Milton Joseph Zmijewski

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.

Selective deprotection of phthalyl protected amines

Abstract Phthalyl amidase selectively deprotects phthalimido groups under very mild aqueous conditions in a one-pot reaction to produce phthalic acid and the free amine. The enzyme has been shown to deprotect several primary amines of distinctly different structure, and exhibits chiral selectivity when the substrate contains extensive β-branching. The enzyme has a definite requirement for ortho positioning of the functional groups on a fixed axis of rotation.

Bioreduction of (R)-carvone and regioselective Baeyer-Villiger oxidations: Application to the asymmetric synthesis of cryptophycin fragment A

Abstract Cryptophycin fragment A ( 1 ) was prepared in high enantiomeric purity in 10 steps from ( R )-carvone. A stereoselective bioreduction of ( R )-carvone to neodihydrocarveol and a regioselective Baeyer-Villiger oxidation of cyclohexanone 8 with pertrifluoroacetic acid were employed in this synthesis.

o-Phthalyl amidase in the synthesis of loracarbef: Process development using this novel biocatalyst

SummaryA 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).

APPLICATION OF BIOCATALYSIS TO DRUG METABOLISM: PREPARATION OF MAMMALIAN METABOLITES OF A BIARYL-BIS-SULFONAMIDE AMPA (α-AMINO-3-HYDROXY-5-METHYLISOXAZOLE-4-PROPIONIC ACID) RECEPTOR POTENTIATOR USING Actinoplanes missouriensis

LY451395 (2-propanesulfonamide, N-[(2R)-2-[4′-[2-[methylsulfonyl)amino]ethyl][1,1′-biphenyl]-4-yl]propyl]-) is a potent and highly selective potentiator of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors. It is a biaryl-bis-sulfonamide and is known to be highly metabolized in preclinical species. In those metabolism studies, the metabolite structures were proposed exclusively by the analysis of mass spectrometric data. Although mass spectrometry is clearly a technique of choice for rapid identification of drug metabolites, occasionally, nuclear magnetic resonance spectroscopy is required to unambiguously assign and characterize, particularly, the regio- and stereochemistry of metabolic changes. Nuclear magnetic resonance spectroscopy, in general, is less sensitive than other detection methods and demands several micrograms of material for the analysis. To support full structure characterization of metabolites by NMR, in this study we demonstrated the application of a microbial-based surrogate biocatalytic system to produce sufficient amounts of the mammalian metabolites of LY451395. The results revealed that incubation of LY451395 with Actinoplanes missouriensis NRRL B3342 generated several metabolites that were previously detected in the in vivo metabolism studies of the preclinical species. Subsequent large-scale bioconversion resulted in the isolation of seven mammalian metabolites in milligram quantities for structural characterization by nuclear magnetic resonance spectroscopy. Furthermore, a selected group of metabolites generated from the microbial conversion served as analytical standards to monitor and quantify drug metabolites during clinical investigations.

IN VIVO METABOLISM OF [14C]RUBOXISTAURIN IN DOGS, MICE, AND RATS FOLLOWING ORAL ADMINISTRATION AND THE STRUCTURE DETERMINATION OF ITS METABOLITES BY LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY AND NMR SPECTROSCOPY

Ruboxistaurin (LY333531), a potent and isoform-selective protein kinase C β inhibitor, is currently undergoing clinical trials as a therapeutic agent for the treatment of diabetic microvascular complications. The present study describes the disposition and metabolism of [14C]ruboxistaurin following administration of an oral dose to dogs, mice, and rats. The study revealed that ruboxistaurin was highly metabolized in all species. Furthermore, the results from the bile duct-cannulated study revealed that ruboxistaurin was well absorbed in rats. The primary route of excretion of ruboxistaurin and its metabolites was through feces in all species. The major metabolite detected consistently in all matrices for all species was the N-desmethyl metabolite 1, with the exception of rat bile, in which hydroxy N-desmethyl metabolite 5 was detected as the major metabolite. Other significant metabolites detected in dog plasma were 2, 3, 5, and 6 and in mouse plasma 2, 5, and 19. The structures of the metabolites were proposed by tandem mass spectrometry with the exception of 1, 2, 3, 5, and 6, which were additionally confirmed either by direct comparison with authentic standards or by nuclear magnetic resonance spectroscopy. To assist identification by nuclear magnetic resonance spectroscopy, metabolites 3 and 5 were produced via biotransformation using recombinant human CYP2D6 and, likewise, metabolite 6 and compound 4 (regioisomer of 3 which did not correlate to metabolites found in vivo) were produced using a microbe, Mortierella zonata. The unambiguous identification of metabolites enabled the proposal of clear metabolic pathways of ruboxistaurin in dogs, mice, and rats.

Discovery, purification, and properties of o-phthalyl amidase from Xanthobacter agilis

Abstract 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.

KINETIC RESOLUTIONS : EVALUATION OF A ONE DIMENSIONAL E VALUE CALCULATION METHOD USING A COMPUTER AND STATISTICAL SOFTWARE

Abstract A recently published curve fitting method of determining E values was put to the test using a computer and statistical software. Using idealized data and curves, the methodology worked reasonably well. Confidence limits generated by the ideal data were still large. However, when real data were employed, this methodology was not satisfactory in generating reliable E values due to inherent errors in the progress curves generated. This approach does not appear to be suitable for determining E values from one dimensional progress curves even when powerful statistical software and a computer are used to evaluate the data.