Regioselective acylation of nucleosides and their analogs catalyzed by Pseudomonas cepacia lipase: enzyme substrate recognition

Article abstract of DOI:10.1016/j.tet.2008.11.045

The substrate recognition of Pseudomonas cepacia lipase in the acylation of nucleosides was investigated by means of rational substrate engineering for the first time. P. cepacia lipase displayed excellent 3′-regioselectivities (96 to >99%) in the lauroyl

Full text of DOI:10.1016/j.tet.2008.11.045

Tetrahedron 65 (2009) 1063–1068
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Regioselective acylation of nucleosides and their analogs catalyzed
by Pseudomonas cepacia lipase: enzyme substrate recognition
,
,
b,
*
Ning Li a b, Min-Hua Zong a , Ding Ma
*
a Lab of Applied Biocatalysis, College of Light Industry and Food Sciences, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2008
Received in revised form 12 November 2008
Accepted 14 November 2008
Available online 24 November 2008
The substrate recognition of Pseudomonas cepacia lipase in the acylation of nucleosides was investigated
by means of rational substrate engineering for the first time. P. cepacia lipase displayed excellent
30-regioselectivities (96 to >99%) in the lauroylation of 20-deoxynucleosides 1a–1e, while low to good
30-regioselectivities (59–89%) in the lauroylation of ribonucleosides 1f–1j. It might be due to the un-
favorable hydrogen bond interaction between 20-hydroxyl group of 1f–1j and phenolic hydroxyl group of
tyrosine residue present in the alternate hydrophobic pocket of the enzyme, which stabilizes the con-
formation of 50-acylation transition state and thus increases the amount of the minor regioisomer. In
addition, various ester derivatives of floxuridine were synthesized successfully by the lipase with high
conversions (99%) and good to excellent 30-regioselectivities under mild conditions. The recognition of
various acyl donors by the enzyme was examined. The enzymatic recognition of acyl groups was ra-
tionalized in terms of the structure of the active site of the lipase, especially the size, shape, and
physicochemical properties.
Keywords:
Floxuridine
Lipase
Nucleosides
Regioselective acylation
Substrate recognition
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
analogs usually exhibit various side effects and suffer from poor
bioavailability in the clinical treatment.7 Moreover, nucleoside
The regioselective acylation of nucleosides remains a tedious,
yet important, task in organic synthesis due to the presence of
multiple hydroxyl groups. In particular, it is difficult to selectively
acylate the 30-hydroxyl of nucleosides in the presence of less hin-
dered 50-hydroxyl, since the former is typically less reactive than
the latter. For example, acyl groups such as pivaloyl and aroyl fa-
vored the primary 50-hydroxyl of nucleosides.1 Hence, the con-
ventional acylation of the 30-hydroxyl involves protecting/
deprotecting steps, which is a time-consuming work-up.2 In the
past 20 years, enzymatic acylation of polyhydroxyl compounds has
attracted increasing synthetic attention, due to the exquisite se-
lectivity, mild reaction conditions, and being environmentally
friendly.3 Among the enzymes, P. cepacia lipase (PSL-C) has proven
to be highly selective toward 30-position in the acylation of nucle-
osides.4 Recently, Lavandera et al. revealed the molecular basis of
the unusual regioselectivity of PSL-C toward the secondary hy-
droxyl of 20-deoxynucleosides by molecular modeling.5
agents are less stable in vivo because of the cleavage of the glycosyl
bond by nucleoside phosphorylase, yielding the compounds of
lower biological activity.8 It has been demonstrated that ester de-
rivatives of parent drugs could effectively circumvent the draw-
backs mentioned above.2b,9 For example, 30-O-retinoyl-FUdR could
remarkably inhibit in vivo solid tumor growth as compared to the
parent drug.10 30-Acetate and amino acid ester derivatives of FUdR
are much more resistant to nucleoside phosphorylase than
FUdR.8,11
Previously, enzymatic regioselective approaches for the acylation
of nucleoside analogs, such as 1-b-D
-arabinofuranosylcytosine,12
5-fluorouridine,13 FUdR and its analogs,14 were developed by our
group. In the present work, we continued to extend our interest to
the enzymatic synthesis of various lipophilic 30-ester derivatives of
nucleosides and their analogs in high conversions and excellent
regioselectivities. Particularly, we focused on the substrate recog-
nition of PSL-C in the acylation of nucleosides by means of rational
substrate engineering (Scheme 1).
Nucleoside analogs, such as floxuridine (FUdR, 1b) and idoxur-
idine (1e) have been acting as effective antitumor or antiviral
agents for many years.6 However, these halogenated nucleoside
2. Results and discussion
Recently, we found that the regioselectivity and reaction rate of
the enzymatic benzoylation of FUdR 1b could be enhanced signif-
icantly by adding ionic liquid.14a Likewise, herein, the ionic
* Corresponding authors. Tel.: þ86 20 87111452; fax: þ86 20 2223 6669 (M.-H.Z.);
tel.: þ86 411 8437 9253; fax: þ86 411 8469 4447 (D.M.).
E-mail addresses: btmhzong@scut.edu.cn (M.-H. Zong), dma@dicp.ac.cn (D. Ma).
0040-4020/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2008.11.045

1064
N. Li et al. / Tetrahedron 65 (2009) 1063–1068
As can be seen in Table 1, 30-laurates of ribonucleosides 1f–1j
were synthesized with high conversions (>98%) and low to good
selectivities (59–89%) in 14–35 h. Interestingly, PSL-C exhibited
similar catalytic behavior in the acylation of the two series of
nucleosides (20-deoxynucleosides 1a–1e and ribonucleosides 1f–
1j). For instance, a poor 30-regioselectivity (59%) and the highest
catalytic activity were observed in the acylation of uridine 1f
(corresponding to 20-deoxyuridine 1a) among 1f–1j, while a good
30-regioselectivity (88%) and the lowest activity were found in the
acylation of 5-iodouridine 1j (corresponding to idoxuridine 1e). It
was suggested that the orientation and binding of the substrates
in the active site of PSL-C might be similar for the acylation of
both 20-deoxynucleosides 1a–1e and ribonucleosides 1f–1j.
However, lower 30-regioselectivities and slower reaction rates
were observed in the acylation of ribonucleosides 1f–1j as com-
pared to those in the acylation of 20-deoxynucleosides 1a–1e, due
to the presence of 20-hydroxyl group. Lavandera et al. proposed
the model of phosphonate analogs for PSL-C-mediated butanoy-
lation of
b
-thymidine 1c by molecular modeling.5 According to
the model, the C20 of sugar moiety is located above the medium-
sized pocket of PSL-C and points into the solvent in the confor-
mation of 30-acylation transition state, while the C20 points into
the alternate hydrophobic pocket of the enzyme in 50-acylation. It
has been proven that there exist several tyrosine residues such as
Tyr23 and Tyr29 in the alternate hydrophobic pocket.16 As we
know, the hydroxyl group is a good acceptor or donor of hydrogen
bond. The lower 30-regioselectivities in the acylation of ribonu-
cleosides 1f–1j might be a result of the unfavorable hydrogen
bond between 20-hydroxyl and the phenolic hydroxyl of the ty-
rosine residue such as Tyr29 of the enzyme, which stabilizes the
conformation of 50-acylation transition state, and thus increases
the amount of the minor regioisomer. Kazlauskas et al. proposed
that the enantioselectivity derived from an extra hydrogen bond
between the phenoxy oxygen of the substrate to the phenolic
hydroxyl of Tyr29 of the enzyme in PSL-C-catalyzed resolution of
2-phenoxy-1-propanol.16 The lower catalytic activity of PSL-C
might partially be attributed to the steric hindrance of 20-hy-
droxyl group in the synthesis of the ester derivatives of ribonu-
cleosides 1f–1j.
Scheme 1. PSL-C-mediated 30-lauroylation of nucleosides.
liquid-containing system was used for the enzymatic acylation of
nucleosides. Upon optimization, [C4MMIm]PF6 (5 mg/mL) turned
out to be the optimal additive (unpublished data).15 The effect of
nucleoside structure, especially R1, R2, and R3 groups, on the en-
zymatic reaction was examined by using vinyl laurate as the acyl
donor (Table 1). It was found that 20-deoxynucleosides 1a–1e could
be transformed to the desired 30-ester derivatives with high
conversions (99%) and excellent regioselectivities (96 to >99%) by
PSL-C in 3–6 h. Furthermore, the 30-regioselectivity increased with
increasing hydrophobicity of R1 group of 20-deoxynucleoside,
which was in agreement with our previous report.14a It might be
due to the favorable remote interaction between R1 group and the
hydrophobic side chain of amino acid residue Leu287 present in the
active site of PSL-C in the conformation of 30-acylation transition
state.5 A more hydrophobic R1 group would result in a stronger
interaction, stabilizing this conformation and thus increasing the
amount of the major regioisomer. In addition, the reaction rate
decreased with increasing bulk of R1 group, possibly due to the
steric strain. For example, 20-deoxyuridine 1a (R1¼H) disappeared
The hypothesis was confirmed by the results from the acylation
of 20-fluoro-20-deoxyuridine 1k and 1-
b-D-arabinofuranosyluracil
1m catalyzed by PSL-C (Table 1). Like 1a, a good 30-regioselectivity
(92%) was achieved in the acylation of 1k, since it is difficult to
make a hydrogen bonding between 20-F and the phenolic hydroxyl
of tyrosine. Besides, 30-laurate of 1m was synthesized with the
selectivity of 69%, which is slightly higher than that (59%) in the
acylation of 1f. The conformation of 20-hydroxyl group of 1m is
different from that of 1f. Perhaps the donor atom (20-OH of 1m) is
too far from the acceptor atom (phenolic OH of tyrosine residue) to
form a hydrogen bond. As a result, a weaker close interaction rather
than a hydrogen bond interaction occurred between 20-hydroxyl of
1m and phenolic hydroxyl of tyrosine residue, resulting in a higher
30-regioselectivity. Previously, a moderate 30-regioselectivity (80%)
was reported in PSL-C-mediated levulinylation of 20-O-alkyl-5-
methyluridine,4a being similar to that (89%) in the lauroylation of 5-
methyluridine 1h. It implies that the oxygen of 20-hydroxyl group of
1f–1j might act as an acceptor of hydrogen bond, while phenolic
hydroxyl of tyrosine as a donor.
in the mixture after the reaction time of 3 h, whereas b-thymidine
1c (R1¼CH3) and idoxuridine 1e (R1¼I) were completely acylated to
the ester derivatives after 3.5 and 6 h, respectively.
Table 1
Effect of substrate structure on PSL-C-mediated lauroylation of nucleosidesa
Nucleoside
Time (h)
Conversionb (%)
30-Regioselectivityc (%)
1a
1b
1c
1d
1e
1f
1g
1h
1i
3
4
3.5
5
6
14
22
16
30
35
12
30
>99
>99
>99
>99
99
99
99
98
98
96
99
>99
>99
>99
59
78
89
86
88
1j
1k
1m
98
98
99
Then, FUdR was used as a model acyl acceptor and its regiose-
lective acylation catalyzed by PSL-C was attempted with vinyl es-
ters as the acyl donors (Scheme 2). Initially, the chain length
specificity (C2–C18) of the lipase was examined (Fig.1). As shown in
Figure 1, PSL-C exhibited the highest acylation activity (48.2 mM/h)
toward vinyl acetate, and the reaction rate decreased with the
elongation of the chain. It is well known that P. cepacia lipase has
a funnel-like binding site.17 Therefore, a longer acyl donor might be
92
69
a
The reaction was carried out at 40 ꢀC, 250 rpm by adding 40 U PSL-C into an-
hydrous THF (2 mL) containing nucleosides (0.04 mmol), vinyl laurate (0.16 mmol)
with [C4MMIm]PF6 (10 mg) as the additive.
Determined by HPLC analysis using SB-C18 column.
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.
b
c

N. Li et al. / Tetrahedron 65 (2009) 1063–1068
1065
more difficult to enter into the active site to form the first tetra-
hedron intermediate (generally considered as the rate-limiting
step), due to the steric strain. Similar phenomenon was observed in
the regioselective acylation of 5-fluorouridine 1g mediated by PSL-
C.18 Interestingly, the 30-regioselectivity increased with the
lengthening chain from C2 to C8, and PSL-C displayed excellent 30-
regioselectivities (>99%) in the enzymatic acylation of FUdR with
fatty acid vinyl esters of more than C8 as acyl donors. According to
the model proposed by Lavandera et al.,5 the increasing 30-regio-
selectivity might be due to the destabilization of the conformation
of 50-acylation transition state by a larger acyl group. The R1 group
of base moiety extends into the large hydrophobic pocket, into
which the acyl group also binds. A larger acyl group might result in
steric clash, and destabilize the conformation of 50-acylation
transition state, thus leading to the decrease of the amount of the
minor regioisomer.
Finally, the effect of the substituent or functional group pres-
ent in the acyl donor on the enzymatic acylation of FUdR was
investigated (Table 2). It was shown that the reaction rate drop-
ped remarkably with branched fatty acid vinyl esters as acyl do-
nors (Table 2, entries 1–4), perhaps due to the steric hindrance.
For example, a conversion of 67% was achieved for the piv-
aloylation after 47 h (entry 1), while FUdR was butanoylated with
a conversion of >99% after 2 h. In addition, the larger the sub-
stituent is, the slower the reaction rate becomes. With vinyl 2-
ethylhexanoate as the acyl donor, a low conversion (10%) was
obtained after 47 h (entry 3). Increasing the reaction temperature
and molar ratio of acyl donor to FUdR could afford improved
results (entries 2 and 4). It is worth noting that no 30,50-diesters
were detected in the pivaloylation and 20-ethylhexanoylation
even at high temperature (50 ꢀC). It could be attributed to the
steric hindrance created by the acyl groups of the monoesters. For
the same reason, Gotor et al.’s effort to synthesize the dibenzoate
from 50-O-protected nucleoside by PSL-C failed.19 When C–C
double bond in the acyl donor is far away from the carbonyl
group, its effect on the enzymatic reaction is marginal (entry 5),
as indicated by the similar catalytic performances, including the
activity and regioselectivity, of PSL-C in the oleoylation and in the
stearoylation. However, the reaction rate decreased substantially
when the double bond conjugated with the carbonyl group, due
to the resonance effect (entries 6–8). Surprisingly, although vinyl
crotonate 3e might be less hindered than vinyl methacrylate 3d
due to the presence of a-methyl group in the latter, the reaction
rate with the latter as the acyl donor was higher than that with
the former (entries 6 and 7). Up to date, the reason still remains
unknown. Watanabe et al. obtained similar results in PSL-C-me-
diated synthesis of 6-esters of
D-glucose: a yield of 39% was
afforded with vinyl methacrylate as the acyl donor, a lower yield
(11%) with vinyl acrylate, and no reaction with vinyl crotonate.20
PSL-C-catalyzed benzoylation of FUdR was more difficult to per-
form (entries 9 and 10), owing to the steric and resonance effects
of phenyl group. Prolonging arm length between phenyl and
carbonyl would reduce the steric strain of the rigid phenyl group.
As a result, vinyl cinnamate 3g is easier to fit into the funnel-like
active site of PSL-C as compared to vinyl benzoate 3f, resulting in
a higher reaction rate (entries 11 and 12).
Scheme 2. Regioselective acylation of FUdR catalyzed by PSL-C.
Table 2
Effect of the substituent or functional group present in the acyl donor on PSL-C-
catalyzed acylation of FUdRa
Entry Acyl
donor (ꢀC)
Temperature Molar Time Conversionc 30-Regioselectivityd
ratiob (h)
(%)
(%)
1
2
3
4
5
6
7
8
3a
40
50
40
50
40
40
40
50
40
50
40
50
4
7
4
7
4
4
4
7
4
7
4
7
47
66
47
90
9
23
33
43
107
66
107
66
67
98
10
41
98
99
78
98
58
99
69
99
96
96
83
82
99
77
86
83
87
88
95
91
3b
3c
3d
3e
9
3f
10
11
12
3g
a
The reaction was carried out at 250 rpm by adding 40 U PSL-C into anhydrous
THF (2 mL) containing FUdR (0.04 mmol), vinyl esters with [C4MMIm]PF6 (10 mg) as
the additive.
b
Figure 1. Effect of acyl chain length on PSL-C-catalyzed acylation of FUdR. The reaction
was carried out at 40 ꢀC, 250 rpm by adding 40 U PSL-C into anhydrous THF (2 mL)
containing FUdR (0.04 mmol), vinyl esters (0.16 mmol) with [C4MMIm]PF6 (10 mg) as
the additive. Symbols: V0 (,); conversion (B); 30-regioseletivity (6).
Molar ratio of vinyl ester to FUdR.
Determined by HPLC analysis using SB-C18 column.
Defined as the ratio of the concentration of the desired product to that of all the
c
d
products, and determined by HPLC analysis using SB-C18 column.

1066
N. Li et al. / Tetrahedron 65 (2009) 1063–1068
3. Conclusions
incubated in a 50 mL Erlenmeyer shaking flask capped with a sep-
tum at 250 rpm and 50 ꢀC. Upon completion of the reaction, the
enzyme was filtered off, and the filtrate was concentrated in vacuo.
The residue was separated and purified through column chroma-
tography using petroleum ether (PE)/ethyl acetate (EA) or
dichloromethane (DCM)/ethyl acetate (EA) as the eluent. The
structures of ester derivatives of FUdR and its analogs were de-
termined by 13C NMR and 1H NMR (Bruker DRX 400 MHz NMR
spectrometer, Germany) at 100.5 and 400 MHz, respectively. All the
ester derivatives of nucleosides are white solid.
In summary, we have developed an efficient enzymatic ap-
proach for 30-acylation of nucleosides and their analogs. The results
revealed that the lower regioselectivities in PSL-C-catalyzed acyl-
ation of ribonucleosides 1f–1j could be attributed to the unfavor-
able hydrogen bond interaction between 20-hydroxyl group and
phenolic hydroxyl group of tyrosine residue present in the alternate
hydrophobic pocket of PSL-C. New interesting information re-
garding substrate–enzyme interactions was obtained from the
study of enzyme substrate recognition in the acylation of nucleo-
sides. These findings would help us in controlling the regiose-
lectivity of the synthetically useful enzyme via chemical
modification and protein engineering.
4.3.1. 30-O-Lauroyl-20-deoxyuridine (2a)
Rf: 0.21 (PE/EA¼4:7). 1H NMR (DMSO-d6)
: 0.85 (t, J¼8.0 Hz, 3H,
d
00
00
00
00
00
00
00
00
00
H12 ),1.24 (br s,16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ),1.53
00
0
00
(t, J¼8.0 Hz, 2H, H3 ), 2.24–2.27 (m, 2H, H2 ), 2.30–2.36 (m, 2H, H2 ),
0
0
0
4. Experimental
4.1. General
3.61 (br s, 2H, H5 ), 3.98 (br s, 1H, H4 ), 5.19–5.23 (m, 2H, H3 þOH),
0
5.68 (d, J¼8.0 Hz,1H, H6), 6.16 (t, J¼8.0 Hz,1H, H1 ), 7.88 (d, J¼8.0 Hz,
1H, H5), 11.35 (s, 1H, H3). 13C NMR (DMSO-d6)
d
: 13.96 (C12 ), 22.11
00
00
00
00
00
00
00
00
00
(C11 ), 24.33 (C3 ), 28.39–29.36 (C4 þC5 þC6 þC7 þC8 þC9 ), 31.30
00
00
0
0
0
0
P. cepacia lipase immobilized on ceramic (PSL-C) was from
Amano Enzyme Inc., Japan. The specific esterification activity of
PSL-C (730 U/g) was assayed according to a previous method.14b
Floxuridine was purchased from Shanghai Hanhong Co., Ltd., China.
(C10 ), 33.43 (C2 ), 36.85 (C2 ), 61.32 (C5 ), 74.67 (C3 ), 84.04 (C4 ),
0
84.84 (C1 ), 102.14 (C5), 140.23 (C6), 150.44 (C2), 163.04 (C4), 172.57
00
(C1 ).
20-Deoxyuridine, idoxuridine,
idine, uridine, 5-fluorouridine, 5-methyluridine, 20-fluoro-20-
deoxyuridine, and 1- -arabinofuranosyluracil were bought from
b
-thymidine, 5-bromo-20-deoxyur-
4.3.2. 30-O-Lauroyl-FUdR (2b)
Rf: 0.24 (PE/EA¼3:2). 1H NMR (DMSO-d6)
d
: 0.84 (br s, 3H, H12 ),
00
00
00
00
00
00
00
00
00
b-
D
1.24 (br s, 16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ), 1.53 (br s,
00
00
0
0
Tuoxin Biotechnology & Science Co., Ltd., China. 5-Iodouridine, 5-
bromouridine, vinyl decanoate, vinyl laurate, vinyl stearate, vinyl
methacylate, vinyl crotonate, vinyl benzoate were obtained from
Sigma–Aldrich Co., USA. Vinyl butyrate, vinyl caproate, vinyl octa-
noate, vinyl myristate, vinyl palmitate, vinyl pivalate, vinyl 2-eth-
ylhexanoate, and vinyl cinnamate were from TCI, Japan. All the
chemicals mentioned above are of high purity (>99%). 1-Butyl-2,3-
dimethylimidazolium hexafluorophosphate ([C4MMIm]PF6) was
purchased from Lanzhou Institute of Chemical Physics, China. Oleic
acid (90%) was from Alfa Aesar, USA. All other chemicals are of the
highest purity commercially available. High performance liquid
chromatography (HPLC) analysis was carried out in an Agilent 1200
chromatograph with a UV detector using Zorbax SB-C18 column
(4.6 mmꢁ250 mm, 5-Micron) under varying conditions depending
on the specific substrate. The absorption wavelength for the anal-
ysis is 260 nm (20-fluoro-20-deoxyuridine), 261 nm (20-deoxyur-
2H, H3 ), 2.27–2.36 (m, 4H, H2 þH2 ), 3.64 (br s, 2H, H5 ), 4.00 (br s,
0
0
0
1H, H4 ), 5.22 (br s, 1H, H3 ), 6.15 (t, J¼6.8 Hz, 1H, H1 ), 8.21 (d,
J¼7.2 Hz, 1H, H6). 13C NMR (DMSO-d6)
00 00
d: 14.17 (C12 ), 22.32 (C11 ),
00
00
00
00
00
00
00
00
24.53 (C3 ), 28.57–29.18 (C4 þC5 þC6 þC7 þC8 þC9 ), 31.51 (C10 ),
00
0
0
0
0
0
33.65 (C2 ), 37.11 (C2 ), 63.45 (C5 ), 74.84 (C3 ), 84.67 (C4 ), 85.19 (C1 ),
124.56, 124.90 (C6), 139.16, 141.45 (C5), 149.19 (C2), 157.06, 157.33
00
(C4), 172.94 (C1 ).
4.3.3. 30-O-Lauroyl-
b-thymidine (2c)
Rf: 0.21 (PE/EA¼1:1). 1H NMR (CDCl3)
d
00
: 0.88 (t, J¼7.2 Hz, 3H,
00
00
00
00
00
00
00
00
H12 ), 1.26 (br s, 16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ),
00
0
00
1.61–1.64 (m, 2H, H3 ), 1.90 (s, 3H, H7), 2.33–2.45 (m, 4H, H2 þH2 ),
0
0
3.27 (br s, 1H, OH), 3.91 (br s, 2H, H5 ), 4.07 (br s, 1H, H4 ), 5.37 (t,
0
0
J¼2.8 Hz, 1H, H3 ), 6.29 (t, J¼7.6 Hz, 1H, H1 ), 7.60 (s, 1H, H6), 9.79 (s,
1H, H3). 13C NMR (CDCl3)
00 00
d: 12.66 (C7), 14.24 (C12 ), 22.79 (C11 ),
00
00
00
00
00
00
00
00
24.91 (C3 ), 29.23–29.71 (C4 þC5 þC6 þC7 þC8 þC9 ), 32.01 (C10 ),
00 0 0 0 0
34.31 (C2 ), 37.44 (C2 ), 62.57 (C5 ), 74.71 (C3 ), 85.42 (C4 ), 85.86
idine and uridine), 263 nm (1-
b
-D-arabinofuranosyluracil), 267 nm
0
00
(b
-thymidine and 5-methyluridine), 269 nm (FUdR and 5-fluoro-
(C1 ), 111.43 (C5), 136.61 (C6), 150.85 (C2), 164.39 (C4), 173.82 (C1 ).
uridine), 279 nm (5-bromo-20-deoxyuridine and 5-bromouridine),
and 280 nm (idoxuridine and 5-iodouridine), respectively. The
regioselectivity was defined as the ratio of the concentration of the
desired product to that of all the products. The initial reaction rate
(V0) and the substrate conversion were calculated from the HPLC
data.
4.3.4. 30-O-Lauroyl-5-bromo-20-deoxyuridine (2d)
Rf: 0.29 (PE/EA¼3:2). 1H NMR (CDCl3)
d
00
: 0.88 (t, J¼7.2 Hz, 3H,
00
00
00
00
00
00
00
00
H12 ), 1.26 (br s, 16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ),
00
0
00
1.59–1.65 (m, 2H, H3 ), 2.33–2.50 (m, 5H, H2 þH2 þOH), 3.94–4.01
0
0
0
(m, 2H, H5 ), 4.12 (d, J¼2.0 Hz, 1H, H4 ), 5.36 (d, J¼4.4 Hz, 1H, H3 ),
0
6.30 (dd, J1¼14.0 Hz, J2¼6.0 Hz, 1H, H1 ), 8.26 (s, 1H, H6), 9.14 (s, 1H,
H3). 13C NMR (CDCl3)
d: 14.36 (C12 ), 22.92 (C11 ), 25.04 (C3 ), 29.34–
00 00 00
4.2. General procedure for enzymatic acylation
00
00
00
00
00
00
00
00
29.83 (C4 þC5 þC6 þC7 þC8 þC9 ), 32.14 (C10 ), 34.41 (C2 ), 38.32
0
0
0
0
0
In a typical experiment, a reaction mixture of FUdR (1b) (10 mg,
0.04 mmol), 4 equiv of vinyl ester and 10 mg [C4MMIm]PF6 in an-
hydrous THF (2 mL) was added to a sealed-cap vial (15 mL) con-
taining PSL-C (40 U). The reaction was carried out at 40 ꢀC, 250 rpm.
Aliquots were withdrawn from the reaction mixture at specified
time intervals, and then diluted by 25-fold with corresponding
mobile phase prior to HPLC analysis.
(C2 ), 62.75 (C5 ), 74.76 (C3 ), 85.83 (C4 ), 86.36 (C1 ), 97.38 (C5),
00
140.39 (C6), 149.89 (C2), 159.19 (C4), 173.90 (C1 ).
4.3.5. 30-O-Lauroyl-idoxuridine (2e)
Rf: 0.36 (PE/EA¼3:2). 1H NMR (CDCl3)
d
00
: 0.88 (t, J¼8.0 Hz, 3H,
00
00
00
00
00
00
00
00
H12 ), 1.26 (br s, 16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ),
00
0
00
1.61–1.64 (m, 2H, H3 ), 2.33–2.49 (m, 4H, H2 þH2 ), 2.73 (br s, 1H,
0
0
OH), 3.93–3.97 (m, 2H, H5 ), 4.12–4.13 (m, 1H, H4 ), 5.36–5.38 (m,
1H, H3 ), 6.26–6.29 (m, 1H, H1 ), 8.34 (s, 1H, H6), 9.39 (s, 1H, H3). 13C
0
0
4.3. Purification and structure determination of the desired
ester derivatives
00 00 00
NMR (CDCl3) d: 14.32 (C12 ), 22.87 (C11 ), 24.99 (C3 ), 29.30–29.98
00
00
00
00
00
00
00
00
0
(C4 þC5 þC6 þC7 þC8 þC9 ), 32.09 (C10 ), 34.37 (C2 ), 38.29 (C2 ),
0
0
0
0
In a typical experiment, THF (8 mL) containing FUdR or its an-
alogs (100 mg), 7 equiv of vinyl ester and PSL-C (300 mg) was
62.67 (C5 ), 68.75 (C5), 74.72 (C3 ), 85.81 (C4 ), 86.35 (C1 ), 145.61
00
(C6), 150.25 (C2), 160.26 (C4), 173.86 (C1 ).

N. Li et al. / Tetrahedron 65 (2009) 1063–1068
1067
4.3.6. 30-O-Lauroyl-uridine (2f)
31.90 (C10 ), 33.68 (C2 ), 60.47 (C5 ), 70.09, 70.23 (C3 ), 82.18 (C4 ),
00
00
0
0
0
Rf: 0.26 (PE/EA¼1:3). 1H NMR (CDCl3)
d
00
: 0.88 (t, J¼8.0 Hz, 3H,
88.44, 88.78 (C1 ), 90.35, 92.24 (C2 ), 102.69 (C5), 141.82 (C6), 150.88
(C2), 164.18 (C4), 172.76 (C1 ).
0
0
00
00
00
00
00
00
00
00
00
H12 ), 1.26 (br s, 16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ),
00
00
1.60–1.65 (m, 2H, H3 ), 2.38–2.48 (m, 2H, H2 ), 3.80–3.93 (m, 2H,
H5 ), 4.19 (br s, 1H, H4 ), 4.60 (t, J¼8.0 Hz, 1H, H2 ), 5.27 (br s, 1H, H3 ),
4.3.12. 30-O-Lauroyl-1-
b-D-arabinofuranosyluracil (2m)
0
0
0
0
Rf: 0.23 (DCM/EA¼1:1). 1H NMR (CDCl3)
: 0.88 (t, J¼7.2 Hz, 3H,
d
0
5.74 (d, J¼8.0 Hz, 1H, H6), 5.83 (d, J¼4.0 Hz, 1H, H1 ), 7.80 (d,
J¼8.0 Hz, 1H, H5), 10.20 (s, 1H, H3). 13C NMR (CDCl3)
d
: 14.32 (C12 ),
H12 ), 1.26 (br s, 16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ),
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
22.89 (C11 ), 25.01 (C3 ), 29.37–29.83 (C4 þC5 þC6 þC7 þC8 þC9 ),
1.60–1.65 (m, 2H, H3 ), 2.38 (t, J¼8.0 Hz, 2H, H2 ), 3.92–3.40 (m, 2H,
00
00
0
0
0
0
0
0
0
32.11 (C10 ), 34.24 (C2 ), 61.99 (C5 ), 72.67 (C3 ), 73.29 (C2 ), 83.68
H5 ), 4.10 (m, 1H, H4 ), 4.44 (br s, 1H, H2 ), 5.14 (br s, 1H, H3 ), 5.65 (d,
0
0
0
(C4 ), 90.75 (C1 ), 102.85 (C5), 141.72 (C6), 151.30 (C2), 164.26 (C4),
J¼8.0 Hz, 1H, H6), 6.07 (d, J¼3.6 Hz, 1H, H1 ), 7.79 (d, J¼8.0 Hz, 1H,
0
0
0
0
174.09 (C1 ).
H5), 9.82 (s, 1H, H3). 13C NMR (CDCl3)
d: 14.32 (C12 ), 22.89 (C11 ),
00
00
00
00
00
00
00
00
00
24.96 (C3 ), 29.32–29.82 (C4 þC5 þC6 þC7 þC8 þC9 ), 32.11 (C10 ),
4.3.7. 30-O-Lauroyl-5-fluorouridine (2g)
34.25 (C2 ), 62.18 (C5 ), 73.41 (C2 ), 79.12 (C3 ), 83.79 (C4 ), 87.15 (C1 ),
00
0
0
0
0
0
Rf: 0.24 (DCM/EA¼3:2). 1H NMR (CDCl3)
d
: 0.88 (t, J¼8.0 Hz, 3H,
100.93 (C5), 142.54 (C6), 150.56 (C2), 164.78 (C4), 173.64 (C1 ).
00
00
00
00
00
00
00
00
00
00
H12 ), 1.26 (br s, 16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ),
For the characterization data of other compounds, see Supple-
mentary data.
00
00
1.59–1.63 (m, 2H, H3 ), 2.38–2.48 (m, 2H, H2 ), 3.83–3.95 (m, 2H,
0
0
0
H5 ), 4.19 (d, J¼4.0 Hz, 1H, H4 ), 4.52 (t, J¼4.0 Hz, 1H, H2 ), 5.26 (br s,
0
0
1H, H3 ), 5.90 (d, J¼4.0 Hz, 1H, H1 ), 8.08 (d, J¼8.0 Hz, 1H, H6), 10.38
Acknowledgements
(d, J¼4.0 Hz, 1H, H3). 13C NMR (CDCl3)
d
00 00
: 14.32 (C12 ), 22.89 (C11 ),
00
00
00
00
00
00
00
00
24.98 (C3 ), 29.37–29.85 (C4 þC5 þC6 þC7 þC8 þC9 ), 32.12 (C10 ),
We wish to thank Ms. Xiu-Mei Liu for the help on NMR. This
research was financially supported by the National Natural Science
Foundation of China (Grant No. 20676043 and 20603036), Science
and Technology Project of Guangdong Province (Grant No.
2006A10602003), Science and Technology Project of Guangzhou
City (Grant No. 2007Z3-E4101), the Open Project Program of the
State Key Laboratory of Catalysis, Dalian Institute of Chemical
Physics, Chinese Academy of Sciences (Grant No. N-06-06).
00
0
0
0
0
0
34.19 (C2 ), 61.85 (C5 ), 72.64 (C3 ), 73.79 (C2 ), 83.76 (C4 ), 89.81 (C1 ),
125.34, 125.69 (C6), 139.59, 141.94 (C5), 149.97 (C2), 157.74, 158.00
00
(C4), 174.25 (C1 ).
4.3.8. 30-O-Lauroyl-5-methyluridine (2h)
Rf: 0.27 (PE/EA¼2:5). 1H NMR (CDCl3)
d
00
: 0.88 (t, J¼8.0 Hz, 3H,
00
00
00
00
00
00
00
00
H12 ), 1.26 (br s, 16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ),
00
00
1.59–1.66 (m, 2H, H3 ), 1.85 (s, 3H, H7), 2.38–2.46 (t, 2H, H2 ), 3.81–
0
0
3.94 (m, 2H, H5 ), 4.19 (d, J¼4.0 Hz, 1H, H4 ), 4.63 (t, J¼4.0 Hz, 1H,
0
0
0
H2 ), 5.27 (t, J¼4.0 Hz 1H, H3 ), 5.79 (d, J¼4.0 Hz, 1H, H1 ), 7.57 (s, 1H,
Supplementary data
H6), 10.11 (s, 1H, H3). 13C NMR (CDCl3)
d
: 12.61 (C7), 14.35 (C12 ),
00
00
00
00
00
00
00
00
00
22.92 (C11 ), 25.04 (C3 ), 29.39–29.86 (C4 þC5 þC6 þC7 þC8 þC9 ),
HPLC analysis conditions, retention time, and characterization
data are available. Supplementary data associated with this article
can be found in the online version, at doi:10.1016/j.tet.2008.11.045.
00
00
0
0
0
32.14 (C10 ), 34.27 (C2 ), 62.13 (C5 ), 72.66 (C3 ), 72.99 (C2 ), 83.60
0
0
(C4 ), 91.05 (C1 ), 111.41 (C5), 137.55 (C6), 151.45 (C2), 164.57 (C4),
00
174.15 (C1 ).
4.3.9. 30-O-Lauroyl-5-bromouridine (2i)
References and notes
Rf: 0.22 (PE/EA¼3:2). 1H NMR (CDCl3)
d
00
: 0.88 (t, J¼8.0 Hz, 3H,
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00
00
1.60–1.65 (m, 2H, H3 ), 2.40–2.48 (m, 2H, H2 ), 3.83–3.99 (m, 2H,
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0
0
0
H5 ), 4.22 (br s, 1H, H4 ), 4.64 (t, J¼4.0 Hz, 1H, H2 ), 5.30 (t, J¼4.0 Hz,
0
0
1H, H3 ), 5.91 (d, J¼8.0 Hz, 1H, H1 ), 8.30 (s, 1H, H6), 10.38 (s, 1H, H3).
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d: 14.32 (C12 ), 22.88 (C11 ), 25.01 (C3 ), 29.36–
00 00 00
00
00
00
00
00
00
00
00
29.84 (C4 þC5 þC6 þC7 þC8 þC9 ), 32.11 (C10 ), 34.24 (C2 ), 61.72
0
0
0
0
0
(C5 ), 72.26 (C3 ), 73.94 (C2 ), 83.65 (C4 ), 90.43 (C1 ), 97.35 (C5),
00
141.04 (C6), 150.69 (C2), 160.02 (C4), 174.25 (C1 ).
4.3.10. 30-O-Lauroyl-5-iodouridine (2j)
Rf: 0.30 (PE/EA¼5:4). 1H NMR (CDCl3)
d
: 0.88 (t, J¼8.0 Hz, 3H,
00
00
00
00
00
00
00
00
00
H12 ), 1.25 (br s, 16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ),
00
00
1.60–1.66 (m, 2H, H3 ), 2.40–2.48 (m, 2H, H2 ), 3.83–3.4.01 (m, 2H,
0
0
0
H5 ), 4.23 (br s, 1H, H4 ), 4.69 (t, J¼4.0 Hz, 1H, H2 ), 5.34 (t, J¼8.0 Hz,
0
0
1H, H3 ), 5.91 (d, J¼4.0 Hz, 1H, H1 ), 8.39 (s, 1H, H6), 10.41 (s, 1H, H3).
13C NMR (CDCl3)
d: 14.33 (C12 ), 22.89 (C11 ), 25.03 (C3 ), 29.36–
00 00 00
00
00
00
00
00
00
00
00
29.83 (C4 þC5 þC6 þC7 þC8 þC9 ), 32.11 (C10 ), 34.27 (C2 ), 61.61
0
0
0
0
0
(C5 ), 69.28 (C5), 72.07 (C3 ), 73.93 (C2 ), 83.49 (C4 ), 90.49 (C1 ),
00
146.19 (C4), 151.04 (C2), 161.08 (C6), 173.85 (C1 ).
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Rf: 0.32 (PE/EA¼1:2). 1H NMR (DMSO-d6)
: 0.85 (t, J¼7.2 Hz, 3H,
d
00
00
00
00
00
00
00
00
00
H12 ), 1.24 (br s, 16H, H4 þH5 þH6 þH7 þH8 þH9 þH10 þH11 ),
00
00
1.51–1.56 (m, 2H, H3 ), 2.39 (t, J¼7.2 Hz, 2H, H2 ), 3.56–3.61 (m, 2H,
0
0
0
H5 ), 4.14 (t, J¼3.2 Hz, 1H, H4 ), 5.18–5.25 (m, 1H, H3 ), 5.33–5.48 (m,
0
1H, H2 ), 5.70 (d, J¼8.0 Hz, 1H, H6), 5.95 (dd, J1¼21.6 Hz, J2¼3.2 Hz,
1H, H1 ), 7.86 (d, J¼8.0 Hz,1H, H5).13C NMR(DMSO-d6)
d
0
00
:14.34(C12 ),
00
00
00
00
00
00
00
00
22.68 (C11 ), 24.91 (C3 ), 28.89–29.58 (C4 þC5 þC6 þC7 þC8 þC9 ),
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