Selective biocatalytic acylation studies on 5-O-(4,4′- Dimethoxytrityl)-2,3-secouridine: An efficient synthesis of UNA monomer

Article abstract of DOI:10.1080/15257770.2012.734424

Lipozyme TL IM (Theremomyces lanuginosus lipase immobilized on silica) in toluene catalyzes the acylation of the 2'-OH over the 3'-OH group in 5'-O-(4,4'-dimethoxytrityl)-2',3'-secouridine (5'-O-DMT-2',3'-secouridine) in a highly selective fashion in moderate to almost quantitative yields. The turn over during benzoyl transfer reactions mediated by vinyl benzoate or benzoic anhydride was faster than in acyl transfer reactions with vinyl acetate or C1 to C5 acid anhydrides; except in the case of butanoic anhydride. The 2'-O-benzoyl-5'-O-DMT-2',3'-secouridine obtained by Lipozyme TL IM catalyzed benzoylation of 5'-O-DMT-2',3'-secouridine was successfully converted into its 3'-O-phosphoramidite derivative in satisfactory yield, which is a building block for the preparation of oligonucleotides containing the uracil monomer of UNA (unlocked nucleic acid).

Full text of DOI:10.1080/15257770.2012.734424

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Selective Biocatalytic Acylation Studies
on 5′-O-(4,4′-Dimethoxytrityl)-2′,3′-
Secouridine: An Efficient Synthesis of
UNA Monomer
Sunil K. Singh a , L. Chandrashekhar Reddy a , Smriti Srivastava a
, Carl E. Olsen b , Yogesh S. Sanghvi c , Niels Langkjær d , Jesper
Wengel d , Virinder S. Parmar a & Ashok K. Prasad a
a Bioorganic Laboratory, Department of Chemistry, University of
Delhi, Delhi, India
b Department of Natural Sciences, University of Copenhagen,
Frederiksberg C, Denmark
c Rasayan Inc., Encinitas, CA, USA
d Department of Physics, Chemistry and Pharmacy, Nucleic Acid
Center, University of Southern Denmark, Odense M, Denmark
Version of record first published: 05 Dec 2012.
To cite this article: Sunil K. Singh , L. Chandrashekhar Reddy , Smriti Srivastava , Carl E. Olsen ,
Yogesh S. Sanghvi , Niels Langkjær , Jesper Wengel , Virinder S. Parmar & Ashok K. Prasad (2012):
Selective Biocatalytic Acylation Studies on 5′-O-(4,4′-Dimethoxytrityl)-2′,3′-Secouridine: An Efficient
Synthesis of UNA Monomer, Nucleosides, Nucleotides and Nucleic Acids, 31:12, 831-840
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Nucleosides, Nucleotides and Nucleic Acids, 31:831–840, 2012
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2012.734424
SELECTIVE BIOCATALYTIC ACYLATION STUDIES ON
5ꢀ-O-(4,4ꢀ-DIMETHOXYTRITYL)-2ꢀ,3ꢀ-SECOURIDINE: AN EFFICIENT
SYNTHESIS OF UNA MONOMER
Sunil K. Singh,1 L. Chandrashekhar Reddy,1 Smriti Srivastava,1
Carl E. Olsen,2 Yogesh S. Sanghvi,3 Niels Langkjær,4 Jesper Wengel,4
Virinder S. Parmar,1 and Ashok K. Prasad1
1Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, India
2Department of Natural Sciences, University of Copenhagen, Frederiksberg C, Denmark
3Rasayan Inc., Encinitas, CA, USA
4Department of Physics, Chemistry and Pharmacy, Nucleic Acid Center, University
of Southern Denmark, Odense M, Denmark
ꢀR
Lipozyme TL IM (Theremomyces lanuginosus lipase immobilized on silica) in toluene
2
catalyzes the acylation of the 2ꢁ-OH over the 3ꢁ-OH group in 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ,3ꢁ-
secouridine (5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine) in a highly selective fashion in moderate to almost quan-
titative yields. The turn over during benzoyl transfer reactions mediated by vinyl benzoate or benzoic
anhydride was faster than in acyl transfer reactions with vinyl acetate or C1 to C5 acid anhydrides;
except in the case of butanoic anhydride. The 2ꢁ-O-benzoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine obtained
ꢀR
by Lipozyme TL IM catalyzed benzoylation of 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine was successfully con-
verted into its 3ꢁ-O-phosphoramidite derivative in satisfactory yield, which is a building block for
the preparation of oligonucleotides containing the uracil monomer of UNA (unlocked nucleic acid).
ꢀR
Keywords UNA; lipozyme TL IM; toluene; regioselective acylation; UNA monomer
INTRODUCTION
Unlocked nucleic acid (UNA) has an incomplete ribose ring open be-
tween the 2ꢁ- and 3ꢁ-carbon atoms and is an acyclic analog of RNA (Figure 1).
Thymine UNA was first introduced in 1995 and it has been shown that the
structural flexibility of UNA monomers destabilizes the duplexes.[1–3] The
Received 29 August 2012; accepted 24 September 2012.
We are thankful to the University of Delhi and DBT, New Delhi for providing financial support
under the DU-DST Purse Grant and under Indo-Danish Collaboration program in Biotechnology. Sunil
K. Singh, L. Chandrashekhar Reddy, and Smriti Srivastava thank CSIR, DBT, and UGC, New Delhi,
respectively, for providing junior/senior research fellowships. Jesper Wengel thanks the Danish National
Research Foundation for funding the Nucleic Acid Center.
Address correspondence to Ashok K. Prasad, Bioorganic Laboratory, Department of Chemistry,
University of Delhi, Delhi 110007, India. E-mail: ashokenzyme@yahoo.com
831

832
S. K. Singh et al.
B
B
B
B
O
O
O
O
O
O
O
O
O
O-
O
P
O
P
H
O
O-
H
O
O-
O
P
O
P
O
O-
O
O
O
O
O
O
O
LNA III
DNA I
RNA II
,
UNA IV
,
,
,
FIGURE 1 Structures of DNA (I); RNA (II); LNA (Locked nucleic acid, III), and UNA (Unlocked
nucleic acid, IV), B = Nucleobase (A, T, G, C, and U).
use of siRNA to knock down gene function has truly revolutionized mam-
malian cell culture studies and holds great promise in therapeutics.[4–6]
Modification of siRNA with UNA nucleotides has shown highly potent gene
silencing activity accompanied by low cell toxicity.[7,8] The biological studies
of UNA-modified antisense oligonucleotides have demonstrated compatibil-
ity with RNase H activity[9] and introduction of UNA monomers in siRNA
duplexes have revealed that UNA is a very attractive modification to improve
the specificity of gene silencing.[10] Notably, strategic placement of UNA
substitution in siRNA has proven to be an important tool for the reduction
of off-target events by >10-fold compared to the unmodified siRNA.[10a] Sim-
ilarly, single incorporation of UNA-U monomer in position 7 has resulted
in a potent design of an aptamer with excellent inhibitory effect on the
fibrin-clot formation.[11]
Synthesis of monomeric building blocks of UNA, that is, phospho-
ramidites of acyclic nucleosides adenosine, guanosine, cytidine, and uridine
requires selective manipulation of one of the two primary hydroxyl groups at
the 2ꢁ- and 3ꢁ-OH positions in the corresponding 5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-
2ꢁ,3ꢁ-seconucleosides (5ꢁ-O-DMT-seconucleosides). The chemical methods
available for the preparation of 2ꢁ-O-benzoylated 5ꢁ-O-DMT-seconucleosides
requires the use of unfriendly chemicals or low temperature and often leads
to the formation of a mixture of benzoylated products, which reduce the
yields of the desired compound.[1,3]
Among the four possible building-blocks of UNA monomers, the util-
ity of UNA-U (uridine analog) for improved siRNA constructs has been
outstanding.[12] Because of the importance of UNA-U monomer for con-
struction of therapeutic oligonucleotides, we elected to develop an im-
proved protocol utilizing an enzymatic protocol. Herein, we report a very
high yielding, selective and environment friendly enzymatic methodol-
ogy for the synthesis of 2ꢁ-O-benzoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine from its
corresponding dihydroxy acyclic nucleoside and conversion of the re-
sulted mono-benzoylated compound to phosphoramidite building block of
UNA-U.

Selective Biocatalytic Acylation Studies on 5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ,3ꢁ-Secouridine 833
RESULTS AND DISCUSSION
There are several reports describing the use of lipases for the selective
manipulations of different hydroxyl groups in sugars or in nucleosides.[13]
Further, lipases have also been used for the separation of the nucleosides
from their anomeric/isomeric mixtures,[14] in the resolution of β-D/L-2ꢁ-
deoxynucleosides,[15] and in the separation of N -7 and N -9 guanine nucleo-
sides.[16] Recently, an efficient biocatalytic methodology has been reported
for the differentiation of two similar acetoxy groups involving primary hy-
droxyl groups in nucleosides resulting in the convenient synthesis of xylo-
LNA monomers.[17]
The acyclic nucleoside monomer of UNA U, that is, 5ꢁ-O-DMT-2ꢁ,3ꢁ-
secouridine (2) was obtained from RiboTask, Denmark. Alternatively, sec-
ouridine 2 can also be obtained from 5ꢁ-O-DMT-uridine (1) following the
literature procedure (Scheme 1).[1,3] For selective benzoylation of the 2ꢁ-
hydroxyl function in 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2) with vinyl benzoate as
acyl donor, five lipases,[18] viz. Candida antarctica lipase-B immobilized on
polyacrylate (NovozymeꢀR -435 or CAL-B), Theremomyces lanuginosus lipase im-
mobilized on silica (LipozymeꢀR TL IM), Amano PS lipase, Candida rugosa
lipase (CRL), and porcine pancreatic lipase (PPL) were screened employ-
ing five organic solvents. We selected tetrahydrofuran (THF), acetonitrile,
toluene, diisopropyl ether (DIPE), and acetone as the solvents for screen-
ing while the reactions were carried out at 40◦C–42◦C and at 150 rpm in
an incubator shaker. Among the different lipases screened, LipozymeꢀR TL
IM (50% w/w of compound 2) in toluene induced selective and efficient
benzoylation of the 2ꢁ-OH group of acyclic nucleoside 2 and furnished the
product 5a in 92% yield (Scheme 1). LipozymeꢀR TL IM catalyzed acylation
reactions in other solvents, that is, acetonitrile, THF, DIPE, and acetone did
not yield any product. Although NovozymeꢀR -435 in toluene initially showed
U
r
DMT
O
U
U
H
U
H
r
DMT
O
r
DMT
O
O
H
O
II
O
O
O
e .
R f 1 3
,
i
R
OCO
O
P
a
5
6
R
H
OCO
O
H
O
O
H
O
O
ri
N
H
H
NP
CC 2C 2O
2
a-
5
g
1
2
6
e® TL IM
H
-
H
R
a
=
-
)
r R
b o CO 2
(
3
(
) (
)
4a-g
Reagents and conditions : i) Lip
ozym
C
)
C OCO
O
,
2
Toluene 40-42 oC
c a o
t
h
N N diisoprop lphosphora-
y
-
,
,
; ii 2 y n e yl
oC
28
-
midochloridite, diisopro
eth l amine, acetonitrile, 25
p
y
yl
Compounds
3a, 4a, 5a, 6 3b, 4b, 5b
4c, 5c
4d,5d
4e, 5e
4f, 5f
4g, 5g
-
3 6
-
-
-
-
-
R
C3H7
-
C5H11
-
CH3
C2H5
iso-
C3H7
C4H9
C6H5
ꢀR
SCHEME 1 Lipozyme TL IM catalyzed regioselective acylation studies on 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine
(2).

834
S. K. Singh et al.
selectivity for the acylation of the 2ꢁ-OH group of 2, the reaction was very
slow and only 10% conversion was observed even after 24 hours of incuba-
tion. The reaction catalyzed by LipozymeꢀR TL IM at higher temperature
than 40◦C–42◦C leads to the enhancement in the rate of the reaction, but
is also accompanied with multiple products formation and cleavage of the
dimethoxytrityl group. PPL, Amano PS and CRL did not show any acylation
reaction on 2 during the test reactions.
In a typical reaction, a solution of 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2) in dry
toluene containing 1.2 equivalents of vinyl benzoate (3a) was incubated with
◦
LipozymeꢀR TL IM in an incubator shaker at 40◦C–42 C and 150 rpm. On
completion of the reaction, as indicated by analytical TLC examination,
enzyme was filtered off and the solvent removed under reduced pressure.
The crude product thus obtained was passed through a small band of silica
gel column, pre-washed with 0.5% triethyl amine in CHCl3 to afford the
pure 2ꢁ-O-benzoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (5a) in 92% yield (Scheme 1,
Table 1).
To study the generality of the biocatalytic acylation reaction, dif-
ferent acylating agents such as vinyl acetate (3b) and benzoic-, acetic-,
propanoic-, butanoic, isobutanoic-, pentanoic-, and hexanoic anhydrides
(4a–g) were evaluated for selective manipulation of the 2ꢁ-OH over the 3ꢁ-OH
group in nucleoside 2 under the above mentioned standardized conditions
ꢀR
TABLE 1 Lipozyme TL IM catalyzed regioselective acylation studies on 5ꢁ-O-DMT-2ꢁ,3ꢁ- secouridine
(2)a in toluene at 40◦C–42◦C
Reaction
time (hour)
S. no.
Acyl donor
Product
% Yieldb
92
1
2
3
4
5
6
7
8
9
Vinyl benzoate (3a)
3
2
4
3
3
1
5
5
3
2ꢁ-O-Benzoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-
secouridine (5a)
Vinyl acetate (3b)
2ꢁ-O-Acetyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-
secouridine (5b)
70
Benzoic anhydride (4a)
Acetic anhydride (4b)
Propanoic anhydride (4c)
Butanoic anhydride (4d)
iso-Butanoic anhydride (4e)
Pentanoic anhydride (4f)
Hexanoic anhydride (4g)
2ꢁ-O-Benzoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-
secouridine (5a)
86
2ꢁ-O-Acetyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-
secouridine (5b)
62
2ꢁ-O-Propanoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-
secouridine (5c)
60
2ꢁ-O-Butanoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-
secouridine (5d)
80
2ꢁ-O-Isobutanoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-
secouridine (5e)
65
2ꢁ-O-Pentanoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-
secouridine (5f)
58
2ꢁ-O-Hexanoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-
secouridine (5g)
65
ꢀR
aAll these reactions, when performed under identical conditions but without adding Lipozyme TL
IM, did not yield any product. bIsolated yield.

Selective Biocatalytic Acylation Studies on 5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ,3ꢁ-Secouridine 835
(Scheme 1, Table 1). The results compiled in Table 1 reveal that LipozymeꢀR
TL IM mediates selective transfer of acyl function on to the 2ꢁ-OH group
of 2 both using active esters 3a and 3b and acid anhydrides 4a--g resulting
in the formation of 2ꢁ-O-acyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridines 5a--g in moderate
to high yields. The turn over in reactions involving the transfer of benzoyl
function from vinyl benzoate 3a or benzoic anhydride 4a is higher than any
of the other acyl transfer reactions (Table 1). Among the two most active
esters, the turn over in benzoylation reaction involving vinyl benzoate (3a)
was 1.3 times larger than with vinyl acetate (3b, entries 1 and 2). Among
the different acid anhydrides (4b--g), the butanoylation reaction involving
butanoic anhydride was the most efficient with respect to the selectivity, yield
and reaction time of the reaction (Table 1, entry 6).
Further, 2ꢁ-O-benzoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (5a) was converted into
the corresponding phosphoramidite 6, a building block for the synthesis
of UNA, by reaction with ClP(OCH2CH2CN)(N(iPr)2) in acetonitrile in
the presence of diisopropyl ethyl amine (DIPEA) following the literature
procedure.[3] The structures of all the acylated nucleosides 5a–g and phos-
1
phoramidite 6 were confirmed from their spectral (IR, H-, 13C-, 31P NMR
spectral and HRMS) data analysis. The structures of known compounds 5a
and 6 were further confirmed on the basis of comparison of their physi-
cal and spectral data with those reported in the literature.[3] All enzymatic
reactions, when performed under identical conditions but without adding
LipozymeꢀR TL IM, did not yield any product.
In summary, it has been demonstrated that LipozymeꢀR TL IM in toluene
can effectively discriminate between the two primary hydroxyl groups present
at the 2ꢁ and 3ꢁ positions in 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2). This has led to the
development of an environment friendly and highly selective methodology
for the efficient synthesis of 2ꢁ-O-benzoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (5a) in
almost quantitative yield. 2ꢁ-O-Benzoyl-5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (5a) ob-
tained by enzymatic benzoylation of 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2) was sub-
sequently used for the synthesis of the 3ꢁ-O-phosphoramidite 6 in satisfactory
yield, which is a building block for the preparation of oligonucleotides con-
taining the uracil monomer of UNA.
EXPERIMENTAL SECTION
General Spectroscopic and Experimental Information
Melting points were determined on a Mettler FP 62 instrument and are
uncorrected. The IR spectra were recorded on a PerkinElmer Spectrum-
100 FTIR instrument by making KBr disc for solid samples and thin film
1
for oils. The H- and 13C NMR spectra were recorded on a 400 MHz
JEOL ECX Spectrospin instrument at 400 and at 100.5 MHz, respectively.
The chemical shift values are reported as δ ppm relative to TMS used

836
S. K. Singh et al.
as internal standard and the coupling constants (J ) are measured in Hz.
All accurate mass measurements were done in ESI positive mode on a
micrOTOF-Q instrument from Bruker Daltonics. The Candida antarctica li-
pase B (NovozymeꢀR -435), LipozymeꢀR TL IM, and Amano PS, PPL, CRL
were used after storing in vacuo over P2O5 for more than 24 hours. THF,
dichloromethane, methanol, ethyl acetate, petroleum ether, and toluene
were dried and distilled prior to use. The spots on analytical TLCs were
detected either under UV light or by charring with 4% alcoholic H2SO4.
Silica gel (particle size 0.040–0.063 mm, Merck) was used for column
chromatography.
General procedure for LipozymeꢀR TL IM Catalyzed Acylation Reaction
on 5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ,3ꢁ-Secouridine (2): Synthesis of 2ꢁ-O-Acyl-5ꢁ-
O-(4,4ꢁ-dimethoxytrityl)-2ꢁ,3ꢁ-secouridine (5a–g). To a solution of nucleoside
2 (100 mg, 18.84 mmol) in anhydrous toluene (5 ml), acyl donor 3a–b, or
4a–g (22.60 mmol) was added followed by the addition of LipozymeꢀR TL
IM (50% w/w of the nucleoside 2). The reaction mixture was incubated
at 40◦C–42◦C at 150 rpm in an incubator shaker and the progress of the
reaction was monitored periodically by analytical TLC. On completion, the
reaction was quenched by filtering off the lipase and the solvent was removed
under reduced pressure. The residue thus obtained was purified by silica gel
column chromatography using 1 to 3% methanol in chloroform as eluent
to afford 2ꢁ-O-acylated nucleosides 5a–g.
2ꢁ-O-Benzoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ,3ꢁ-secouridine (5a).[3] The
product was obtained by LipozymeꢀR TL IM catalyzed benzoylation of
5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2) using vinyl benzoate (3a) and benzoic
anhydride (4a) as white foam-like solid in 92% and 86% yields, respectively.
The physical and spectral data of the compound were found identical to
those reported in the literature.[3]
2ꢁ-O-Acetyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ,3ꢁ-secouridine (5b). The prod-
uct was obtained by LipozymeꢀR TL IM catalyzed regioselective acylation
of 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2) using vinyl acetate (3b) and acetic an-
hydride (4b) as acetylating reagent as a white foam-like solid in 70% and
62% yields, respectively. IR (thin film) νmax: 3451, 3202, 3061, 1746, 1693,
1511, 1251 cm−1; 1H NMR (CDCl3, 400 MHz): δ 7.98 (s, 1H, NH), 7.43–7.21
(m, 2H, ArH), 7.98 (d, 1H, J = 7.32 Hz, H6), 7.70–7.63 (m, 1H, ArH),
7.56–7.48 (m, 2H, ArH), 7.35–7.17 (m, 4H, ArH), 6.89–6.81 (m, 4H, ArH),
6.20 (t, 1H, J = 5.7 Hz, H1ꢁ), 5.56 (d, 1H, J = 8.0 Hz, H5), 4.83 (t, 1H,
J = 5.2 Hz, 3ꢁ-OH), 4.71–4.45 (m, 2H, H2ꢁ), 3.73 (s, 6H, –OCH3), 3.70–3.62
(m, 1H, H4ꢁ), 3.50–3.40 (m, 2H, H3ꢁ), 3.11–2.96 (m, 2H, H5ꢁ), 2.10 (s, 3H,
–COCH3); 13C NMR (CDCl3, 100.6 MHz): δ 170.29, 163.08, 158.50, 150.72,
144.32, 139.51, 135.39, 29.88, 127.92, 127.82, 126.94, 113.09, 102.91, 86.61,
81.58, 80.21, 63.59, 63.37, 62.32, 55.19, 20.57; HR-ESI-TOF-MS: m/z 613.2120
([M+Na]+), calcd. for [C32H34N2O9+Na]+ 613.2157.

Selective Biocatalytic Acylation Studies on 5ꢁ-O-(4,4ꢁ-Dimethoxytrityl)-2ꢁ,3ꢁ-Secouridine 837
2ꢁ-O-Propanoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ,3ꢁ-secouridine (5c). The
product was obtained by LipozymeꢀR TL IM catalyzed regioselective
propanoylation of 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2) using propanoic anhy-
dride (4c) as a viscous oil in 60% yield. IR (thin film) νmax: 3464, 3200,
1
3060, 1693, 1509, 1251 cm−1; H NMR (CDCl3, 400 MHz): δ 7.98 (s, 1H,
NH), 7.40–7.32 (m, 10H, ArH and H6), 6.81–6.79 (d, 4H, J = 8.4 Hz, ArH),
6.09 (brs, 1H, H1ꢁ), 5.60 (d, 1H, J = 8.1 Hz, H5), 4.32–4.23 (m, 2H, H2ꢁ),
3.78 (s, 7H,OCH3 and H4ꢁ), 3.37–3.65 (m, 2H, H3ꢁ), 3.21 (brs, 2H, H5ꢁ),
2.32 (q, 2H, J = 7.2 Hz, –CH2CH3), 1.10 (t, 3H, J = 6.0 Hz, –CH2CH3); 13
C
NMR (CDCl3, 100.6 MHz): δ 173.70, 162.82, 158.53, 150.53, 144.33, 139.48,
135.43, 135.38, 129.89, 127.92, 127.85, 126.96, 113.11, 102.85, 86.65, 81.72,
80.34, 63.47, 63.34, 62.37, 55.21, 27.23, 8.88; HR-ESI-TOF-MS: m/z 627.2283
([M+Na]+), calcd. for [C33H36N2O9+Na]+ 627.2313.
2ꢁ-O-Butanoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ,3ꢁ-secouridine (5d). The pro-
duct was obtained by LipozymeꢀR TL IM catalyzed regioselective butanoyla-
tion of 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2) using butanoic anhydride (4d) as a
viscous oil in 80% yield. IR (thin film) νmax: 3431, 1689, 1489, 1251 cm−1.
1H NMR (CDCl3, 400 MHz): δ 9.29 (s, 1H, NH), 7.41–7.33 (m, 10H, ArH
and H6), 6.80 (d, 4H, J = 7.8 Hz, ArH), 6.11 (brs, 1H, H1ꢁ), 5.60 (d,
1H, J = 7.5 Hz, H5), 4.26 (m, 2H, H2ꢁ), 3.77 (brs, 7H, –OCH3 and H4ꢁ),
3.68–3.65 (m, 2H, H3ꢁ), 3.21 (brs, 2H, H5ꢁ), 2.46 (brs, 1H, OH), 2.27 (t,
2H, J = 6.3 Hz, –CH2CH2CH3), 1.61 (m, 2H, –CH2CH2CH3), 0.90 (t, 3H, J
= 6.9 Hz, –CH2CH2CH3); 13C NMR (CDCl3, 100.6 MHz): δ 173.05, 162.42,
158.57, 150.25, 144.33, 139.41, 135.62, 129.90, 127.93, 127.87, 126.98, 113.14,
102.81, 86.72, 81.84, 80.51, 63.32, 63.32, 62.45, 55.23, 35.73, 18.18, 13.57;
HR-ESI-TOF-MS: m/z 641.2434 ([M+Na]+), calcd. for [C34H38N2O9+Na]+
641.2470.
2ꢁ-O-Isobutanoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ,3ꢁ-secouridine (5e). The
product was obtained by LipozymeꢀR TL IM catalyzed regioselective isobu-
tanoylation of 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2) using isobutanoic anhydride
(4e) as a viscous oil in 65% yield. IR (thin film) νmax: 3459, 3202, 1693, 1510,
1251 cm−1. 1H NMR (CDCl3, 400 MHz): δ 9.29 (s, 1H, NH), 7.41 (d, 1H, J =
7.5 Hz, H6), 7.35–7.21 (m, 9H, ArH), 6.80 (d, 4H, J = 8.68 Hz, ArH), 6.13 (t,
1H, J = 5.04 Hz, H1ꢁ), 5.60 (d, 1H, J = 8.24 Hz, H5), 4.26–4.24 (m, 2H, H2ꢁ),
3.78 (brs, 8H, –OCH3, H4ꢁ and OH), 3.68–3.65 (m, 2H, H3ꢁ), 3.21 (brs, 2H,
H5ꢁ), 2.53 (septet, 1H, J = 7.36 Hz, –CH(CH3)2), 1.12 (d, 6H, J = 6.88 Hz,
–CH(CH3)2); 13C NMR (CDCl3, 100.6 MHz): δ 176.31, 163.26, 158.52,150.88,
144.34, 139.54, 135.44, 129.89, 127.93, 127.82, 126.94, 113.10, 102.82, 86.62,
81.64, 80.17, 63.35, 63.32, 62.32, 55.19, 33.78, 18.81, 18.73; HR-ESI-TOF-MS:
m/z 641.2437 ([M+Na]+), calcd. for [C34H38N2O9+Na]+ 641.2470.
2ꢁ-O-Pentanoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ,3ꢁ-secouridine (5f). The
product was obtained by LipozymeꢀR TL IM catalyzed regioselective pen-
tanoylation of 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2) using pentanoic anhydride

838
S. K. Singh et al.
(4f) as a viscous oil in 58% yield. IR (thin film) νmax: 3463, 3200, 3059,
1
1690, 1608, 1509, 1251 cm−1. H NMR (CDCl3, 400 MHz): δ 9.29 (s, 1H,
NH), 7.41 (d, 1H, J = 7.5 Hz, H6), 7.33–7.20 (m, 9H, ArH), 6.79 (d, 4H,
J = 8.72 Hz, ArH), 6.10 (t, 1H, J = 4.12 Hz, H1ꢁ), 5.60 (d, 1H, J = 8.24 Hz,
H5), 4.23 (m, 2H, H2ꢁ), 3.76 (brs, 8H,-OCH3, H4ꢁ and OH), 3.66–3.63 (m,
2H, H3ꢁ), 3.19 (brs, 2H, H5ꢁ), 2.28 (t, 2H, J = 7.32 Hz, –CH2CH2CH2CH3),
1.54 (quintet, 2H, J = 7.32 Hz, –CH2CH2CH2CH3), 1.28–1.26 (m, 2H,
–CH2CH2CH2CH3), 0.85 (t, 3H, J = 7.36 Hz, –CH2CH2CH2CH3); 13C
NMR (CDCl3, 100.6 MHz): δ 173.08, 163.11, 158.50, 150.75, 144.21, 139.49,
135.42, 129.88, 127.93, 127.81, 126.92, 113.09, 102.85, 86.60, 81.62, 80.18,
63.36, 63.34, 62.29, 55.18, 33.54, 26.67, 22.07, 13.58; HR-ESI-TOF-MS: m/z
655.2579 ([M+Na]+), calcd. for [C35H40N2O9+Na]+ 655.2626.
2ꢁ-O-Hexanoyl-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2ꢁ,3ꢁ-secouridine (5g). The pro-
duct was obtained by LipozymeꢀR TL IM catalyzed regioselective hexanoyla-
tion of 5ꢁ-O-DMT-2ꢁ,3ꢁ-secouridine (2) using hexanoic anhydride (4g) as a
viscous oil in 65% yield. IR (thin film) νmax: 3451, 3202, 1742, 1692, 1510,
1
1330 cm−1. H NMR (CDCl3, 400 MHz): δ 9.29 (s, 1H, NH), 7.41 (d, 1H,
J = 7.5 Hz, H6), 7.35–7.21 (m, 9H, ArH), 6.79 (d, 4H, J = 8.68 Hz, ArH),
6.08 (t, 1H, J = 5.04 Hz, H1ꢁ), 5.60 (d, 1H, J = 7.8 Hz, H5), 4.26–4.24
(m, 2H, H2ꢁ), 3.78 (brs, 8H, –OCH3, H4ꢁ and OH), 3.68–3.65 (m, 2H,
H3ꢁ), 3.21 (brs, 2H, H5ꢁ), 2.30 (t, 2H, J = 7.36 Hz, –CH2CH2CH2CH2CH3),
1.59 (quintet, 2H, J = 7.32 Hz, –CH2CH2CH2CH2CH3), 1.27–1.25 (m, 4H,
–CH2CH2CH2CH2CH3), 0.86 (t, 3H, J = 6.88 Hz, –CH2CH2CH2CH2CH3),
13C NMR (CDCl3, 100.6 MHz): δ 173.09, 162.60, 158.53, 150.95, 144.33,
139.48, 135.43, 129.89, 127.93, 127.86, 126.97, 113.13, 102.83, 86.67, 81.76,
80.42, 63.35, 63.34, 62.40, 55.18, 33.82, 31.11, 24.35, 22.21, 13.84, 9.89;
HR-ESI-TOF-MS: m/z 669.2742 ([M+Na]+), calcd. for [C36H42N2O9+Na]+
669.2783.
2ꢁ-O-Benzoyl-3ꢁ-O-(2-cyanoethoxy(diisopropylamino)phosphino)-5ꢁ-O-(4,
4ꢁ-dimethoxy-trityl)-2ꢁ,3ꢁ-secouridine (6). The product was prepared as
per the literature procedure in 65% yield.[3] Structure of the synthesized
compound 6 was established on the basis of its 31P NMR spectrum and its
comparison with the reported data.[3]
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ꢀR
18. Lipase NovozymeꢀR -435 and Lipozyme TL IM were obtained as gift from Novozyme A/S Denmark.
Amino PS was obtained as gift from ISIS pharmaceuticals, USA. CRL and PPL were purchased from
Sigma-Aldrich chemical company, USA.