Study On Whole Cells-catalyzed Regioselective Synthesis Of1-β-D-Arabinofuranosylcytosine Monoesters

1-β-D-Arabinofuranosylcytosine (ara-C) is one of the major drug in the therapy treamentof acute leukemia. However, ara-C has major clinical shortfalls in the treatment of solidtumors, since they cannot easily transfer across the cell membrane by passive diffusion andmight undergo a rapid enzymatic deactivation in plasma. To improve the clinical efficiency ofthose analogs, lipophilic modification has gained much attention as a promising strategy,which is also valuable for new nucleoside drug/prodrug discovery anddevelopment.Lipase-catalyzed synthesis of nucleoside monoesters has gained much attentionin the past decade, due to their high regioselectivity, simplicity, mild reaction conditions andenvironmental friendliness. However, the main hurdle to the industrial application ofenzymatic approaches in synthesis of nucleoside esters is the high cost of the procedure andlow stability of some free enzymes in organic solvents. Therefore, the development of newbiocatalysts has recently gained much attention. Whole cells are preferred to isolated enzymesbecause they eliminate the needs for enzyme purification and immobilization which accountfor a large part in the enzyme cost. In addition, the whole cells can provide a naturalenvironment for enzyme location which protects the enzymes from a rapid deactivation innon-aqueous solvents. Till then the biocatalytic synthesis of5′-monoester of ara-C byfreeze-dried microbial cells has so far remained unexplored. In this dissertation, thepossibility of biocatalytic synthesis of ara-C monoesters by whole cells from different sourceswere investigated. The influences of the culture conditions on the biomass and syntheticactivities of the cells in ara-C acylation reaction were discussed and the cultivation conditionsfor whole cells with high catalytic activity were optimized. In addition, the catalyticperformances of two kinds of whole-cell biocatalysts in synthsis of the nucleoside estersdifferent organic sovents were compared and the reaction conditions for the whole-cellcatalyzed synthesis of nucleoside esters were also optimized.Pseudomonas fluorescens was screened from five different Pseudomonas strains due toits high catalytic ativity and ability to catalyze the regioselective acylation towards the5’-OHof ara-C, while Aspergillus oryzae was selected for selectively catalyzing the acylationtowards3’-OH of ara-C.The catalytic performances of the bacterial cells were significantly enhanced bycultivation with a mixed carbon sources containing yeast extract and additional lipid-relatedsubstrates, especially the supplement of soybean oil. The cultivation of the cells supplementedwith glucose, however, resulted in both low biomass and catalytic activities in the acylation. Taking into account the yield and5′-regioselectivity of the cell-mediated reaction, yeastextract appeared to be the most suitable for cell cultivation, of all the tested nitrogen sources.Due to the fact that the nutrient concentrations and culture time are also crucial factorsaffecting the corresponding cell-catalyzed reaction, their effect on the catalytic performanceof the cells were also investigated. The best soybean oil concentration, yeast extractconcentration and culture time were0.5%(w/v),0.1%(w/v) and48h, respectively, underwhich the yield and5′-regioselectivity of the reaction catalyzed by the cell biocatalyst inorganic solvent reached75.4%and96.8%after120-hour reaction.Compared with pure organic solvents, the reaction catalyzed by whole cells fromPseudomonas fluorescens was greatly improved by using co-solvent mixtures of pyridine andhydrophobic solvents. In the case of the whole cells form P. fluorescens catalyzed-acylationof ara-C with vinyl acetate (VA), the optimal co-solvent, biocatalyst dosage, the molar ratioof VA to ara-C, reaction temperature and shaking speed were30%hexane-pyridine,50mg/mL、30:1、30℃and140rpm, respectively, under which the initial rate and the yieldwere1.6mmol/L·h and83.1%.When vinyl propionate (VP) as acyl donor, although theoptimum reaction conditions had some differences, the profiles of the effects of variousvariables on the reaction were similar. Under the optimal conditions, the initial rate and thesubstrate conversion were2.8mmol/L·h and77.9%, respectively. The5’-regioselectivity ofthe reaction showed little variation within the scope assayed and was higher than92%.Investigation of the whole cells form Aspergillus oryzae catalyzed acylation of ara-Cwith VP showed that compared with P. fluorescens, the reaction more inclined to thesynthesis of ara-C3’-ester. Both the initial rate and the yield were closely relatied with thepolarity of the solvent mixtures. For the reactions, the most suitable co-solvent, molar ratio ofVP to ara-C,biocatalyst dosage, reaction temperature and shaking speed were30%isopropylether-pyridine,90:1,60mg/mL,30℃,180rpm, under which the initial rate and the yield were10.7mmol/L·h and88.3%. Both the water content and the reaction temperature showed acrucial effect on the regioselectivity of whole cells from A. oryzae, while the other reactionconditions, had little effect on the regioselectivity.This study not only expands the scope of regioselective acylation of nucleoside bybiocatalysis, enriches our knowledge of whole cells-catalyzed synthesis of nucleoside ester innon aqueous media, but also provides a novel and efficient route to prepare the monoesterof1-β-D-arabinofuranosylcytosine.