Enzymatic Synthesis of Enantiopure α‐ and β‐Amino Acids by Phenylalanine Aminomutase‐Catalysed Amination of Cinnamic Acid Derivatives

Abstract

The phenylalanine aminomutase (PAM) from Taxus chinensis catalyses the conversion of alpha-phenylalanine to beta-phenylalanine, an important step in the biosynthesis of the N-benzoyl phenylisoserinoyl side-chain of the anticancer drug taxol. Mechanistic studies on PAM have suggested that (E)-cinnamic acid is an intermediate in the mutase reaction and that it can be released from the enzyme's active site. Here we describe a novel synthetic strategy that is based on the finding that ring-substituted (E)-cinnamic acids can serve as a substrate in PAM-catalysed ammonia addition reactions for the biocatalytic production of several important beta-amino acids. The enzyme has a broad substrate range and a high enantioselectivity with cinnamic acid derivatives; this allows the synthesis of several non-natural aromatic alpha- and beta-amino acids in excellent enantiomeric excess (ee >99 %). The internal 5-methylene-3,5-dihydroimidazol-4-one (MIO) cofactor is essential for the PAM-catalysed amination reactions. The regioselectivity of amination reactions was influenced by the nature of the ring substituent.

Molecular Cloning and Characterization of the Phenylalanine Aminomutase Gene From Taxus baccata L.

“…Recent mechanistic studies on the aminomutases (PAM and TAM) revealed that 4-hydroxycinnamic acid (in the case of TAM) and cinnamic acid (in the case of PAM) are intermediates in the aminomutase reaction that can be released from the active site of the enzyme. These observations indicate that TAM and PAM might exhibit ammonia lyase activity and suggest the possibility of using the second half of the aminomutase reaction to synthesize β-amino acids from cinnamates (12). Comparison of the amino acid sequence of TbPAM with PAL and TAM indicate that the enzyme also contain a signature Ala-Ser-Gly motif which rearranges auto-catalytically to the MIO moiety in the active site of the enzyme.…”

Discovery and Investigation of Mutase-like Activity in a Phenylalanine Ammonia Lyase from Anabaena variabilis

“…The main difference between the two enzyme classes in this respect is that enzymes with only PAL activity show production of α-phenylalanine derivatives, [14,15] whereas characterised PAMs allow formation of both regioisomers as a mixture in most cases [16][17][18]. These findings imply that, in the synthetic direction, PAL enzymes accommodate cinnamate derivatives only in the α-productive binding mode, whereas (R)-PAMs (e.g., TwPAM from Taxus wallichiana) are able to sample both predicted conformers more equally.…”

Chiral Integrated Catalysts Composed of Bifunctional Thiourea and Arylboronic Acid: Asymmetric Aza-Michael Addition of α,β-Unsaturated Carboxylic Acids

“…From the perspective of atom and step economy, achieving a direct Michael reaction of α,β-unsaturated carboxylic acids without the use of any activating templates is highly desirable. Despite strong synthetic utility, the direct Michael addition of α,β-unsaturated carboxylic acids has never been reported, except for an enzyme-catalyzed azaMichael addition 13,14) ; very few strategies have been exploited to catalytically activate the hydroxy group of carboxylic acids via transient covalent bond formation.By contrast, since the discovery by Yamamoto and Ishihara that electron-deficient arylboronic acids are efficient activators of carboxylic acids in direct amidation reactions, [15][16][17][18] 24,25) Hall reported that different types of arylboronic acids could be used for the cycloaddition of α,β-unsaturated carboxylic acids to dienes and 1,3-dipoles. 20,21) Recently, we also revealed that aminoarylboronic acid was the best catalyst for the intramolecular hetero-Michael addition of α,β-unsaturated carboxylic acids bearing sulfonamides or phenols.…”

“…From the perspective of atom and step economy, achieving a direct Michael reaction of α,β-unsaturated carboxylic acids without the use of any activating templates is highly desirable. Despite strong synthetic utility, the direct Michael addition of α,β-unsaturated carboxylic acids has never been reported, except for an enzyme-catalyzed azaMichael addition 13,14) ; very few strategies have been exploited to catalytically activate the hydroxy group of carboxylic acids via transient covalent bond formation.…”