William L. Parker

Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius.

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO(2) using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH,98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces98% ee.

Influence of Formaldehyde Impurity in Polysorbate 80 and PEG‐300 on the Stability of a Parenteral Formulation of BMS‐204352: Identification and Control of the Degradation Product

The purpose of this study was to identify a degradation product formed in the clinical parenteral formulation of BMS‐204352, investigate the role of excipients in its formation, and develop a strategy to minimize/control its formation. The degradant was identified as the hydroxy methyl derivative (formaldehyde adduct, BMS‐215842) of the drug substance based upon liquid chromatography/mass spectroscopy (LC/MS), liquid chromatography/mass spectroscopy/mass spectroscopy (LC/MS/MS), nuclear magnetic resonance (NMR), and chromatographic comparison to an authentic sample of hydroxymethyl degradation product, BMS‐215842. An assay method for the detection of formaldehyde based on HPLC quantitation of formaldehyde dinitrophenylhydrazone was developed to quantitate its levels in various Polysorbate 80 and PEG 300 excipient lots. A direct relationship between the levels of formaldehyde in the excipients and the formation of the hydroxymethyl degradant was found. To confirm the hypothesis that the formaldehyde impurity in these two excipients contributed to the formation of the hydroxymethyl degradant, several clinical formulation lots were spiked with formaldehyde equivalent to 1, 10, and 100 mg/g of BMS‐204352. A correlation was found between the formaldehyde level and the quantity of the hydroxymethyl degradant formed upon storage at 5 and 25°C. From these experiments, a limit test on the formaldehyde content in polysorbate 80 and PEG 300 can be set as part of a strategy to limit the formation of the degradation product.

The “hydrophobic collapse” conformation of paclitaxel (Taxol®) has been observed in a non-aqueous environment: Crystal structure of 10-deacetyl-7-epitaxol

Abstract Together with the crystal structure of paclitaxel, this single-crystal X-ray diffraction study of 10-deacetyl-7-epitaxol provides examples of the “hydrophobic collapse” conformation of paclitaxel in solid state. More importantly, it gives the first evidence that the “hydrophobic collapse” conformation exists in a non-aqueous environment. This study demonstrates that in solid state, a bioactive molecule could adopt a conformation which is usually held only in an aqueous medium through the process of hydrophobic collapse. The special arrangement of molecules and large solvent channels found in the crystal structure suggest a similarity of the molecular environment between the solid state and the aqueous solution. Comparison to the crystal structure of docetaxel (Taxotere®) reveals that the flexibility around C1′–C2′ and C2′–C3′ appears to be fully responsible for the orientation of the side chain. Moreover, a careful comparison of the crystal structures has indicated that the non hydrophobic collapse conformation found in the crystal of paclitaxel is probably caused by molecular packing. It is a common feature for molecules of docetaxel, paclitaxel and 10-deacetyl-7-epitaxol that the 2′-hydroxyl and the 4′-carbonyl groups are involved in molecular interactions by hydrogen bonding.

Enzymatic C-4 deacetylation of 10-deacetylbaccatin III

Second‐generation paclitaxel analogues that require replacement of the C‐4 acetate by other substituents are in development. An enzyme able to specifically remove the C‐4 acetate from paclitaxel could simplify preparation of the analogues. Several strains were isolated from soil samples that contain enzyme activities able to 4‐deacetylate 10‐DAB (10‐deacetylbaccatin III). Selection was made using plates containing 10‐DAB as the sole carbon source and screening colonies for deacetylation of 10‐DAB. Two strains initially isolated were identified as Rhodococcus sp. and deposited with the A.T.C.C. (Manassas, VA, U.S.A.) as strains 202191 and 202192. Whole cells were able to convert 10‐DAB into 4,10‐DDAB (4‐deacetyl‐10‐deacetylbaccatin III) in 90% yield. The enzyme activity in these strains was not effective with paclitaxel and 10‐deacetylpaclitaxel, although 4,10‐DDAB was produced from baccatin III. The activity in these strains was associated with an insoluble fraction of cell extracts. Several additional isolates were obtained that were identified as variants of Stenotrophomonas maltophilia, and a soluble C‐4 deacetylase was purified approx. 218‐fold from one of them. The activity of this enzyme was limited to 10‐DAB, and the enzyme was not effective with paclitaxel or baccatin III.

Enzymatic preparation of 5-hydroxy-l-proline, N-Cbz-5-hydroxy-l-proline, and N-boc-5-hydroxy-l-proline from (α-N-protected)-l-ornithine using a transaminase or an amine oxidase

N-Cbz-4,5-dehydro-L-prolineamide or N-Boc-4,5-dehydro-L-prolineamide are alternative key intermediates for the synthesis of saxagliptin, a dipeptidyl peptidase IV (DPP4) inhibitor recently approved for treatment of type 2 diabetes mellitus. An efficient biocatalytic method was developed for conversion of L-ornithine, N-α-benzyloxycarbonyl (Cbz)-L-ornthine, and N-α-tert-butoxycarbonyl (Boc)-L-ornithine to 5-hydroxy-L-proline, N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline, respectively. Rec. Escherichia coli expressing lysine-ε-aminotransferase and rec Pichia pastoris expressing L-ornithine oxidase were used for these conversions. N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline were chemically converted to key intermediates N-Cbz-4,5-dehydro-L-prolineamide and N-Boc-4,5-dehydro-L-prolineamide, respectively.

Microbial N-demethylation: biotransformation and recovery of a drug metabolite.

A total of 39 microbes were screened for the ability to selectively N‐demethylate (3R,5S,E)‐7‐(4‐(4‐fluorophenyl)‐6‐isopropyl‐2‐(methyl(1‐methyl‐1H‐1,2,4‐triazol‐5‐yl)aminopyrimidin‐5‐yl)‐3,5‐dihydroxy‐hept‐6‐enoic acid (I), a potential drug for lowering blood cholesterol levels. Two Streptomyces species were found to carry out the desired N‐demethylation. Bioconversion by Streptomyces griseus A.T.C.C. 13273 and product recovery were scaled up to the multi‐gram level.