Christopher Roberge

Chiral bio-resolution of racemic indene oxide by fungal epoxide hydrolases

Eighty fungal strains were evaluated for their production of an epoxide hydrolase capable of catalyzing the kinetic resolution of racemic indene oxide into 1(S),2(R) indene oxide. This screen identified Diplodia gossipina ATCC 16391 as the best catalyst. Process development studies of this bio-resolution demonstrated that the optical purity of the 1(S),2(R) indene oxide produced was dependent upon pH and substrate concentration. A shake-flask-scale bio-resolution process supported the production of preparative quantities of optically pure (ee=100%) 1(S),2(R) indene oxide.

Asymmetric bioreduction of a keto ester to its corresponding (S)-hydroxy ester by Microbacterium sp. MB 5614

A bioprocess employing Microbacterium sp. MA 5614 was developed for the production of the MK-0476 (S)-hydroxy ester by asymmetric bioreduction of the corresponding keto ester. The cysteinyl leukotriene 1 receptor antagonist MK-0476 [3-[(1S)-[3(E)-[2-(7-chloroquinolinyl)ethenyl]phenyl]-3-(acetylphenyl)-propylthiol]-2(S)-methylpropanoic acid] is currently undergoing clinical evaluation for the treatment of asthma. Optimization of the bioconversion medium composition and the time of keto ester addition, as well as the development of a keto ester feeding strategy yielded improvements in the bioreduction rate and final (S)-hydroxy ester titer, by 20- and 25-fold respectively. This asymmetric bioreduction process supported the production of preparative quantities of (S)-hydroxy ester with an enantiomeric excess greater than 95%.

Bioconversion of indene to trans-2S,1S-bromoindanol and 1S,2R-indene oxide by a bromoperoxidase/dehydrogenase preparation from Curvularia protuberata MF5400

Abstract 1S,2R-Indene oxide is the precursor of cis -1S,2R-aminoindanol, a key intermediate for the Merck HIV–1 protease inhibitor, Crixivan®. As an alternative to the challenging chemical synthesis of this chiral epoxide from indene, the biotransformation route using an enzyme catalyst was examined. Approximately 3% of the 400 fungal cultures isolated from high salt environments were found to possess neutral haloperoxidase activities. Subsequent studies revealed that indene conversion by these positive cultures could only be obtained when both hydrogen peroxide and bromide ions were present. The products were generally racemic trans -bromoindanols which upon basification yielded racemic epoxides. Finally, it was found that a crude enzyme preparation from the fungal culture Curvularia protuberata MF5400 converted indene to the chiral 2S,1S-bromoindanol which can be chemically converted to the desired 1S,2R-epoxide through basification or used directly in the asymmetric synthesis of cis -1S,2R-aminoindanol. The bioconversion rate and the enantiomeric excess (ee) achieved with this cell-free system were heavily pH dependent. An initial 1.5-h reaction at pH 7.0 gave ∼10% yield of the chiral bromoindanol or epoxide from indene, and the yield was rapidly improved to >30% for trans -2S,1S-bromoindanol with an ee of 80%. Reaction mechanistic studies revealed that the stereoselectivity observed was apparently due to a specific dehydrogenase activity present in MF5400 which was also found to resolve chemically synthesized racemic trans -2-bromoindanols.

Asymmetric Biosynthesis of Key Aromatic Intermediates in the Synthesis of an Endothelin Receptor Antagonist

The feasibility of five potential biocatalytic routes were investigated for the chiral synthesis of key intermediates of an experimental endothelin receptor antagonist. Two asymmetric bioreductions of a ketoester and a chlorinated ketone to their corresponding chiral alcohol yielded very encouraging leads. Pichia delftensis (strain MY 1569) and Rhodotorula piliminae (ATCC 32762) were found to respectively bioreduce the esterified ketone and chlorinated substrate to their corresponding (S) alcohol with enantiomeric excesses > 98% and > 99% respectively. When scaled up in laboratory bioreactors (23-liter scale), both processes produced the desired (S) alcohol intermediate with elevated yield, about 88% and 97% for the ketoester and chloroketone respectively. Investigative chemical syntheses employing the (S) ester alcohol showed that unfavorable racemization occurred during the subsequent synthetic steps. However, the use of the (S) chloroalcohol as chiral synthon for the production of the endothelium receptor antagonist was successfully demonstrated at a preparative scale.

Process development for the production of the (S)-acid precursor of a novel elastase inhibitor (L-694,458) through the lipase-catalyzed kinetic resolution of a β-lactam benzyl ester

Abstract A limited screen of several commercially-available and internally-produced lipases and esterases identified the lipase PS-800 as a suitable biocatalyst for the resolution of a racemic β-lactam benzyl ester to the ( S )-acid. This β-lactam is a precursor to the elastase inhibitor L-694,458, an experimental drug targeted for the treatment of cystic fibrosis. Key to the development of a scalable process was the optimization of β-lactam and surfactant (Triton X-100) charges. These optimization studies yielded a 21-fold increase in the volumetric production of the ( S )-acid (from 0.38 g/ l to 8.0 g/ l ) and a 10-fold improvement in the initial bioconversion rate (from 17 mg/( l ·h) to 170 mg/( l ·h)). Additionally, these studies achieved the control of the ( S )-acid enantiomeric excess (e.e.) which was improved from less than 65% to greater than 90%. Keys to an economical and high yielding process, both the recycling of the lipase (at levels of 90%) and the use of re-racemized unreacted ( R )-benzyl ester in multiple reaction cycles were successfully demonstrated. This process was scaled up in 2.0- l reactors and afforded gram quantities of greater than 90% e.e ( S )-acid, which upon purification was successfully used to synthesize the elastase inhibitor L-694,458.

The highly stereospecific enzyme catalysed transamination of 4-fluorophenylglyoxylic acid to 4-(S)-fluorophenylglycine

Cell free extracts from a number of bacterial strains belonging to the Pseudomonas and Bacillus genera were found to catalyse the conversion of 4-fluorophenylglyoxylic acid to 4-(S)-fluorophenylglycine in high optical purity (99% ee).