The Interaction of Nitrophenylalanines with Wild Type and Mutant 4‐Methylideneimidazole‐5‐one‐less Phenylalanine Ammonia Lyase

Abstract

Racemic nitrophenylalanines and (E)‐nitrophenylacrylates are synthesized from the corresponding aldehydes. Both products are important for the examination of the mechanism of action of phenylalanine ammonia lyase (PAL). For the reaction of the rac‐nitrophenylalanines with both wild type (wt) PAL and an 4‐methylideneimidazole‐5‐one (MIO)‐less mutant, the kinetic constants Km and Vmax are determined and compared with those of the natural substrate L‐phenylalanine: the Km values for the racemic nitrophenylalanines with wt PAL are up to 9 times higher, however, the Vmax values are up to 5 times lower. Compared to wt PAL, the catalytic activity of MIO‐less PAL mutant for the deamination of L‐phenylalanine is approximately 400 times, while that for 3‐nitrophenylalanine is approximately 50 times lower. Both wt and MIO‐less PALs are enantioselective for L‐nitrophenylalanines. Thus, enantiopure D‐nitrophenylalanines can be biosynthesized from racemic substrates. The biocatalytic synthesis of the corresponding L‐enantiomers is achieved by the reverse reaction, starting from (E)‐nitrophenylacrylates. Both enantiomeric products obtained with wt and MIO‐less PAL are spectroscopically and chromatographically characterized and their optical rotations measured.

Synthetic and Therapeutic Applications of Ammonia-lyases and Aminomutases

“…More recently, Retey and coworkers demonstrated the conversion of nitrophenylalanine with PcPAL, in both the deamination and the amination directions, showing also that the MIO-less mutant PcPAL-Ser202Ala is active on such substrates, even with considerably higher specific activities than the natural substrates. 153 This was thought to be further evidence of a Friedel−Crafts-like mechanism, since the strong electron-withdrawing effect of the nitro group provides the same activation as that provided by the MIO moiety after the Friedel−Crafts-type attack. Very recently, however, we showed evidence of a MIO-independent competing reaction pathway with highly activated substrates (i.e., those carrying strong EWGs) that proceeds nonstereoselectively and more slowly.…”

“…237 Nitro-substituted phenylalanines were also obtained in very good yields. 139,153 Access to such a broad variety of compounds through one simple biocatalytic method (with excellent enantiomeric purity in all cases) opened up potential sustainable routes to the manufacture of countless non-natural amino acid derivatives for the pharmaceutical and agrochemical industry. PcPAL showed remarkable thermal stability compared to the free enzyme: no significant loss of activity at 70°C for 38 h, with an optimum temperature found to be around 60°C (1.5-fold increase in temperature tolerance compared to the free enzyme).…”

“…Rétey and co-workers 153,236,237 and later Poppe and co-workers 149,242 employed isolated PcPAL for the production of a very broad panel of enantiomerically pure D-heteroarylalanines via the consumption of the L-enantiomer (Scheme 35). Furthermore, Zhu et al demonstrated the large scale resolution of racemic phenylalanine in a packed-bed reactor using covalently immobilized RgPAL on a mesoporous silica support, to give >99% conversion with a space-time yield of 7.2 g L -1 h -1 and perfect enantioselectivity.…”

“…237 Nitro-substituted phenylalanines were also obtained in very good yields. 139,153 Access to such a broad variety of compounds through one simple biocatalytic method (with excellent enantiomeric purity in all cases) opened up potential sustainable routes to the manufacture of countless non-natural amino acid derivatives for the pharmaceutical and agrochemical industries. Furthermore, such a broad substrate scope facilitated the development of PAL-specific assay methods using modified or functionalized cinnamic acid analogues as molecular probes.…”

“…Retey and co-workers 153,236,237 and later Poppe and coworkers 149,242 of racemic phenylalanine in a packed-bed reactor using covalently immobilized RgPAL on a mesoporous silica support, to give >99% conversion with a space-time yield of 7.2 g L −1 h −1 and perfect enantioselectivity. 246 To overcome the 50% theoretical yield limitation of kinetic resolution approaches, Parmeggiani et al recently proposed a chemoenzymatic amination−deracemization strategy for the synthesis of D-phenylalanines directly from cinnamic acids (Scheme 36).…”