (d, J=4.3 Hz, 1H), 3.81 (d, J=11.3 Hz, 1H), 3.76 (d, J=8.2 Hz, 1H), 3.71-3.74 (m, 1H), 3.69 (d, J=8.2 Hz, 1H), 3.22 (qd, J=10.7, 2.5 Hz, 2H)

N-{(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}-N-methylacetamide (I-o-1)

[0469]

[0470] To a stirred mixture of N-((1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-yl)acetamide (I-n-2) (19 mg, 0.038 mmol) and N,N-dimethylformamide (1.5 mL) was added sodium hydride (60% suspension in mineral oil) in one portion at room temperature and the mixture was stirred for 30 minutes. Iodomethane (16 mg, 0.11 mmol) was added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate (3 mL), brine (2 mL), and water (2 mL). The organic extract was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified on a silica gel column, eluting with a gradient from 10% to 50% of ethyl acetate in heptane to obtain the title product as a colorless gum (19 mg, 97%). Mixture of rotamers (3:1). Rotamer 1: ¹H NMR (CHLOROFORM-d) δ: 7.21-7.43 (m, 15H), 5.37 (s, 1H), 4.93 (d, J=10.9 Hz, 1H), 4.65 (d, J=11.7 Hz, 1H), 4.54 (d, J=11.3 Hz, 1H), 4.50 (d, J=11.7 Hz, 1H), 4.37-4.45 (m, 2H), 4.15 (d, J=9.8 Hz, 1H), 4.09 (d, J=3.5 Hz, 1H), 3.93 (d, J=8.6 Hz, 1H), 3.85 (dd, J=10.1, 3.5 Hz, 1H), 3.78 (d, J=8.2 Hz, 1H), 3.64 (d, J=8.2 Hz, 1H), 3.45 (d, J=8.6 Hz, 1H), 2.80 (s, 3H), 2.19 (s, 3H); rotomer2: ¹H NMR (CHLOROFORM-d) δ: 7.21-7.43 (m, 15H), 5.27 (s, 1H), 5.12 (d, J=10.9 Hz, 1H), 4.94-4.98 (m, 1H), 4.74 (d, J=12.1 Hz, 1H), 4.56 (d, J=11.7 Hz, 1H), 4.46 (d, J=11.3 Hz, 1H), 4.37-4.46 (m, 2H), 4.14-4.17 (m, 1H), 3.97 (d, J=8.6 Hz, 1H), 3.82-3.89 (m, 2H), 3.79 (d, J=8.2 Hz, 1H), 3.60-3.63 (m, 1H), 2.71 (s, 3H), 2.08 (s, 3H); ¹³C NMR (CHLOROFORM-d) δ: 172.2, 138.0, 137.3, 137.2, 128.6, 128.5, 128.5, 128.4, 128.1, 128.0, 127.9, 127.9, 127.9, 127.8, 103.2, 83.2, 75.2, 74.1, 73.8, 73.7, 73.0, 72.4, 70.1, 69.2, 61.2, 28.0, 22.2; LCMS (AP+): 1.99 min, 518.0 (M+H)⁺.

N-{(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}-N-methylmethanesulfonamide (I-o-2)

[0471]

[0472] To a stirred mixture of N-((1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-yl)methanesulfonamide (1-n-4) (19 mg, 0.035 mmol) and N,N-dimethylformamide (1.5 mL) was added sodium hydride (60% suspension in mineral oil) in one portion at room temperature and the mixture was stirred for 30 min. Iodomethane (16 mg, 0.11 mmol) was added in one portion at room temperature. The reaction mixture was stirred at room temperature for 16 hours and partitioned between ethyl acetate (3 mL), brine (2 mL), and water (2 mL). The organic extract was washed with brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified on a silica gel column, eluting with a gradient from 10% to 50% of ethyl acetate in heptane to obtain the title product as a colorless gum (11 mg, 56%). ¹H NMR (CHLOROFORM-d) δ: 7.25-7.39 (m, 15H), 5.39 (d, J=0.8 Hz, 1H), 4.93 (d, J=11.7 Hz, 1H), 4.80 (d, J=11.3 Hz, 1H), 4.58 (d, J=11.3 Hz, 1H), 4.45 (d, J=11.7 Hz, 1H), 4.43 (d, J=11.7 Hz, 1H), 4.41 (d, J=11.7 Hz, 1H), 4.21 (d, J=3.5 Hz, 1H), 4.18 (d, J=10.5 Hz, 1H), 3.98 (d, J=8.6 Hz, 1H), 3.87 (dd, J=10.5, 3.9 Hz, 1H), 3.82 (d, J=8.6 Hz, 1H), 3.62 (d, J=8.2 Hz, 1H), 3.45 (d, J=9.0 Hz, 1H), 2.83 (s, 3H), 2.68 (s, 3H); ¹³C NMR (CHLOROFORM-d) δ: 138.1, 137.3, 136.8, 128.7, 128.5, 128.4, 128.4, 128.1, 128.1, 128.0, 127.8, 104.6, 82.9, 74.9, 73.8, 73.5, 73.3, 71.6, 70.3, 69.4, 59.7, 37.5, 29.5; LCMS (AP+): 2.08 min, 575.8 (M+Na)⁺.

tert-butyl {(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}methylcarbamate (I-o-3)

[0473]

[0474] To a stirring solution of tert-butyl {(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}carbamate (I-n-8) (100 mg, 0.178 mmol) in N,N-dimethylformamide (1.5 mL) was added sodium hydride (60% dispersion in mineral oil, 8.55 mg, 0.214 mmol) at room temperature. The reaction was stirred for 1 hour before the addition of iodomethane (0.055 mL, 0.89 mmol). The reaction was allowed to stir overnight at room temperature. After 24 hours, the reaction was quenched with water and extracted three times ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 12 g silica gel column) and eluting with a gradient of 0-100% ethyl acetate/dichloromethane yielding the title compound (78 mg, 76%). Method C: 3 minute run LRMS [M+Na=598]. ¹H NMR (compound is a mixture of two rotamers ˜1:1)

[0475] Rotamer 1: ¹H NMR (METHANOL-d₄) δ: 7.08-7.48 (m, 15H), 5.26 (s, 1H), 4.83-4.90 (m, 1H), 4.73 (d, J=11.7 Hz, 1H), 4.47-4.58 (m, 3H), 4.36-4.47 (m, 2H), 4.21 (d, J=3.5 Hz, 1H), 3.98 (dd, J=10.7, 3.3 Hz, 1H), 3.92 (d, J=8.2 Hz, 2H), 3.61 (d, J=7.8 Hz, 1H), 3.47 (dd, J=8.6, 3.9 Hz, 1H), 2.75 (s, 3H), 1.42 (s, 9H)

[0476] Rotamer 2: ¹H NMR (METHANOL-d₄) δ: 7.08-7.48 (m, 15H), 5.21 (s, 1H), 4.83-4.90 (m, 1H), 4.73 (d, J=11.7 Hz, 1H), 4.47-4.58 (m, 3H), 4.36-4.47 (m, 2H), 4.21 (d, J=3.5 Hz, 1H), 3.98 (dd, J=10.7, 3.3 Hz, 1H), 3.92 (d, J=8.2 Hz, 2H), 3.61 (d, J=7.8 Hz, 1H), 3.47 (dd, J=8.6, 3.9 Hz, 1H), 2.70 (s, 3H), 1.44-1.52 (m, 9H)

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]-N-methylacetamide (29)

[0477]

[0478] A mixture of N-((1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-yl)-N-methylacetamide (I-o-1) (19 mg, 0.037 mmol), 1-methyl-1,4-cyclohexadiene (0.093 mL, 0.81 mmol), 10% Pd on activated carbon (20 mg), and 2-propanol (2.5 mL) was stirred at 80° C. for 3 hours. Water (0.2 ml) was added and the whole mixture was loaded on silica gel and dried on a rotary evaporator. The material was purified on a silica gel column, eluting with a gradient from 4% to 15% of methanol in dichloromethane gave the title compound as a colorless gum (4.3 mg, 47%). ¹H NMR (mixture of rotomers ˜1:1)

[0479] Rotamer 1: ¹H NMR (METHANOL-d₄) δ: 5.20 (s, 1H), 4.65 (d, J=10.5 Hz, 1H), 4.02-4.09 (m, 1H), 3.89-3.98 (m, 2H), 3.84 (s, 1H), 3.78-3.82 (m, 1H), 3.68 (s, 1H), 3.11 (s, 3H), 2.15 (s, 3H)

[0480] Rotamer 2: ¹H NMR (METHANOL-d₄) δ: 5.37 (s, 1H), 4.02-4.09 (m, 1H), 3.89-3.98 (m, 3H), 3.84-3.87 (m, 1H), 3.79-3.83 (m, 1H), 3.71 (d, J=8.2 Hz, 1H), 2.98 (s, 3H), 2.15 (s, 3H)

[0481] ¹³C NMR (METHANOL-d₄) δ: 175.4, 104.6, 85.8, 71.3, 69.7, 66.2, 62.3, 59.1, 28.8, 22.7; LCMS (ES−): 0.41 min, 246.2 (M−H)⁻.

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]-N-methylmethanesulfonamide (30)

[0482]

[0483] A mixture of N-((1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-((benzyloxy)methyl)-6,8-dioxabicyclo[3.2.1]octan-4-yl)-N-methylmethanesulfonamide (I-o-2) (11 mg, 0.020 mmol), 1-methyl-1,4-cyclohexadiene (0.093 mL, 0.81 mmol), 10% Pd on activated carbon (20 mg), and 2-propanol (2.5 mL) was stirred at 80° C. for 3 hours. Water (0.2 ml) was added and the whole mixture was loaded on silica gel and dried on a rotary evaporator. Chromatography on a silica gel column, eluting with a gradient from 4% to 15% of methanol in dichloromethane gave the title compound as a colorless gum (2.9 mg, 51%). ¹H NMR (METHANOL-d₄) δ: 5.26 (d, J=1.2 Hz, 1H), 4.00-4.05 (m, 1H), 3.92-3.96 (m, 1H), 3.89-3.91 (m, 1H), 3.84 (d, J=1.2 Hz, 1H), 3.77-3.83 (m, 2H), 3.68 (d, J=7.8 Hz, 1H), 3.35 (s, 3H), 2.93 (s, 3H); ¹³C NMR (METHANOL-d₄) δ: 106.1, 85.5, 71.5, 69.7, 65.9, 62.6, 62.2, 37.8, 30.4; LCMS (ES−): 0.42 min, 282.0 (M−H)⁻.

tert-butyl [(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]methylcarbamate (31)

[0484]

[0485] To a solution of tert-butyl {(1S,2R,3R,4R,5S)-2,3-bis(benzyloxy)-1-[(benzyloxy)methyl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}methylcarbamate (I-o-3)(75 mg, 0.13 mmol) in 2-propanol (1.0 mL) and tetrahydrofuran (0.5 mL) in a 5 mL microwave vial was added 1-methyl-1,4-cyclohexadiene (0.18 mL, 1.56 mmol) followed by the addition of 10% palladium on carbon (50% wet wt/wt., 20.0 mg). The vial was sealed and heated to 80° C. for 4 hours. After 4 hours, the TLC (10% methanol/dichloromethane) showed that the reaction was not complete but there was formation of the desired product. An additional 1-methyl-1,4-cyclohexadiene (0.18 mL, 1.6 mmol) was added and the reaction was resealed and heated to 80° C. overnight (18 hours). After 22 total hours, the reaction was diluted with methanol and filtered through a Life Sciences Acrodisc 25 mm syringe filter. The filtrate was concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 4 g silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound (29.0 mg, 73%) as a solid. Method C: 3 minute run

[0486] LRMS [M+Na=328]. Compound is a mixture of two rotamers ˜1:1:

[0487] Rotamer 1: ¹H NMR (METHANOL-d₄) δ: 5.22 (br. s., 1H), 4.19 (d, J=10.6 Hz, 1H), 4.00 (d, J=10.6 Hz, 1H), 3.90-3.95 (m, 2H), 3.77-3.82 (m, 2H), 3.67 (d, J=7.6 Hz, 1H), 2.94 (s, 3H), 1.47 (s, 9H)

[0488] Rotamer 2: ¹H NMR (METHANOL-d₄) δ: 5.21 (br. s., 1H), 4.05-4.10 (m, 1H), 4.00 (d, J=10.6 Hz, 1H), 3.90-3.95 (m, 2H), 3.77-3.82 (m, 2H), 3.67 (d, J=7.6 Hz, 1H), 2.94 (s, 3H), 1.47 (s, 9H) (1S,2R,3R,4R,5S)-1-(hydroxymethyl)-4-(methylamino)-6, 8-dioxabicyclo[3.2.1]octane-2,3-diol hydrochloride (32)

[0489] To a solution of tert-butyl [(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]methylcarbamate (31) (25.3 mg, 0.0829 mmol) in dichloromethane (5.0 mL) was added 4.0M hydrogen chloride in dioxane (0.518 mL, 2.07 mmol) and the reaction was allowed to stir for 48 hours at room temperature. After 48 hours, the reaction was concentrated under reduced pressure. The crude material was washed with ethyl acetate (5 mL) which created a solid and diluted with heptane (10 mL) and concentrated under reduced pressure yielding the title compound as a solid (30.0 mg, 130%). Method C: 3 minute run LRMS [M+1=206]. ¹H NMR (METHANOL-d₄) δ: 5.63 (s, 1H), 3.90-3.97 (m, 3H), 3.84 (s, 2H), 3.78 (d, J=8.2 Hz, 1H), 3.10-3.20 (m, 1H), 2.84 (s, 3H)

N-[(1S,2R,6R,7R,8S)-4,4-dimethyl-1-(15-phenyl-2,5,8,11,14-pentaoxapentadec-1-yl)-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-3)

[0490]

[0491] To a solution of N-[(1S,2R,6R,7R,8S)-1-(hydroxymethyl)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-1) (1200 mg, 4.39 mmol) and 13-iodo-1-phenyl-2,5,8,11-tetraoxatridecane

(see Synthetic Metals, 162(23), 2163-2170; 2012; 7000 mg, 17.76 mmol) in dichloromethane (30.0 mL) was added tetrabutylammonium hydrogen sulfate (2290 mg, 6.60 mmol) followed by the addition of 12.5M Sodium hydroxide aqueous (30.0 mL, 380 mmol). The reaction was allowed to stir at room temperature for 64 hours. After 64 hours, the reaction was diluted with water and dichloromethane. The layers were separated and the aqueous layer was extracted two additional times with dichloromethane. The combined organic layers were washed with 1N hydrochloric acid, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting crude material was added ethyl acetate (50 mL) and stirred for 30 minutes. The resulting precipitate was filtered. The filtrate was concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (ISCO RediSep Gold 80 g silica gel column) and eluting with a gradient of 0-100% ethyl acetate/heptane immediately followed by eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound (1267 mg, 53.5%). Method C: 1.5 minute run LRMS [M+Na=562]. ¹H NMR (METHANOL-d₄) δ: 7.13-7.45 (m, 5H), 5.22 (d, J=1.6 Hz, 1H), 4.55 (s, 2H), 4.30 (d, J=5.9 Hz, 1H), 4.15 (t, J=6.4 Hz, 1H), 3.89-3.97 (m, 2H), 3.85 (d, J=7.8 Hz, 1H), 3.73-3.79 (m, 2H), 3.58-3.71 (m, 16H), 1.98 (s, 3H), 1.48 (s, 3H), 1.33 (s, 3H)

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(15-phenyl-2,5,8,11,14-pentaoxapentadec-1-yl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (33)

[0492]

[0493] A solution of N-[(1S,2R,6R,7R,8S)-4,4-dimethyl-1-(15-phenyl-2,5,8,11,14-pentaoxapentadec-1-yl)-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-3) (60.0 mg, 0.11 mmol) in acetic acid (4.0 mL), methanol (1.0 mL) and water (1.0 mL) was heated to 70° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted with toluene a second time and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 4 g Gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound as a gum (42.5 mg, 77%). Method C: 3 minute run LRMS [M+1=500]. ¹H NMR (METHANOL-d₄) δ: 7.14-7.45 (m, 5H), 5.21 (s, 1H), 4.55 (s, 2H), 3.92-4.01 (m, 2H), 3.88 (d, J=4.3 Hz, 1H), 3.77 (d, J=7.8 Hz, 1H), 3.70 (dd, J=9.8, 3.9 Hz, 1H), 3.58-3.68 (m, 18H), 1.98 (s, 3H)

N-[(1S,2R,6R,7R,8S)-1-(13-azido-2,5,8,11-tetraoxatridec-1-yl)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-2)

[0494]

[0495] To a stirred solution of N-[(1S,2R,6R,7R,8S)-1-(hydroxymethyl)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-1) (10.0 g, 36.59 mmol, 1.0 eq) in N,N-dimethylformamide (200 ml) was added potassium hydroxide (8.21 g, 146.37 mmol, 4 eq) at 5° C. (ice/water). After addition, the reaction mixture was stirred at 5° C. for 30 min. Then 1-azido-2-{2-[2-(2-iodoethoxy)ethoxy]ethoxy}ethane (36.13 g, 109.78 mmol, 3.0 eq) was added to the reaction mixture at 5° C. (ice/water). The reaction mixture was stirred at 5° C. (ice/water) for 30 minutes and the reaction mixture was heated to 27° C. and stirred at 27° C. for 18 hours. After 18 hours, the reaction mixture was poured into ice/water and extracted three times with dichloromethane (400 ml). The combined organic layers were washed three times with water (400 ml), brine (500 ml), dried over sodium sulfate, filtered and concentrated to give crude product. The crude product was purified by silica gel chromatography eluted with dichloromethane:methanol=100:1-40:1 to the title compound (10.0 g, 57.6%) as colorless oil. Method C: 3 minute run LRMS [M+45 (formic acid)=519]. ¹H NMR (METHANOL-d₄) δ: 5.23 (d, J=2.0 Hz, 1H), 4.31 (d, J=5.9 Hz, 1H), 4.16 (t, J=6.6 Hz, 1H), 3.93-3.97 (m, 1H), 3.90-3.93 (m, J=2.0 Hz, 1H), 3.86 (d, J=7.8 Hz, 1H), 3.78 (d, J=3.9 Hz, 1H), 3.75 (d, J=1.6 Hz, 1H), 3.61-3.71 (m, 14H), 3.37 (t, J=4.9 Hz, 2H), 1.98 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H)

N-[(1S,2R,3R,4R,5S)-1-(13-azido-2,5,8,11-tetraoxatridec-1-yl)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (34)

[0496]

[0497] A solution of N-[(1S,2R,6R,7R,8S)-1-(13-azido-2,5,8,11-tetraoxatridec-1-yl)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-2) (82.0 mg, 0.17 mmol) in acetic acid (6.0 mL), methanol (1.45 mL) and water (1.45 mL) was heated to 70° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted with toluene a second time and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 4 g Gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound as an oil (43.3 mg, 58%). Method C: 3 minute run LRMS [M−1=433]. ¹H NMR (METHANOL-d₄) δ: 5.21 (d, J=0.8 Hz, 1H), 3.98 (d, J=9.8 Hz, 1H), 3.94 (d, J=9.8 Hz, 1H), 3.89 (d, J=4.3 Hz, 1H), 3.78 (d, J=7.8 Hz, 1H), 3.72 (dd, J=10.1, 4.3 Hz, 1H), 3.61-3.69 (m, 16H), 3.38 (t, J=4.9 Hz, 2H), 1.99 (s, 3H)

N-[(1S,2R,6R,7R,8S)-4,4-dimethyl-1-(2,5,8,11-tetraoxatetradec-13-en-1-yl)-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-4)

[0498]

[0499] To a solution of N-[(1S,2R,6R,7R,8S)-1-(hydroxymethyl)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-1) (50.0 mg, 0.18 mmol) and 3-{2-[2-(2-iodoethoxy)ethoxy]ethoxy}prop-1-ene

(see Organic Letters, 5(11), 1887-1890; 2003, 139.0 mg, 0.463 mmol) in dichloromethane (1.5 mL) was added tetrabutylammonium hydrogen sulfate (95.3 mg, 0.275 mmol) followed by the addition of 12.5M sodium hydroxide aqueous (0.75 mL, 9.4 mmol). The reaction was allowed to stir at room temperature overnight. After 18 hours, the reaction was diluted with water and dichloromethane. The layers were separated and the aqueous layer was extracted two additional times with dichloromethane. The combined organic layers were washed with 1N hydrochloric acid, water, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting crude material was diluted with ethyl acetate (5 mL) and the resulting precipitate was stirred at room temperature for 30 minutes. The precipitate was filtered and the filter cake was washed with ethyl acetate (2×5 mL). The filtrate was concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (ISCO RediSep Gold 4 g silica gel column) and eluting with a gradient of 0-100% ethyl acetate/heptane. The column was then eluted with a gradient of 0-20% methanol/dichloromethane yielding the title compound (13.6 mg, 17%). Method C: 1.5 minute run LRMS [M+Na=468]. ¹H NMR (METHANOL-d₄) δ: 5.92 (ddt, J=16.8, 10.9, 5.7 Hz, 1H), 5.28 (dd, J=17.4, 1.4 Hz, 1H), 5.23 (d, J=1.6 Hz, 1H), 5.16 (dd, J=10.3, 1.0 Hz, 1H), 4.31 (d, J=5.9 Hz, 1H), 4.15 (t, J=6.4 Hz, 1H), 4.02 (d, J=5.5 Hz, 2H), 3.89-3.97 (m, 2H), 3.86 (d, J=7.8 Hz, 1H), 3.73-3.80 (m, 2H), 3.56-3.72 (m, 12H), 1.98 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H).

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(2,5,8,11-tetraoxatetradec-13-en-1-yl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (35)

[0500]

[0501] A solution of N-[(1S,2R,6R,7R,8S)-4,4-dimethyl-1-(2,5,8,11-tetraoxatetradec-13-en-1-yl)-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-4) (13.0 mg, 0.029 mmol) in acetic acid (1.0 mL), methanol (0.25 mL) and water (0.25 mL) was heated to 70° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted with toluene a second time and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 4 g Gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound (6.5 mg, 55%). Method C: 3 minute run LRMS [M+1=406]. ¹H NMR (METHANOL-d₄) δ: 5.80-6.09 (m, 1H), 5.28 (dd, J=17.2, 1.6 Hz, 1H), 5.21 (d, J=0.8 Hz, 1H), 5.16 (dd, J=10.5, 1.2 Hz, 1H), 4.02 (d, J=5.5 Hz, 2H), 3.98 (d, J=9.8 Hz, 1H), 3.95 (d, J=10.1 Hz, 1H), 3.89 (d, J=3.9 Hz, 1H), 3.78 (d, J=8.2 Hz, 1H), 3.71 (dd, J=10.0, 4.5 Hz, 1H), 3.57-3.68 (m, 14H), 1.99 (s, 3H).

N-[(1S,2R,6R,7R,8S)-4,4-dimethyl-1-(2,5,8,11-tetraoxatetradec-13-yn-1-yl)-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-5)

[0502]

[0503] To a solution of N-[(1S,2R,6R,7R,8S)-4,4-dimethyl-1-(15-phenyl-2,5,8,11,14-pentaoxapentadec-1-yl)-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-1) (100.0 mg, 0.366 mmol) and 3-{2-[2-(2-iodoethoxy)ethoxy]ethoxy}prop-1-yne

(see Synthesis, (10), 1639-1644; 2010, 425.0 mg, 1.43 mmol) in dichloromethane (3 mL) was added tetrabutylammonium hydrogen sulfate (191 mg, 0.550 mmol) followed by the addition of 12.5M sodium hydroxide aqueous (1.5 mL, 19 mmol). The reaction was allowed to stir at room temperature overnight. After 18 hours, the reaction was diluted with water and dichloromethane. The layers were separated and the aqueous layer was extracted two additional times with dichloromethane. The combined organic layers were washed with 1N hydrochloric acid, water, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The resulting crude material was diluted with ethyl acetate (20 mL) and the resulting precipitate was stirred at room temperature for 30 minutes. The precipitate was filtered and the filter cake was washed with ethyl acetate (2×15 mL). The filtrate was concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (ISCO RediSep Gold 12 g silica gel column) and eluting with a gradient of 0-100% ethyl acetate/heptane. The column was then eluted with a gradient of 0-20% methanol/dichloromethane yielding the title compound (70.0 mg, 43%). Method C: 1.5 minute run LRMS [M+Na=466]. ¹H NMR (METHANOL-d₄) δ: 5.23 (d, J=1.6 Hz, 1H), 4.31 (d, J=5.9 Hz, 1H), 4.19 (d, J=2.3 Hz, 2H), 4.16 (t, J=6.4 Hz, 1H), 3.90-3.97 (m, 2H), 3.86 (d, J=7.8 Hz, 1H), 3.74-3.79 (m, 2H), 3.60-3.72 (m, 12H), 2.85 (t, J=2.3 Hz, 1H), 1.98 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H)

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(2,5,8,11-tetraoxatetradec-13-yn-1-yl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (36)

[0504]

[0505] A solution of N-[(1S,2R,6R,7R,8S)-4,4-dimethyl-1-(2,5,8,11-tetraoxatetradec-13-yn-1-yl)-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-5) (70.0 mg, 0.16 mmol) in acetic acid (4.0 mL), methanol (1.0 mL), and water (1.0 mL) was heated to 70° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted with toluene a second time and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 4 g Gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound as a gum (57.6 mg, 90%). Method C: 3 minute run LRMS [M+1=404]. ¹H NMR (METHANOL-d₄) δ: 5.22 (s, 1H), 4.19 (d, J=1.8 Hz, 2H), 3.98 (d, J=10.0 Hz, 1H), 3.94 (d, J=10.0 Hz, 1H), 3.89 (d, J=4.1 Hz, 1H), 3.78 (d, J=8.2 Hz, 1H), 3.71 (dd, J=10.0, 4.1 Hz, 1H), 3.60-3.69 (m, 14H), 2.86 (s, 1H), 1.99 (s, 3H)

N-[(1S,2R,3R,4R,5S)-1-(13-amino-2,5,8,11-tetraoxatridec-1-yl)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (37)

[0506]

[0507] A solution of N-[(1S,2R,3R,4R,5S)-1-(13-azido-2,5,8,11-tetraoxatridec-1-yl)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (34) (40.0 mg, 0.092 mmol) in ethanol (2 mL) was passed through the H-cube (conditions: catalyst (10% palladium on carbon (30×4 mm), flow rate: 1 mL/min., temperature: room temperature, pressure=Full H₂). After passing through the H-cube, the solution was collected and concentrated under reduced pressure yielding the title compound as a gum (17.2 mg, 46%). Method C: 3 minute run LRMS [M+1=409]. ¹H NMR (METHANOL-d₄) δ: 5.21 (s, 1H), 3.92-4.00 (m, 2H), 3.89 (d, J=3.9 Hz, 1H), 3.78 (d, J=8.2 Hz, 1H), 3.69-3.74 (m, 1H), 3.61-3.69 (m, 14H), 3.56 (t, J=5.1 Hz, 2H), 2.85 (t, J=5.1 Hz, 2H), 1.99 (s, 3H)

N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(13-hydroxy-2,5,8,11-tetraoxatridec-1-yl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (38)

[0508]

[0509] N-[(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-(15-phenyl-2,5,8,11,14-pentaoxapentadec-1-yl)-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide (33) (43 mg, 0.086 mmol) was dissolved in methanol (2 mL) was passed through the H-cube (conditions: catalyst (20% palladium hydroxide carbon (30×4 mm), flow rate: 1 mL/min., temperature: 60 C, pressure=Full H₂). After passing through the H-cube, the solution was collected and concentrated under reduced pressure yielding the title compound (32.2 mg, 91%). ¹H NMR (METHANOL-d₄) δ: 5.21 (s, 1H), 3.98 (d, J=9.4 Hz, 1H), 3.95 (d, J=10.1 Hz, 1H), 3.89 (d, J=4.3 Hz, 1H), 3.78 (d, J=8.2 Hz, 1H), 3.71 (dd, J=9.8, 4.3 Hz, 1H), 3.61-3.69 (m, 16H), 3.54-3.59 (m, 2H), 1.99 (s, 3H). ¹³C NMR (METHANOL-d₄) δ: 174.1, 102.6, 84.3, 73.8, 72.5, 71.7, 71.7(2), 71.6, 71.5, 71.4, 70.5, 70.2, 69.0, 62.4, 56.4, 22.7

N-[(1S,2R,6R,7R,8S)-1-(13-hydroxy-2,5,8,11-tetraoxatridec-1-yl)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-6)

[0510]

[0511] N-[(1S,2R,6R,7R,8S)-4,4-dimethyl-1-(15-phenyl-2,5,8,11,14-pentaoxapentadec-1-yl)-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-3) (2.897 g, 5.37 mmol) was dissolved in methanol (150 mL) was passed through the H-cube (conditions: catalyst (10% palladium on carbon (30×4 mm), flow rate: 1 mL/min., temperature: 60° C., pressure=Full H₂). After passing through the H-cube, the solution was collected and concentrated under reduced pressure yielding the title compound as a gum (2.5 g, 100%). Method C: 1.5 minute run LRMS [M+1=450]. ¹H NMR (METHANOL-d₄) δ: 5.23 (d, J=1.6 Hz, 1H), 4.31 (d, J=5.9 Hz, 1H), 4.16 (t, J=6.4 Hz, 1H), 3.89-3.97 (m, 2H), 3.86 (d, J=7.8 Hz, 1H), 3.74-3.80 (m, 2H), 3.60-3.71 (m, 14H), 3.53-3.59 (m, 2H), 1.98 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H)

N-[(1S,2R,6R,7R,8S)-4,4-dimethyl-1-(13-oxo-2,5,8,11-tetraoxatridec-1-yl)-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-6a)

[0512]

[0513] To a solution of N-[(1S,2R,6R,7R,8S)-1-(13-hydroxy-2,5,8,11-tetraoxatridec-1-yl)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-6) (175 mg, 0.389 mmol) in dichloromethane (5.0 mL) was added Dess-Martin reagent (354 mg, 0.584 mmol) which resulted in a mixture. After ˜30 minutes, the reaction became almost homogeneous. After 3 hours, the reaction mixture was diluted with dichloromethane and filtered through a plug of celite and washed with dichloromethane. The filtrate was concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 24 g gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane. The tubes containing the desired product were concentrated under reduced pressure. The resulting material was diluted with dichloromethane (4 mL) and diluted with ethyl ether (10 mL) which resulted in a white precipitate. The solution was decanted and the solid was diluted with dichloromethane (2 mL) and ethyl ether (8 mL) and decanted a second time. The decanted solution was passed through a Life Science Acrodisc 25 mm syringe filter with 0.45 um Nylon membrane. The collected solution was concentrated under reduced pressure yielding the title compound as a gum (65.0 mg, 37%). Method C: 3 minute run LRMS [M+1=448]. ¹H NMR (CHLOROFORM-d) δ: 9.74 (s, 1H), 5.63 (d, J=9.0 Hz, 1H), 5.34 (d, J=1.6 Hz, 1H), 4.23 (d, J=5.9 Hz, 1H), 4.17 (s, 2H), 4.09-4.15 (m, 1H), 4.01 (t, J=6.2 Hz, 1H), 3.97 (d, J=10.1 Hz, 1H), 3.77-3.85 (m, 3H), 3.68-3.76 (m, 5H), 3.61-3.68 (m, 7H), 2.03 (s, 3H), 1.56 (s, 3H), 1.36 (s, 3H).

1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-oic acid (38A)

[0514]

[0515] To a solution of N-[(1S,2R,6R,7R,8S)-4,4-dimethyl-1-(13-oxo-2,5,8,11-tetraoxatridec-1-yl)-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-6a) (60.0 mg, 0.13 mmol) in tetrahydrofuran/t-butanol (1.5 mL/1.5 mL) was treated with 2-methyl-2-butene (1.0 mL) followed by a solution of sodium chlorite (169.4 mg, 2.01 mmol) and sodium phosphate (250.0 mg, 2.58 mmol) (monobasic and monohydrate, 250 mg, 2.58 mmol) in water (1.5 mL) via glass pipet. The reaction was allowed to stir at room temperature for 24 hours. After 24 hours, the reaction mixture was poured into water and extracted with ethyl acetate (three times). The organic layer was discarded. The aqueous layers were concentrated under reduced pressure and the resulting crude was dissolved in methanol (10 mL) and dichloromethane (100 mL) and the resulting mixture was filtered. The filtrate was concentrated under reduced pressure. The resulting material was dissolved in methanol (5 mL) and dichloromethane (50 mL) and the resulting mixture was filtered. The filtrate was purified using the CombiFlash Rf (RediSep 4 g silica gel column) and eluting with a gradient of 0-100% methanol/dichloromethane yielding the title compound as a gum as a sodium salt (40 mg, None, 67%). LRMS [M+1=424]; ¹H NMR (METHANOL-d₄) δ: 5.24 (s, 1H), 4.14 (s, 2H), 3.97 (d, J=10.1 Hz, 2H), 3.90 (d, J=3.9 Hz, 1H), 3.81 (d, J=7.8 Hz, 1H), 3.63-3.77 (m, 15H), 2.01 (s, 3H)

1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl methanesulfonate (I-e-7)

[0516]

[0517] To a solution of 1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl methanesulfonate (I-e-6) (1117 mg, 2.49 mmol) in dichloromethane (12.4 mL) was added triethylamine (1.05 mL, 7.45 mmol) and cooled to 0° C. using an ice bath followed by the addition of methane sulfonyl chloride (0.232 mL, 2.98 mmol). The reaction was allowed to warm slowly to room temperature and stirred at room temperature for 1.5 hours. After 1.5 hours, the reaction was quenched with water and extracted. The layers were separated and the aqueous layer was extracted an additional time with dichloromethane. The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure yielding the title compound which was carried on crude (1300.0 mg, 99.2%). Method C: 3 minute run LRMS [M+Na=550]. ¹H NMR (METHANOL-d₄) δ: 5.23 (d, J=2.0 Hz, 1H), 4.34-4.40 (m, 2H), 4.31 (d, J=5.9 Hz, 1H), 4.15 (t, J=6.4 Hz, 1H), 3.89-3.97 (m, 2H), 3.86 (d, J=7.8 Hz, 1H), 3.72-3.81 (m, 4H), 3.59-3.71 (m, 12H), 3.11 (s, 3H), 1.98 (s, 3H), 1.48 (s, 3H), 1.34 (s, 3H)

S-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}ethanethioate (I-e-8)

[0518]

[0519] To a solution of 1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-ylmethanesulfonate (I-e-7) (125.0 mg, 0.237 mmol) in N,N-dimethylformamide (2 mL) was added potassium thioacetate (135 mg, 1.18 mmol) and the reaction was stirred at room temperature for 64 hours. After 64 hours, the reaction was diluted with water and extracted with ethyl acetate three times. The combined organic layers washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 4 g silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound as a gum (95.2 mg, 79.2%). Method C: 3 minute run LRMS [M+Na=530]. ¹H NMR (METHANOL-d₄) δ: 5.23 (d, J=1.6 Hz, 1H), 4.31 (d, J=5.9 Hz, 1H), 4.16 (t, J=6.4 Hz, 1H), 3.90-3.97 (m, 2H), 3.86 (d, J=7.8 Hz, 1H), 3.74-3.79 (m, 2H), 3.55-3.72 (m, 14H), 3.08 (t, J=6.6 Hz, 2H), 2.32 (s, 3H), 1.98 (s, 3H), 1.49 (s, 3H), 1.34 (s, 3H)

S-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl} ethanethioate (39)

[0520]

[0521] A solution of S-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}ethanethioate (I-e-8) (81.0 mg, 0.16 mmol) in acetic acid (6.0 mL), methanol (1.45 mL) and water (1.45 mL) was heated to 70° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted with toluene a second time and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 4 g Gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound as a gum (53.7 mg, 72%). Method C: 3 minute run LRMS [M+1=468]. ¹H NMR (METHANOL-d₄) δ: 5.23 (s, 1H), 4.00 (d, J=9.8 Hz, 1H), 3.97 (d, J=9.8 Hz, 1H), 3.91 (d, J=4.3 Hz, 1H), 3.80 (d, J=7.8 Hz, 1H), 3.73 (dd, J=10.1, 4.3 Hz, 1H), 3.63-3.70 (m, 14H), 3.60 (t, J=6.6 Hz, 2H), 3.10 (t, J=6.4 Hz, 2H), 2.34 (s, 3H), 2.01 (s, 3H)

N-{(1S,2R,3R,4R,5S)-2,3-dihydroxy-1-[13-(pyridin-2-yldisulfanyl)-2,5,8,11-tetraoxatridec-1-yl]-6,8-dioxabicyclo[3.2.1]oct-4-yl}acetamide (40)

[0522]

[0523] To a solution of S-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl} ethanethioate (39) (50 mg, 0.11 mmol) in methanol (3 mL) followed by the addition of a 0.5M sodium methoxide solution in methanol (1.28 mL, 0.642 mmol) and the reaction was allowed to stir for 45 minutes at room temperature. After 45 minutes, acetic acid (42 mg, 0.70 mmol, 0.040 mL) was added and stirred for 10 minutes. The methanol solution was then added drop wise to a stirring solution of 2,2′-disulfanediyldipyridine (35.3 mg, 0.160 mmol) in a mixture of methanol (2 mL) and acetic acid (1 mL). The reaction was allowed to stir for 2 hours at room temperature. After 2 hours, the reaction was concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep Gold 4 g silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound (31.4 mg, 55%). Method C: 3 minute run LRMS [M+Na=557]. ¹H NMR (METHANOL-d₄) δ: 8.39 (d, J=4.3 Hz, 1H), 7.94 (d, J=8.2 Hz, 1H), 7.83 (td, J=7.8, 1.6 Hz, 1H), 7.22 (dd, J=6.8, 5.3 Hz, 1H), 5.21 (s, 1H), 3.92-4.00 (m, 2H), 3.88 (d, J=4.3 Hz, 1H), 3.77 (d, J=7.8 Hz, 1H), 3.71 (t, J=6.0 Hz, 3H), 3.59-3.67 (m, 12H), 3.52-3.58 (m, 2H), 3.02 (t, J=6.0 Hz, 2H), 1.99 (s, 3H) tert-butyl {1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-yl}carbamate (I-f-3)

See Journal of Organic Chemistry, 73(14), 5602-5605; 2008 for synthesis of (I-f-3).

1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-amine hydrochloric acid (I-p-1)

[0524]

[0525] To a solution of tert-butyl {1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-yl}carbamate (I-f-3) (3000 mg, 8.945 mmol) in dichloromethane (45 mL) was added 4.0M hydrogen chloride in dioxane (20 mL, 89.4 mmol) and the reaction was stirred for 18 hours at room temperature. After 18 hours, the reaction was concentrated under reduced pressure yielding of an oil. Ethyl acetate (20 mL) was added to the crude mixture and the resulting mixture was stirred. Heptane (20 mL) was added and the mixture was stirred for 2 hours at room temperature. The material was filtered and the filter cake was washed with ethyl acetate and dried by pulling vacuum for 2 hours yielding the title compound (2140 mg, 88%). ¹H NMR (METHANOL-d₄) δ: 4.25 (s, 6H), 3.72 (s, 6H), 2.97 (s, 3H).

benzyl [6-({1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-yl}amino)-6-oxohexyl]carbamate (I-q-1)

[0526]

[0527] To a solution of 6-{[(benzyloxy)carbonyl]amino}hexanoic acid

(2910 mg, 11.0 mmol) in N,N-dimethylformamide (4 mL) and tetrahydrofuran (20.0 mL) was added N-(3-dimethylaminopropyl)-N′-ethylcarbodimiimide hydrochloride (I-p-1) (2150 mg, 11.0 mmol) and 1-hydroxybenzotriazole (1480 mg, 11.0 mmol) and the reaction was allowed to stir at room temperature for 1 hour during which time the reaction became homogeneous. 1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-amine hydrochloric acid (2130 mg, 7.84 mmol) was added neat in one portion to the stirring reaction mixture followed by the addition of N,N-diisopropylethylamine (5.46 mL, 31.4 mmol) and the reaction was heated to 60° C. for 24 hours. The reaction was allowed to cool to room temperature and was stirred for 24 hours. The reaction was quenched with water (150 mL) and extracted with ethyl acetate. The aqueous layer was washed an additional time with ethyl acetate. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 80 g silica gel column) and eluting with a gradient of 0-100% ethyl acetate/heptane yielding the title compound as an oil which solidified upon standing (3250 mg, 86%). Method C: MassLynx\Acid_3.0Min.olp—LRMS [M+1=483]. ¹H NMR (METHANOL-d₄) δ: 7.10-7.46 (m, 5H), 5.06 (s, 2H), 4.14 (d, J=2.0 Hz, 6H), 3.79 (s, 6H), 3.03-3.20 (m, 2H), 2.83 (t, J=2.1 Hz, 3H), 2.18 (t, J=7.2 Hz, 2H), 1.59 (quin, J=7.3 Hz, 2H), 1.44-1.54 (m, 2H), 1.28-1.40 (m, 2H)

6-(pyridin-2-yldisulfanyl)hexanoic acid (I-r-1)

[0528]

[0529] To a solution of 2,2′-disulfanediyldipyridine

(1490 mg, 6.75 mmol) in a mixture of ethanol (12.0 mL) and acetic acid (0.291 mL) was stirred under nitrogen followed by the drop wise addition of 6-sulfanylhexanoic acid

(1000.0 mg, 6.75 mmol) in ethyl acetate (6.0 mL). The reaction was allowed to stir for 2 hours at room temperature. After 2 hours, the reaction was concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep Gold 40 g silica gel column) and eluting with a gradient of 0-100% ethyl acetate (2% acetic acid modifier)/heptane yielding crude title compound (1170 mg). The crude material was purified again using the CombiFlash Rf (RediSep Gold 40 g silica gel column) and eluting with a gradient of 0-100% ethyl acetate (2% acetic acid modifier)/heptane yielding the title compound as an oil (544 mg, 31%).

1-{[6-(pyridin-2-yldisulfanyl)hexanoyl]oxy}pyrrolidine-2,5-dione (I-s-1)

[0530]

[0531] To a solution of 6-(pyridin-2-yldisulfanyl)hexanoic acid (I-r-1) (705 mg, 2.2 mmol) in N,N-dimethylformamide (4 mL) was added N-Hydroxysuccinimide (306 mg, 2.66 mmol) followed by N-(3-dimethylaminopropyl)-N-ethylcarbodimiimide hydrochloride (520 mg, 2.66 mmol). The reaction was allowed to stir at room temperature overnight. The following morning, the reaction was quenched with water and extracted three times with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 40 g gold column) and eluting with a gradient of 0-100% ethyl acetate/heptane yielding the title compound (364 mg, 47%). Method C: 1.5 minute run LRMS [M+1=355]. ¹H NMR (METHANOL-d₄) δ: 8.39 (d, J=4.7 Hz, 1H), 7.85-7.90 (m, 1H), 7.77-7.84 (m, 1H), 7.21 (dd, J=6.6, 5.5 Hz, 1H), 2.77-2.90 (m, 6H), 2.61 (t, J=7.2 Hz, 2H), 1.63-1.83 (m, 4H), 1.46-1.59 (m, 2H)

N-{1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-yl}-6-(pyridin-2-yldisulfanyl)hexanamide (I-t-1)

[0532]

[0533] To a solution of 1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-amine hydrochloric acid (I-p-1) (100.0 mg, 0.324 mmol) in N,N-dimethylformamide (2.0 mL) was added N,N-diisopropylethylamine (0.339 mL, 1.95 mmol) and was allowed to stir for 10 minutes before adding 1-{[6-(pyridin-2-yldisulfanyl)hexanoyl]oxy}pyrrolidine-2,5-dione (I-s-1) (138 mg, 0.389 mmol) in one portion and the reaction was then heated to 60° C. for 16 hours. After 16 hours, the reaction was diluted with water and extracted with three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 12 g silica gel column) and eluting with a gradient of 0-100% ethyl acetate/heptane yielding the title compound as a gum (66.7 mg, 43%). Method C: 1.5 minute run LRMS [M+Na=497]. ¹H NMR (METHANOL-d₄) δ: 8.39 (d, J=4.7 Hz, 1H), 7.85-7.90 (m, 1H), 7.78-7.84 (m, 1H), 7.19-7.25 (m, 1H), 4.06-4.23 (m, 6H), 3.72-3.84 (m, 6H), 2.78-2.87 (m, 5H), 2.12-2.20 (m, 2H), 1.71 (quin, J=7.3 Hz, 2H), 1.57 (quin, J=7.3 Hz, 2H), 1.36-1.50 (m, 2H)

1-{[4-(benzyloxy)butanoyl]oxy}pyrrolidine-2,5-dione (I-u-1)

[0534]

[0535] To a solution of 4-(Benzyloxy)butanoic acid (1000 mg, 3.77 mmol) in N,N-dimethylformamide (7.54 mL) was added N-Hydroxysuccinimide (521 mg, 4.52 mmol) followed by N-(3-dimethylaminopropyl)-N-ethylcarbodimiimide hydrochloride (885 mg, 4.52 mmol). The reaction was allowed to stir at room temperature overnight. The following morning, the reaction was quenched with water and extracted three times with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 40 g gold column) and eluting with a gradient of 0-100% ethyl acetate/heptane yielding the title compound (1098 mg, 100%). Method C: 1.5 minute run LRMS [M+Na=314]. ¹H NMR (METHANOL-d₄) δ: 7.11-7.50 (m, 5H), 4.51 (s, 2H), 3.56 (t, J=6.0 Hz, 2H), 2.81 (s, 4H), 2.73 (t, J=7.2 Hz, 2H), 1.99 (quin, J=6.6 Hz, 2H)

4-(benzyloxy)-N-{1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-yl}butanamide (I-v-1)

[0536]

[0537] To a solution of 1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-amine trifluoroacetic acid (I-p-1) (750.0 mg, 1.62 mmol) in N,N-dimethylformamide (5 mL) was added N,N-diisopropylethylamine (1.69 mL, 9.71 mmol) and was allowed to stir for 10 minutes before the addition of 1-{[4-(benzyloxy)butanoyl]oxy}pyrrolidine-2,5-dione (I-u-1) (566 mg, 1.94 mmol) in N,N-dimethylformamide (1 mL) and the reaction was then heated to 60° C. for 72 hours. After 72 hours, the reaction was diluted with water and extracted with three times with ethyl acetate. The combined organic layers were washed with water, brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 24 g silica gel column) and eluting with a gradient of 0-100% ethyl acetate/heptane yielding the title compound as a gum (495 mg, None, 74%). Method C: 1.5 minute run LRMS [M+1=412]. ¹H NMR (METHANOL-d₄) δ: 7.21-7.41 (m, 5H), 4.51 (s, 2H), 4.12 (d, J=2.3 Hz, 6H), 3.78 (s, 6H), 3.51 (t, J=6.2 Hz, 2H), 2.82 (t, J=2.3 Hz, 3H), 2.28 (t, J=7.2 Hz, 2H), 1.79-1.94 (m, 2H)

tert-butyl (1,3-bis[(1-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}propan-2-yl)carbamate (I-w-1)

[0538]

[0539] A 50 mL round bottom flask equipped with stir bar was charged with tert-butyl {1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-yl}carbamate (I-f-3) (305.0 mg, 0.909 mmol) was added N-[(1S,2R,6R,7R,8S)-1-(13-azido-2,5,8,11-tetraoxatridec-1-yl)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (1-e-2) (1433.0 mg, 3.020 mmol) in t-butanol (12 mL) followed by the addition of water (5 mL) followed by the addition of sodium ascorbate (1840 mg, 9.09 mmol) neat and the reaction was purged with nitrogen for 10 minutes. Copper (II) sulfate (147 mg, 0.909 mmol) was added in 1 mL of water (deionized) and stirred at room temperature for 24 hours. After 24 hours, the reaction was quenched by adding the reaction mixture to a saturated ammonium chloride (30 mL) and conc. ammonium hydroxide (3 mL) and extracted three times with dichloromethane (20 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 80 g gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound as a white foam (789.0 mg, None, 49.3%). Method C: 1.5 minute run LRMS [M+45−1=1804]. ¹H NMR (METHANOL-d₄) δ: 8.00 (s, 3H), 5.23 (d, J=1.6 Hz, 3H), 4.51-4.63 (m, 12H), 4.29 (d, J=5.9 Hz, 3H), 4.16 (t, J=6.4 Hz, 3H), 3.87-3.96 (m, 12H), 3.84 (d, J=7.8 Hz, 3H), 3.73-3.80 (m, 6H), 3.64-3.72 (m, 12H), 3.54-3.63 (m, 30H), 1.98 (s, 9H), 1.48 (s, 9H), 1.40 (s, 9H), 1.33 (s, 9H)

N-(1,3-bis[(1-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}propan-2-yl)-6-(pyridin-2-yldisulfanyl)hexanamide (I-x-1)

[0540]

[0541] To a solution of N-{1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-yl}-6-(pyridin-2-yldisulfanyl)hexanamide (I-t-1) (66.0 mg, 0.14 mmol) and N-[(1S,2R,6R,7R,8S)-1-(13-azido-2,5,8,11-tetraoxatridec-1-yl)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-2) (219 mg, 0.459 mmol) in t-butanol (2 mL) was added water (0.5 mL, deionized water). Sodium ascorbate (84.3 mg, 0.417 mmol) was added as a solid and the reaction mixture was purged with nitrogen for 5 minutes before the addition of copper (II) sulfate (6.73 mg, 0.0417 mmol) in water (0.5 mL, deionized water) and stirred at room temperature for 24 hours. The reaction was quenched by adding the reaction mixture to a saturated ammonium chloride (20 mL) and conc. ammonium hydroxide (2 mL) and extracted three times with dichloromethane (15 mL). The combined organic layers were washed with brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 12 g gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the impure title compound (105.0 mg, None, 40%). The crude (105.0 mg) was purified again using the CombiFlash Rf (RediSep 4 g Gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound as a gum (94.5 mg, 36%). Method C: MassLynx\Acid_3.0Min.olp—LRMS [M+Na=1921]. ¹H NMR (METHANOL-d₄) δ: 8.38 (d, J=4.7 Hz, 1H), 7.97 (s, 3H), 7.82-7.88 (m, 1H), 7.78-7.81 (m, 1H), 7.20 (t, J=5.9 Hz, 1H), 5.23 (s, 3H), 4.52-4.62 (m, 12H), 4.29 (d, J=5.9 Hz, 3H), 4.15 (t, J=6.4 Hz, 3H), 3.86-3.96 (m, 12H), 3.81-3.85 (m, 3H), 3.72-3.80 (m, 12H), 3.54-3.71 (m, 36H), 2.79 (t, J=7.2 Hz, 2H), 2.16 (t, J=7.2 Hz, 2H), 1.98 (s, 9H), 1.64-1.73 (m, 2H), 1.50-1.57 (m, 2H), 1.48 (s, 9H), 1.42 (d, J=6.6 Hz, 2H), 1.32 (s, 9H)

benzyl {6-[(1,3-bis[(1-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}propan-2-yl)amino]-6-oxohexyl}carbamate (I-y-1)

[0542]

[0543] A 250 mL round bottom flask equipped with stir bar was charged with benzyl [6-({1,3-bis(prop-2-yn-1-yloxy)-2-[(prop-2-yn-1-yloxy)methyl]propan-2-yl}amino)-6-oxohexyl]carbamate (I-q-1) (880.0 mg, 1.82 mmol) was added N-[(1S,2R,6R,7R,8S)-1-(13-azido-2,5,8,11-tetraoxatridec-1-yl)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-7-yl]acetamide (I-e-2) (3075.0 mg, 6.8 mmol) in t-butanol (26 mL) followed by the addition of water (12 mL) followed by the addition of sodium ascorbate (3690 mg, 18.2 mmol) neat and the reaction was purged with nitrogen for 10 minutes. Copper (II) sulfate (294 mg, 1.82 mmol) was added in 1 mL of water and stirred at room temperature for 24 hours. After 24 hours, the reaction was quenched by adding the reaction mixture to a saturated ammonium chloride (50 mL) and conc. ammonium hydroxide (5 mL) and extracted three times with dichloromethane (45 mL). The combined organic layers were dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude material was purified using the CombiFlash Rf (RediSep 80 g gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound as a solid (1890.0 mg, 54.4%) and impure title compound. The crude (1270.0 mg, 36.5%) was purified using the CombiFlash Rf (RediSep 80 g gold silica gel column) and eluting with a gradient of 0-20% methanol/dichloromethane yielding the title compound (607.0 mg, 17.5%). Total yield of title compound 2.497 g (72%). Method C: 3 minute run LRMS [M+1=1907]. ¹H NMR (METHANOL-d₄) δ: 7.99 (s, 3H), 7.21-7.47 (m, 5H), 5.25 (d, J=1.6 Hz, 3H), 5.07 (s, 2H), 4.53-4.62 (m, 12H), 4.31 (d, J=5.9 Hz, 3H), 4.18 (t, J=6.4 Hz, 3H), 3.88-3.98 (m, 12H), 3.85 (d, J=7.8 Hz, 3H), 3.74-3.81 (m, 12H), 3.53-3.71 (m, 36H), 3.10 (q, J=6.2 Hz, 2H), 2.18 (t, J=7.2 Hz, 2H), 2.00 (s, 9H), 1.53-1.65 (m, 2H), 1.50 (s, 11H), 1.34 (s, 11H)

N-(1,3-bis[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}propan-2-yl)-6-(pyridin-2-yldisulfanyl)hexanamide (41)

[0544]

[0545] A solution of N-(1,3-bis[(1-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}propan-2-yl)-6-(pyridin-2-yldisulfanyl)hexanamide (I-x-1) (94.5 mg, 0.0498 mmol) in acetic acid (4 mL), methanol (1 mL) and water (1.0 mL) was heated to 70° C. for 64 hours. After 64 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and concentrated under reduced pressure. The crude material was diluted with toluene a second time and concentrated under reduced pressure yielding impure title compound as a gum (85.3 mg). The crude material was purified using reverse phase chromatography using the conditions below yielding the title compound as a gum (47.6 mg, 53.8%).

Purification Conditions:

[0546] The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reversed-phase HPLC. Column: Waters Sunfire C18 19×100, 5 u; Mobile phase A: 0.05% TFA in water (v/v); Mobile phase B: 0.05% TFA in acetonitrile (v/v); Gradient: 80.0% H20/20.0% Acetonitrile linear to 65% H20/35% Acetonitrile in 8.5 min to 0% H2O/100% MeCN to 9.0 min, Hold at 0% H20/100% Acetonitrile from 9.0 to 10.0 min. Flow: 25 mL/min. Yielding 47.6 mg of the title compound as a gum (retention time 2.87, mass observed=890.4376).

QC Conditions:

[0547] Column: Waters Atlantis dC18 4.6×50, 5 u; Mobile phase A: 0.05% TFA in water (v/v); Mobile phase B: 0.05% TFA in acetonitrile (v/v); 95.0% H20/5.0% Acetonitrile linear to 50% H20/50% Acetonitrile in 3.75 min, to 5% H20/95% Acetonitrile to 4.0 min, Hold at 5% H20/95% Acetonitrile from 4.0 min to 5.0 min. Flow: 2 mL/min. Retention time=2.87; Mass observed=890.4376. Method C: 3 minute run LRMS [½M=889]. ¹H NMR (METHANOL-d₄) δ: 8.41 (d, J=4.7 Hz, 1H), 7.99 (s, 3H), 7.84-7.91 (m, 2H), 7.26 (t, J=5.9 Hz, 1H), 5.21 (s, 3H), 4.58 (t, J=5.0 Hz, 6H), 4.56 (s, 6H), 3.95 (t, J=8.8 Hz, 6H), 3.89 (t, J=5.0 Hz, 6H), 3.86-3.88 (m, 3H), 3.74-3.78 (m, 9H), 3.71 (dd, J=9.4, 4.1 Hz, 3H), 3.54-3.67 (m, 42H), 2.80 (t, J=7.0 Hz, 2H), 2.16 (t, J=7.3 Hz, 2H), 1.99 (s, 9H), 1.68 (quin, J=7.3 Hz, 2H), 1.50-1.57 (m, 2H), 1.35-1.44 (m, 2H)

N-[(1S,2R,3R,4R,5S)-1-(13-{4-[(3-[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}-2-aminopropoxy)methyl]-1H-1,2,3-triazol-1-yl}-2,5,8,11-tetraoxatridec-1-yl)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide-hydrochloric acid salt (42)

[0548]

[0549] A solution of tert-butyl (1,3-bis[(1-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,6R,7R,8S)-7-(acetylamino)-4,4-dimethyl-3,5,9,11-tetraoxatricyclo[6.2.1.0˜2,6˜]undec-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}propan-2-yl)carbamate (I-w-1) (210 mg, 0.119 mmol) in acetic acid (8.0 mL), methanol (2.0 mL) and Water (2.0 mL) was heated to 70° C. overnight. After 18 hours, the reaction was cooled to room temperature and concentrated under reduced pressure. The crude material was diluted with toluene and methanol and concentrated under reduced pressure. The crude material was diluted with toluene a second time and concentrated under reduced pressure. The crude material was diluted with dichloromethane (10 mL) and methanol (4 mL) to which was added 4.0M hydrogen chloride in dioxane (2.0 mL, 8 mmol). The reaction mixture was stirred at room temperature overnight After 18 hours, the reaction was concentrated under reduced pressure. The crude material was diluted with ethyl acetate (1 mL) and to which was added heptane (10 mL) and concentrated under reduced pressure. The material was then placed under high vacuum for 18 hours yielding the title compound as a solid (198.8 mg, 106%). Method C: 3 minute run LRMS [M+Na=1561]. ¹H NMR (METHANOL-d₄) δ: 8.13-8.21 (m, 3H), 5.22 (s, 3H), 4.71 (s, 9H), 4.65 (d, J=4.7 Hz, 6H), 3.92-4.00 (m, 12H), 3.90 (d, J=4.3 Hz, 3H), 3.58-3.80 (m, 51H), 2.02 (s, 9H) 6-azido-N-(1,3-bis[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}propan-2-yl)hexanamide (43)

[0550] To a solution of N-[(1S,2R,3R,4R,5S)-1-(13-{4-[(3-[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}-2-aminopropoxy)methyl]-1H-1,2,3-triazol-1-yl}-2,5,8,11-tetraoxatridec-1-yl)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide-hydrochloric acid salt (42) (25 mg, 0.016 mmol) in N,N-dimethylformamide (0.5 mL) was added N,N-diisopropylethylamine (0.0111 mL, 0.0635 mmol) and was allowed to stir for 10 minutes before being added to neat 1-[(6-azidohexanoyl)oxy]pyrrolidine-2,5-dione

(see PCT Int. Appl., 2011034951, 24 Mar. 2011, 6.05 mg, 0.0238 mmol) and the reaction was allowed to stir at room temperature for 18 hours. The reaction was then heated to 60° C. for 32 hours. After 32 hours, the reaction was concentrated under reduced pressure. The crude material was diluted with dimethylsulfoxide (1 mL) and passed through a syringe filter and the crude material was purified using reverse-phase chromatography using the conditions seen below yielding the title compound as a gum (6.2 mg, 23%).

Purification Conditions

[0551] The residue was dissolved in dimethyl sulfoxide (1 mL) and purified by reversed-phase HPLC (Column: Waters Sunfire C18 19×100, 5 u; Mobile phase A: 0.05% TFA in water (v/v); Mobile phase B: 0.05% TFA in acetonitrile (v/v); 90.0% H20/10.0% Acetonitrile linear to 70% H20/30% Acetonitrile in 10.5 min, 70% H20/30% Acetonitrile linear to 0% H20/100% MeCN in 0.5 min, Hold at 0% H20/100% Acetonitrile from 11.0 min to 12.0 min. Flow: 25 mL/min.

QC Conditions

[0552] Column: Waters Atlantis dC18 4.6×50, 5 u; Mobile phase A: 0.05% TFA in water (v/v); MobCe phase B: 0.05% TFA in acetonitrile (v/v); 95.0% H20/5.0% Acetonitrile linear to 5% H20/95% Acetonitrile in 4.0 min, Hold at 5% H20/95% Acetonitrile from 4.0 min to 5.0 min. Flow: 2 mL/min.; Retention time=1.77 minutes; Mass observed=839.7097. Method C: 3 minute run LRMS [M+1=1678]. ¹H NMR (METHANOL-d₄) δ: 8.00 (s, 3H), 5.21 (s, 3H), 4.58 (t, J=4.7 Hz, 6H), 4.57 (s, 6H), 3.95 (t, J=10.0 Hz, 6H), 3.85-3.92 (m, 9H), 3.74-3.80 (m, 9H), 3.71 (dd, J=10.0, 4.1 Hz, 3H), 3.55-3.68 (m, 42H), 3.25 (t, J=6.5 Hz, 2H), 2.19 (t, J=7.3 Hz, 2H), 1.99 (s, 9H), 1.52-1.62 (m, 4H), 1.33-1.41 (m, 2H)

N-(1,3-bis[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}propan-2-yl)hept-6-enamide (44)

[0553]

[0554] To a solution of N-[(1S,2R,3R,4R,5S)-1-(13-{4-[(3-[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]-2-{[(1-{1-[(1S,2R,3R,4R,5S)-4-(acetylamino)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-1-yl]-2,5,8,11-tetraoxatridecan-13-yl}-1H-1,2,3-triazol-4-yl)methoxy]methyl}-2-aminopropoxy)methyl]-1H-1,2,3-triazol-1-yl}-2,5,8,11-tetraoxatridec-1-yl)-2,3-dihydroxy-6,8-dioxabicyclo[3.2.1]oct-4-yl]acetamide-hydrochloric acid salt (42) (25 mg, 0.016 mmol) in N,N-dimethylformamide (0.5 mL) was added N,N-diisopropylethylamine (0.0111 mL, 0.0635 mmol) and was allowed to stir for 10 minutes before being added to neat 1-(hept-6-enoyloxy)pyrrolidine-2,5-dione

(see Angewandte Chemie, International Edition, 51(25), 6144-6148, S6144/1-S6144/53; 2012, 5.36 mg, 0.0238 mmol) and the reaction was allowed to stir at room temperature for 18 hours. The reaction was then heated to 60° C. for 32 hours. After 32 hours, the reaction was concentrated under reduced pressure. The crude material was diluted with dimethylsulfoxide (1 mL) and passed through a syringe filter and the crude material was purified using reverse-phase chromatography using the conditions seen below yielding the title compound as a gum (4.9 mg, 19%).