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 1f1j as com-
pared to those in the acylation of 2 -deoxynucleosides 1a1e. The higher reaction rates and excellent
5 -regioselectivities might derive from a favorable hydrogen bonding between the 2 -hydroxyl group of
1f1j 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 1f1j as compared to those in the acylation of 2 -
as a model reaction In a previous report, it was demon- deoxynucleosides 1a1e (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 1f1j closely resembled those in the acylation of 2 -
and DMSO (Therefore, four less polar solvents deoxynucleosides 1a1e. 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- 1f1j (corresponding to 2 -deoxyuridine 1a with R1 = H among
ity. Unfortunately, TLL exhibited unsatisfactory 5 -regioselectivities 1a1e), 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 1a1e and
Next, the substrate recognition of TLL was studied with the 1f1j 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 1a1e, and high substrate conversions (>99%)
sides 1f1j. 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 1f1j 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
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