Journal of Biotechnology 140 (2009) 250–253
lipase-catalyzed regioselective acylation ofnucleosides: Enzyme substrate recognition
a Laboratory of Applied Biocatalysis, South China University of Technology, Guangzhou 510640, China
b 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
pared to those in the acylation of 2 -deoxynucleosides 1a
. The higher reaction rates and excellent
5 -regioselectivities might derive from a favorable hydrogen bonding between the 2 -hydroxyl group of
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
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-
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.
(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.
N. Li et al. / Journal of Biotechnology 140 (2009) 250–253
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
as compared to those in the acylation of 2 -
as a model reaction In a previous report, it was demon-
(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
in highly polar solvents such as pyridine, DMF
closely resembled those in the acylation of 2 -
and DMSO (Therefore, four less polar solvents
. 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-
(corresponding to 2 -deoxyuridine 1a
with R1 = H among
ity. Unfortunately, TLL exhibited unsatisfactory 5 -regioselectivities
), 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
Next, the substrate recognition of TLL was studied with the
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-
and high substrate conversions (>99%)
. 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-
with R1 = I (49%, entry 5). The reason might be that the increas-
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
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
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Coordination or Opposition in the Human SoulBetween body/flesh [soma/sarx], mind [nous, etc.], soul/life [psyche], spirit/Ghost [pneuma]Acts 2:26 Therefore did my heart rejoice, and my tongue was Mt 6:25 Therefore I say unto you, Take no thought for your life, glad; moreover also my flesh shall rest in hope:what ye shall eat, or what ye shall drink; nor yet for your body, what ye shall put on