Kenichi Mochida

Chemo-enzymatic synthesis of optically pure l-leucovorin, an augmentor of 5-fluorouracil cytotoxicity against cancer

Optically pure l-leucovorin was synthesized on a large scale by the combination of chemical and enzymatic processes. After reduction of folate with zinc, dihydrofolate was reduced asymmetrically to (6)-tetra-hydrofolate by use of dihydrofolate reductase from E. coli C600/pTP600, with simultaneous NADPH cofactor recycling using glucose dehydrogenase from Gluconobacter scleroideus KY3613. Calcium l-leucovorin.4H2O (113 g) was obtained from (6S)-tetrahydrofolate via 5,10-methyenyltetrahydrofolate by formylation, reflux, addition of calcium ions and floricil column chromatography, with an overall yield of 50% based on folate. The l-leucovorin showed optical purity of 99.9% de as (6S)-form.

Lipase-catalyzed diacylation of 1,3-butanediol

SummaryA kinetic resolution of 1,3-butanediol was accomplished by lipase-catalyzed enantioselective diacylations in organic solvent. Diacylation of 1,3-butanediol was carried out using immobilized lipase SP382 (from Candida sp.) to produce (R) -1,3-diacetoxybutane with 85.8% e.e.. And then, this optically active product was chemically hydrolyzed to diol, and re-acylated with lipase SP382 to (R) -1,3-diacetoxybutane with over 98% e.e..

A Facile One-Pot Synthesis of Vinpocetine

Abstract A one-pot synthesis of vinpocetine from vincamine was established. Lewis acids caused transesterification and/or dehydration of vincamine in EtOH. FeCl3 catalyzed both transesterification and dehydration while Ti(OEt)4 selectively catalyzed transesterification.

Large-scale chemoenzymic synthesis of calcium (6S)-5-formyl-5,6,7,8-tetrahydrofolate [(–)-leucovorin] using the NADPH recycling method

Chemoenzymic large-scale synthesis of the calcium salt of (6S)-5-formyltetrahydrofolic acid [(–)-leucovorin, (6S)-5] was achieved from folic acid 1via(6S)-tetrahydrofolic acid [(6S)-3] by using dihydrofolate reductase (DHFR) produced by Escherichia coil, harbouring a high-expression plasmid, pTP64–1. On the other hand, for the diastereoselective reduction of 7,8-dihydrofolic acid 2 to tetrahydrofolate (6S)-3, a new NADPH recycling system was constructed by coupling with glucose dehydrogenase from Gluconobacter scieroides. Having these enzymic systems to hand, compound 1 was reduced by zinc powder in alkaline solution to give compound 2 which, without isolation, was reduced enzymatically to afford tetrahydrofolate (6S)-3(94% de). The pH adjustment of the reaction mixture containing dihydrofolate 2 was done with phosphoric acid in order to remove zinc ion which inhibited the following enzymic reduction. The formed tetrahydrofolate (6S)-3 was converted into entirely optically pure N-formyl compound (6S)-5 on a large scale. The specific rotation value of (–)-leucovorin was [α]D20–13.3 (c 1, water). For the comparison of pharmacological effects, a completely optically pure form of (+)-leucovorin [(6R)-5] was also prepared on a preparative scale. Compound (6S)-5 was 300-fold more active compared with the (6R)-diastereoisomer.

Enzymatic peptide synthesis by proline iminopeptidase

SummaryProline iminopeptidase was screened for the enzymatic synthesis of peptides containing Pro. The enzyme was purified from the best strainBacillus brevis, and the partially purified enzyme was used for peptide synthesis from Pro-benzylester and Phe. The dipeptide Pro-Phe was obtained in a 40% yield.

Microbial Hydroxylation of 2-Phenylpropionic Acid

The hydroxylation of 2-phenylpropionic acid (PPH) to 2-(p-hydroxyphenyl)propionic acid (HPPH) was investigated using various microorganisms. The highest hydroxylating activity was found in Streptomyces rimosus ATCC 10907. The reaction product was isolated and identified as 2-(p-Miydroxyphenyl)propionic acid, which had no specific rotation, indicating that the reaction was not stereoselective at the benzylic position. In a 5-1 jar fermentor containing a medium consisting of 2% sorbitol, 2% soybean meal, and 1% malt extract (pH 7.2), the conversion rate from 0.2% of PPH reached 60% using a PPH-resistant mutant, KY-661-18.

Chemoenzymatic synthesis of four diastereomers of (6-fluoro-2-chromanyl) oxirane: An intermediate of a potent β-blocker

The synthesis of optically active 5-acetoxy-3-(p-fluorophenoxy)-1-pentanol 4, for the synthesis of the potent β-blocker R-67555, bis[2-(2-chromanyl-6-fluoro)-2-hydroxyethyl]amine 1, was investigated. The acetylation of 3-(p-fluorophenoxy)-1,5-pentanediol 5a using lipozyme and the hydrolysis of 1,5-diacetoxy-3-(p-fluorophenoxy)pentane 5b using lipase Amano P yielded (3S)- and (3R)-5-acetoxy-3-(p-fluorophenoxy)-1-pentanol 4, respectively, with high enantiomeric excess. Four diastereomers of (6-fluoro-2-chromanyl)oxirane 2, important intermediates for the synthesis of R-67555, were synthesized by chemical methods using (S)-4 and (R)-4.