Kinetic Resolution of β-Amino Esters by Acylation Using Immobilized Penicillin Amidohydrolase

Abstract

Penicillin amidohydrolase [EC 3.5.1.11] was used to resolve stereoisomers of a β-amino acid ester (ethyl 3-amino-5-(trimethylsilyl)-4-pentynoate) by phenylacetylation. After screening commercially available sources of the immobilized enzyme, one was found to be significantly more efficient, and this was developed at 1-L scale reaction. The effects of phenylacetic acid concentration, β-amino acid ester concentration, and pH on bioconversion rates and side reactions were examined. The enzymatic reaction was monitored off-line by naphthoylation of samples and chiral analytical chromatography. The best conditions for the bioconversion were pH 5.7, 28 °C, and 14 000 units of enzyme activity per liter. The phenylacetic acid concentration was set at 50 g/L (0.37 M), and the amine at 100 g/L (0.47 M). Under these conditions, yields of the desired (S)amino acid ester were on the order of 90% with ee's of 95% or greater in less than 12 h. This process, along with a slight modification, was tested through 15 cycles at 0.4-L scale, and was scaled to 70 L. Recycle results extrapolated to approximately 25 reaction cycles before the enzyme lost 50% of its initial activity. Through three runs at 70 L, overall yield of (S)-amine was 42.7 ( 0.6%, overall yield of (R)-phenylacetyl amide was 47.2 ( 1.8%. The average ee of the amine (two runs) was 98.1 ( 0.4%, for the amide the ee was 99.5 ( 0.2%. Experimental Section I. Analytical. ReVersed Phase (RP-HPLC) Chromatography. Aqueous samples for RP-chromatography were diluted into an equal volume of acetonitrile, except for amine

Improved β-lactam acylases and their use as industrial biocatalysts

“…Highly pure free amine and amide was obtained using 50 g/L phenylacetic acid as free acid, 100 g/L ethyl-3-amino-5-(trimethylsilyl)-4-pentynoate as the amine and 14 000 U/L PGA-450 (Roche) as enzyme at pH 5.7, 288C. Moreover, the reaction could be scaled up to 70L batches, with a half-life of the enzyme of approximately 20 runs [41 ]. In addition, enantioselective hydrolysis of an ester lacking a PAA moiety by E. coli PGA was reported [42], as well as enantioselective hydrolysis of the amide bond or ester bond of several glutaryl coupled substrates by a cephalosporin acylase [43].…”

6.11 N-Acylation Reactions of Amines

“…422 BcL, E >100 Figure 73 A representative range of chiral amides prepared in high enantioselectivity via lipase-catalyzed N-acylation of their parent (rac)-amines using esters or acids as acyl donors. [429][430][431][432] BcL, Burkholderia cepacia lipase; PaL, Pseudomonas aeruginosa lipase; Sub, subtilisin; Pen A, penicillin acylase. range of amides, whose N-acylation reactions often proceed with excellent levels of enantioselectivity (Figures 74(a) and (b)).…”

Effects of maintaining temperature and time on properties and structures of poly(dimethyldiallylammonium chloride)

“…This fact has prevented the study of such reactions, except in the most favorable cases. e.g., aliphatic amines with moderately favorable p K value (around 6) (Landis et al, 2002). In this paper, this requirement has been fulfilled by taking advantage of the high resistance of the new PGA hydrophilized preparation to the deleterious effects of the organic cosolvents (Abian et al, 2001).…”