Efficient Asymmetric Synthesis Of D-phenylglycine And Its Derivatives Via Nitrilase Catalyzed Resolution

Unnatural amino acids play a significant role in the fine chemical and pharmaceutical industries. Among them, D-phenylglycine and its derivatives are essential chiral building blocks for the synthesis of antibiotics, such as ampicillin, cefalexin, and amoxicillin. A conventional process for manufacture of D-phenylglycine is crystallization of the diastereomeric salts of its racemate via (+)-camphor-8-sulfonic acid. Biocatalysis, the use of microbial cells or isolated enzymes in the production of fine chemicals, is becoming one of the greenest technologies for industrial purpose. Therefore, many chemo-enzymatic approaches involving aminoacylase, amidase, hydantoinases, amino acid dehydrogenases, lipase and penicillin G acylase have been developed to prepare enantiopure unnatural amino acids. Previously, immobilized D-hydantoinase and D-N-carbamolase have been widely used in a process known as "hydantoinase process" to convert racemic5-(p-hydroxyphenyl)-hyda-ntoin to D-p-hydroxyphenylglycine. However, D-phenylglycine has not been produced through this process at an industrial scale.Nitrilases (EC3.5.5.1) constitute an important class of hydrolase that convert nitriles to corresponding carboxylic acids and ammonia. In recent years, nitrilases have attracted tremendous attentions in the preparation of optically pure carboxylic acids. Especially, many nitrilases found in bacteria have been reported to enantioselectively hydrolyze mandelonitrile to (R)-mandelic acid. However, only few reaserch have been reported for asymmetric synthesis of a-amino acid. The nitrilase route is different from the other enzyme routes mentioned above with two advantages:(i) raw material a-aminonitrile can be obtained more easily than a-amino acids derivatives;(ii) only single enzyme is required. Therefore, the strategy of nitrilase mediated dynamic kinetic resolution for D-phenylglycine and its derivatives was developed in this thesis.First, a nitrilase with high (R)-enantioselectivity from Sphingomonas wittichii RW1was discovered for the enantioselective hydrolysis of phenylglycinonitrile. A mini nitrilase library was constructed and function based screen was measured. The gene encoding a nitrilase from S. wittichii RW1with high (R)-enantioselectivity on phenylglycinonitrile was cloned and heterologously expressed. The nitrilase ReSWRW1was purified with a molecular mass of about40kDa. The maximum activity was observed around pH8.0and40℃, respectively. The nitrilase preferred aliphatic nitriles, and the maximum activity was obtained for succinonitrile, which was approximately30-fold higher than that for phenylglycinonitrile. The E. coli BL21/ReSWRW1dry cells were used to catalyze kinetic resolution of phenylglycinontrile, affording (R)-phenylglycine with46%yield,95%ee. No amide by-product was observed throughout the hydrolysis process.Second, a strategy for preparing D-phenylglycine and its derivatives with nitrilase mediated dynamic kinetic resolution in aqueous system or biphasic system was developed, respectively. For accelerating the racemization, different alkaline conditions (pH9.5-11) were compared. The nitrilase mediated dynamic kinetic resolution for D-phenylglycine was achieved under pH11.0, however, only a yield of68%was obtained. In order to suppress the decomposition of the substrate and improve the yield, an aqueous-1-octanol biphasic reaction system was investigated. Several important parameters were optimized in detail, and the water content of10%was essential for the suppression of the decomposition of substrate. The maximum yield of81%was obtained under the optimized conditions, which is1.2times greater than that in aqueous system. These results indicated that the nitrialse mediated dynamic kinetic resolution was a promising method for D-phenylglycine and its derivatives.Third, a nitrilase-CLEAs mediated dynamic kinetic resolution for asymmetric synthesis of D-N-formyl-phenylglycine was developed. From the point of substrate engineering, different N-acyl-phenylglycinonitriles were examined for the stabilization and racemization. And the N-formyl-phenylglycinonitrile was chosen for the nitrilase-CLEAs mediated dynamic kinetic resolution. The CLEAs of nitrilase from S. wittichii RW1were successfully prepared with the maximum activity recovery of45.6%. And the CLEAs showed higher stability than the whole cells under various conditions. Through optimization of a series of important factors, D-N-formyl-phenylglycine was synthesized with yield of92%and97%ee at pH8.5and30℃. The nitrilase-CLEAs could be reused at least6batches without dramatic yield loss. This nitrilase-CLEAs mediated dynamic kinetic resolution may be potential for industrial production of D-N-formyl-phenylglycine.Fourth, inversion of nitrilase enantioselectivity by directed evolution. A model of nitrilase from S. wittichii RW1was built by ab initio method. And base on the3D structure, four sites of amino acids near the substrate-binding pocket were selected for iterative saturation mutagenesis (ISM). A "small but smart" mutation library was constructed and screened. The best variant4D239(W165A, L168W, W188Y, A190S, Y194V) with S-selectivity (E=21) was found in the mutation library. The docking results indicated that the binding free energy and hydrogen bond played key roles in the interaction between substrate and nitrilase. Moreover, the analysis also provides the theoretical basis on the inversion enantioselectivity of nitrilase.