Substrate Report for: N-(3-Carboxy-4-nitrophenyl)phthalamidic-acid

A dephthalylation step utilizing a novel enzyme, o-phthalyl amidase, was developed. This step was part of a potentially new large scale synthetic route for a novel beta-lactam antibiotic Loracarbef. The enzyme was isolated from the organism Xanthobacter agilis. Purification of the enzyme to near homogeneity was accomplished by a 3-step procedure. Studies indicated that the phthalimido group can be opened chemically to generate the o-phthalyl derivative. This enzyme then can remove the phthalyl group from o-phthalylated amides. Optimization of the process was achieved by combining these two hydrolysis steps. Conversion yields of 85-97.8% (mol/mol) were obtained from reactions at substrate concentrations of 5-10% (w/v).

A selective screen for organisms that would metabolize o-phthalyl protected beta-lactams resulted in the discovery of a Xanthobacter agilis strain that contains an o-phthalyl amidase. The low level of enzyme expression in this organism could be enhanced by growing it on medium containing o-phthalate. The enzyme was purified to near homogeneity by a 6-step procedure. The phthalyl amidase was characterized for its molecular mass, amino acid composition, internal sequences, catalytic and kinetic properties. No metal ion was required by or stimulatory to the enzyme. The amidase catalyzed conversion could be complete with a reaction stoichiometry of 1:1. The pH and temperature stability of the enzyme is improved significantly by increasing ionic strength of the buffer. The enzyme exhibits a broad substrate specificity for o-phthalylated amides; however, it demonstrates an absolute requirement for the o-phthalyl protecting group. The broad substrate acceptance, high catalytic activity, and stability at high salt or substrate concentration of the enzyme indicates that it can serve as a gentle method for deprotecting phthalimido and o-phthalyl protected amides in new chemo-enzymatic synthetic routes.