The regioselectivity of the addition of N-nucleophiles to N-carbonyl protected dehydroalanine derivatives, P-conjugate addition vs. wimine capture, can be controlled completely by varying the N-protecting group, or, the carboxy protecting group thereby providing a model for the chemistry of dehydroalanine residues within enzymes.
In addition to ammonia, 3-methylaspartase is able to catalyse the conjugate addition of hydrazine, hydroxylamine, methoxylamine and alkylamines to various alkyl-and halogenofumaric acids to give single enantiomers of the corresponding N-su bstituted aspartic acids.The efficient stereospecific synthesis of small polar biologically active compounds for use as probes in elucidating enzyme mechanism or, receptor site specificity, remains a major synthetic challenge. Commonly problems are associated with the formation of single enantiomers and with the laborious protection and deprotection of key functional groups, Methylaspartase (EC 4.3.1.2) catalyses the deamination of (2S,3S)-3-methylaspartic acid (Scheme 1) .1Previously we had demonstrated that the enzyme was useful in providing access to various 3-substituted aspartic acids via the direct amination of substituted fumaric acids.* Here we show that a range of N-nucleophiles can serve as surrogates for ammonia. We report on the scope and stereospecificity of the conjugate addition reactions and also comment on the geometry of the active site space.In common with some other ammonia-lyases, the active site of methylaspartase is believed to contain a dehydroalanine residue, derived from Ser-173, to which the amino group of amino acid substrates adds [to form covalent C-N enzymesubstrate adducts (see the reverse of Scheme 2)] prior to the deamination of these substrates.3-5Previous studies in our laboratory had indicated that hydrazine , hydroxylamine and phenylhydrazines could also react with this dehydroalanine residue to cause irreversible inhibition. However, when 14C-p-nitrophenylhydrazinet was reacted with the enzyme, the radiolabelled fragment slowly dissociated from the protein during dialysis. This result indicated that the inhibition of the enzyme was not completely irreversible and that for small hydrazines, which would allow the simultaneous cooccupation of the active site by Michael acceptors, it might be possible to synthesise hydrazino acids.
The title addition of hydrazine to substituted fumaric acids occurs in excellent conversion but lower yields. Each addition is found to follow the same course as that for ammonia, the natural nucleophile for the enzyme. -(GULZAR, M. S.; AKHTAR, M.; GANI, D.; J.
Control of the Regioselectivity of N-Nucleophile Addition to N-Carbonyl Protected Dehydroalanines: A Model for the Ammonia-lyase Enzymes.-The N-alkyloxycarbonyldehydroalanine ethylamide (I) reacts with propylamine (II) to generate exclusively the diaminal (III). In contrast, similar reaction of the unsaturated compounds (IV) affords only the conjugate addition products (V). The formation of (III) supports an enamine trapping mechanism in enzymic deamination reactions. -(GULZAR, M. S.; MORRIS, K. B.; GANI, D.; J.
biochemical syntheses, microbiological syntheses biochemical syntheses, microbiological syntheses O 0035
-053Enantiospecific Conjugate Addition of N-Nucleophiles to Substituted Fumaric Acids Using Methylaspartase.-Starting with prochiral fumaric acid (I) an efficient microbiological reaction to the optically active acids (III) using the title enzyme is achieved. It is noteworthy that the reaction fails if R' is larger than ethyl. -(GULZAR, M. S.; AKHTAR, M.; GANI, D.; J.
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