Transesterification Synthesis of Chloramphenicol Esters with the Lipase from Bacillus amyloliquefaciens

Fengying Dong, Lingmeng Li, Lin Lin, Dannong He, Jingwen Chen, Wei Wei, Dongzhi Wei
2017 Molecules  
This work presents a synthetic route to produce chloramphenicol esters by taking advantage the high enantio-and regio-selectivity of lipases. A series of chloramphenicol esters were synthesized using chloramphenicol, acyl donors of different carbon chain length and lipase Lip BA (lipase cloned from Bacillus amyloliquefaciens). Among acyl donors with different carbon chain lengths, vinyl propionate was found to be the best. The influences of different organic solvents, reaction temperature,
more » ... n temperature, reaction time, enzyme loading and water content on the synthesis of the chloramphenicol esters were studied. The synthesis of chloramphenicol propionate (0.25 M) with 4.0 g L −1 of Lip BA loading gave a conversion of~98% and a purity of~99% within 8 h at 50 • C in 1,4-dioxane as solvent. The optimum mole ratio of vinyl propionate to chloramphenicol was increased to 5:1. This is the first report of B. amyloliquefaciens lipase being used in chloramphenicol ester synthesis and a detailed study of the synthesis of chloramphenicol propionate using this reaction. The high enzyme activity and selectivity make lipase Lip BA an attractive catalyst for green chemical synthesis of molecules with complex structures. Chloramphenicol is a natural antibiotic with a wide spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria [2] [3] [4] . Recently, chloramphenicol has been administered in increasing dosages due to the increased incidence of antibiotic resistance [5] . Unfortunately, it can also produce liver and kidney function inhibition, grey baby syndrome, aplastic anemia and so on. Meanwhile the bitterness of chloramphenicol cannot be accepted by most people [6], so different chloramphenicol derivatives have been produced minimize this bitterness [1] such as chloramphenicol succinate or chloramphenicol palmitate esters produced by means of regioor stereoselective chemical [7] and enzymatic methods [8] . Among all catalytic enzymes, lipases catalyze transesterification reactions on hydroxyl groups with high regioselectivity and mild reaction conditions [9-11]. Daugs et al. reported the lipase-mediated esterification of chloramphenicol palmitate in toluene and DMF [12]. Bizerra et al. reported that the Candida antarctica lipase type B (CAL-B) catalyzed the synthesis of chloramphenicol palmitate (0.15 M) to reach 99% conversion in 24 h at 50 • C [8]. Using the non-imprinted lipase nanogel and the lipase from Thermomyces lanuginosus [13], Wang et al. produced chloramphenicol palmitate with a conversion of 99% within 20 h at 20 • C. Ottolina et al. reported lipase G was the best biocatalytic agent, giving an excellent conversion to the corresponding esters at 45 • C after 24-72 h [14]. Further, Ottolina et al. reported the lipase-mediated esterification of chloramphenicol for the synthesis of several derivatives in anhydrous acetone to explore the effect of different trifluoroethyl esters. In this research most studies have focused on the synthesis of a single chloramphenicol ester, performed at an undesirable low temperature, with low substrate concentration and long reaction times. Previously, our group reported lipase Lip BA (accession number: KF040967) cloned from Bacillus amyloliquefaciens and the detailed enzymatic properties of the recombinant enzyme [15] . Furthermore, in our previous report, this enzyme was used in biocatalysis and cinnamyl esters were synthesized using lipase (Lip BA ) through the transesterification route in a non-aqueous system with vinyl propionate as the best acyl donor [16] . In this study, we have developed a chloramphenicol ester synthesis by using different carbon chain length acyl donors [17] . Among the acyl donors with different carbon chain lengths, vinyl propionate was chosen to act as the best acyl donor. Through single factor experiments, the enzymatic synthesis of chloramphenicol propionate ester was studied in detail for the first time, with different factors affecting the conversion efficiency. Furthermore, the transesterification reaction yield at high substrate concentrations (0.25 M) was the highest, and the reaction time (8 h) was the shortest at this concentration (0.25 M) in the previous reported literature. Using 0.25 M substrate concentrations, the chloramphenicol propionate provided excellent yield (98%) and purity (99%) within 8 h at 50 • C in 1,4-dioxane as solvent. Owing to the enzymatic properties such as high substrate concentration, high conversion rate, high product yield and purity, this lipase has potential value in industrial applications, particularly for chloramphenicol propionate synthesis. Results and Discussion Synthesis of Chloramphenicol Propionate Esters The transesterification reaction of chloramphenicol with vinyl propionate was performed using Lip BA in 1,4-dioxane. The products were purified by silica gel chromatography and characterized by 13 C-NMR and 1 H-NMR analysis. As Yoshimoto [18] established, acylation of a sugar hydroxyl group results in a downfield shift of the peak corresponding to the O-acylated carbon and an upfield shift of the peak corresponding to the neighboring carbon. Characterization of the chloramphenicol ester by 13 C-NMR revealed that the signals for R 1 of the chloramphenicol ester were shifted downfield and the C-2 positions shifted upfield ( Figure 1A ), compared with chloramphenicol ( Figure 1C ), indicating chloramphenicol was substituted at the R 1 position. Almost no shifting of the peak corresponding to the secondary alcohol in the product was observed, compared with chloramphenicol ( Figure 1C) . Furthermore, the 1 H-NMR spectrum showed the two hydrogens on the methylene groups of the chloramphenicol ester groups as characteristic peaks i and j at 4.36 and 4.24 ppm ( Figure 1B ), Molecules 2017, 22, 1523 3 of 11 respectively, with a significant downfield shift, compared with 3.80, 3.71 ppm for chloramphenicol ( Figure 1D ). 1 H-NMR also showed that the hydrogen on the methine group and secondary alcohol of the chloramphenicol ester groups give the characteristic peaks f and e at 5.267 and 5.368 ppm ( Figure 1B) , and these undergo a minor shift, compared with chloramphenicol at 5.137 and 5.373 ppm ( Figure 1D ). 1 H-NMR also confirmed the regioselective acylation at the primary hydroxyl group (R 1 ). These results all confirm that Lip BA showed effective regioselectivity in the transesterification of chloramphenicol with vinyl propionate. Molecules 2017, 22, 1523 3 of 11 the chloramphenicol ester groups give the characteristic peaks f and e at 5.267 and 5.368 ppm ( Figure 1B) , and these undergo a minor shift, compared with chloramphenicol at 5.137 and 5.373 ppm ( Figure 1D ). 1 H-NMR also confirmed the regioselective acylation at the primary hydroxyl group (R1). These results all confirm that LipBA showed effective regioselectivity in the transesterification of chloramphenicol with vinyl propionate.
doi:10.3390/molecules22091523 pmid:28925953 fatcat:krqt3vjrdjgv3kx3sk2sciql34