Biotransformation reactions acylation

In biotransformation reactions, ILs can act as tunable solvents or immobilizing agents or additives. They can also be coupled to substrates or other reagents (e.g., acylating agents in Hpase-catalyzed transesterifications [62]). Recent examples of chosen applications are presented below. 

Fig. 9. Biotransformation of dihydroquinidine (and acylated derivatives) to the corresponding (3S)-3-hydroxy compounds (as % observed at the reaction plateau), using various collection strains of Mucor plumbeus (ATCC American Type Culture Collection CBS Centraalbureau voor Schimmelcultures DSM Deutsche Sammlung von Mikroorganismen MMP Mycotheque of the Paris Museum of National History)...

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

In recent years biotransformations have also shown their potential when applied to nucleoside chemistry [7]. This chapter will give several examples that cover the different possibiUties using biocatalysts, especially lipases, in order to synthesize new nucleoside analogs. The chapter will demonstrate some applications of enzymatic acylations and alkoxycarbonylations for the synthesis of new analogs. The utQity of these biocatalytic reactions for selective transformations in nucleosides is noteworthy. In addition, some of these biocatalytic processes can be used not only for protection or activation of hydroxyl groups, but also for enzymatic resolution of racemic mixtures of nucleosides. Moreover, some possibilities with other biocatalysts that can modify bases, such as deaminases [8] or enzymes that catalyze the synthesis of new nucleoside analogs via transglycosylation [9] are also discussed. 

Hepatic damage related to isoflurane anesthesia has very occasionally been described (9,10), including one report of hepatic necrosis and death (11). Hepatitis or hepatocellular injury has been described with all current volatile anesthetics. Among these, halothane-associated hepatitis has been best characterized and is probably caused by an immune reaction induced by hepatocyte proteins that have been covalently trifluoroacetylated by the trifluoro-acetyl metabolite of halothane. The reactive acyl-halide metabolite of trifluoroacetic acid can trifluoroacetylate liver proteins, resulting in immune-mediated hepatic necrosis (12). However, isoflurane biotransformation to trifluoroacetate is less than 0.2%, compared with 15-20% for halothane. 

Hydratases that add water to unsaturated fatty acids coupled to coenzyme A (CoA) or acyl carrier protein (ACP) cannot be used in vitro, and consequently have to be applied in whole-cell biotransformations. Prohibitive as this may seem to production on a commercial scale, Kanegafuchi has developed a process, making use of whole cells of Candida rugosa, to produce (R)-2-hydroxybutanoic acid (31) from butanoic acid (30) (Scheme 11.5-5). The series of reactions catalyzed by these cells include coupling of butanoic acid to CoA, desaturation of butyryl-CoA to 2-butenyl-CoA and water addition catalyzed by enolyl-CoA hydratase (enoylase, unsaturated enoyl-... 

Some new approaches to suppress competitive reactions in protease-catalyzed peptide synthesis have been developed in our group [14], namely leaving group manipulations at the acyl donor in kinetically controlled reactions, enzymatic synthesis in organic solvent-free microaqueous systems, cryoenzymatic peptide synthesis, and biotransformations in frozen aqueous systems using the reverse hydrolysis potential of proteases and other hydrolases... 

Biotransformations are now firmly established in the synthetic chemist s armoury, especially reactions employing inexpensive hydrolytic enzymes for the resolution of racemates and for the desymmetrization of prochiral substrates. From a practical viewpoint, biocatalytic resolution is arguably the simplest method available to obtain synthetically useful quantities of chiral synthons. As an illustration of this point, many racemic secondary alcohols ROH can be resolved without prior derivatization by combining with a lipase and a volatile acyl donor (usually vinyl acetate) in an organic solvent, to effect irreversible transesterification once the desired degree of conversion has been reached, routine filtration to remove the enzyme and concentration of the filtrate affords the optically enriched products ROAcyl and ROH directly. 

Some reviews have described elegant examples for the enantioselective acylation of racemic alcohols [14-18]. Here, we have selected some reactions of considerable importance due to different factors attending to the substrate structure, the possibility to scale up the biotransformations, and the enz5mie recycling. 

A trypsin-related bacterial enzyme that was tested in coupling reactions is the lysine-specific serine protease I from Achromobacter lyticus. The enzyme is secreted and widely used in sequence analysis of proteins [55]. It was also used in a chemo-enzymatic route for the production of human insulin from porcine insulin. Since this endopeptidase cleaves only after Lys, it could be applied for replacing the C-terminus of the insulin B-chain from -Lys-Ala (porcine C-terminal sequence) to -Lys-Thr (human C-terminus) in a two-step or a single-step reaction. Using the B-chain as the acyl donor and Thr-OBu in DMF-ethanol mixtures as the nucleophile, a high conversion (85-90%) was obtained [9,56]. Trypsin could also be applied in this biotransformation but required higher enzyme loading. 

In order for the prcx ess to be more practical at large scale a number of improvements were made. As described previously, vinyl butyrate was found to be the most selective acyl donor. However, in consideration of scale-up costs, vinyl butyrate was much more expensive than vinyl propionate. Furthermore, the smell associated with butyrates was an added process consideration. Since vinyl propionate was only slightly less selective than vinyl butyrate (around 5% d.e.), on balance it was decided to scale up the biotransformation with vinyl propionate. In addition, MTBE was replaced with heptane as the reaction solvent. This prevented the solvent swap required for the diol/ester partition. The change in solvent gave no loss in selectivity. This was thus a very volume-efficient resolution that could be performed in standard chemical vessels without the need for any specialized equipment. After flie reaction was complete the workup was performed as outlined previously. This process was scaled up to 40-kg input batches of racemic/meso diol without complication. 

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