Jeffrey T. Vicenzi

Large-scale stereoselective enzymatic ketone reduction with in situ product removal via polymeric adsorbent resins

Abstract 3,4-methylene-dioxyphenyl acetone was stereoselectively reduced to the corresponding S-3,4-methylene-dioxyphenyl isopropanol in >95% isolated yield and >99.9% enantiomeric excess (ee). The NAD(P)H-dependent enzymatic activity within living Zygosaccharomyces rouxii was utilized to accomplish this reaction. Since the substrate and product were both toxic to Z. rouxii, polymeric hydrophobic resins were used to both supply substrate to and remove the product from the reaction mixture as it formed. This approach allowed the reaction concentration to be increased from 6 to 40 g l−1. The reaction was scaled-up to a volume of 300 l by utilizing a commercially available agitated filter as a reactor. This reactor design allowed the reaction, product isolation, and resin recycle to be accomplished within a single piece of equipment. The overall reactor productivity was 75 g l−1 day−1.

Enantioselective Syntheses of 1-Carbacephalosporins from Chemoenzymically Derived β-Hydroxy-α-Amino Acids: Applications to the Total Synthesis of Carbacephem Antibiotic Loracarbef

Abstract Serine hydroxymethyltransferase (SHMT) derived from recombinant E. coli was found to be able to catalyze the condensation between glycine and 4-pentenaldehyde, affording enantiopure l -erythro-2-amino-3-hydroxy-6-heptenoic acid (AHHA) in high yield and throughput. Conversion of this chiral intermediate of biosynthetic origin to the oral carbacephalosporin antibiotic loracarbef (Lorabid®) via β-lactam forming reactions and subsequent Dieckmann cyclization was achieved.

High yielding culture conditions for the biosynthesis of D-amino acid oxidase byTrigonopsis variabilis

SummaryA process was developed for the production ofTrigonopsis variabilis and D-amino acid oxidase. Yields for the yeast were in excess of 220 g/l wet weight and 62 g/l dry weight. Using cephalosporin C as a substrate the enzyme concentration was 7000 units per liter.

Enzymatic synthesis of diastereospecific carbacephem intermediates using serine hydroxymethyltransferase

The serine hydroxymethyltransferase (SHMT) gene glyA was over-expressed in Escherichia coli and the enzyme was purified to near homogeneity. Reaction conditions for E. coli and rabbit liver SHMTs were optimized using succinic semialdehyde methyl ester (SSAME) and glycine. The catalytic efficiency (kcat/Km) of E. coli SHMT for SSAME was 2.8-fold higher than that of rabbit liver enzyme. E. coli SHMT displayed a pH-dependent product distribution different from that of rabbit liver enzyme. For the pyridoxal-5′-phosphate (PLP)-dependent reaction, E. coli and rabbit liver SHMTs showed a high product diastereospecificity. The stoichiometric ratio of PLP to the dimeric E. coli SHMT was 0.5–0.7, indicating a requirement for external PLP for maximal activity. Using SSAME or its analog at a high temperature, E. coli SHMT mediated efficient condensation via a lactone pathway. In contrast, at a low temperature, the enzyme catalyzed efficient conversion of 4-penten-1-al via a non-lactone mechanism. Efficient conversion of either aldehyde type to a desirable diastereospecific product was observed at a pilot scale. E. coli SHMT exhibited a broad specificity toward aldehyde substrates; thus it can be broadly useful in chemo-enzymatic synthesis of a chiral intermediate in the manufacture of an important carbacephem antibiotic.