No abstract

The current enzymatic production of semisynthetic β-lactam antibiotics requires isolation and purification of the intermediate 6-aminopenicillanic acid which adds cost and complexity to the manufacturing process. In this work, we took advantage of the unique substrate specificity of aamino ester hydrolases to perform a purely aqueous one-pot production of ampicillin from penicillin G and D-phenylglycine methyl ester, catalyzed by α-amino ester hydrolase and penicillin G acylase. The synthesis was performed in both a one-pot, one-step synthesis resulting in a maximum conversion of 39%, and a one-pot, two-step process resulting in a maximum conversion of 47%. The two-enzyme cascade reported in this paper is a promising alternative to the current enzymatic two-step, two-pot manufacturing process for semisynthetic β-lactam antibiotics which requires intermittent isolation of 6-aminopenicillanic acid.

Semi-synthetic β-lactam antibiotics are synthesized enzymatically with the use of penicillin G acylase (PGA). Currently, PGA only exhibits weak diastereoselectivity with respect to the alpha amino group of rac-phenylglycine methyl ester (rac-PGME) when it is coupled with 6-aminopenicillanic acid to synthesize ampicillin. Therefore, we sought to improve the diastereoselectivity of PGA by targeting residues for site-saturation based on the proximity to the substrate's chiral center. Four variants with improved selectivity for (R)-ampicillin synthesis were identified, all resulting from a mutation at the β24 position. βPhe24Ala, while not identified from our library screening, was obtained with site-directed mutagenesis because it has been previously shown to be selective for (R)-enantiomers with substituents other than an amino group. The diastereomeric excess (d.e.(R)) value of 37% for the wild-type enzyme was improved to a d.e.(R) value of 98% for our most selective mutant, βPhe24Ala. Also, four mutations at the α146 position that resulted in (S)-selective PGA variants were identified. βPhe24 and αPhe146 are on opposite sides of the alpha carbon of the substrate, and we have shown that altering these residues results in enhanced selectivity in opposite directions. All variants that showed selectivity for (S)-ampicillin synthesis showed decreased synthetic activity for pure substrates and a decreased synthesis-to-hydrolysis ratio. In contrast, the mutants that were selective for (R)-ampicillin showed significantly decreased primary and secondary hydrolysis when synthesizing ampicillin from pure (R)-PGME, resulting in up to 4-fold decrease in the synthesis to hydrolysis ratio and up to 2-fold increase in the yield achieved. Finally, it was discovered that the selective PGA variants have racemase or epimerase activity, a fascinating phenomenon that has never been reported.

The penicillins, cephalosporins, carbapenems, and monobactams, make up the β‐lactam family of antibiotics. There are currently more than 25 antibiotics in this family that are approved by the United States Food and Drug Administration and over 50 companies that manufacture them. Their four‐membered β‐lactam ring, which is the part of the antibiotic that is responsible for their bactericidal capabilities, is sensitive to acids, bases, and heat. Therefore, the original chemical synthesis processes utilized to make these compounds required subzero temperatures, hazardous chemicals for protecting groups, and large volumes of organic solvents, rendering them both environmentally harmful and economically taxing. The original syntheses are increasingly being replaced with biocatalytic syntheses which are carried out at ambient temperatures, in aqueous medium, and without the use of auxiliary chemicals, rendering them environmentally benign and economically advantageous processes. The most important enzymes utilized for these processes are penicillin G acylases, penicillin V acylases, cephalosporin acylases, and expandases.

Ethylene glycol has the ability to act as a reactant when employed as a co-solvent for the enzymatic synthesis of ampicillin from (R)-phenylglycine methyl ester and 6-aminopenicillanic acid. The side reaction positively affects yield because its product, (R)-phenylglycine hydroxyethyl ester, is an intermediate for ampicillin synthesis.