Doi:10.1016/j.jbiotec.2009.02.003

Journal of Biotechnology 140 (2009) 250–253 Thermomyces lanuginosus lipase-catalyzed regioselective acylation ofnucleosides: Enzyme substrate recognition a Laboratory of Applied Biocatalysis, South China University of Technology, Guangzhou 510640, Chinab State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Substrate recognition of Thermomyces lanuginosus lipase in the acylation of nucleosides was revealed through rational substrate engineering for the first time. T. lanuginosus lipase displayed higher catalytic activities and excellent 5 -regioselectivities (94–>99%) in the acylation of ribonucleosides 1f–1j as com-
pared to those in the acylation of 2 -deoxynucleosides 1a–1e. The higher reaction rates and excellent
5 -regioselectivities might derive from a favorable hydrogen bonding between the 2 -hydroxyl group of
1f–1j and phenolic hydroxyl group of Tyr21 present in the hydrophilic region of the lipase.
Crown Copyright 2009 Published by Elsevier B.V. All rights reserved.
NucleosideSubstrate recognitionSubstrate engineeringEnzymatic acylationRegioselectivity 1. Introduction
reactivity in the nucleosides. Enzymatic regioselective acylationof the nucleosides has received increasing attentions in synthetic Natural nucleosides serve as the building blocks for the bio- chemistry, due to its simplicity, exquisite selectivity, high efficiency logical synthesis of DNA or RNA in the cells. Their analogs such as 1-␤-d-arabinofuranosylcytosine (and halo- Thermomyces lanuginosus lipase (TLL) is a glycosylated hydrolase display antitumor or antiviral bioactivities. How- with a molecular weight of 30 kDa and an optimum pH of 11–12 ever, most of nucleoside drugs exhibited low oral bioavailability in the clinical treatment, due to the low lipid solubility and poor have revealed that the active site of TLL comprises two subsites: (a) a hydrophobic region, into which the acyl moiety binds; (b) a Additionally, various side effects of these drugs were associated hydrophilic region, into which the alcohol moiety fits with its clinical application (Many efforts have Although enzymatic regioselective acylation of nucleo- been made to overcome these limitations by chemists. Chemical modification of sugar moiety is one of the successful strategies are few reports regarding TLL-catalyzed acylation of nucleosides in the literatures (Previously, enzymatic regios- has been demonstrated that the ester derivatives displayed higher elective approaches for the acylation of nucleoside analogs such chemotherapeutic efficacy than the parent drugs ( are two classical examples which act as the antiviral alter- natives to ganciclovir and acyclovir, respectively. Nevertheless, it is the present work, we continued to focus our interest on the sub- difficult to selectively acylate the desired hydroxyl group of nucle- strate recognition of TLL in the acylation of nucleosides by means osides through traditional organic synthesis methods owing to the presence of two or three hydroxyl groups with similar chemical The solvent is one of the key factors on the reaction in nonaque- ous biocatalysis. The catalytic activity and selectivity of the enzymesuch as the enantioselectivity (and regioselectivity (as well as the thermodynamic Corresponding author. Fax: +86 411 8469 4447.
Corresponding author. Fax: +86 20 2223 6669.
E-mail addresses: (M.-H. Zong), (D. Ma).
could be manipulated by the reaction medium, which is 0168-1656/$ – see front matter. Crown Copyright 2009 Published by Elsevier B.V. All rights reserved.
doi: N. Li et al. / Journal of Biotechnology 140 (2009) 250–253 Fig. 1. TLL-catalyzed lauroylation of nucleosides.
known as ‘solvent engineering’. Because of the polar nature, nucle- the conformation of 5 -acylation transition state and increasing the osides are poorly soluble in hydrophobic organic solvents, a group activation energy of the enzymatic reaction.
of friendly media for the enzyme, while hydrophilic organic sol- Interestingly, TLL displayed higher catalytic activities and vents usually inactivate the enzyme. The optimal reaction medium excellent 5 -regioselectivities (94–>99%) in the acylation of was thus screened with TLL-mediated lauroylation of floxuridine 1b
ribonucleosides 1f–1j as compared to those in the acylation of 2 -
as a model reaction In a previous report, it was demon- deoxynucleosides 1a–1e (1–5 and 6–10). Moreover,
strated that TLL displayed no catalytic activity in the acylation of the effects of R1 group on the 5 -regioselectivity in the acylation of 5-fluorouridine 1g in highly polar solvents such as pyridine, DMF
ribonucleosides 1f–1j closely resembled those in the acylation of 2 -
and DMSO (Therefore, four less polar solvents deoxynucleosides 1a–1e. For example, the enzymatic lauroylation
were examined. As shown in catalytic activities were of uridine 1f afforded the highest 5 -regioselectivity (>99%) among
observed in the tested solvents as well as good substrate solubil- 1f–1j (corresponding to 2 -deoxyuridine 1a with R1 = H among
ity. Unfortunately, TLL exhibited unsatisfactory 5 -regioselectivities 1a–1e), while the lowest (94%) for the acylation of 5-iodouridine 1j
(51–71%) in the lauroylation of floxuridine. Among the four solvents, (corresponding to idoxuridine 1e with R1 = I). The unique difference
the best result was achieved in THF.
of the structure between the two groups of nucleosides 1a–1e and
Next, the substrate recognition of TLL was studied with the 1f–1j lies in the 2 -substituent. As shown in 2 -hydroxyl
lauroylation as a model reaction (As shown in group might be the origin of the higher reaction rates and excellent TLL displayed high catalytic activities in the acylation of 2 - 5 -regioselectivities in TLL-mediated lauroylation of ribonucleo- deoxynucleosides 1a–1e, and high substrate conversions (>99%)
sides 1f–1j. The X-ray crystallographic study has revealed that TLL
were achieved within the reaction time of 1.5–3.0 h (entries 1–5).
has a tyrosine residue Tyr21 in the hydrophilic region of the active Nevertheless, the 5 -regioselectivities of the enzymatic reactions site, into which the alcohol moiety binds, corresponding to Tyr28 were low to moderate (49–77%). In addition, the reaction rate and in Rhizomucor miehei lipase In addition, the the 5 -regioselectivity showed a clear dependence on the R1 group regions of the active sites and the lids are closely similar in the two of 2 -deoxynucleosides. Both the enzymatic reaction rate and the 5 - regioselectivity decreased with increasing bulk of R1 group molecular dynamic simulations of R. miehei lipase have revealed entries 1–5). For example, the highest selectivity was obtained for that the phenolic hydroxyl group of Tyr28 of the enzyme con- the enzymatic acylation of 2 -deoxyuridine 1a with R1 = H (77%,
tributes to the stabilization of the transition state entry 1), lower for the acylation of floxuridine 1b with R1 = F (71%,
It is well known that the hydroxyl group is a good hydrogen entry 2), even lower for the acylation of 1c with R1 = CH3 (50%, entry
bond acceptor or donor. As a result, it is easy to make a hydrogen 3) and 1d with R1 = Br (59%, entry 4), and the lowest for the acylation
bond interaction between the 2 -hydroxyl group of ribonucleo- of 1e with R1 = I (49%, entry 5). The reason might be that the increas-
sides 1f–1j and phenolic hydroxyl group of Tyr21 of TLL. The extra
ing size of R1 group results in unfavorable steric strain, destabilizing hydrogen bond might be responsible for the higher reaction rates Table 1
Effect of organic solvents on TLL-catalyzed lauroylation of floxuridine 1b
a The reaction was initiated by adding 60 U TLL (433 U/g) into anhydrous organic solvent (2 mL) containing 0.04 mmol floxuridine and 0.24 mmol vinyl laurate and then the mixture incubated at 40 ◦C, 250 rpm.
c Determined by HPLC analysis using SB-C18 column.
d Defined as the ratio of the concentration of the desired product to that of all the products, and determined by HPLC analysis using SB-C18 column.
N. Li et al. / Journal of Biotechnology 140 (2009) 250–253 2006A10602003), Science and Technology Project of Guangzhou Effect of substrate structure on TLL-catalyzed lauroylation of nucleosides Appendix A. Supplementary data
HPLC analysis conditions, retention time and characterization data and NMR spectra of the compounds are available as supple- mentary data. Supplementary data associated with this article can References
Arcos, J.A., Hill Jr., C.G., Otero, C., 2001. Kinetics of the lipase-catalyzed synthesis of glucose esters in acetone. Biotechnol. Bioeng. 73, 104–110.
Berezovskaya, Y.V., Chudinov, M.V., 2005. Ester derivatives of nucleoside inhibitors a The reaction was initiated by adding 60 U TLL (433 U/g) into anhydrous THF of reverse transcriptase. 1. Molecular transport systems for 3’-azido-3’- (2 mL) containing 0.04 mmol nucleoside and 0.24 mmol vinyl laurate and then the deoxythymidine and 2 ,3 -didehydro-3 -deoxythymidine. Russ. J. Bioorg. Chem.
mixture incubated at 40 ◦C, 250 rpm.
b Determined by HPLC analysis using SB-C18 column.
Brady, L., Brzozowski, A.M., Derewenda, Z.S., Dodson, E., Dodson, G., Tolley, S., Turken- c Defined as the ratio of the concentration of the desired product to that of all the burg, J.P., Christiansen, L., Huge-Jensen, B., Norskov, L., 1990. A serine protease products, and determined by HPLC analysis using SB-C18 column.
triad forms the catalytic centre of a triacylglycerol lipase. Nature 343, 767–770.
Chabner, B.A., Ryan, D.P., Paz-Area, L., Garcia-Carbonero, R., Calabresi, P., 2001. Anti- neoplastic agents. In: Hardman, G., Limbird, L.E. (Eds.), Goodman & Gilman’s The and excellent 5 -regioselectivities in the enzymatic acylation of Pharmacological Basis of Therapeutics. Mc-Graw Hill, New York, pp. 1389–1459.
ribonucleosides 1f–1j. Likewise, Gotor and co-workers have pro-
Crooks, R.J., 1995. Valaciclovir: a review of its potential in the management of genital herpes. Antiviral Chem. Chemother. 6, 39–44.
posed that the excellent regioselectivity of Candida antarctica lipase De Clercq, E., 2004. Antiviral drugs in current clinical use. J. Clin. Virol. 30, 115–133.
B toward 5 -hydroxyl group of thymidine comes from an extra De Clercq, E., Field, H.J., 2006. Antiviral prodrugs – the development of successful remote interaction between the base moiety of thymidine and the prodrug strategies for antiviral chemotherapy. Br. J. Pharmacol. 147, 1–11.
Derewenda, U., Swenson, L., Wei, Y., Green, R., Kobos, P.M., Joerger, R., Haas, M.J., Derewenda, Z.S., 1994. Conformational lability of lipases observed in the absence Kazlauskas and co-workers attributed the high enantioselectivity of an oil-water interface: crystallographic studies of enzymes from the fungi of Pseudomonas cepacia lipase toward 2-phenoxy-1-propanol to an Humicola lanuginosa and Rhizopus delemar. J. Lipid Res. 35, 524–534.
extra hydrogen bond between the phenoxy oxygen of the substrate Diaz-Rodriguez, A., Fernandez, S., Lavandera, I., Ferrero, M., Gotor, V., 2005. Novel and efficient regioselective enzymatic approach to 3 -, 5 - and 3 ,5 -di-O-crotonyl to the phenolic OH of Tyr29 of the enzyme ( 2 -deoxynucleoside derivatives. Tetrahedron Lett. 46, 5835–5838.
Ferrero, M., Gotor, V., 2000. Biocatalytic selective modifications of conventional A slightly lower, yet good 5 -regioselectivity (89%) was achieved nucleosides, carbocyclic nucleosides, and C-nucleosides. Chem. Rev. 100,4319–4347.
in the enzymatic acylation of 1-␤-d-arabinofuranosyluracil 1m
Green, E.A., Rosenstein, R.D., Shiono, R., Abraham, D.J., Trus, B.L., Marsh, R.E., 1975.
y 12) as compared to that in the acylation of uridine 1f.
The crystal structure of uridine. Acta Cryst. B 31, 102–107.
The X-ray crystallographic studies have indicated that the confor- Grem, J.L., 2000. 5-Fluorouracil: forty-plus and still ticking. A review of its preclinical and clinical development. Invest. New Drugs 18, 299–313.
mation of 2 -hydroxyl group of 1m differs from that of 1f (
Gunji, H., Kharbanda, S., Kufe, D., 1991. Induction of internucleosomal DNA frag- mentation in human myeloid leukemia cells by 1-␤-d-arabinofuranosylcytosine.
group of 1m and phenolic hydroxyl group of Tyr21 of the enzyme
Heidelberger, C., Ansfield, F.J., 1963. Experimental and clinical use of fluorinated might be beyond the range required for a hydrogen bond interac- pyrimidines in cancer chemotherapy. Cancer Res. 23, 1226–1243.
tion. As a result, a weaker close interaction rather than hydrogen Landowski, C.P., Song, X.Q., Lorenzi, P.L., Hilfinger, J.M., Amidon, G.L., 2005. Floxuri- bond interaction occurred, thus leading to a lower regioselectivity.
dine amino acid ester prodrugs: enhancing Caco-2 permeability and resistanceto glycosidic bond metabolism. Pharm. Res. 22, 1510–1518.
As can be seen in entry 11, in spite of the absence of an Lavandera, I., Fernandez, S., Magdalena, J., Ferrero, M., Kazlauskas, R.J., Gotor, V., extra hydrogen bond between F atom and phenolic hydroxyl group 2005. An inverse substrate orientation for the regioselective acylation of 3’,5’- of Tyr21 of the enzyme, TLL still showed a good 5 -regioselectivity diaminonucleosides catalyzed by Candida antarctica lipase B? ChemBioChem 6, (92%) in the acylation of 2 -fluoro-2 -deoxyuridine 1k. The unex-
Lawson, D.M., Brzozowski, A.M., Rety, S., Verma, C., Dodson, G.G., 1994. Probing the pected fact could be attributed to the inductive effect of 2 -F nature of substrate binding in Humicola lanuginosa lipase through X-ray crystal- atom. The F atom with strong electronegativity would withdraw lography and intuitive modelling. Protein Eng. 7, 543–550.
the electrons of the adjacent 3 -hydroxyl group, which decreases Li, X.F., Lou, W.Y., Smith, T.J., Zong, M.H., Wu, H., Wang, J.F., 2006a. Efficient regiose- lective acylation of 1-␤-d-arabinofuranosylcytosine catalyzed by lipase in ionic remarkably the nucleophilicity of 3 -hydroxyl group ( liquid containing systems. Green Chem. 8, 538–544.
In contrast, the reactivity of 5 -hydroxyl group was Li, X.F., Zong, M.H., Wu, H., Lou, W.Y., 2006b. Markedly improving Novozym 435- affected marginally due to its position far away from the F atom.
mediated regioselective acylation of 1-␤-d-arabinofuranosylcytosine by usingco-solvent mixtures as the reaction media. J. Biotechnol. 124, 552–560.
In summary, the catalytic activities and regioselectivities of TLL Li, X.F., Zong, M.H., Yang, R.D., 2006c. Novozym 435-catalyzed regioselective acyla- showed a clear dependence on the substrate structure, especially tion of 1-␤-d-arabinofuranosylcytosine in a co-solvent mixture of pyridine and R1, R2 and R3 group in the acylation of nucleosides. A deep insight isopropyl ether. J. Mol. Catal. B: Enzym. 38, 48–53.
Li, N., Zong, M.H., Liu, X.M., Ma, D., 2007. Regioselective synthesis of 3’-O-caproyl- was casted into the interactions between the enzyme and the sub- floxuridine catalyzed by Pseudomonas cepacia lipase. J. Mol. Catal. B: Enzym. 47, strates (nucleosides) in the catalytic pathway. The findings would provide a guide to control and maximize the regioselectivity of Li, N., Ma, D., Zong, M.H., 2008a. Enhancing the activity and regioselectivity of lipases for 3’-benzoylation of floxuridine and its analogs by using ionic liquid-containing the synthetically useful enzyme via the chemical modification or systems. J. Biotechnol. 133, 103–109.
Li, N., Zong, M.H., Ma, D., 2008b. Regioselective acylation of nucleosides catalyzed by Candida antarctica lipase B: enzyme substrate recognition. Eur. J. Org. Chem., Acknowledgements
Li, N., Zong, M.H., Ma, D., 2009. Regioselective acylation of nucleosides and their analogs catalyzed by Pseudomonas cepacia lipase: enzyme substrate recognition.
We wish to thank Ms. Xiu-Mei Liu for the help on NMR. This research was financially supported by the National Natural Sci- Liu, B.K., Wu, Q., Xu, J.M., Lin, X.F., 2007. N-Methylimidazole significantly improves lipase-catalysed acylation of ribavirin. Chem. Commun., 295–297.
ence Foundation of China (Grant Nos. 20676043 and 20603036), Neves Petersen, M.T., Fojan, P., Petersen, S.B., 2001. How do lipases and esterases Science and Technology Project of Guangdong Province (Grant No.
work: the electrostatic contribution. J. Biotechnol. 85, 115–147.
N. Li et al. / Journal of Biotechnology 140 (2009) 250–253 Norin, M., Haeffner, F., Achour, A., Norin, T., Hult, K., 1994. Computer modeling of Tawaki, S., Klibanov, A.M., 1992. Inversion of enzyme enantioselectivity mediated by substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and the solvent. J. Am. Chem. Soc. 114, 1882–1884.
Candida rugosa. Protein Sci. 3, 1493–1503.
Tollin, P., Wilson, H.R., Young, D.W., 1973. The crystal and molecular structure of Otero, C., Arcos, J.A., Berrendero, M.A., Torres, C., 2001. Emulsifiers from solid and uracil-␤-d-arabinofuranoside. Acta Cryst. B 29, 1641–1647.
liquid polyols: different strategies for obtaining optimum conversions and selec- Tuomi, W.V., Kazlauskas, R.J., 1999. Molecular basis for enantioselectivity of lipase tivities. J. Mol. Catal. B: Enzym. 11, 883–892.
from Pseudomonas cepacia toward primary alcohols. Modeling, kinetics, and Prusoff, W.H., 1959. Synthesis and biological activities of iododeoxyuridine, an ana- chemical modification of Tyr29 to increase or decrease enantioselectivity. J. Org.
log of thymidine. Biochim. Biophys. Acta 32, 295–296.
Rubio, E., Fernandez-Mayorales, A., Klibanov, A.M., 1991. Effect of the solvent on Wang, H., Zong, M.H., Wu, H., Lou, W.Y., 2007. Novel and highly regioselective route enzyme regioselectivity. J. Am. Chem. Soc. 113, 695–696.
for synthesis of 5-fluorouridine lipophilic ester derivatives by lipozyme TL IM. J.
Smith, M.B., March, J., 2007. Localized chemical bonding. In: March’s Advanced Organic Chemistry: Reactions, Mechanisms and Structure. John Wiley & Sons,Inc., Hoboken, New Jersey, pp. 16–22.

Source: http://fruit.dicp.ac.cn/publication/papers/lining20092.pdf