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Sep 19, 2017 - corresponding to the secondary alcohol in the product was observed, ... excellent selectivity for the acylation of the primary alcohol [8,17].
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Transesterification Synthesis of Chloramphenicol Esters with the Lipase from Bacillus amyloliquefaciens Fengying Dong 1 , Lingmeng Li 1 , Lin Lin 2 , Dannong He 2 , Jingwen Chen 3 , Wei Wei 1, * and Dongzhi Wei 1, * 1

2 3

*

State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China; [email protected] (F.D.); [email protected] (L.L.) Research Laboratory for Functional Nanomaterial, National Engineering Research Center for Nanotechnology, Shanghai 200241, China; [email protected] (L.L.); [email protected] (D.H.) Department of Pathology, Microbiology and Immunology, School of medicine, University of South Carolina, 6311 Garners Ferry Rd., Columbia, SC 29209, USA; [email protected] Correspondence: [email protected] (W.W.); [email protected] (D.W.); Tel./Fax: +86-21-6425-1803 (W.W.); Tel./Fax: +86-21-6425-2078 (D.W.)

Received: 24 July 2017; Accepted: 4 September 2017; Published: 19 September 2017

Abstract: 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 LipBA (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, 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 LipBA 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 LipBA an attractive catalyst for green chemical synthesis of molecules with complex structures. Keywords: enzymatic catalysis; regioselectivity; chloramphenicol esters; green chemistry; Bacillus amyloliquefaciens

1. Introduction In recent years, enzymes have been applied to synthesize chloramphenicol derivatives, which has aroused widespread interest because of their high activity, moderate catalytic conditions, and environmental friendliness. This due to the fact that the enzymatic approach avoids the need for protection and deprotection procedures to discriminate between the different available hydroxyl groups in chloramphenicol, as chloramphenicol substituents on the primary hydroxyl group are rapidly hydrolyzed in vivo to the biologically active drug [1]. In addition, the separation steps to remove impurities that have close physical and chemical properties to the target compound results in lower final conversion, higher costs, time-and energy-consumption and excess discharge of waste. In this sense, enzymatic catalysis with its high enantio- and regioselectivity is attractive for the green synthesis of chemical compounds.

Molecules 2017, 22, 1523; doi:10.3390/molecules22091523

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Chloramphenicol is a natural antibiotic with a wide spectrum of antimicrobial activity against Gram-positive and Gram-negative bacteria [2–4]. 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 LipBA (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 (LipBA ) 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. 2. Results and Discussion 2.1. Synthesis of Chloramphenicol Propionate Esters The transesterification reaction of chloramphenicol with vinyl propionate was performed using LipBA 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 R1 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 R1 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),

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respectively, with a significant downfield shift, compared with 3.80, 3.71 ppm for chloramphenicol 3 of 11 (Figure 1D). 1 H-NMR also showed that the hydrogen on the methine group and secondary alcohol of the chloramphenicol groups give the characteristic peaks and and e at 5.368 5.267ppm and(Figure 5.368 ppm the chloramphenicol ester ester groups give the characteristic peaks f and e atf5.267 1B), (Figure 1B), and these undergo a minor shift, compared with chloramphenicol at 5.137 and 5.373 ppm and these undergo a minor shift, compared with chloramphenicol at 5.137 and 5.373 ppm (Figure 1D).1H1 H-NMR also confirmed the regioselective acylation at the primary hydroxyl group (R ). (Figure 1D). 1 NMR also confirmed the regioselective acylation at the primary hydroxyl group (R1). These results These results confirm thateffective LipBA showed effectivein regioselectivity in the transesterification of all confirm thatallLip BA showed regioselectivity the transesterification of chloramphenicol chloramphenicol with vinyl propionate. with vinyl propionate. Molecules 2017, 22, 1523

Figure spectra of chloramphenicol propionate ester and chloramphenicol. (A) 13C-NMR spectrum Figure1.1.NMR NMR spectra of chloramphenicol propionate ester and chloramphenicol. (A) 13 C-NMR 1 1 3)2CO; (B) H-NMR spectrum of chloramphenicol propionate of chloramphenicol propionate ester in (CD spectrum of chloramphenicol propionate ester in (CD3 )2 CO; (B) H-NMR spectrum of chloramphenicol 1 1 ester in (CDester 3)2CO; (C) 13C-NMR spectrum of chloramphenicol in (CD3)2CO;in(D) spectrum of propionate in (CD3 )2 CO; (C) 13 C-NMR spectrum of chloramphenicol (CDH-NMR 3 )2 CO; (D) H-NMR chloramphenicol in (CD 3 ) 2 CO. spectrum of chloramphenicol in (CD3 )2 CO.

2.2. Acyl Donors Donors 2.2. Transesterification Transesterification of of Chloramphenicol Chloramphenicol with with Different Different Acyl Single thethe transesterification of Single factor factor experiments experiments were were carried carriedout outininour ourstudy. study.Firstly, Firstly, transesterification chloramphenicol with different acyl donors was investigated. These were vinyl acetate, vinyl of chloramphenicol with different acyl donors was investigated. These were vinyl acetate, propionate, vinyl vinyl butyrate, vinylvinyl neononanoate, vinyl decanoate and vinyl vinyl propionate, butyrate, neononanoate, vinyl decanoate and vinyllaurate, laurate,respectively. respectively. Recent experimental studies on chloramphenicol ester synthesis by enzymatic catalysis are Recent experimental studies on chloramphenicol ester synthesis by enzymatic catalysis are listed listed in in Table 1. The preparation of chloramphenicol esters through transesterification is shown in Figure Table 1. The preparation of chloramphenicol esters through transesterification is shown in Figure2A. 2A. These revealed that that lipase lipasecan canbe beapplied appliedininchloramphenicol chloramphenicolester estersynthesis synthesis with acyl donors These data data revealed with acyl donors as as transesterification substrates. Among the different acyl donors vinyl propionate led to quantitative transesterification substrates. Among the different acyl donors vinyl propionate led to quantitative and and high high conversion conversion (81%) (81%) to to the the corresponding corresponding chloramphenicol chloramphenicol ester ester under under the the same same reaction reaction −1; chloramphenicol 0.25 M in ethanol; conditions (Table 2, reaction conditions: enzyme loading 4.0 g L − 1 conditions (Table 2, reaction conditions: enzyme loading 4.0 g L ; chloramphenicol 0.25 M in ethanol; acyl that Lip LipBA gives ◦ C;44hh and acyl donor/chloramphenicol donor/chloramphenicol==5:1; 5:1;TT==50 50°C; and 200 200 rpm). rpm). This This trend trend showes showes that BA gives higher conversion with short chain rather than medium and long chain donors. Chloramphenicol higher conversion with short chain rather than medium and long chain donors. Chloramphenicol gave gave a lower conversion (≤55%) the esters otherdonors acyl donors because of the reactivity poorer reactivity a lower conversion (≤55%) to theto esters using using other acyl because of the poorer of these of these acyl donors. It should be noted that the diacylated compounds b (R 3 = COOCH2CH3, R1 = H, acyl donors. It should be noted that the diacylated compounds b (R3 = COOCH2 CH3 , R1 = H, Figure 2) Figure 2) and c (R1 = R3 = COOCH2CH3, Figure 2) were not detected after 8 h. These experimental and c (R 1 = R3 = COOCH2 CH3 , Figure 2) were not detected after 8 h. These experimental results results were consistent the reports from Bizerra and[8,12]. DaugsThis [8,12]. This effect can be explained were consistent with thewith reports from Bizerra and Daugs effect can be explained because because diacylated compound b and c could not correctly fit in the active site of LipBAachievements . The above diacylated compound b and c could not correctly fit in the active site of LipBA . The above achievements suggest LipBA lipase is an ideal catalyst for the regioselective acylation of chloramphenicol, showing an excellent selectivity for the acylation of the primary alcohol [8,17]. The molar ratio of chloramphenicol to vinyl propionate had a significant effect on the conversion of the product. A poor conversion (69%) was obtained at the molar ratio of chloramphenicol to vinyl propionate of 1:1. When

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suggest LipBA lipase is an ideal catalyst for the regioselective acylation of chloramphenicol, showing an excellent selectivity for the acylation of the primary alcohol [8,17]. The molar ratio of chloramphenicol to vinyl propionate had a significant effect on the conversion of the product. A poor conversion (69%) was obtained at the molar ratio of chloramphenicol to vinyl propionate of 1:1. When the molar ratio of vinyl propionate to chloramphenicol was increased to 5:1, the conversion of chloramphenicol was 81% (Table 2). There was no significant further increase in the product yield with a further increase in the amount of vinyl propionate used, therefore the molar ratio of vinyl propionate to chloramphenicol used was 5:1. Table 1. Recent experimental studies in chloramphenicol esters synthesis by enzymatic catalysis. Chloramphenicol Esters

Enzyme Resource

Concentration t (h)

Solvent

Reference

Chloramphenicol acetate

CAL-B CAT

C. antarctica lipase S. aureus

0.15 M 0.0015 M

40 12

1,4-dioxane, phosphate buffer

[8] [19]

Chloramphenicol propionate

CAL-B

C. antarctica lipase

0.15 M

6

1,4-dioxane,

[8]

CAT

S. aureus

0.0015 M

12

phosphate buffer

[19]

Chloramphenicol butyrate

CAT

S. aureus

0.0015 M

12

phosphate buffer

[19]

Chloramphenicol succinate

NR











Chloramphenicol pivalate

NR











Chloramphenicol decanoate

NR











Chlor5amphenicol laurate

CAL-B

C. antarctica lipase

0.15 M

24

1,4-dioxane

[8]

Chloramphenicol cinnamate Molecules 2017, 22, 1523 Chloramphenicol palmitate

NR











CAL-B

C. antarctica lipase

0.15 M

24

1,4-dioxane

[8]

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nanogel T. lanuginosus 0.15 M was increased 20 acetonitrile [13] the molar ratio of vinyl propionate to chloramphenicol to 5:1, the conversion of lipase – 70 mM 120 toluene [17] chloramphenicol was 81% (Table 2). There was no significant further increase in the product yield Chloramphenicol B.amyloliquefaciens 0.25 M used, 8therefore 1,4-dioxane with a further propionate increase in Lip theBAamount of vinyl propionate the molar ratio– of vinyl NR: not reported. propionate to chloramphenicol used was 5:1.

Figure Lipase-mediated esterification of chloramphenicol usingusing vinyl vinyl propionate in organic solvents. Figure2. 2. Lipase-mediated esterification of chloramphenicol propionate in organic (A) Preparation of chloramphenicol propionatepropionate through transesterification; (B) TLC analysis product; solvents. (A) Preparation of chloramphenicol through transesterification; (B) TLCofanalysis (C) of chloramphenicol and chloramphenicol-3-propionate. The assay conditions were of Chromatogram product; (C) Chromatogram of chloramphenicol and chloramphenicol-3-propionate. The assay described inwere material and methods. conditions described in material and methods. Table 1. Recent experimental studies in chloramphenicol esters synthesis by enzymatic catalysis. Chloramphenicol esters Chloramphenicol acetate Chloramphenicol propionate

Enzyme CAL-B CAT CAL-B

Resource C. antarctica lipase S. aureus C. antarctica lipase

Concentration 0.15 M 0.0015 M 0.15 M

t (h) 40 12 6

Solvent 1,4-dioxane, phosphate buffer 1,4-dioxane,

Reference [8] [19] [8]

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Molecules 2017, 22, 1523 5 of 11 Molecules 2017, 2017, 22, 22, 1523 1523 5 of of 11 11 Molecules Table 2. The acyl donors’ effect on the yield of chloramphenicol. Reaction conditions: enzyme loading Molecules 2017, 22, 1523 555 of 11 Molecules 2017, 22, 1523 of 11 Molecules 2017, 22, 1523 5 of − 1 ◦ g L22, chloramphenicol 0.25 M in ethanol; acyl donor/chloramphenicol = 5:1; T = 50 C; 4 h and Molecules4.0 2017, 22, ;1523 1523 5 of of 11 11 Molecules 2017, 5 11 Table 2. The acyl donors’ effect on the yield of chloramphenicol. Reaction conditions: enzyme loading Molecules 2017, Table 2. 22, The1523 acyl donors’ donors’ effect effect on on the the yield yield of of chloramphenicol. chloramphenicol. Reaction Reaction conditions: conditions: enzyme enzyme loading loading5 of 11 Table 2. The acyl

200 −1 rpm. Table 2. acyl donors’ effect on the yield of chloramphenicol. Reaction conditions: enzyme loading Table 2. The acyl donors’ effect the yield of chloramphenicol. Reaction conditions: loading 4.0 g L ;; The chloramphenicol 0.25 M in ethanol; acyl donor/chloramphenicol == 5:1; T 50 °C; h and 200 rpm. −1 4.0 gL L−1 chloramphenicol 0.25 Mon in ethanol; ethanol; acyl donor/chloramphenicol 5:1; T === 50 50 °C; °C; 444enzyme h and and 200 200 rpm. −1 Table 2. The acyl donors’ effect on the yield of chloramphenicol. Reaction conditions: enzyme loading 4.0 g ; chloramphenicol 0.25 M in acyl donor/chloramphenicol = 5:1; T h rpm. Table 2. The acyl donors’ effect on the yield of chloramphenicol. Reaction conditions: enzyme loading 4.0 g L ; chloramphenicol 0.25 M in ethanol; acyl donor/chloramphenicol = 5:1; T = 50 °C; 4 h and 200 rpm. −1 Table 2. The acyl donors’ effect on the yield of chloramphenicol. Reaction conditions: enzyme loading 4.0 g L ; chloramphenicol 0.25 M in ethanol; acyl donor/chloramphenicol = 5:1; T = 50 °C; 4 h and 200 rpm. a Table 2. The acyl donors’ effect on the yield of chloramphenicol. Reaction conditions: enzyme loading −1 4.0 ethanol; donor/chloramphenicol = 5:1; T == 50 4h and 200 Compound 0.25 Structure Ratio Time (h)°C; −1; chloramphenicol 4.0 g gL LCompound chloramphenicol 0.25 M M in inChemical ethanol; acyl acyl donor/chloramphenicol 5:1; TTime 50 °C; hConversion and 200 rpm. rpm. aaa Ratio (h) Conversion −1; chloramphenicol Chemical Structure Compound Ratio=== 5:1; Time (h) 444 h Conversion 4.0 g L 0.25 M in ethanol; acyl donor/chloramphenicol 5:1; T = 50 50 °C; h and 200 rpm. −1;; chloramphenicol Chemical Structure Compound Ratio Time (h) Conversion a 4.0 g L 0.25 M in ethanol; acyl donor/chloramphenicol T = °C; and 200 rpm. Chemical Structure Compound Ratio Time (h) Conversion Chemical Structure a Compound Ratio Time (h) Conversion Chemical Structure a Compound Ratio Time (h) Conversion a Chemical Structure Compound Ratio Time (h) Conversion a Chemical Structure Vinyl acetate 5:1 4 55% Vinyl acetate 5:1 4 55% Compound Ratio Time (h) Conversion a Chemical Structure Vinyl acetate 5:1 4 55% Compound Ratio Time (h) Conversion Chemical Structure Vinyl acetate acetate 5:1 4 55% Vinyl 5:1 4 55% Vinyl acetate 5:1 44 55% Vinyl 5:1 55% O O Vinyl acetate acetate 5:1 4 55% O Vinyl acetate 5:1 4 55% O Vinyl acetate 55% Vinyl propionate 5:1 4 81% O Vinyl propionate 5:1 4 81% Vinyl propionate 5:1 4 81% O Vinyl propionate propionate 5:1 81% Vinyl 5:1 444 81% O O O O Vinyl propionate 5:1 81% O O Vinyl propionate 5:1 44 81% O Vinyl propionate 5:1 81% O Vinyl propionate 5:1 4 81% O Vinyl propionate 81% Vinyl butyrate 5:1 4 30% O Vinyl butyrate 5:1 4 30% O VinylVinyl butyrate 5:1 30% butyrate 5:1 5:1 4 44 30% O Vinyl butyrate 30% Vinyl butyrate 5:1 44 30% Vinyl butyrate 5:1 30% Vinyl butyrate 5:1 4 30% Vinyl butyrate butyrate 5:1 4 30% Vinyl 5:1 4 30% Vinyl neononanoate neononanoate 5:1 4 23% Vinyl 5:1 4 23% VinylVinyl neononanoate 5:1 23% Vinyl neononanoate 23% neononanoate 5:1 5:1 4 444 23% Vinyl neononanoate 5:1 23% Vinyl neononanoate 5:1 4 23% Vinyl neononanoate neononanoate 5:1 4 23% Vinyl 5:1 4 23% Vinyl neononanoate 5:1 4 23% Vinyl decanoate decanoate 20% Vinyl 5:1 4 20% Vinyl decanoate 5:1 4 20% Vinyl decanoate 5:1 4 20% Vinyl decanoate 20% 5:1 5:1 4 4 20% Vinyl decanoate Vinyl decanoate 5:1 44 20% Vinyl decanoate 5:1 20% Vinyl decanoate 20% Vinyldecanoate laurate 5:1 4 8% Vinyl 20% Vinyl laurate 5:1 4 8% Vinyl laurate 5:1 4 8% Vinyl laurate 5:1 4 8% Vinyl laurate 5:1 4 8% Vinyl laurate laurate 5:1 5:1 4 44 8%8% VinylVinyl laurate 5:1 8% Vinyl laurate 5:1 4 8% Vinyl propionate 5:1 4 69% Vinyl laurate 8% Vinyl propionate propionate 5:1 69% Vinyl 5:1 44 69% Vinyl propionate 5:1 4 69% Vinyl propionate 5:1 4 69% Vinyl propionate 69% VinylVinyl propionate 5:1 69% 5:1 5:1 4 444 69% propionate Vinyl propionate 5:1 69% 5:1 69% Vinyl propionate 10:1 4 82% Vinyl propionate 10:1 4 82% Vinyl propionate propionate 10:1 82% Vinyl 10:1 444 82% Vinyl propionate 10:1 82% Vinyl propionate 10:1 44 82% Vinyl propionate 10:1 82% VinylVinyl propionate 10:1 82% 10:115:1 4 44 82% propionate 10:1 82% Vinyl propionate 84% Vinyl propionate 15:1 4 84% Vinyl propionate propionate 15:1 84% Vinyl 15:1 444 84% Vinyl propionate 15:1 84% Vinyl propionate 15:1 4 84% a Determined Vinyl propionate propionate 15:1 84% by HPLC and referred to the initial concentration of chloramphenicol. Conversions a Determined Vinyl 15:1 44 84% by HPLC and referred to the initial concentration of chloramphenicol. Conversions a Determined Vinyl propionate 15:1 4 84% Vinyl propionate 15:1 4 84% by HPLC and referred to the initial concentration of chloramphenicol. Conversions a Determined by HPLC and referred to the initial concentration of chloramphenicol. Conversions a Determined by HPLC and referred to the initial concentration of chloramphenicol. Conversions determined by HPLC after 4 h of reaction. a Determined determined byby HPLC after 4h hreferred of reaction. reaction. HPLC and to a Determined determined by HPLC after of by HPLC and referred to the the initial initial concentration concentration of of chloramphenicol. chloramphenicol. Conversions Conversions a Determined determined by HPLC after 444 h of reaction. by HPLC and referred to the initial concentration of chloramphenicol. Conversions adetermined by HPLC after h of reaction. a Determined Determined by HPLC and referred to the initial concentration of chloramphenicol. Conversions by HPLC and referred to the initial concentration of chloramphenicol. Conversions determined by determined by HPLC after 4 h of reaction. determined by HPLC after 4 h of reaction. determined by HPLC after 44 h h of of reaction. reaction. 2.3. Effect Effect of Different Different Solvents HPLC afterby 4 hHPLC ofSolvents reaction. determined after 2.3. of 2.3. Effect of Different Solvents

2.3. Effect of Different Solvents 2.3. Effect of Different Solvents 2.3. Effect of Different Solvents The effect of solvents on enzymatic reactions is critical for non-aqueous medium [20]. The solvent 2.3. Effect of Different Solvents The effect of solvents on enzymatic enzymatic reactions reactions is is critical critical for for aaa non-aqueous non-aqueous medium medium [20]. [20]. The The solvent solvent 2.3. Effect of Different Solvents The effect of solvents on 2.3. Effect of Different Solvents 2.3. Effect of Different Solvents The effect of solvents on enzymatic reactions is critical for a non-aqueous medium [20]. The solvent The effect of solvents on enzymatic reactions is critical for a non-aqueous medium [20]. The solvent affects the catalytic power of enzymes by changing the three-dimensional conformation of proteins, affects theeffect catalytic poweron ofenzymatic enzymes by by changing the three-dimensional three-dimensional conformation of proteins, The of reactions is for medium The solvent affects the catalytic power of enzymes changing the conformation of proteins, The effect of solvents solvents on enzymatic reactions is critical critical for aaa non-aqueous non-aqueousconformation medium [20]. [20].of The solvent affects the catalytic power of enzymes by changing the three-dimensional proteins, The effect of solvents on enzymatic reactions is critical for non-aqueous medium [20]. The solvent affects the catalytic power of enzymes by changing the three-dimensional conformation of proteins, and therefore significantly alters the conversion [21]. It is reported that enzymes had a better stability The effect of solvents on enzymatic reactions is critical for a non-aqueous medium [20]. The solvent The effect of solvents on enzymatic reactions is critical for a non-aqueous medium [20]. The solvent and therefore significantly alters the conversion [21]. It is reported that enzymes had a better stability affects the catalytic power of enzymes by changing the three-dimensional conformation of proteins, and therefore significantly alters the conversion [21]. It is reported that enzymes had a better stability affects the catalytic power of enzymes by changing the three-dimensional conformation of proteins, and therefore significantly alters the conversion [21]. It is reported that enzymes had a better stability affects the catalytic power of enzymes by changing the three-dimensional conformation of proteins, and therefore significantly alters the conversion [21]. It is reported that enzymes had a better stability affects thethe catalytic of enzymes bythan changing the three-dimensional of of proteins, compatibility inpower non-polar solvents than in[21]. polar solvents, due toenzymes theconformation fact polar solvents with affects catalytic power of enzymes by changing the three-dimensional conformation proteins, and compatibility in non-polar solvents in polar solvents, due to the fact polar solvents with therefore significantly alters the It reported that had aa better stability and compatibility in non-polar solvents than in in polar solvents, due to the fact polar solvents with therefore significantly alterssolvents the conversion conversion [21]. It is is reporteddue thatto enzymes had better stability and compatibility in non-polar than polar solvents, the fact polar solvents with therefore significantly alters the conversion [21]. It is reported that enzymes had a better better stability compatibility in non-polar solvents than in polar solvents, due to the fact polar solvents with and therefore significantly alters the conversion [21]. It is reported that enzymes had a stability and therefore significantly alters the conversion [21]. It is reported that enzymes had a better stability E T values show bad compatibility with enzyme molecules that leads to reduced activity [22]. high and in solvents than in solvents, the fact polar ETT values values show show bad compatibility compatibility with enzyme molecules that to leads to reduced activity with [22]. high and compatibility compatibility in non-polar non-polar solvents with than enzyme in polar polarmolecules solvents, due due to the to fact polar solvents solvents with E bad with enzyme molecules that leads to reduced activity [22]. high and compatibility in non-polar solvents than in polar solvents, due to the fact polar solvents with E T values show bad compatibility that leads reduced activity [22]. high and compatibility in non-polar solvents than polar solvents, due to the fact polar solvents with and compatibility in non-polar solvents in polar solvents, due to the fact polar solvents with high E T values show bad compatibility with enzyme molecules that leads to reduced activity [22]. high In this study, the polarity of the reaction medium was selected according to the empirical polarity T values show bad compatibility enzyme that to reduced activity [22]. high In thisE study, the polarity of the reaction reactionwith medium wasmolecules selected according according to the empirical polarity In this study, the polarity of the medium was selected to the empirical polarity E T values show bad of compatibility with enzyme molecules that leads leadsto tothe reduced activity [22]. high In this study, the polarity the reaction medium was selected according empirical polarity E T values show bad compatibility with enzyme molecules that leads to reduced activity [22]. high Ethis bad bad compatibility withwith enzyme that leads to reduced activity In this In the polarity ofby the reaction medium was selected to the empirical polarity ETstudy, values show compatibility molecules that leads toCastillo reduced [22]. high parameter E T (30) described Reichardt for pure solvents and adapted by et al. [23–25]. T values parameter Eshow T(30) (30) described by Reichardt for enzyme puremolecules solvents andaccording adapted by Castillo etactivity al.[22]. [23–25]. In the polarity the reaction was selected according to the polarity parameter E T described by Reichardt for pure solvents and adapted by Castillo et al. [23–25]. In this this study, study, the described polarity of ofby theReichardt reaction medium medium was selected according to Castillo the empirical empirical polarity parameter E T (30) for pure solvents and adapted by et al. [23–25]. In this study, the polarity of the reaction medium was selected according to the empirical polarity study, the polarity of the reaction medium was selected according to the empirical polarity parameter parameter E T (30) described by Reichardt for pure solvents and adapted by Castillo et al. [23–25]. E T(30) (30) takes into account solvent–solute interactions at the the molecular level [26]. [26]. Theempirical effect of different In thistakes study, theaccount polarity ofby the reaction medium was selected according to the polarity E T into solvent–solute interactions at molecular level The effect of different parameter T(30)account described for and by et al. [23–25]. E T (30) takes takesE into solvent–solute interactions at the molecular molecular level [26]. The effect of different parameter Einto T(30) described by Reichardt Reichardt for pure pure solvents solvents and adapted adapted by Castillo Castillo et of al.different [23–25]. E T (30) solvent–solute interactions at the level [26]. The effect parameter E T(30) (30)account described by Reichardt for pure solvents and adapted by Castillo et al. [23–25]. (30) described by Reichardt for pure solvents and adapted by Castillo et al. [23–25]. E (30) takes into E TE (30) takes into account solvent–solute interactions at the molecular level [26]. The effect of different parameter E T described by Reichardt for pure solvents and adapted by Castillo et al. [23–25]. media (toluene, dichloromethane, tertrahydrofuran, 1,4-dioxane, acetonitrile, acetone and ethanol) T T media (toluene, dichloromethane, tertrahydrofuran, 1,4-dioxane, acetonitrile, acetone and ethanol) E T(30) takes into account solvent–solute interactions at the molecular level [26]. The effect of different media (toluene, dichloromethane, tertrahydrofuran, 1,4-dioxane, acetonitrile, acetone and ethanol) ETT(30) (30) takes takes into dichloromethane, account solvent–solute solvent–solute interactions at at1,4-dioxane, the molecular molecular level [26]. [26]. acetone The effect effect of different different media (toluene, tertrahydrofuran, acetonitrile, and ethanol) E into account interactions the level The of solvent–solute interactions at the molecular level [26]. The effect of different media (toluene, media (toluene, dichloromethane, tertrahydrofuran, 1,4-dioxane, acetonitrile, acetone and ethanol) E Taccount (30) takes into account solvent–solute interactions at the molecular level [26]. The effect of different ranging from E T (30) 33.9 (non-polar) to E T (30) 51.9 (polar) were studied with vinyl propionate as acyl ranging from E ETT(30) (30) 33.9 (non-polar) (non-polar)tertrahydrofuran, to E ETT(30) (30) 51.9 51.9 (polar) (polar) were studied studied with vinyl vinyl propionate as acyl media dichloromethane, 1,4-dioxane, acetonitrile, acetone and ranging from 33.9 to were with propionate as acyl media (toluene, (toluene, dichloromethane, tertrahydrofuran, 1,4-dioxane, acetonitrile, acetone and ethanol) ethanol) ranging from E T(30) 33.9 (non-polar) to E T(30) 51.9 (polar) were studied with vinyl propionate as acyl media (toluene, dichloromethane, tertrahydrofuran, 1,4-dioxane, acetonitrile, acetone and ethanol) −1 dichloromethane, tertrahydrofuran, 1,4-dioxane, acetonitrile, acetone and ethanol) ranging from E (30) 33.9 ranging from E T (30) 33.9 (non-polar) to E T (30) 51.9 (polar) were studied with vinyl propionate as acyl media (toluene, dichloromethane, tertrahydrofuran, 1,4-dioxane, acetonitrile, and ethanol) donor (Table 3). The reaction conditions were as follows: follows: enzyme loading 4.0vinyl g acetone L−1 ; chloramphenicol chloramphenicol −1propionate T donor (Table 3). The reaction conditions were as enzyme loading 4.0 g L ; ranging from E T (30) 33.9 (non-polar) to E T (30) 51.9 (polar) were studied with as donor (Table 3). The 33.9 reaction conditions were as follows: follows: enzyme loading 4.0vinyl gL L−1 ;propionate chloramphenicol ranging from3). ETT(30) (30) 33.9 (non-polar) to E Ewere T(30) 51.9 (polar) enzyme were studied studied with vinyl as acyl acyl donor (Table The reaction conditions as loading 4.0 g ; chloramphenicol −1 ranging from E (non-polar) to T (30) 51.9 (polar) were with propionate as acyl (non-polar) to 51.9 (polar) were studied with vinyl propionate as acyl donor (Table donor 3). The reaction conditions as follows: enzyme loading 4.0 gT ;; chloramphenicol ranging ETE (30) 33.9 (non-polar) to ETwere (30) 51.9 (polar) were studied with vinyl propionate asreaction acyl 0.25 M in different organic solvents; vinyl propionate/chloramphenicol == 5:1; =−1 50 °C; 44 h and 200 T (30) 0.25 M(Table infrom different organic solvents; vinyl propionate/chloramphenicol 5:1; TL 50 °C;3). hThe and 200 donor (Table 3). reaction conditions were as enzyme 4.0 L chloramphenicol −150 0.25 M in different organic solvents; vinyl propionate/chloramphenicol 5:1; T °C; h and 200 donor (Table 3). The The reaction conditions were as follows: follows: enzyme loading loading 4.0 g gT L===−1 chloramphenicol −150 −1 ; chloramphenicol 0.25 M in different organic solvents; vinyl propionate/chloramphenicol ===aM 5:1; °C; 444 h and 200 donor (Table 3). The reaction conditions were as follows: enzyme loading 4.0 g L ;; conversion chloramphenicol conditions were as follows: enzyme loading 4.0 g L 0.25 in different organic solvents; 0.25 M in different organic solvents; vinyl propionate/chloramphenicol 5:1; T = 50 °C; h and 200 donor (Table 3). The reaction conditions were as follows: enzyme loading 4.0 g L ; chloramphenicol rpm. From the results, 1,4-dioxane was the best organic solvent and gave highest (89%) rpm. From the results, 1,4-dioxane was the best organic solvent and gave a highest conversion (89%) 0.25 M in different organic solvents; vinyl propionate/chloramphenicol = 5:1; T = 50 °C; 4 h and 200 rpm. From the results, results, 1,4-dioxane was the propionate/chloramphenicol best organic organic solvent and and gave gave==aa5:1; highest conversion (89%) 0.25 M M in different different organic solvents; vinyl propionate/chloramphenicol 5:1; T == 50 50 °C; 44 h h and and 200 ◦ C; 4 h solvent rpm. From the 1,4-dioxane was the best highest conversion (89%) 0.25 in organic solvents; vinyl T °C; 200 vinyl propionate/chloramphenicol = 5:1; T = 50 and 200 rpm. From the results, 1,4-dioxane was rpm. From the results, 1,4-dioxane was the best organic solvent and gave a highest conversion (89%) of chloramphenicol ester (chloramphenicol propionate) (Table 3, Figure 3). The results in Table 0.25 M in different organic solvents; vinyl propionate/chloramphenicol = 5:1; T = 50 °C; 4 h and 200 of chloramphenicol ester (chloramphenicol propionate) (Table 3, Figure 3). The results in Table 33 rpm. From the results, 1,4-dioxane was the best organic solvent and gave aa3). highest conversion (89%) of chloramphenicol ester (chloramphenicol propionate) (Table 3, Figure The results in Table rpm. From the results, 1,4-dioxane was the best organic solvent and gave highest conversion (89%) of chloramphenicol ester (chloramphenicol propionate) (Table 3, Figure 3). The results in Table 333 rpm. From the results, 1,4-dioxane was the best organic solvent and gave a highest conversion (89%) the best organic solvent and gave a highest conversion (89%) of chloramphenicol ester (chloramphenicol of chloramphenicol ester (chloramphenicol propionate) (Table 3, Figure 3). The results in Table indicate that Lip BA had better operational stability and tolerance (67% residual enzyme activity after 12 h) rpm. From the results, 1,4-dioxane was the best organic solvent and gave a highest conversion (89%) indicate that Lip LipBA BA had hadester better operational stability stability and tolerance tolerance (67% residual enzyme activityin after 12 h) h)33 of (chloramphenicol propionate) (Table 3, Figure The Table indicate that better operational and (67% residual enzyme activity after 12 of chloramphenicol chloramphenicol ester (chloramphenicol propionate) (Table 3, residual Figure 3). 3). The results results in Table indicate that Lip BA had better operational stability and tolerance (67% enzyme activity after 12 h) of chloramphenicol ester (chloramphenicol propionate) (Table 3, Figure 3). The results in Table propionate) (Table 3, Figure 3). Thechloramphenicol results inpropionate) Table 3 indicate that Lip better operational stability indicate that Lip BA had better operational stability and tolerance (67% residual enzyme activity after 12 h) of chloramphenicol ester (chloramphenicol (Table 3, Figure 3). The results in Table 33 in 1,4-dioxane. Furthermore, both chloramphenicol and vinyl propionate had good solubility and BA had in 1,4-dioxane. Furthermore, both and vinyl propionate had good solubility and indicate that Lip BA had better operational stability and tolerance (67% residual enzyme activity after 12 h) in 1,4-dioxane. Furthermore, both chloramphenicol chloramphenicol and vinyl vinyl propionate had good good solubility and indicate that Lip LipFurthermore, BA had better operational stability and and tolerance tolerance (67% residual enzyme enzyme activity after 12 12 h) in 1,4-dioxane. both and propionate had solubility and indicate that BA had better operational stability (67% residual activity after h) and tolerance residual enzyme activity afterand 12to h) in 1,4-dioxane. Furthermore, both chloramphenicol in 1,4-dioxane. Furthermore, both and vinyl propionate had solubility and indicate that Lip BA had better operational stability tolerance (67% residual enzyme activity after 12 h) compatibility in 1,4-dioxane. This was favorable the organic reactants near the active sites of compatibility in(67% 1,4-dioxane. This chloramphenicol was favorable to the organic reactants neargood the active sites of in Furthermore, both chloramphenicol and vinyl propionate had good solubility and compatibility in 1,4-dioxane. This was favorable to the organic reactants near the active sites of in 1,4-dioxane. 1,4-dioxane.in Furthermore, both chloramphenicol and vinyl propionate had good solubility and compatibility 1,4-dioxane. This was favorable to the organic reactants near the active sites of in 1,4-dioxane. Furthermore, both chloramphenicol and vinyl propionate had good solubility and and vinyl propionate had good solubility and compatibility in 1,4-dioxane. This was favorable to the compatibility in 1,4-dioxane. This was favorable to the organic reactants near the active sites of in 1,4-dioxane. Furthermore, both chloramphenicol and vinyl propionate had good solubility and catalyst (Lip BA ), which effectively promoted the occurrence of the reaction. Therefore 1,4-dioxane was catalyst (Lip BA ), which effectively promoted the occurrence of the reaction. Therefore 1,4-dioxane was compatibility in 1,4-dioxane. This was favorable to the organic reactants near the active sites of catalyst (LipBA BA), ),in which effectively promoted the occurrence occurrence of the the reaction. reaction. Therefore 1,4-dioxane was compatibility inwhich 1,4-dioxane. This was favorable favorable to the the organic organic reactants near the the1,4-dioxane active sites sites of catalyst (Lip effectively promoted the of Therefore was compatibility 1,4-dioxane. This was to reactants near active of organic reactants near the active sites of catalyst (Lip ), which effectively promoted the occurrence catalyst (Lip BA ), which effectively promoted the occurrence of the reaction. Therefore 1,4-dioxane was compatibility in 1,4-dioxane. This was favorable to BA theunder organic reactants near conditions, the1,4-dioxane active sites of of chosen as the optimum reaction solvent. Inthe thisoccurrence study, under the same reaction reaction conditions, 0.15was M chosen as the optimum reaction solvent. In this study, the same 0.15 M catalyst (Lip BA), which promoted of reaction. Therefore chosen as the optimum reaction solvent. In this study, under the same reaction conditions, 0.15 M catalystas (Lip BA), which effectively effectively promoted the occurrence of the the reaction. Therefore 1,4-dioxane was chosen the optimum reaction solvent. In this study, under the same reaction conditions, 0.15 M catalyst (Lip BA ), which effectively promoted the occurrence of the reaction. Therefore 1,4-dioxane was the reaction. Therefore 1,4-dioxane was chosen asstudy, the optimum reaction solvent. this study, under chosen as the optimum reaction solvent. In this under the same reaction 0.15 Mthe catalyst (Lip BA), which effectively promoted the occurrence of the reaction. Therefore 1,4-dioxane was chloramphenicol can be fully converted into chloramphenicol propionate. 0.25 M chloramphenicol chloramphenicol can be be fully converted converted into chloramphenicol propionate. 0.25 In Mconditions, chloramphenicol chosen reaction solvent. In study, same conditions, 0.15 chloramphenicol can fully into chloramphenicol propionate. 0.25 M chloramphenicol chosen as as the the optimum optimum reaction solvent.into In this this study, under under the the same reaction reaction conditions, 0.15 M M chloramphenicol can be fully converted chloramphenicol propionate. 0.25 M chloramphenicol chosen as the optimum reaction solvent. In this study, under the same reaction conditions, 0.15 M same reaction conditions, 0.15 M chloramphenicol can be fully converted into chloramphenicol propionate. chloramphenicol can be fully converted into chloramphenicol propionate. 0.25 M chloramphenicol can be fully dissolved in 1,4-dioxane and gave a higher conversion compared with 0.5 M (Figure 3), chosen as the optimum reaction solvent. In this study, under the same reaction conditions, 0.15 M can be fully fully dissolved dissolved in fully 1,4-dioxane andinto gavechloramphenicol a higher higher conversion conversion compared with 0.5 M M (Figure (Figure 3), 3), chloramphenicol can be converted propionate. 0.25 M chloramphenicol can be in 1,4-dioxane and gave a compared with 0.5 chloramphenicol can be fully converted into chloramphenicol propionate. 0.25 M chloramphenicol can be fully dissolved in 1,4-dioxane gave aain higher conversion compared with 0.5 3), chloramphenicol can be fully converted into chloramphenicol propionate. 0.25 M chloramphenicol 0.25 M can converted be fullyand dissolved 1,4-dioxane and gave a higher conversion compared can be fully dissolved in 1,4-dioxane and gave higher conversion compared with 0.5 M M (Figure (Figure 3), so 0.25 Mchloramphenicol was thecan ideal concentration for further reaction condition optimization. chloramphenicol be fully into chloramphenicol propionate. 0.25 M chloramphenicol so 0.25 M was the ideal concentration for further reaction condition optimization. can be dissolved in 1,4-dioxane gave conversion compared with so 0.25 M was the ideal concentration for further reaction condition optimization. can0.25 be fully fully dissolved inconcentration 1,4-dioxane and and gave aaa higher higher conversion compared with 0.5 0.5 M M (Figure (Figure 3), 3), so M was the ideal for further reaction condition optimization. can be fully dissolved in 1,4-dioxane and gave higher conversion compared with 0.5 M (Figure 3), with 0.5 M (Figure 3), so 0.25 M was the ideal concentration for further reaction condition optimization. so 0.25 M was the ideal concentration for further reaction condition optimization. can be fully dissolved in 1,4-dioxane and gave a higher conversion compared with 0.5 M (Figure 3), so 0.25 M was the ideal concentration for further reaction condition optimization. so 0.25 0.25 M M was was the the ideal ideal concentration concentration for for further further reaction reaction condition condition optimization. optimization. so so 0.25 M was the ideal concentration for further reaction condition optimization.

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Figure 3. 3. Effect Effect of of the the reaction reaction conditions conditions on on the the conversion conversion of of chloramphenicol. chloramphenicol. Reaction Reaction conditions: conditions: Figure Chloramphenicol concentration: 0.15, 0.25, 0.5 M; acyl donor: 0: control, 1: vinyl acetate, 2: vinyl Chloramphenicol concentration: 0.15, 0.25, 0.5 M; acyl donor: 0: control, 1: vinyl acetate, 2: vinyl propionate, propionate, 3: acetone, 4: vinyl neononanoate, 5: vinyl decanoate, 6: vinyl laurate; Reaction solvent: 3: acetone, 4: vinyl neononanoate, 5: vinyl decanoate, 6: vinyl laurate; Reaction solvent: 1: ethanol 2: 1: ethanol, 2: 1,4-dioxane, 3: acetone, 4: acetonitrile, 5: tetrahydrofuran, 6: dichloromethane, 7: toluene; 1,4-dioxane, 3: acetone, 4: acetonitrile, 5: tetrahydrofuran, 6: dichloromethane, 7: toluene; temperature: 0: temperature: 0: control, T = 10, 20, 30, 40, 50, 60 ◦ C; enzyme loading = 0, 0.5, −11.0, 2.0, 4.0, 8.0 g L−1 ; control, T = 10, 20, 30, 40, 50, 60 °C; enzyme loading = 0, 0.5, 1.0, 2.0, 4.0, 8.0 g L ; water content = 0%, water content = 0%, 0.5%, 1.0%, 2.0%, 4.0%; 8 h and 200 rpm. 0.5%, 1.0%, 2.0%, 4.0%; 8 h and 200 rpm. Table 3. Single experiments experiments in in the the chloramphenicol chloramphenicol ester Table 3. Single ester synthesis synthesis reaction. reaction. Solvent ETE(30) Solvent T(30) Control Control - Toluene 33.9 Toluene 33.9 1,4-Dioxane 36.0 1,4-Dioxane 36.0 THF 37.4 THF 37.4 Dichloromethane Dichloromethane 40.7 40.7 Acetone 42.2 Acetone 42.2 Acetonitrile 45.6 Acetonitrile 45.6 Ethanol 51.9 Ethanol 51.9 Temperature 1,4-Dioxane 36.0 Temperature 1,4-Dioxane 36.0 1,4-Dioxane 36.0 1,4-Dioxane 36.0 1,4-Dioxane 36.0 1,4-Dioxane 36.0 1,4-Dioxane 36.0 1,4-Dioxane 36.0 1,4-Dioxane 36.0 1,4-Dioxane 36.0 Time 1,4-Dioxane 36.0 Time 1,4-Dioxane 36.0 1,4-Dioxane 36.0 1,4-Dioxane 36.0 1,4-Dioxane 36.0 1,4-Dioxane 36.0 b Factor Factor Solvent Solvent

Temperature Temperature 40◦ C °C 40 40◦ C °C 40 40 40◦ C °C ◦ 40 40 C °C ◦ 40 40 C °C 40 ◦ C 40◦ °C 40 C 40 °C 40 ◦ C 40 °C 20 ◦ C 20 °C 30 ◦ C 30◦ °C 40 C 40◦ C °C 50 50◦ C °C 60 60◦ °C 50 C 50◦ C °C 50 50◦ C °C 50 50 °C

All reactions were performed in 10 mL 1,4-dioxane containing 4.0 g L

Time Time 44 hh 44 hh 44 hh 44 hh 44 hh 4h 4h 4h 4h 4h 4h 4h 4h 4h 4h 4h 44 hh 44 hh 4h 8h 8h 12 h 12 hh 16 −116 h

Conversionbb(%) (%) Conversion 00 50 50±±1.2 1.2 89±±2.1 2.1 89 70±±2.3 2.3 70 60±±1.5 1.5 60 72 ± 0.7 72 ± 0.7 80 ± 1.4 80 ± 1.4 81 ± 0.9 81 ± 0.9 85 ± 1.7 85 ± 1.7 87 ± 2.3 87 ± 2.3 89 ± 1.5 89 91±±1.5 2.2 91 90±±2.2 1.9 90 ± 1.9 98 ± 0.6 98 96±±0.6 1.8 96 92±±1.8 2.1 92 ± 2.1

Purity(%) (%) Purity 00 50 50±±0.7 0.7 95±±2.5 2.5 95 85±±2.6 2.6 85 70±±1.5 1.5 70 75 ± 1.2 75 ± 1.2 85 ± 0.9 85 ± 0.9 87 ± 0.5 87 ± 0.5 93 ± 1.3 93 ± 1.3 95 ± 2.1 95 ± 2.1 95 ± 2.8 95 99±±2.8 1.9 99 93±±1.9 2.4 93 ± 2.4 99 ± 1.5 99 90±±1.5 1.2 90 85±±1.2 0.7 85 ± 0.7

LipBA , 0.25 M chloramphenicol, and 1.25 M

All reactions were performed in 10 mL 1,4-dioxane containing L−1 Lip BA, 0.25 M of chloramphenicol, propionate. Conversion was determined by HPLC and referred to4.0 the ginitial concentration chloramphenicol. Purity was determined by flash chromatography. and 1.25 M Vinyl propionate. Conversion was determined by HPLC and referred to the initial concentration of chloramphenicol. Purity was determined by flash chromatography. b Vinyl

2.4. Effect of Reaction Temperature

2.4. Effect of Reaction Temperature Enzyme stability decreased with the increase of temperature out of a certain range. The effect of reaction temperature on esterification studied at 20, 30, 40,out 50 and 60 ◦ C for 4 h. The reaction Enzyme stability decreased with the was increase of temperature of a certain range. effect of − 1 conditions were as follows: enzyme was loading 4.0 gatL20,; 30, chloramphenicol 0.25 in The 1,4-dioxane; reaction temperature on esterification studied 40, 50 and 60 °C forM 4 h. reaction −1; chloramphenicol vinyl propionate/chloramphenicol 5:1; reaction 4 h and 200 rpm. The indicatedvinyl that conditions were as follows: enzyme =loading 4.0 g Ltime 0.25 Mresults in 1,4-dioxane; ◦ the conversion (91%) and purity reached maximum value 50 C (Table 3, Figure 3). propionate/chloramphenicol = 5:1;(99%) reaction timetheir 4 h and 200 rpm. Theatresults indicated that the With further(91%) increasing temperature, the conversion and purityvalue declined gradually. results revealed conversion and purity (99%) reached their maximum at 50 °C (TableThe 3, Figure 3). With further increasing temperature, the conversion and purity declined gradually. The results revealed that the increasing temperature facilitated the nucleophilic substitution reaction when the temperature

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that the increasing temperature facilitated the nucleophilic substitution reaction when the temperature was withina acertain certainrange, range,thus thusenhancing enhancing the the reaction reaction rate. rate. However, was within However, aa higher highertemperature temperaturegradually gradually inactivated the enzyme and degraded the product due to the poor product stability. On the inactivated the enzyme and degraded the product due to the poor product stability. On the otherother hand, it was also possible that the elevated temperature promoted reactivity of the secondary alcohol it hand, was also possible that the elevated temperature promoted reactivity of the secondary alcohol group group and thus brought a negative impact on the lipase efficient [27]. and thus brought a negative impact on the lipase efficient [27]. 2.5.Effect EffectofofReaction ReactionTime Time 2.5. Reactiontime timestudies studies indicated indicated the the performance performance of Reaction of the the enzyme enzyme as asthe thereaction reactionprogressed. progressed. Furthermore, the reaction time helped to determine the shortest time necessary to obtain Furthermore, the reaction time helped to determine the shortest time necessary to obtainaahigh high conversion. The effect of reaction time on esterification was studied at 4, 6, 12 and 16 h. The reaction conversion. The effect of reaction time on esterification was studied at 4, 6, 12 and 16 h. The reaction conditions were as follows: enzyme loading 4.0 g L−1; chloramphenicol 0.25 M in 1,4-dioxane; vinyl conditions were as follows: enzyme loading 4.0 g L−1 ; chloramphenicol 0.25 M in 1,4-dioxane; vinyl propionate/chloramphenicol = 5:1; reaction temperature 50 °C. As shown in Table 3, when the time propionate/chloramphenicol = 5:1; reaction temperature 50 ◦ C. As shown in Table 3, when the time was increased to 8 h, chloramphenicol was almost fully converted to chloramphenicol propionate was increased to 8 h, chloramphenicol was almost fully converted to chloramphenicol propionate with with a conversion of 98% and a purity over 99% after 8 h reaction at 50 °C (Table 3, Figure 3). a conversion of 98% and a purity over 99% after 8 h reaction at 50 ◦ C (Table 3, Figure 3). Increasing the Increasing the reaction time up to 16 h led to a decrease in both conversion (92%) and purity (85%). reaction time up to 16 h led to a decrease in both conversion (92%) and purity (85%). This might be due This might be due to the poor stability of the chloramphenicol propionate and the elevated reactivity to the poor stability of the chloramphenicol propionate and the elevated reactivity of the secondary of the secondary alcohol group at the longer time. alcohol group at the longer time. 2.6. Effect of Enzyme Loading 2.6. Effect of Enzyme Loading The enzyme loading is crucial for any bioconversion process. Thus, the effect of enzyme loading The enzyme loading is crucial for any bioconversion process. Thus, the effect of−1enzyme loading was studied for different enzyme concentrations such as 0.5, 1.0, 2.0, 4.0 and 8.0 g L of the reaction was studied for different enzyme concentrations such as 0.5, 1.0, 2.0, 4.0 and 8.0 g L−1 of the reaction volume. The reaction conditions were as follows: chloramphenicol 0.25 M in 1,4-dioxane; vinyl volume. The reaction conditions were as follows: chloramphenicol 0.25 M in 1,4-dioxane; vinyl propionate/chloramphenicol = 5:1; T = 50 °C; 8 h and 200 rpm. As shown in Figure 4, the conversion propionate/chloramphenicol = 5:1; T =4.0 50 g◦ C; and 200loading rpm. Asover shown the conversion increased with LipBA loading up to L−18. hEnzyme 4.0 in g Figure L−1 not4,only led to no − 1 − 1 increased with Lip loading up to 4.0 g L . Enzyme loading over 4.0 g L not only to no BA improvement in the conversion rate, but also caused lower conversion and purity (Figure led 4). This improvement in the conversion rate, but also caused lower conversion and purity (Figure 4). This may may be due to the formation of enzyme aggregates which led to a drop in the exposed surface area beofdue to the formation of enzyme aggregates which led to a drop in the exposed surface area the catalyst to the reactants and accordingly a decreased mass transfer. Enzyme particles presentof the to the and accordingly a decreased masswith transfer. Enzyme which particles present on oncatalyst the inner sidereactants of aggregates were not available to react the substrate reduced the −1 the inner side of aggregates were not available to react with the substrate which reduced the overall overall interfacial area [20,28]. These data suggested that 4.0 g L of enzyme loading was optimal for interfacial area [20,28]. These data suggested that 4.0 g L−1 of enzyme loading was optimal for the the conversion and purity. conversion and purity.

Figure4. 4. Enzyme Enzyme loading loading effect effect on on the the conversion conversion of of chloramphenicol. chloramphenicol. Reaction Figure Reactionconditions: conditions: chloramphenicol 0.25 M in 1,4-dioxane; vinyl propionate/chloramphenicol = 5:1; T = 50 °C; chloramphenicol 0.25 M in 1,4-dioxane; vinyl propionate/chloramphenicol = 5:1; T = 508 ◦hC;and 8h 200200 rpm. and rpm.

2.7. Influence of the Water Content Water content is essential in any organic biosynthesis for enzyme flexibility and for optimal catalytic activity [29]. The water content effect on LipBA initial activity was examined using water Molecules 2017, 22, 1523 8 of 11 concentrations ranging from 0% to 4.0% (w/w) in 1,4-dioxane. As shown in Figure 5, the less water in the medium, the more the transesterification reaction was favored. The synthesis activity of chloramphenicol with LipBA was significantly reduced at water contents up to 2.0% (w/w), 2.7. Influence of thepropionate Water Content which was consistent with some previous studies indicating that the synthetic activity of most lipases Water content is essential in any organic biosynthesis for enzyme flexibility and for optimal was optimal at relatively low water content in organic systems (typically below 1% (w/w)) [22]. In this catalytic activity [29]. The water content effect on LipBA initial activity was examined using water reaction, the reduction of synthesis activity would be due to a larger amount of water in the organic concentrations ranging from 0% to 4.0% (w/w) in 1,4-dioxane. As shown in Figure 5, the less water medium. Water would reduce the solubility and the diffusibility of the substrates and the ester in the medium, the more the transesterification reaction was favored. The synthesis activity of product. Therefore, the conversion would be decreased with the increasing water content [15,17,30]. chloramphenicol propionate with LipBA wasexperiment significantlyresults, reduced water contents up to 2.0% (w/w), Finally, according to the single factor theatoptimum reaction condition was which was consistent with some previous studies that the synthetic activity of most lipases as follows: acyl donor: vinyl propionate; organicindicating solvent: 1,4-dioxane; reaction temperature: 50 °C; was optimal at 8relatively lowloading: water content in; substrate organic systems (typicallychloramphenicol below 1% (w/w))0.25 [22]. this reaction time: h; enzyme 4.0 g L−1 concentration: M,Invinyl reaction, the reduction of synthesis activity would be due to a larger amount of water in the organic propionate/chloramphenicol = 5:1. Under this condition, chloramphenicol was almost fully converted medium. Water would reduce thewith solubility and theof diffusibility the substrates to chloramphenicol propionate a conversion 98% and aofpurity over 99%.and the ester product. Therefore, the conversion would be decreased with the increasing water content [15,17,30].

Figure 5. Water content effect on the conversion of chloramphenicol. Reaction conditions: enzyme Figure 5. Water content effect on the conversion of chloramphenicol. Reaction conditions: enzyme loading loading−1 4.0 g L−1 ; chloramphenicol 0.25 M in 1,4-dioxane; vinyl propionate/chloramphenicol = 5:1; 4.0 g L ; chloramphenicol 0.25 M in 1,4-dioxane; vinyl propionate/chloramphenicol = 5:1; T = 50 °C; 8 T = 50 ◦ C; 8 h and 200 rpm. h and 200 rpm.

Finally, according to the single factor experiment results, the optimum reaction condition was 3. Materials and Methods as follows: acyl donor: vinyl propionate; organic solvent: 1,4-dioxane; reaction temperature: 50 ◦ C; reaction time: 8 h; enzyme loading: 4.0 g L−1 ; substrate concentration: chloramphenicol 0.25 M, vinyl 3.1. Materials propionate/chloramphenicol = 5:1. Under this condition, chloramphenicol was almost fully converted The lipase LipBA was obtained from B. amyloliquefaciens Nsic8 as previously reported in our research to chloramphenicol propionate with a conversion of−198% and a purity over 99%. team, and the specific activity was 1750 ± 153 U mg [15]. One unit (U) of lipase activity was defined asMaterials the amount ofMethods enzyme required to produce 1 μmol of product per min under the defined assay 3. and conditions [31]. Chloramphenicol and various vinyl esters were purchased from Sigma-Aldrich 3.1. (St. Materials Louis, MO, USA). 1,4-Dioxane, ethyl acetate and other chemicals were analytical reagent grade and were used without further purification. The lipase Lip was obtained from B. amyloliquefaciens Nsic8 as previously reported in our BA

research team, and the specific activity was 1750 ± 153 U mg−1 [15]. One unit (U) of lipase activity was defined as the amount of enzyme required to produce 1 µmol of product per min under the defined assay conditions [31]. Chloramphenicol and various vinyl esters were purchased from Sigma-Aldrich (St. Louis, MO, USA). 1,4-Dioxane, ethyl acetate and other chemicals were analytical reagent grade and were used without further purification.

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3.2. Enzymatic Synthesis of Cinnamyl Acetate and Single Factor Experiment The whole-cell catalyst containing lipase LipBA were centrifuged, washed once with 100 mM Tris–HCl (pH 8.0), and then lyophilized by vacuum freezing. The transesterification reaction was carried out in a 50 mL Eppendorf tube and 1,4-dioxane was used as solvent. To determine the optimum reaction parameters in the chloramphenicol ester synthesis reaction, single factor experiments were carried out in this study. Firstly, different acyl donors (vinyl acetate, vinyl propionate, vinyl butyrate, vinyl decanoate, vinyl laurate and vinyl palmitate) were used. Secondly, the effect of different organic solvents (toluene, dichloromethane, tertrahydrofuran, 1,4-dioxane, acetonitrile, acetone and ethanol) with varying ET (30) values, were studied. Thirdly, the effect of reaction temperature was studied at 20, 30, 40, 50 and 60 ◦ C. The reactor was shaken at 200 rpm and immersed in a thermostatic water bath to keep the system within ±2 ◦ C of the desired temperature. Fourthly, the effect of reaction time (4, 8, 12 and 16 h) was detected. Fifthly, the enzyme loading was studied in the range from 0.5 to 8.0 g L−1 . The effect of water concentration (ranging from 0% to 4.0% (w/w)) was analysis. All experiments were performed under a nitrogen atmosphere. Aliquots were regularly analyzed by HPLC and the reaction was stopped after complete consumption of the starting material. Finally the enzyme was filtered off. The solvent was evaporated under reduced pressure to get the total products. A parallel reaction under the same conditions without the addition of the enzyme was used as a control. All experiments were performed three times. 3.3. Analysis Method The progress of reactions was by TLC using a ZF-1 UV analyzer to visualize the spots (254 nm) [19]. The prepared samples were spotted onto the silica gel TLC plate, and then the plate was placed in a chamber containing a solvent system of n-hexane and ethyl acetate (7:3). After the plate was dried, the products bands were dyed by solid iodine (Figure 2B). The conversion was calculated by HPLC (Waters 2690 Separations Module; Waters 2487 UV/Vis Dual Absorbance Detector; Waters, Milford, MA, USA; readings were made at 280 nm, Figure 2C) using a silica gel column (Waters Symmetry TM, C8, 3.9 × 150 mm) eluted with methanol/water (70:30), flow rate, 1 mL/min [17]. The crude reaction product was purified by silica gel chromatography and characterized by NMR analysis. The position of acylation in the enzyme-prepared chloramphenicol esters was established by 1 H-NMR (400 MHz, (CD3 )2 CO) and 13 C-NMR (101 MHz, (CD3 )2 CO) on a RESONANCE ECZ 400S spectrometer (JEOL Resonance Inc., Tokyo, Japan). Deuterated acetone was used as solvent; trimethylsilane was used as an internal reference. 4. Conclusions In this study, the effects of various parameters on the lipase (LipBA ) catalyzed chloramphenicol transesterification with different acyl donor in organic solvents were studied. Among different acyl donors, vinyl propionate had the highest conversion efficiency in 1,4-dioxane as solvent. Overall a conversion of 98% and a purity of 99% were achieved in 8 h using 4.0 g L−1 of catalyst at 50 ◦ C. The optimum mole ratio of vinyl propionate to chloramphenicol was 5:1. In the present work, we have shown that lipase LipBA could easily be used as an efficient whole-cell biocatalyst for the synthesis of short-chain chloramphenicol esters. In organic media, the LipBA showed high conversion and regio-selectivity. Furthermore, the biocatalyst displayed high versatility toward short-chain precursors typical of chloramphenicol esters. These results demonstrate that lipase LipBA with a high catalytic activity and regio- and stereoselectivity, holds great promise for the green synthesis of chemicals with complex structures in organic media. Acknowledgments: This research was financially supported by the National Natural Science Foundation of China (No. C31570795), the Shanghai International Science and Technology Cooperation Project (No. 14520720500), the Minhang District Leading Talent Project (No. 201541), and the Shanghai Talent Development Project (No. 201531).

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Author Contributions: Fengying Dong and Wei Wei designed the research and wrote the paper; Fengying Dong, Lingmeng Li and Lin Lin performed the experiments; Dannong He and Jingwen Chen contributed reagents and analysis tools; Dongzhi Wei supervised research. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds chloramphenicol propionate and chloramphenicol were available from the authors. © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).