Derivatives of .Alpha.-Difluoromethylornithine Useful in Analysis

The present invention relates to novel derivatives of .alpha.-difluoromethylornithine (.alpha.-dFMO) which are useful in cytochemical analysis. .alpha.-dFMO is an enzyme activated irreversible inhibitor of ornithine decarboxylase (ODC) and its localization is of importance in various tests and especially in cytochemical localizations of enzymes. According to the present invention .alpha.-dFMO is coupled to specific molecules, and thus it is rendered visible. Amongst the molecules to which it may be coupled without loss of its enzymatic inhibitory activity there may be mentioned rhodamine, in which case the rhodamine-labelled inhibitor may be used for direct visualization in fluorescence microscopy or by similar techniques; or it may be coupled to biotin, and the biotin-labelled inhibitor can be used for indirect detection of the enzyme following its binding to avidin conjugated to a suitable peroxidase, such as horseradish peroxidase and visualization of the peroxidase reaction product.

The .alpha.-difluoromethylornithine can be labelled with the different marker molecules, while maintaining its activity as an inhibitor.

The following examples illustrate the tagging of .alpha.-dFMO, with rhodamine-B-isothiocyanate and with biotin, respectively.

Rhodamine-labelled inhibitor: The inhibitor was dissolved in H.sub.2 O and reacted with excess cupric carbonate to mask the .alpha.-amino group from reacting with the rhodamine label, leaving the .alpha.-amino group free. Rhodamine-B-isothiocyanate (Sigma) was mixed, using a 100:1 (inhibitor:rhodamine) molar ratio, with the inhibitor-copper chelate in a 0.4 M sodium carbonate-bicarbonate buffer, pH 8.4, overnight at 4.degree. C. The mixture was then brought to pH 3 with HCl and the precipitate formed, washed and lyophilized. The preparation was dissolved in Tris-HCl buffer, pH 7.2, filtered, passed through a column of Sepharose coupled to lysine and the eluate, cleared from excess free rhodamine, used for biochemical and cytochemical studies.

The obtained rhodamine-labelled .alpha.-dFMO has the formula: ##STR1##

Biotinyl-N-hydroxysuccinimide synthesized according to Bayer and Wilchek, (Meth. Enzymol. 46, 613, (1977)) was dissolved in dimethyl-formamide and added slowly in a 1:1 ratio to an inhibitor-copper chelate in a carbonate-bicarbonate buffer, pH 8.6, and mixed for 2 hours at room temperature. The supernatant was decanted, H.sub.2 S was added and the precipitate formed was lyophilized and then redissolved in Tris-HCl buffer, pH 7.2, and used for cytochemical studies. The obtained biotin-labelled .alpha.-dFMO has the formula: ##STR2##

The final reaction products from both Example 1 and Example 2 were separated by high voltage paper electrophoresis according to Degani and Patchornik (Biochem, 13 (1):1, (1974)), and identified by characteristic color reactions. The inhibitor .alpha.-dFMO identified by the ninhydrin reaction, was not present either in the rhodamine-labelled inhibitor preparation (IR, identified by the rhodamine color and fluorescence) or in the biotin-labelled preparation (IB, identified by a specific color reaction for biotin). The labelled reagents could be extracted with water.

Following the synthesis of the conjugated inhibitor and its isolation according to the methods outlined, the inhibition of ODC activity by the product was examined in tissue homogenates. Rhodamine-labelled .alpha.-dFMO inhibited 43% and biotin-labelled 64% of the enzyme activity present in 7 day old rat cerebellum. Rhodamine or biotin alone were completely devoided of inhibitory activity.

Fixation reduced ODC activity to a certain extent. However, in spite of this reduction, substantial enzyme activity still remained in the tissues examined.

The present invention shows that the specific enzyme activated irreversible inhibitor of the enzyme ODC-.alpha.-dFMO-, can be labelled, and still retain its inhibitory activity, with specific molecules (tags) which render it visible by light and fluorescence microscopy. Rhodamine-B-isothiocyanate served as the fluorescent tag and made it possible to directly visualize the tissue localization of the labelled inhibitor by fluorescence microscopy. Biotin-labelled inhibitor was visualized indirectly via the brown reaction product catalyzed by HRP-conjugated avidin which binds specifically and strongly to biotin. Using this procedure ODC has been localized in tissue sections of rat brain and liver.