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Highly Enantioselective Hydrolysis of (R,S) ‐Phenylalanine Isopropyl Ester by Subtilisin Carlsberg. Continuous Synthesis of (S) ‐Phenylalanine in a Hollow Fiber/Liquid Membrane Reactor
Author(s) -
Ricks Edith E.,
EstradaValdés Maria C.,
Mclean Thomas L.,
Iacobucci Guillermo A.
Publication year - 1992
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp00015a004
Subject(s) - chemistry , phenylalanine , hydrolysis , racemization , enantioselective synthesis , enantiomer , subtilisin , isopropyl , enantiomeric excess , kinetic resolution , racemic mixture , reaction rate constant , catalysis , organic chemistry , medicinal chemistry , stereochemistry , chromatography , enzyme , kinetics , amino acid , biochemistry , physics , quantum mechanics
Abstract The resolution of racemic phenylalanine esters with esterases has been reexamined in relation to the development of a continuous process based on the use of hollow fiber/liquid membrane (SLM) reactors. The requirement for high enantioselectivity was met by phenylalanine isopropyl ester, whose (R) ‐enantiomer was found refractory to the action of subtilisin Carlsberg, in water, pH 7.5, at 25 °C. The continuous feeding of (R, S) ‐Phe‐O‐iPr‐HCl at 2 mm/h to 0.7 g/L subtilisin resulted in steady‐state reaction conditions that gave (S) ‐Phe‐OH of 95% enantiomeric excess (ee) at the theoretical rate. The unreacted (R) ‐Phe‐O‐iPr, which permeated out continuously, had 80% ee and was racemized quantitatively by heating with a salicylaldehyde catalyst in refluxing toluene. Doubling the feeding rate (4 mm/h) created excessive accumulation of (R) ‐Phe‐O‐iPr in the reactor, which inhibited the enzyme through nonproductive binding, lowering the (S) ‐Phe‐OH resolution rate to 68% of the theoretical rate. Similar experiments with (R, S) ‐Phe‐OMe‐HCl gave (S) ‐Phe‐OH of 9–48% ee, due to the enzymatic hydrolysis of (R)‐Phe‐OMe experienced during the runs. When α‐chymotrypsin was used for the continuous hydrolysis of (R, S)‐Phe‐O‐iPr, the isolated (S) ‐Phe‐OH showed 67% ee, indicating that substantial hydrolysis of the (‐R) ‐ester had occurred.