Lipase-Catalyzed One-Step and Regioselective Synthesis of

Article pubs.acs.org/OPRD

Lipase-Catalyzed One-Step and Regioselective Synthesis of Clindamycin Palmitate Zhixian Li,† Yifei Zhang,† Mengmeng Lin,† Pingkai Ouyang,†,‡ Jun Ge,*,† and Zheng Liu*,† †

Department of Chemical Engineering, Tsinghua University, Beijing, Haidian District 100084, P.R. China College of Life Science and Pharmaceutical Engineering, Nanjing University of Technology, Hongqiao Bridge, Gulou, Nanjing 21009, Jiangsu, P.R. China

‡

S Supporting Information *

ABSTRACT: Chemical synthesis of clindamycin palmitate, a prodrug with taste greatly improved more than that of clindamycin, involves laborious steps of protection and deprotection to achieve the monoacylation only at 2-hydroxyl group of clindamycin and gives an overall yield below 50%. Here we report the first example of one-step synthesis of clindamycin palmitate with high regioselectivity using immobilized Candida antarctica lipase B (Novozym 435) as the catalyst. The lipasecatalyzed synthesis reached a conversion above 90% in 12 h using toluene as solvent and, moreover, a highly regioselective acylation at the 2-hydroxyl of clindamycin. The significantly improved conversion achieved at an excellent regioselectivity makes this enzymatic process attractive for the synthesis of clindamycin ester derivatives.

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INTRODUCTION Enzymatic catalysis, with the advantages of high chemo-, regio-, and enantioselectivity, is one of the greenest developing technologies to improve a wide range of production processes in the chemical and pharmaceutical industries.1 The use of enzymatic processes reduces energy consumption, avoids laborious chemical synthetic steps, and generates less waste and toxic side products. The appreciation of the advantages of enzymatic catalysis encouraged us to explore the possibility of one-step enzymatic synthesis of clindamycin palmitate, an established antibiotic prodrug of clindamycin with proved effectiveness against Gram-positive organisms and Gramnegative anaerobes.2 It is widely applied to treat diseases including infections of the respiratory tract,3 skin,4 and softtissue5 and could be used as a first-line treatment option for the methicillin-resistant Staphylococcus aureus (MRSA).6 All these attract growing interest in the clinical practice of this antibiotic. However, the currently employed chemical synthesis of clindamycin palmitate has a selectivity limitation in which protection and deprotection procedures have to be involved to achieve the monoacylation of clindamycin at the 2-hydroxyl position (Scheme 1). This results in a yield being lower than 50% and produces various wastes and byproducts which impose a heavy burden onto the downstream processing.7 Lipase has been reported to catalyze esterification reactions with high regioselectivity,8 and thus holds promise for the synthesis of clindamycin palmitate. Here, we describe the first effort towards one-step synthesis of clindamycin palmitate by an enzymatic regioselective acylation of clindamycin with vinyl palmitate (Scheme 1).

was chosen as the substrate to avoid the effect of chemical equilibrium on the final conversion. And excess vinyl palmitate was added in order to accelerate the reaction rate and increase the conversion of clindamycin. The results are listed in Table 1, from which it is suggested that immobilized Candida antarctica lipase (Novozym 435) gives the highest yield (∼88%) after a 9h reaction. The lipases from Thermomyces lanuginosus (Lipozyme TL IM), Rhizomucor miehei (Lipozyme RM IM) and Candida rugosa gave moderate yields. Lipozym TL 100L and porcine pancreas lipase did not show desired activity. Thus Novozym 435 was chosen for the synthesis of clindamycin palmitate. The influence of solvents with a wide range of log P (from −1.0 to 3.9) on the reaction catalyzed by Novozym 435 was investigated. Table 2 indicates that high conversions (88% and 90%) were achieved in more hydrophobic solvents (toluene and hexane), while poor conversions (0% and 5%) were achieved in very hydrophilic solvents (DMF and dioxane); this might be attributed to the stripping of essential water from the enzyme molecules by these hydrophilic solvents, which led to enzyme denaturation. The conversions in THF and dichloromethane were lower than that in acetonitrile and acetone. This is probably due the high polarity of THF and dichloromethane which leads to the distortion of protein configuration and the deactivation of the enzyme. Taking into account both conversion and the solubility of the substrate, toluene was selected as the solvent for the following study. The effects of temperature and reaction time on the synthesis of clindamycin palmitate were also examined. As shown in Figure 1, the conversion was greatly enhanced (above 80%) when the temperature was above 40 °C. The thermal stability of Novozym 435 has been investigated previously, showing the activity and conversion can be largely retained even in a high

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RESULTS AND DISCUSSION The present study started by screening the suitable lipase catalyst from commercially available lipase catalysts from different resources, in terms of their activities for the acylation of clindamycin with vinyl palmitate in toluene. Vinyl palmitate © 2013 American Chemical Society

Received: May 22, 2013 Published: August 12, 2013 1179

dx.doi.org/10.1021/op400135y | Org. Process Res. Dev. 2013, 17, 1179−1182

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Scheme 1. (A) Enzyme-Catalyzed Acylation of Clindamycin Palmitate in Organic Solvent and (B) Current Chemical Synthesis Process of Clindamycin Palmitate

Table 1. Synthesis of clindamycin palmitate with different types of lipase enzyme catalysta

origin of lipase

conversion (%)b,c

Novozym 435 Lipozyme TL IM Lipozyme TL 100L Lipozyme RM IM Candida rugosa lipase porcine pancreas lipase without lipase

Candida antarctica Thermomyces lanuginosus Thermomyces lanuginosus Rhizomucor miehei Candida rugosa porcine pancreas −

88 57 0 17 14 0 0

Figure 1. (A) Effect of reaction temperature on the conversion. Experimental conditions: 0.1 mmol clindamycin free base, 0.3 mmol vinyl palmitate, 30 mg/mL Novozym 435, 2 mL of toluene, 9 h. (B) Effect of reaction time on the conversion. Experimental conditions: 0.1 mmol clindamycin free base, 0.3 mmol vinyl palmitate, 30 mg/mL Novozym 435, 2 mL of toluene, 40 °C.

a

The full experimental details and the detailed information about enzymes are available in the SI. bConversion was determined by HPLC. cExperimental conditions: 0.1 mmol clindamycin free base, 0.3 mmol vinyl palmitate, 30 mg/mL enzyme catalyst, 2 mL toluene, 200 rpm, 40 °C, 9 h.

purified (see Experimental Section) with a yield of ∼90% (1.57g) and a purity of 97% (calculated from the HPLC data, see the Supporting Information [SI], Figure S3; the 3% of impurity was the unreacted clindamycin and palmitate). Compared with clindamycin palmitate hydrochloride (USP reference standard, Rockville, MD, U.S.A.), the HPLC, NMR, and FT-IR analyses suggest no new impurity was introduced in the new enzymatic process (see SI, Figures S14−S16). The FT-IR spectrum of the product revealed characteristic absorption bands at 3450 (OH stretch), 2850−2920 (aliphatic CH stretch), 1683 (amide CO stretching) cm−1, discernible in that of clindamycin (Table 3). An extra IR signal of the product at 1735 cm−1, ascribable to the palmitoyl carbonyl group, indicated that clindamycin palmitate had been generated. Moreover, the result of ESI-MS [m/z 663.9 (M+)] demonstrated the monoacylation of clindamycin (see details in SI). To examine the regioselectivity of the Novozym 435catalyzed acylation towards three hydroxyl groups in the clindamycin structure, the initial reactant clindamycin and product clindamycin palmitate were characterized by 13C, 1H NMR, and DEPT, respectively (see details in the SI). In addition, the structure of product was further confirmed by the

Table 2. Novozym 435-catalyzed synthesis of clindamycin palmitate in various solvents entry

solvent

log Pa

conversion (%)b

1 2 3 4 5 6 7 8

DMF dioxane acetonitrile acetone THF dichloromethane toluene hexane

−1.0 −0.5 −0.39 −0.23 0.46 0.6 2.6 3.9

0 5 80 56 17 24 88 90

a

Laane et al.9 bExperimental conditions: 0.1 mmol clindamycin free base, 0.3 mmol vinyl palmitate, 30 mg/mL Novozym 435, 2 mL solvent, 200 rpm, 40 °C, 9 h.

temperature (about 70 °C).10 We selected 40 °C as the optimal temperature on the basis of both conversion and energy saving. In a larger-scale experiment, 1.06 g (2.5 mmol) of clindamycin free base, 2.11g (7.5 mmol) of vinyl palmitate, and 30 mg/mL of Novozym 435 were added in 50 mL of toluene under the conditions of 200 rpm, 40 °C, and 12 h; the product was 1180

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Table 3. FT-IR spectrum of clindamycin palmitatea

a

cmpd

wave number (cm−1)

clindamycin product

3450 (OH stretch), ∼2850, ∼2920 (aliphatic CH stretch), 1683 (amide CO stretching) 3450 (OH stretch), ∼2850, ∼2920 (aliphatic CH stretch), 1683 (amide CO stretching), 1735 (ester, CO stretch)

FT-IR spectrum is available in SI.

indicated the occurrence of the expected acylation. The C-2 (δ 69.5) of clindamycin shifted downfield by 1.5 ppm, and the resonances corresponding to its neighboring carbon atoms C-1 (δ 88.4) and C-3 (δ 71.0) showed an upfield shift of 4.0 and 3.1 ppm, respectively. The other carbons of the transformed product gave signals at positions almost identical with those of the starting substrate, suggesting the acylation occurred only at the 2-OH position. In the 1H NMR spectrum of the acylated product, the H-2 signal at δ 5.01 was shifted downfield by 1.09 ppm from that of clindamycin (Table 5). Furthermore, the resonances arising

HMQC NMR spectra in which the signals for key carbon and hydrogen atoms were identified (Figure 2).

Table 5. Characteristic 1H NMR spectral data of clindamycin and clindamycin palmitate in DMSO-d6a hydrogen 1 2 3 4 5 acyl a1

It is established that the acylation of a hydroxyl group results in a downfield shift of the peak corresponding to the O-acylated carbon atom and an upfield shift of the peak corresponding to the neighbouring carbon atom.11 The 13C NMR spectrum of the product exhibited additional carbon signals at δ 173.2 (C O) and 21.0−35.0 (aliphatic CH chain) (Table 4), which

1 2 3 4 5

clindamycin C (δ, ppm)/DEPT 88.4, CH 69.5, CH 71.0, CH 68.1, CH 67.7, CH

clindamycin palmitate 5.42 (1H, d, J = 5.6 Hz) 5.01 (1H, dd, J = 5.6, 10.5 Hz) 3.61 (1H, dd, J = 2.8, 10.5 Hz) 4.14 (1H, d, J = 9.9 Hz) 3.86 (1H, s) 2.31−0.87 (15H)

H NMR spectra are available in SI.

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Table 4. Comparative 13C NMR and DEPT assignments for clindamycin and clindamycin palmitatea 13

(1H, d, J = 5.5 Hz) (1H, dd, J = 5.5, 10.0 Hz) (1H, dd, J = 3.0, 10.0 Hz) (1H, d, J = 9.9 Hz) (1H, s)

from H-1 and H-3 moved slightly downfield without any changes in the splitting pattern, whereas characteristic signals of H-4 and H-5 remained almost unchanged. The chemical shift deviation of neighbouring protons also provided evidence for the freshly introduced 2-O-acyl group. These results confirm that the Novozym 435-catalyzed acylation of clindamycin generated exclusively the 2-O-acyl derivative in a regioselective manner. In the three-step chemical manufacture for clindamycin palmitate, the yield of each step was 85%, 83%, and 70%, respectively, which results in a low overall yield.7 The spacetime yield for the chemical synthesis route was around 1.9 g/ (L·h) similar to the enzymatic route in this study which is around 2.1 g/(L·h). In addition, to test the stability of the lipase-catalyzed synthesis in a practical process, the catalyst Novozyme 435 was reused for seven cycles without loss of any efficiency, and a productivity of 10.1 g product per gram of Novozym 435 was accomplished after the repeated cycles of using the biocatalyst.

Figure 2. HMQC NMR spectra of clindamycin palmitate.

carbon

clindamycin 5.21 3.92 3.39 4.11 3.80 −

CONCLUSION In conclusion, a lipase (Novozyme 435)-catalyzed one-step and regioselective synthesis of clindamycin palmitate is proposed, which simplifies the synthesis by excluding the group protection/deprotection procedures. One-step enzymatic synthesis reached ∼100% selectivity at the 2-OH position. Also demonstrated is a higher yield (∼90% vs 50%) than the established three-step chemical procedure. These encouraging results indicate the potential of the lipase-catalyzed one-step synthesis for the facile and efficient production of clindamycin palmitate.

clindamycin palmitate 13 C (δ, ppm)/DEPT 84.4, CH 71.0, CH 67.9, CH 69.5, CH 68.2, CH

a13

C NMR spectra are available in SI. 1181

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(7) (a) Zhao, P.; Cheng, X.; Lan, W.; Tian, B. Appl. Chem. Ind. 2008, 37, 651. (b) Long, D.; Cheng, X.; Mo, Q.; Guo, Q.; Peng, L. China Patent, CN 1810821, 2006. (8) (a) Junior, I. I.; Flores, M. C.; Sutili, F. K.; Leite, S. G. F.; Miranda, L. S. D.; Leal, I. C. R.; de Souza, R. O. M. A. Org. Process Res. Dev. 2012, 16, 1098. (b) Korupp, C.; Weberskirch, R.; Muller, J. J.; Liese, A.; Hilterhaus, L. Org. Process Res. Dev. 2010, 14, 1118. (c) Chen, Z. G.; Tan, R. X.; Cao, L. Green Chem. 2009, 11, 1743. (d) Wang, R.; Zhang, Y.; Huang, J.; Lu, D.; Ge, J.; Liu, Z. Green Chem. 2013, 15, 1155. (e) Zhu, J.; Zhang, Y.; Lu, D.; Zare, R. N.; Ge, J.; Liu, Z. Chem. Commun. 2013, 49, 6090. (f) Ge, J.; Lu, D.; Wang, J.; Liu, Z. Biomacromolecules 2009, 10, 1612. (g) Ge, J.; Lu, D.; Yang, C.; Liu, Z. Macromol. Rapid Commun. 2011, 32, 546. (h) Barnwell, N.; Cheema, L.; Cherryman, J.; Dubiez, J.; Howells, G.; Wells, A. Org. Process Res. Dev. 2006, 10, 644. (9) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol. Bioeng. 1987, 30, 81. (10) (a) Cruz, J. C.; Pfromm, P. H.; Rezac, M. E. Process Biochem. 2009, 44, 62. (b) Yu, D. H.; Wang, Z.; Chen, P.; Jin, L.; Cheng, Y. M.; Zhou, J. G.; Cao, S. G. J. Mol. Catal. B: Enzym. 2007, 48, 51. (11) (a) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol. Bioeng. 1987, 30, 81. (b) Yoshimoto, K.; Itatani, Y.; Tsuda, Y. Chem. Pharm. Bull. 1980, 28, 2065.

EXPERIMENTAL SECTION All enzymatic processes were carried out in a temperaturecontrolled incubator shaken at 200 rpm. Clindamycin free base 1.06 g (2.5 mmol) and vinyl palmitate 2.11 g (7.5 mmol) were dissolved in 50 mL of toluene. After the mixture stirred for 30 min at room temperature, 1200 mg of Novozym 435 was added, and the mixture incubated at a certain temperature. After reaction, the mixture was filtered, followed by evaporation of the solvent. Then dichloromethane was added, followed by the addition of hydrochloric acid to adjust the pH to 1−2, and then the solvent was evaporated. The product was obtained after solubilizing in a small amount of ethanol and then precipitation in acetonitrile. The purified product was obtained by filtration (∼1.57 g, ∼90% yield) and subjected to NMR analysis which indicated no 3- and 4-palmitoyl regioisomers were formed. The purity of ∼97% was obtained by HPLC analysis (Figure S2 in SI; the impurities are mainly reactants including clindamycin and palmitate). The product was characterized by NMR analysis. 1H, 13C, DEPT, HMQC NMR spectra were recorded on a JEOL instrument at 600 MHz, using DMSO-d6 as the solvent and TMS as the internal reference.

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ASSOCIATED CONTENT

S Supporting Information *

Materials; purity assessment of clindamycin palmitate; analytical methods; 1H, 13C and HMQC; ESI-MS; FT-IR. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] *E-mail: [email protected] Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China under the Grant Numbers of 21036003 and 21206082, Tsinghua University Initiative Scientific Research Program, and Doctoral Fund of Ministry of Education of China under the Grant Numbers of 2012000 and 2120046.

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