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Highly efficient regioselective acylation of 6-azauridine catalyzed by Thermomyces lanuginosus lipase
S380
Abstracts / Journal of Biotechnology 136S (2008) S356–S401
V3-P-065
V3-P-066
Highly efficient regioselective acylation of 6-azauridine catalyzed by Thermomyces lanuginosus lipase
Enzymatic synthesis of acyclovir–galactose conjugate with liver targeting and anti-HBV activity
Min-Hua Zong ∗ , Zhao-Yu Wang
Bo Jiang, Jing Quan, Yan Zheng, Li-Min Zhu ∗
Laboratory of Applied Biocatalysis, South China University of Technology, Guangzhou 510640, China
College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China
E-mail address: [email protected] (M.-H. Zong).
E-mail address: [email protected] (L.-M. Zhu).
6-Azauridine is a kind of nucleoside drug extensively applied in the treatment of psoriasis, dengue and herpes viral infections (Zinni et al., 2007). However, it exhibits various side effects due to the deribosylation forming a less active and much more toxic compound, 6-azauricil. Fortunately, fatty ester derivatives of 6-azauridine can avoid the drawbacks and act as prodrugs with improved pharmacokinetic profile and therapeutic efficacy. Regioselective acylation of nucleosides possessing several hydroxyl groups of similar chemical reactivity, such as 6-azauridine, is a fundamental challenge to organic chemists (Li et al., 2008). It is usually extremely difficult to selectively acylate the desired hydroxyl of unprotected 6-azauridine via conventional chemical approaches. Enzymatic acylation of nucleosides in nonaqueous media, however, is a practicable option for this purpose owing to the high specificity of enzymes. In order to prepare the fatty ester derivatives of 6-azauridine efficiently, attempt was made to use Lipozyme TL IM, an inexpensive lipase from Thermomyces lanuginosa, as a catalyst for the regioselective acylation of 6-azauridine with a rang of vinyl fatty acid esters (from C2 to C18) in different organic solvents. Acetone, THF, 1,4-dioxane or tert-butanol were found to be the good solvents for the substrates while maintaining the activity of the enzyme. And the end products of the acylation were exclusively 5 -O-monoesters of 6-azauridine as confirmed by 1 H NMR and 13 C NMR analysis. For an in-depth understanding of the reaction, the effects of some crucial factors on enzymatic acylation of 6-azauridine with vinyl palmitate were further examined. As a result, the optimal reaction media, initial water activity, enzyme dosage, 6-azauridine concentration, molar ratio of 6-azauridine to vinyl palmitate and reaction temperature proved to be acetone, 0.07, 450 U/mL, 13 mM/L, 1:7 and 40 ◦ C, respectively, under which the substrate conversion and the regioselectivity were both as high as >99% after a reaction time of 6.5 h.
Galactose (Gal) can specifically bind to the asyaloglycoprotein receptor present on the hepatocyte membrane (Wadhwa and Rice, 1995; De la Fuente and Penades, 2006). Therefore galactose can be potentially used to target drugs to liver. Most of the galactose–drug conjugates are chemically synthesized by four steps at least (Takakura and Hashida, 1996; Roy and Baek, 2002). In this study acyclovir (ACV) was used as the parent drug and a two-step enzyme-catalyzed strategy has been used to synthesize Gal–ACV conjugate in organic solvents. Firstly, transesterification of ACV with divinyl adipate was performed under the catalysis of lipase acrylic resin from Candida antarctica (CAL-B) in anhydrous acetone at 50 ◦ C and stirred at 250 rpm. O-Acylacyclovir derivative was obtained successfully. And then, protease-catalyzed highly regioselective acylation of galactose with vinyl ester of acyclovir gave ACV–Gal derivative. The obtained compound was characterized with IR, NMR. The compound was then evaluated for the anti-HBV activity in the HepG2.2.15 cell line in vitro and liver targeting in mice in vivo. In an in vitro HBV inhibition assay, ACV–Gal conjugate effectively suppressed the secretion of the HBV antigens comparing with ACV. IC50 values of 0.007 mg/mL and 0.15 mg/mL were obtained for ACV–Gal conjugate and ACV, respectively, at day 7 for both HBsAg and HBeAg. Furthermore, using intravenous injection in mice, 75% of the dose was recovered in the liver. These results indicated that ACV–Gal conjugate with good anti-HBV activity and strong liver targeting property can be obtained by enzymatic synthesis. Moreover, enzymatic synthesis promoted an novel approach for development a series of acyclovir derivates.
References Li, N., Ma, D., Zong, M.H., 2008. Enhancing the activity and regioselectivity of lipases for 3 -benzoylation of floxuridine and its analogs by using ionic liquidcontaining systems. J. Biotechnol. 133, 103–109. Zinni, M.A., Iglesias, L.E., Iribarren, A.M., 2007. Preparation of potential 3deazauridine and 6-azauridine prodrugs through an enzymatic alcoholysis. J. Mol. Catal. B: Enzym. 47, 86–89.
doi:10.1016/j.jbiotec.2008.07.874
References De la Fuente, J.M., Penades, S., 2006. Glyconanoparticles: types, synthesis and applications in glycoscience, biomedicine and material science. Biochim. Biophys. Acta 1760, 636–651. Roy, R., Baek, M.G., 2002. Glycodendrimers: novel glycotope isosteres unmasking sugar coding. case study with T-antigen markers from breast cancer MUC1 glycoprotein. J. Biotechnol. 90, 291–309. Takakura, Y., Hashida, M., 1996. Macromolecular carrier systems for targeted drug delivery: pharmacokinetic considerations on biodistribution. Pharm. Res. 13, 820–831. Wadhwa, M.S., Rice, K.G., 1995. Receptor mediated glycotargeting. J. Drug Target. 11, 255–268.
doi:10.1016/j.jbiotec.2008.07.875 V3-P-067 Enzymatic synthesis of 6-chloropurine riboside by whole cell of Lactobacillus helveticus Yan Zheng, Bo Jiang, Jing Quan ∗ , Li-Min Zhu ∗ College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China E-mail address: [email protected] (L.-M. Zhu). In recent years, the synthesis of 6-chloropurine riboside has received much attention because of its potential function. For example, it can be used as a significant agent for antitumor and anti-virus. 6-Chloropurine riboside has been prepared by con-
Abstracts / Journal of Biotechnology 136S (2008) S356–S401
V3-P-065
V3-P-066
Highly efficient regioselective acylation of 6-azauridine catalyzed by Thermomyces lanuginosus lipase
Enzymatic synthesis of acyclovir–galactose conjugate with liver targeting and anti-HBV activity
Min-Hua Zong ∗ , Zhao-Yu Wang
Bo Jiang, Jing Quan, Yan Zheng, Li-Min Zhu ∗
Laboratory of Applied Biocatalysis, South China University of Technology, Guangzhou 510640, China
College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China
E-mail address: [email protected] (M.-H. Zong).
E-mail address: [email protected] (L.-M. Zhu).
6-Azauridine is a kind of nucleoside drug extensively applied in the treatment of psoriasis, dengue and herpes viral infections (Zinni et al., 2007). However, it exhibits various side effects due to the deribosylation forming a less active and much more toxic compound, 6-azauricil. Fortunately, fatty ester derivatives of 6-azauridine can avoid the drawbacks and act as prodrugs with improved pharmacokinetic profile and therapeutic efficacy. Regioselective acylation of nucleosides possessing several hydroxyl groups of similar chemical reactivity, such as 6-azauridine, is a fundamental challenge to organic chemists (Li et al., 2008). It is usually extremely difficult to selectively acylate the desired hydroxyl of unprotected 6-azauridine via conventional chemical approaches. Enzymatic acylation of nucleosides in nonaqueous media, however, is a practicable option for this purpose owing to the high specificity of enzymes. In order to prepare the fatty ester derivatives of 6-azauridine efficiently, attempt was made to use Lipozyme TL IM, an inexpensive lipase from Thermomyces lanuginosa, as a catalyst for the regioselective acylation of 6-azauridine with a rang of vinyl fatty acid esters (from C2 to C18) in different organic solvents. Acetone, THF, 1,4-dioxane or tert-butanol were found to be the good solvents for the substrates while maintaining the activity of the enzyme. And the end products of the acylation were exclusively 5 -O-monoesters of 6-azauridine as confirmed by 1 H NMR and 13 C NMR analysis. For an in-depth understanding of the reaction, the effects of some crucial factors on enzymatic acylation of 6-azauridine with vinyl palmitate were further examined. As a result, the optimal reaction media, initial water activity, enzyme dosage, 6-azauridine concentration, molar ratio of 6-azauridine to vinyl palmitate and reaction temperature proved to be acetone, 0.07, 450 U/mL, 13 mM/L, 1:7 and 40 ◦ C, respectively, under which the substrate conversion and the regioselectivity were both as high as >99% after a reaction time of 6.5 h.
Galactose (Gal) can specifically bind to the asyaloglycoprotein receptor present on the hepatocyte membrane (Wadhwa and Rice, 1995; De la Fuente and Penades, 2006). Therefore galactose can be potentially used to target drugs to liver. Most of the galactose–drug conjugates are chemically synthesized by four steps at least (Takakura and Hashida, 1996; Roy and Baek, 2002). In this study acyclovir (ACV) was used as the parent drug and a two-step enzyme-catalyzed strategy has been used to synthesize Gal–ACV conjugate in organic solvents. Firstly, transesterification of ACV with divinyl adipate was performed under the catalysis of lipase acrylic resin from Candida antarctica (CAL-B) in anhydrous acetone at 50 ◦ C and stirred at 250 rpm. O-Acylacyclovir derivative was obtained successfully. And then, protease-catalyzed highly regioselective acylation of galactose with vinyl ester of acyclovir gave ACV–Gal derivative. The obtained compound was characterized with IR, NMR. The compound was then evaluated for the anti-HBV activity in the HepG2.2.15 cell line in vitro and liver targeting in mice in vivo. In an in vitro HBV inhibition assay, ACV–Gal conjugate effectively suppressed the secretion of the HBV antigens comparing with ACV. IC50 values of 0.007 mg/mL and 0.15 mg/mL were obtained for ACV–Gal conjugate and ACV, respectively, at day 7 for both HBsAg and HBeAg. Furthermore, using intravenous injection in mice, 75% of the dose was recovered in the liver. These results indicated that ACV–Gal conjugate with good anti-HBV activity and strong liver targeting property can be obtained by enzymatic synthesis. Moreover, enzymatic synthesis promoted an novel approach for development a series of acyclovir derivates.
References Li, N., Ma, D., Zong, M.H., 2008. Enhancing the activity and regioselectivity of lipases for 3 -benzoylation of floxuridine and its analogs by using ionic liquidcontaining systems. J. Biotechnol. 133, 103–109. Zinni, M.A., Iglesias, L.E., Iribarren, A.M., 2007. Preparation of potential 3deazauridine and 6-azauridine prodrugs through an enzymatic alcoholysis. J. Mol. Catal. B: Enzym. 47, 86–89.
doi:10.1016/j.jbiotec.2008.07.874
References De la Fuente, J.M., Penades, S., 2006. Glyconanoparticles: types, synthesis and applications in glycoscience, biomedicine and material science. Biochim. Biophys. Acta 1760, 636–651. Roy, R., Baek, M.G., 2002. Glycodendrimers: novel glycotope isosteres unmasking sugar coding. case study with T-antigen markers from breast cancer MUC1 glycoprotein. J. Biotechnol. 90, 291–309. Takakura, Y., Hashida, M., 1996. Macromolecular carrier systems for targeted drug delivery: pharmacokinetic considerations on biodistribution. Pharm. Res. 13, 820–831. Wadhwa, M.S., Rice, K.G., 1995. Receptor mediated glycotargeting. J. Drug Target. 11, 255–268.
doi:10.1016/j.jbiotec.2008.07.875 V3-P-067 Enzymatic synthesis of 6-chloropurine riboside by whole cell of Lactobacillus helveticus Yan Zheng, Bo Jiang, Jing Quan ∗ , Li-Min Zhu ∗ College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai, 201620, China E-mail address: [email protected] (L.-M. Zhu). In recent years, the synthesis of 6-chloropurine riboside has received much attention because of its potential function. For example, it can be used as a significant agent for antitumor and anti-virus. 6-Chloropurine riboside has been prepared by con-