Roger Arthur Sheldon

Penicillin acylase-catalyzed resolution of amines in aqueous organic solvents

Abstract Penicillin acylase from Alcaligenes faecalis catalyzes the enantioselective acylation of amines with phenylacetamide in a kinetically controlled reaction in water at pH 11. Addition of cosolvent to the reaction mixture significantly improved the enantioselectivity in most cases. Penicillin acylase from E. coli also catalyzed the phenylacetylation of amines, but an order of magnitude less efficiently than with the enzyme of A. faecalis . Amine resolution via kinetically controlled acylation competes effectively with hydrolysis of N -acylated compounds and constitutes a synthetically useful alternative to existing lipase-based methods.

KINETIC STUDY OF PENICILLIN ACYLASE FROM ALCALIGENES FAECALIS

Penicillin acylase from Alcaligenes faecalis has a very high affinity for both natural (benzylpenicillin, K m=0.0042 mM) and colorimetric (6‐nitro‐3‐phenylacetamidobenzoic acid, K m=0.0045 mM) substrates as well as the product of their hydrolysis, phenylacetic acid (K i=0.016 mM). The enzyme is partially inhibited at high benzylpenicillin concentrations but the triple SES complex formed still retains 43% of the maximal catalytic activity; the affinity of benzylpenicillin for the second substrate molecule binding site is much lower (K S′=54 mM) than for the first one. Phenylmethylsulfonyl fluoride was shown to be a very effective irreversible inhibitor, completely inactivating the penicillin acylase from A. faecalis in a few minutes at micromolar concentrations; this compound was used for enzyme active site titration. The absolute values of the determined kinetic parameters for enzymatic hydrolysis of 6‐nitro‐3‐phenylacetamidobenzoic acid (k cat=95 s−1 and k cat/K m=2.1×10−7 M−1 s−1) and benzylpenicillin (k cat=54 s−1 and k cat/K m=1.3×10−7 M−1 s−1) by penicillin acylase from A. faecalis were shown to be highest of all the enzymes of this family that have so far been studied.

Highly efficient and enantioselective enzymatic acylation of amines in aqueous medium

Abstract A new strategy based on the unique catalytic properties, stability and enantioselectivity of the relatively unknown penicillin acylase from Alcaligenes faecalis has been developed for the effective and enantioselective acylation of amines in aqueous medium. In contrast to lipase-catalyzed acylations in organic solvents, the penicillin acylase-catalyzed acylation of amines in aqueous solution is a rapid and chemoselective process leading to a product which can subsequently be deacylated by the same enzyme, imposing secondary enantiocontrol and leading to effective resolution.

Penicillin acylase-catalyzed synthesis of ampicillin in "aqueous solution-precipitate" systems. High substrate concentration and supersaturation effect

Abstract Penicillin acylase-catalyzed ampicillin synthesis via acyl group transfer in aqueous solution is highly dependent on the initial substrate concentration. The solubility of one substrate, 6-aminopenicillanic acid (6-APA), can be advantageously enhanced by the presence of acyl donor, the second substrate. Furthermore, a comparison of enzymatic synthesis in homogeneous solution with synthesis in a heterogeneous system having partially undissolved reactants, reveals major advantages for the latter approach. In this “aqueous solution–precipitate” system, accumulation of both products, ampicillin and d -(−)-phenylglycine, proceeds through the formation of their supersaturated solutions. Subsequent precipitation of the product ampicillin positively influences the efficiency of the biocatalytic process. As a result, ampicillin synthesis proceeds in 93% conversion on 6-APA and in 60% conversion on d -(−)-phenylglycine methyl ester.

Penicillin acylase-catalyzed peptide synthesis: a chemo-enzymatic route to stereoisomers of 3,6-diphenylpiperazine-2,5-dione

Abstract Chiral dipeptides of phenylglycine were synthesized using immobilized Escherichia coli penicillin acylase. The high selectivity of penicillin acylase for l -amino acids as the nucleophile resulted in the efficient acylation of l -phenylglycine by d -phenylglycine amide at pH 9.7 to give d -phenylglycyl- l -phenylglycine in 69% yield. No isomers or tripeptides were formed. The low enantiospecificity of the enzyme for the acyl donor provided the possibility of preparing the corresponding l , l -dipeptides, starting from l -phenylglycine methyl ester as both donor and acceptor at pH 7.5, resulting in a 63% yield of l -phenylglycyl- l -phenylglycine methyl ester. The product precipitated under the reaction conditions; this effectively prevented the formation of oligomers as well as chemical transformation of the product. The dipeptide esters of phenylglycine easily cyclized to diketopiperazines in aqueous methanol. l -Phenylglycyl- l -phenylglycine methyl ester formed l , l -3,6-diphenylpiperazine-2,5-dione ( cis ); the achiral trans isomer was obtained from d -phenylglycyl- l -phenylglycine methyl ester.

A green, fully enzymatic procedure for amine resolution, using a lipase and a penicillin G acylase

A green procedure for the kinetic resolution of chiral amines via enzymatic acylation and deacylation has been demonstrated. The fully enzymatic approach obviates the common, waste-generating deacylation under strongly alkaline conditions. The acylating agent was (R)-phenylglycine propyl ester in combination with Candida antarctica lipase B as acylation catalyst. The enantiomerically enriched amides were subsequently deacylated in the presence of the penicillin G acylase from Alcaligenes faecalis. The degree of enantiomer recognition by CaLB in the acylation of aliphatic amines was unexpectedly modest, but a considerable further enantiomeric enrichment could be accomplished in the course of the subsequent enzymatic hydrolysis step.