AUTHOR=Eppinger Erik , Gröning Janosch Alexander David , Stolz Andreas 

TITLE=Chemoenzymatic enantioselective synthesis of phenylglycine and phenylglycine amide by direct coupling of the Strecker synthesis with a nitrilase reaction

JOURNAL=Frontiers in Catalysis

VOLUME=2

YEAR=2022

URL=https://www.frontiersin.org/journals/catalysis/articles/10.3389/fctls.2022.952944

DOI=10.3389/fctls.2022.952944

ISSN=2673-7841

ABSTRACT=<p>The conversion of <italic>rac</italic>-phenylglycinonitrile by different variants of the nitrilase from <italic>Pseudomonas fluorescens</italic> EBC191 (EC 3.5.5.1) was studied and the amounts and chiral composition of the formed phenylglycine and phenylglycine amide compared. Muteins that converted <italic>rac</italic>-phenylglycinonitrile to extraordinarily high amounts of phenylglycine or phenylglycine amide were tested for the chemoenzymatic enantioselective one-pot synthesis of (<italic>R</italic>)- and (<italic>S</italic>)-phenylglycine and (<italic>R</italic>)- and (<italic>S</italic>)-phenylglycine amide. The chemoenzymatic synthesis combined the initial step in the traditional chemical Strecker synthesis which results in the formation of <italic>rac</italic>-phenylglycinonitrile from benzaldehyde, cyanide, and ammonia with the enzymatic conversion of the formed nitrile by the nitrilase variants. The aminonitrile synthesis was optimized in order to obtain conditions which allowed under mildly alkaline conditions (pH 9.5) maximal yields of phenylglycinonitrile and the <italic>in-situ</italic> racemization of the compound. The racemic phenylglycinonitrile was directly converted under the alkaline conditions without any interposed purification step by cells of <italic>Escherichia coli</italic> overexpressing recombinant nitrilase variants. The application of a mutant of <italic>E. coli</italic> defect in a (<italic>S</italic>)-phenylglycine amide hydrolysing peptidase (<italic>E. coli</italic> JM109Δ<italic>pepA</italic>) expressing a highly reaction- and (<italic>R</italic>)-specific nitrilase variant allowed the synthesis of (<italic>R</italic>)-phenylglycine with <italic>ee</italic>-values ≥ 95% in yields up to 81% in relation to the initially added benzaldehyde. These yields indicated a dynamic kinetic resolution which involved the racemization of (<italic>S</italic>)- to (<italic>R</italic>)-phenylglycinonitrile under the used alkaline conditions with the concurrent hydrolysis of (<italic>R</italic>)-phenylglycinonitrile to (<italic>R</italic>)-phenylglycine. The addition of resting cells of <italic>E. coli</italic> JM109Δ<italic>pepA</italic> synthesizing an amide forming nitrilase variant to the final product of the Strecker synthesis and/or using <italic>E. coli</italic> strains with an intact aminopeptidase gene resulted in the preferred formation of (<italic>S</italic>)-phenylglycine amide, (<italic>R</italic>)-phenylglycine amide or (<italic>S</italic>)-phenylglycine.</p>