Chemoenzymatic synthesis of 3'-deoxy-3'-(4-substituted-triazol-1-YL)-5- methyluridine

Article abstract of DOI:10.1080/15257770.2013.847957

An efficient protocol has been developed for the synthesis of a small library of 3'-deoxy-3'- (4-substituted-triazol-1-yl)-5-methyluridine using Cu(I)-catalyzed Huisgen-Sharpless-Meldal 1,3- dipolar cycloaddition reaction of 3'-azido-3'-deoxy-5-methyluridine with different alkynes under optimized condition in an overall yields of 76%-92%. Here, the azido precursor compound, i.e., 3'-azido-3'-deoxy-5-methyluridine was chemoenzymatically synthesized from D-xylose in good yield. Some of the alkynes used in cycloaddition reaction were synthesized by the reaction of hydroxycoumarins or naphthols with propargyl bromide in acetone using K₂CO₃ in excellent yields. All synthesized compounds were unambiguously identified on the basis of their spectral (IR, ¹H-, ¹³C NMR spectra, and high-resolution mass spectra) data analysis.

Full text of DOI:10.1080/15257770.2013.847957

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Chemoenzymatic Synthesis of 3′-
Deoxy-3′-(4-Substituted-Triazol-1-YL)-5-
Methyluridine
Anu Arya a , Divya Mathur a , Abhilash Tyagi a , Rajesh Kumar a ,
Vinod Kumar b , Carl E. Olsen c , Rajendra K. Saxena b & Ashok K.
Prasad a
a Bioorganic Laboratory, Department of Chemistry , University of
Delhi , Delhi , India
b Department of Microbiology , University of Delhi South Campus ,
New Delhi , India
c Department of Natural Sciences , University of Copenhagen ,
Frederiksberg C , Denmark
Published online: 11 Dec 2013.
To cite this article: Anu Arya , Divya Mathur , Abhilash Tyagi , Rajesh Kumar , Vinod Kumar , Carl
E. Olsen , Rajendra K. Saxena & Ashok K. Prasad (2013) Chemoenzymatic Synthesis of 3′-Deoxy-3′-
(4-Substituted-Triazol-1-YL)-5-Methyluridine, Nucleosides, Nucleotides and Nucleic Acids, 32:12,
646-659, DOI: 10.1080/15257770.2013.847957
To link to this article: http://dx.doi.org/10.1080/15257770.2013.847957
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Nucleosides, Nucleotides and Nucleic Acids, 32:646–659, 2013
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.847957
CHEMOENZYMATIC SYNTHESIS
OF 3ꢀ-DEOXY-3ꢀ-(4-SUBSTITUTED-TRIAZOL-1-YL)-5-METHYLURIDINE
Anu Arya,1 Divya Mathur,1 Abhilash Tyagi,1 Rajesh Kumar,1 Vinod Kumar,2
Carl E. Olsen,3 Rajendra K. Saxena,2 and Ashok K. Prasad1
1Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi, India
2Department of Microbiology, University of Delhi South Campus, New Delhi, India
3Department of Natural Sciences, University of Copenhagen, Frederiksberg C, Denmark
An efficient protocol has been developed for the synthesis of a small library of 3ꢁ-deoxy-3ꢁ-
2
(4-substituted-triazol-1-yl)-5-methyluridine using Cu(I)-catalyzed Huisgen–Sharpless–Meldal 1,3-
dipolar cycloaddition reaction of 3ꢁ-azido-3ꢁ-deoxy-5-methyluridine with different alkynes under op-
timized condition in an overall yields of 76%–92%. Here, the azido precursor compound, i.e.,
3ꢁ-azido-3ꢁ-deoxy-5-methyluridine was chemoenzymatically synthesized from D-xylose in good yield.
Some of the alkynes used in cycloaddition reaction were synthesized by the reaction of hydroxy-
coumarins or naphthols with propargyl bromide in acetone using K2CO3 in excellent yields. All
1
synthesized compounds were unambiguously identified on the basis of their spectral (IR, H-, 13C
NMR spectra, and high-resolution mass spectra) data analysis.
Keywords Azidonucleosides; bio-conjugates; click chemistry; enzymatic acylation; tria-
zolylnucleosides
INTRODUCTION
Bioconjugation has recently emerged as a fast-growing technology
that affects almost every discipline of life sciences. It aims at the liga-
tion of two or more molecules to form a new complex with the com-
bined properties of its individual components.[1] The copper(I)-catalyzed
Huisgen–Sharpless–Meldal 1,3-dipolar cycloaddition reaction to form 1,4-
disubstituted triazole bridges is a very popular and unique reaction for the
Received 24 March 2013; accepted 17 September 2013.
We are thankful to the University of Delhi (DU) and Department of Science and Technology
(DST), New Delhi for providing financial support under DU-DST Purse Grant for the execution of the
work. A.A., D.M., A.T., R.K., and V.K. thank Department of Biotechnology, University Grant Commission,
DU-DST, Council of Scientific & Industrial Research and Ministry of New and Renewable Energy, New
Delhi, for providing junior/senior research fellowships.
Address correspondence to Ashok K. Prasad, Bioorganic Laboratory, Department of Chemistry,
University of Delhi, Delhi 110007, India. E-mail: ashokenzyme@yahoo.com
646

Chemoenzymatic Synthesis of 3ꢁ-Triazolylnucleosides
647
O
O
N
O
N
NH
R
N
O
O
O
NH
NH
O
HO
Ph
N
O
N
N
O
O
N
N
N
N
O
O
N
OH
OH OH
3
N
2
1
R
OCH3
NH2
N
N
N
N
N
N
O
HO
N
N
NH
O
N
HO
HO
N
O
N
O
O
OH
N
N
N
Ph
OH OH
5
OH OH
6
4
FIGURE 1 Examples of various 1,2,3-triazole conjugated nucleosides.
preparation of bioconjugates[2,3], drug like scaffolds[4] and other useful ma-
terials.[5–8] Triazolylnucleosides, which can be generated by Cu-catalyzed
1,3-dipolar cycloaddition reaction of azidonucleoside with alkyne using click
reaction, are of special interest because of their pronounced biological ac-
tivities.[9–12]
The Cu-catalyzed azide-alkyne condensation (CuAAC) reaction has been
applied for the functionalization of both, sugar and base moieties for the
discovery of novel triazolylnucleoside derivatives potentially endowed with
biological activity. Thus, triazole can be tethered to nucleoside at vari-
ous positions such as 2ꢁ, 3ꢁ, 5ꢁ and also at anomeric position of sugar
as well as on the base. Zhou et al.[13] have reported 1,2,3-triazole func-
tionalized thymidines 1 and 2 as antiviral agents (Figure 1). Triazole-
linked coumarin nucleoside conjugates 3 have been reported as fluorescent
probes and signaling units.[14] Mahony et al.[15] have reported 2ꢁ-triazolyl-2ꢁ-
deoxyadenosines 4 as t-RNA synthetase inhibitor. Synthesis of 4-substituted
1,2,3-triazolylnucleosides 5 and their in vitro cytostatic activity have been
documented.[16] Montague et al.[17] have presented C5-(1-substituted-1,2,3-
triazol-4-yl)-2ꢁ-deoxyuridines 6 as antiviral agents. Encouraged by the useful-
ness of triazolylnucleosides, we have, for the first time, synthesized a series of
3ꢁ-deoxy-3ꢁ-(4-substituted-triazol-1-yl)-5-methyluridine by the cyclocondensa-
tion of 3ꢁ-azido-3ꢁ-deoxy-5-methyluridine with different alkynes. Further the
azidonucleoside, precursor for the synthesis of triazolylnucleosides has been

648
A. Arya et al.
HO
BzO
(i) H2SO4, CuSO4,
acetone, rt, 24hr
Novozyme®-435, THF
vinyl benzoate, 70 min,
40oC, 96 %
O
O
OH
D-Xylose
OH
O
O
(ii) 1M HCl solution in
MeOH, 40oC
O
O
7
8
9
90 %
BzO
BzO
Tf2O, Py, CH2Cl2
O
NaN3, DMF
24hr, 100oC
45 %
O
AcOH, Ac2O, H2SO4
6hr, rt, 75 %
OTf
1 hr, -10oC
85 %
O
O
O
N3
O
10
11
O
O
N
NH
NH
BzO
O
HO
N
BzO
O
O
BSA, Thymine,
K2CO3/MeOH
rt, 1hr, 79 %
O
O
OAc
TMSOTf, CH3CN
12hr, 50-70oC
83 %
N3 OH
14
N3 OAc
12
N3 OAc
13
SCHEME 1 Synthesis of 3ꢁ-azido-3ꢁ-deoxy-5-methyluridine.
efficiently prepared by following a novel chemoenzymatic approach using
NovozymeꢀR -435 in tetrahydrofuran (THF).[18]
RESULTS AND DISCUSSION
Nine 3ꢁ-deoxy-3ꢁ-(4-substituted-triazol-1-yl)-5-methyluridine have been
synthesized by the CuAAC reaction of 3ꢁ-azido-3ꢁ-deoxy-5-methyluridine
(14) with different alkynes, i.e., phenylacetylene, propargyl alcohol,
5-chloro-1-pentyne, 7-coumaryloxy-methylacetylene, 4-methyl-7-coumarylo
xymethylacetylene, 3-ethyl-4-methyl-7-coumaryloxymethylacetylene, 4-coum
aryloxymethylacetylene, 2-naphthyloxymethylacetylene and 1-naphthy
loxymethylacetylene. The precursor 3ꢁ-azido-3ꢁ-deoxy-5-methyluridine (14)
was chemoenzymatically synthesized starting from D-xylose following the
modified procedure of Gadthula et al.[19] (Scheme 1). Here, D-xylose (7) was
selectively converted to a monoketal, 1,2-O-isopropylidene-α-D-xylofuranose
(8) in a two-step process in 90% yield. The selective benzoylation of pri-
mary hydroxyl group over the secondary hydroxyl group in compound 8
was probed by the mediation of different lipases in organic solvents. Thus,
five different lipases, viz., NovozymeꢀR -435 or CAL-B, LipozymeꢀR TL IM,
Amano PS, CRL, and PPL were screened for the selective benzoylation of
1,2-O-isopropylidene-α-D-xylofuranose (8) with vinyl benzoate in different

Chemoenzymatic Synthesis of 3ꢁ-Triazolylnucleosides
649
organic solvents, i.e., THF, toluene, diisopropyl ether (DIPE), acetonitrile,
and acetone at 40◦C, 50◦C, and 60◦C and at 200 rpm in an incubator shaker.
Among different lipases NovozymeꢀR -435 (50% w/w of the substrate) in
THF and toluene at 40◦C selectivity and efficiently benzoylated the primary
hydroxyl group over the secondary hydroxyl group in compound 8 to af-
ford monobenzoylated sugar derivative 9 (Scheme 1). However, the rate
of NovozymeꢀR -435-catalyzed benzoylation reaction in THF was much faster
than the same reaction in toluene.
In a typical reaction, a solution of 1,2-O-isopropylidene-α-D-xylofuranose
(8, 200 mg) and vinyl benzoate (1 equiv.) in tetrahydrofuran (20 mL) was
incubated with NovozymeꢀR -435 (50% w/w of the substrate) in an incu-
bator shaker at 40◦C and at 200 rpm. The reaction mixture was shaken
for 70 minutes until the completion of the reaction as indicated by thin
layer chromatography (TLC) examination. The progress of reaction was
also monitored by high performance liquid chromatography (HPLC) and
it was quenched on completion by filtering off the enzyme (Figure 2). The
solvent was removed under reduced pressure and the crude product thus
obtained was purified by silica gel column chromatography using ethyl ac-
etate in petroleum ether as gradient solvent system to exclusively afford the
pure 5-O-benzoyl-α-D-xylofuranose (9) in 96% yield (Scheme 1).
In order to introduce an azido group at 3ꢁ-position, com-
pound 9 was first converted into 1,2-O-isopropylidene-5-O-benzoyl-3-O-
trifluoromethanesulfonyl-α-D-xylofuranose (10) by reaction with trifluo-
romethanesulfonic anhydride in 85% yield followed by azidation of the re-
sulting triflate with sodium azide in dimethylformamide (DMF) to afford
FIGURE 2 NovozymeꢀR -435 catalyzed regioselective benzoylation of 1,2-O-isopropylidene-α-D-
xylofuranose (8) using vinyl benzoate as acyl donor in THF at 40◦C. (Color figure available online).

650
A. Arya et al.
O
O
NH
NH
HO
HO
CuI
N
O
N
O
R
O
O
EtOH/H2O/THF
(1:1:1)
60 °C, 15 hrs
15 a-i
N3
OH
N
OH
N
76-92 %
N
14
R
16 a-i
Compds.
15 and 16
c
e
d
a
b
O
O
O
O
O
O
R
CH2OH
CH2CH2CH2Cl
Ph
g
h
f
i
O
O
O
O
O
O
O
O
SCHEME 2 Synthesis of 3ꢁ-triazole bridged nucleoside conjugates via Cu(I)-catalyzed 1,3-dipolar cy-
cloaddition.
3-azido-1,2-O-isopropylidene-5-O-benzoyl-3-deoxy-α-D-xylofuranose (11) in
relatively low yield (45%). The azide 11 was converted into 3-azido-1,2-di-O-
acetyl-5-O-benzoyl-3-deoxy-β-D-ribofuranose (12) in 75% yield using acetic
acid, acetic anhydride, and sulphuric acid. The azido ribofuranoside 12 was
then coupled with thymine in the presence of trimethylsilyl triflate and
N ,O-bis(trimethylsilyl)acetamide using Vo¨rbruggen coupling protocol[20] to
give the corresponding azido nucleoside, 3ꢁ-azido-2ꢁ-O-acetyl-5ꢁ-O-benzoyl-3ꢁ-
deoxy-5-methyluridine (13) in 83% yield, which upon deprotection led to
the formation of key intermediate 3ꢁ-azido-3ꢁ-deoxy-5-methyluridine (14) in
79% yield (Scheme 1).
Further, the optimization of CuAAC reaction between azidonucleoside
14 and phenylacetylene was carried out using various reaction condi-
tions. Thus, molar ratios of the reagents and solvents were optimized
for the click reaction, i.e., CuSO4-sodium ascorbate in t-BuOH:H2O
(1:1), t-BuOH:H2O:THF (1:1:1), and EtOH:H2O:THF (1:1:1), CuI in
t-BuOH:H2O (1:1), t-BuOH:H2O:THF (1:1:1), and EtOH:H2O:THF
(1:1:1) were used. The best result was obtained by using 0.15 molar
equiv. of CuI in EtOH:H2O:THF in (1:1:1) ratio at 60◦C. Thus, the
high fidelity of Cu (I)-catalyzed process allowed the 1,3-dipolar cy-
cloaddition reaction of 3ꢁ-azidonucleoside 14 with different alkynes

Chemoenzymatic Synthesis of 3ꢁ-Triazolylnucleosides
651
K2CO3, acetone
R1
OH
+
O
R1
Br
reflux, 60 °C,12hr
17 d-i
18
15d-i
Compds.
15 and 17
f
g
h
i
d
e
O
O
O
O
O
O
O
O
R
SCHEME 3 Synthesis of propargyloxy-coumarins and –naphthalenes.
15a-i in the presence of 0.15 molar equiv. of CuI in a solution of
EtOH:H2O:THF (1:1:1) at 60◦C to afford 3ꢁ-deoxy-3ꢁ-(4-phenyl-1,2,3-triazol-
1-yl)-5-methyluridine (16a), 3ꢁ-deoxy-3ꢁ-(4-hydroxymethyl-1,2,3-triazol-1-
yl)-5-methyluridine (16b), 3ꢁ-deoxy-3ꢁ-[4-(3-chloropropyl)-1,2,3-triazol-1-yl]
-5-methyluridine (16c), 3ꢁ-deoxy-3ꢁ-[4-(7-coumaryloxymethyl)-1,2,3-triazol-
1-yl]-5-methyluridine (16d), 3ꢁ-deoxy-3ꢁ-[4-(4-methyl-7-coumaryloxymethyl)-
1,2,3-triazol-1-yl]-5-methyluridine (16e), 3ꢁ-deoxy-3ꢁ-[4-(3-ethyl-4-methyl
-7-coumaryloxymethyl)-1,2,3-triazol-1-yl]-5-methyluridine (16f), 3ꢁ-deoxy-
3ꢁ-[4-(4-coumaryloxymethyl)-1,2,3-triazol-1-yl]-5-methyluridine (16g), 3ꢁ-
deoxy-3ꢁ-[4-(2-naphthyloxymethyl)-1,2,3-triazol-1-yl]-5-methyluridine (16h),
and 3ꢁ-deoxy-3ꢁ-[4-(1-naphthyloxymethyl)-1,2,3-triazol-1-yl]-5-methyluridine
(16i) in yields ranging from 76% to 92% (Scheme 2).
The acetylene derivatives 15a–c are commercially available. The other
six acetylene derivatives 15d–i were prepared by the reaction of propar-
gyl bromide with appropriately substituted hydroxycoumarins or naph-
thols under anhydrous condition in acetone–K2CO3 at 60◦C in quantitative
yields (Scheme 3). Thus, reaction of 7-hydroxycoumarin (17d), 7-hydroxy-
4-methylcoumarin (17e), 3-ethyl-4-methyl-7-hydroxycoumarin (17f), and
4-hydroxycoumarin (17g) with propargyl bromide (18) afforded 7-
propargyloxycoumarin (15d), 4-methyl-7-propargyloxycoumarin (15e), 3-
ethyl-4-methyl-7-propargyloxycoumarin (15f), and 4-propargyloxycoumarin
(15g) in 81%–95% yields. Similarly, reaction of β-naphthol (17h)
and α-naphthol (17i) with propargyl bromide (18) afforded 2-
propargyloxynaphthalene (15h) and 1-propargyloxynaphthalene (15i) in
91%–93% yields, respectively. The hydroxycoumarins 17d–f were synthe-
sized using Pechmann condensation[21] of resorcinol with malic acid, ethyl
acetoacetate and ethyl 2-ethylacetoacetate in sulfuric acid in 68%–80%
yields.
The structures of all the synthesized compounds 8–14, 15d–i, 16a–i, and
17d–f were unambiguously identified on the basis of their spectral (IR, 1H-,
13C NMR spectra, and high-resolution mass spectra (HRMS)) data analysis.

652
A. Arya et al.
The structures of known compounds 8–14, 15d, 15e, 15h, and 15i were fur-
ther confirmed on the basis of comparison of their spectral and/or physical
data with those reported in the literature.[19]
CONCLUSION
A chemoenzymatic protocol has been developed for the efficient synthe-
sis of 3ꢁ-azido-3ꢁ-deoxy-5-methyluridine, which was then used for the synthe-
sis of nine new 3ꢁ-deoxy-3ꢁ-(4-substituted-triazol-1-yl)-5-methyluridine using
Cu(I)-catalyzed Huisgen—Sharpless–Meldal 1,3-dipolar cycloaddition reac-
tion under optimized condition in nearly quantitative yield. The antiviral
activity evaluation of the synthesized novel triazolylnucleosides is under
progress.
EXPERIMENTAL SECTION
General Spectroscopic and Experimental Information
Melting points were determined on Buchi M-560 (Buchi Labortechnik
AG, Flawil, Switzerland) instrument and are uncorrected. The IR spectra
were recorded on a Perkin-Elmer model 2000 FTIR spectrometer (Perkin
Elmer Co., Massachusetts, USA) by making KBr disk for solid samples and
1
thin film for oils. The H- and 13C NMR spectra were recorded on a Jeol
alpha-400 spectrometer (Jeol Ltd, Tokyo, Japan) at 400 and 100.6 MHz, re-
spectively, using tetramethylsilane (TMS) as internal standard. The chemical
shift values are on δ scale and the coupling constants (J ) are in Hz. Signals
1
from OH group(s) in H NMR spectra recorded in CDCl3 were verified by
removing them by D2O exchange method. The HRMS were done on micro
TOF-Q instrument from Bruker Daltonics, Bremen (Germany) and were run
in electrospray ionization (ESI) positive mode. Reactions were conducted un-
der an atmosphere of nitrogen when anhydrous solvents were used. Analyti-
cal TLCs were performed on precoated Merck silica-gel 60F254 plates (Merck
KGaA, Darmstadt, Germany); the spots were detected under UV light. Sil-
ica gel (100–200 mesh) was used for column chromatography. Chemicals
were obtained from commercial suppliers (Sigma-Aldrich Chemical Private
Ltd., Bangalore, India and S.G. Enterprises, Delhi, India) and were used
without any further purification unless otherwise noted. Thermomyces lanug-
inosus lipase immobilized on silica (LipozymeꢀR TL IM), Candida antarctica
lipase B immobilized on polyacrylate (lewatite, CAL-B, or NovozymeꢀR -435),
and Amano PS lipase were provided by Novozymes Inc. (Copenhagen, Den-
mark) and ISIS pharmaceuticals (Carlsbad, CA, USA) as a gift, respectively,
whereas Candida rugosa lipase (CRL) and porcine pancreatic lipase (PPL)
were purchased from Sigma Chemical Co. (USA). All enzymes were used af-
ter storing in vacuum over P2O5 for more than 24 hours. The HPLC system

Chemoenzymatic Synthesis of 3ꢀ-Triazolylnucleosides
653
(Waters Corp., Milford, MA, USA) equipped with a quaternary pump system
and a refractive index detector (RID-10A, Shimadzu, Kyoto, Japan) for sugar
analysis has been used to monitor lipase-catalyzed benzoylation reaction. Re-
action aliquot (10–25 μL) were filtered through a 0.22 μm nylon membrane
and were analyzed by HPLC using EMPOWER 2 software (Waters Corp.,
Milford, MA, USA) and 1 mL min−1 solvent flow rate with mobile phase of
70% acetonitrile and 30% water.
General Procedure for the Synthesis of Propargyloxycoumarins (15d–g) and
–Naphthalenes (15h--i)
To a solution of coumarins 17d–g/naphthols 17h–i (100 mmol) in dry
acetone (10 mL), anhydrous potassium carbonate (150 mmol), and propar-
gyl bromide 18 (150 mmol) were added. The resultant mixture was stirred
and refluxed at 50◦C for 12 hours, then the mixture was cooled and fil-
tered, and the solvent was removed under reduced pressure. The residue
was treated with 15 mL of water and extracted with ethyl acetate. The com-
bined organic phase was washed with water, dried over anhydrous sodium
sulfate, and evaporated in vacuum. The crude product thus obtained was
purified by crystallization from ethyl acetate/hexane mixture to give propar-
gyloxycoumarins 15d–g and propargyloxynaphthalenes 15h–i in 81%–95%
yield.
7-Propargyloxycoumarin (15d)
It was obtained as a white solid (yield = 95%). M. P. 118◦C–120◦C. IR
(KBr) νmax: 3249, 2880, 2100, 1699, 1611, 1447, 1249, 1021, and 832 cm−1;
1H NMR (400 MHz, DMSO-d6): δ 3.63 (t, 1H, J = 2.56 Hz, C CH), 4.91
(d, 2H, J = 2.20 Hz, -OCH2C CH), 6.29 (d, 1H, J = 9.52 Hz, C-3H), 6.96
(dd, 1H, J = 2.20 and 8.80 Hz, C-6H), 7.02 (d, 1H, J = 2.20 Hz, C-8H),
7.63 (d, 1H, J = 8.80 Hz, C-5H), 7.97 (d, 1H, J = 9.52 Hz, C-4H); 13C NMR
(100.6 MHz, CDCl3): δ 56.12, 76.45, 77.36, 102.10, 112.35, 112.66, 114.20,
125.59, 152.39, 154.97, 160.29, and 161.07; HR-ESI-TOF-MS: m/z 201.0507
([M+H]+), calcd. for [C12H8O3+H]+ 201.0207.
4-Methyl-7-propargyloxycoumarin (15e)
It was obtained as a creamy solid (yield = 93%). M. P. 130◦C–134◦C.
IR (KBr) νmax: 3222, 2980, 2120, 1701, 1458, 1225, 1069, and 779 cm−1;
1H NMR (400 MHz, CDCl3): δ 2.40 (s, 3H, C-4 CH3), 2.58 (t, 1H, J =
2.92 Hz, C CH), 4.76 (d, 2H, J = 2.20 Hz, -OCH2C CH), 6.16 (s, 1H,
C-3H), 6.93–6.94 (m, 2H, C-6H, and C-8H), 7.52 (d, 1H, J = 8.80 Hz, C-5H);
13C NMR (100.6 MHz, CDCl3): δ 18.63, 56.12, 76.46, 77.36, 102.10, 112.35,
112.66, 114.20, 125.59, 152.40, 154.97, 160.29, and 161.07; HR-ESI-TOF-MS:
m/z 215.0663 ([M+H]+), calcd. for [C13H10O3+H]+ 215.0763.

654
A. Arya et al.
3-Ethyl-4-methyl-7-propargyloxycoumarin (15f)
It was obtained as a light yellow solid (yield = 89%). M. P. 74◦C–78◦C.
IR (KBr) νmax: 3200, 2942, 2109, 1699, 1474, 1277, 1093, and 732 cm−1; 1H
NMR (400 MHz, DMSO-d6): δ 1.01 (t, 3H, J = 7.32 Hz, CH2CH3), 2.34 (s,
3H, C-4 CH3), 2.51 (q, 2H, J = 7.32 Hz, CH2CH3), 3.61 (t, 1H, J = 2.20 Hz,
C CH), 4.89 (d, 2H, J = 2.92 Hz, -OCH2C CH), 6.94–6.96 (m, 2H, C-6H,
and C-8H), 7.66 (d, 1H, J = 8.80 Hz, C-5H); 13C NMR (100.6 MHz, DMSO-
d6): δ 12.97, 14.37, 20.26, 56.03, 78.82, 79.19, 101.52, 112.47, 114.26, 124.14,
126.37, 146.41, 152.88, 159.16, and 160.75; HR-ESI-TOF-MS: m/z 243.0976
([M+H]+), calcd. for [C15H14O3+H]+ 243.0976.
4-Propargyloxycoumarin (15g)
It was obtained as a light yellow solid (yield = 81%). M. P. 127◦C–130◦C.
IR (KBr) νmax: 3255, 2800, 2098, 1700, 1500, 1269, 1066, and 776 cm−1; 1H
NMR (400 MHz, CDCl3): δ 2.69 (t, 1H, J = 2.20 Hz, C CH), 4.88 (d, 2H,
J = 2.20 Hz, -OCH2C CH), 5.84 (s, 1H, C-3H), 7.27–7.31 (m, 2H, C-6H,
and C-8H), 7.63–7.65 (m, 1H, C-5H), and 7.83 (d, 1H, J = 7.32 Hz, C-5H);
13C NMR (100.6 MHz, CDCl3): δ 65.91, 73.65, 77.00, 87.41, 118.79, 119.75,
126.69, 128.31, 129.04, 149.04, 158.24, and 160.01; HR-ESI-TOF-MS: m/z
201.0507 ([M+H]+), calcd. for [C12H8O3+H]+ 201.0208.
2-Propargyloxy naphthalene (15h)
It was obtained as white solid (yield = 91%). M. P. 70◦C–73◦C. IR (KBr)
νmax: 3245, 2789, 2103, 1449, 1230, 1090, and 782 cm−1; 1H NMR (400 MHz,
CDCl3): δ 2.58 (t, 1H, J = 1.8 Hz, C CH), 4.82 (d, 2H, J = 2.2 Hz, -
OCH2C CH), 7.21–7.26 (m, 2H, C-1H, and C-7H), 7.37–7.41 (m, 2H, C-
4H), 7.46–7.50 (m, 1H, C-5H), 7.78–7.81 (m, 3H, C-2H, C-6H, and C-3H);
13C NMR (100.6 MHz, CDCl3): δ 55.74, 75.64, 78.43, 107.31, 118.68, 123.97,
126.43, 126.88, 1267.62, 129.23 (C-9), 129.54 (C-10), 134.19, and 155.36; HR-
ESI-TOF-MS: m/z 183.0765 ([M+H]+), calcd. for [C13H10O+H]+ 183.0778.
1-Propargyloxy naphthalene (15i)
It was obtained as white solid (yield = 93%). M. P. 69◦C–74◦C. IR
1
(KBr) νmax: 3257, 2809, 2176, 1431, 1271, 1100, and 788 cm−1; H NMR
(400 MHz, CDCl3): δ 2.61 (t, 1H, J = 2.2 Hz, C CH), 4.91 (d, 2H, J =
2.2 Hz, -OCH2C CH), 6.98 (d, 1H, J = 8.1 Hz, C-1H), 7.45 (t, 1H, J =
8.1 Hz, C-2H), 7.54–7.58 (m, 3H, C-5H, C-6H, and C-3H), 7.87–7.89 (m,
1H, C-4H), and 8.37–8.39 (m, 1H, C-7H); 13C NMR (100.6 MHz, CDCl3): δ
55.93, 75.53, 78.52, 105.32, 121.06, 121.92, 125.31, 125.49, 126.42, 127.37,
134.42, and 153.17; HR-ESI-TOF-MS: m/z 183.0765 ([M+H]+), calcd. for
[C13H10O+H]+ 183.0778.

Chemoenzymatic Synthesis of 3ꢁ-Triazolylnucleosides
655
General Procedure for the Synthesis of 3ꢀ-Substituted Triazolylnucleosides
(16a–i)
To a solution of 3ꢁ-azido-3ꢁ-deoxy-5-methyluridine (14, 3.60 mmol) and
alkynes 15a–i (4.32 mmol) in EtOH:H2O:THF (3 mL, 1:1:1), copper (I)
iodide (0.54 mmol) was added. Thereafter, the reaction mixture was stirred
at 60◦C and the reaction was monitored by TLC. On completion of the
reaction the solvent was evaporated under reduced pressure and the crude
product thus obtained was purified by silica gel column chromatography
using methanol/chloroform as eluting solvent to afford the desired products
16a–i.
3ꢀ-Deoxy-3ꢀ-(4-phenyl-1,2,3-triazol-1-yl)-5-methyluridine (16a)
It was obtained as creamy solid (yield = 88%). M. P. 265◦C–269◦C.
IR (KBr) νmax: 3447, 3260, 2375, 1740, 1675, 1465, 1385, 1254, 1102, and
716 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 1.81 (s, 3H, C-5 CH3), 3.38 (brs,
1H, OH), 3.60–3.64 (m, 1H, C-5ꢁHa), 3.76–3.79 (m, 1H, C-5ꢁHb), 4.09–4.16
(m, 2H, OH, and C-4ꢁH), 4.51 (d, 1H, J = 5.12 Hz, C-2ꢁH), 4.69 (s, 1H, C-3ꢁH),
5.99 (s, 1H, C-1ꢁH), 7.37 (t, 1H, J = 7.32 Hz, ArH), 7.45 (t, 2H, J = 7.32 Hz,
ArH), 7.85–7.88 (m, 3H, 2 x ArH and C-6H), 8.64 (s, 1H, C-5ꢁꢁH), 11.38 (s,
1H, NH); 13C NMR (100.6 MHz, DMSO-d6): δ 12.27, 60.61, 60.84, 72.81,
81.30, 86.22, 109.60, 122.74, 125.12, 127.87, 128.95, 130.79, 136.30, 145.92,
150.71, and 163.78; HR-ESI-TOF-MS: m/z 386.1420 ([M+H]+), calcd. for
[C18H19N5O5+H]+ 386.1433.
3ꢀ-Deoxy-3ꢀ-(4-hydroxymethyl-1,2,3-triazol-1-yl)-5-methyluridine (16b)
It was obtained as white solid (yield = 92%). M. P. 196◦C–200◦C. IR
(KBr) νmax: 3381, 3298, 1762, 1750, 1745, 1684, 1473, 1384, 1257, 1106,
1
and 766 cm−1; H NMR (400 MHz, DMSO-d6): δ 1.79 (s, 3H, C-5 CH3),
3.55–3.58 (m, 1H, C-5ꢁHa), 3.72–3.75 (m, 1H, C-5ꢁHb), 4.46–4.52 (m, 4H,
OH, C-4ꢁH, C-2ꢁH, and C-3ꢁH), 4.61 (brs, 1H, OH), 5.19–5.22 (m, 2H, C-4ꢁꢁ
CH2OH), 5.41 (s, 1H, C-4ꢁꢁ CH2OH), 5.95 (s, 1H, C-1ꢁH), 7.86 (s, 1H, C-6H),
7.99 (s, 1H, C-5ꢁꢁH), 11.33 (s, 1H, NH); 13C NMR (100.6 MHz, DMSO-d6): δ
12.28, 55.04, 60.59, 60.67, 72.61, 81.43, 88.36, 109.54, 124.06, 136.29, 147.45,
150.72, and 163.77; HR-ESI-TOF-MS: m/z 340.1212 ([M+H]+), calcd. for
[C13H17N5O6+H]+ 340.1207.
3ꢀ-Deoxy-3ꢀ-[4-(3-chloropropyl)-1,2,3-triazol -1-yl]-5-methyluridine (16c)
It was obtained as white solid (yield = 80%). M. P. 387◦C–390◦C. IR
(KBr) νmax: 3437, 2926, 1743, 1689, 1456, 1272, 1111, and 771 cm−1; 1H NMR
(400 MHz, DMSO-d6): δ 0.83–0.85 (m, 2H, C-4ꢁꢁ CH2CH2CH2), 1.20–1.31
(m, 4H, C-4ꢁꢁ CH2CH2CH2 and C-4ꢁꢁ CH2CH2CH2), 1.79 (s, 3H, C-5 CH3),
3.67–3.76 (m, 2H, C-5ꢁHa and C-5ꢁHb), 4.10–4.12 (m, 1H, C-4ꢁH), 4.45–4.67
(m, 4H, C-2ꢁH, 2 x OH and C-3ꢁH), 5.93 (s, 1H, C-1ꢁH), 7.87 (s, 1H, C-6H),

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A. Arya et al.
8.35 (s, 1H, C-5ꢁꢁH), 11.35 (s, 1H, NH); 13C NMR (100.6 MHz, CDCl3):
δ 14.12, 31.69, 31.74, 49.13, 51.70, 56.25, 60.86, 61.35, 103.65, 111.09,
121.35, 121.59, 135.98, 151.35, and 164.37; HR-ESI-TOF-MS: m/z 387.1123
([M+H]+), calcd. for [C15H20N5O5Cl+H]+ 387.1133.
3ꢀ-Deoxy-3ꢀ-[4-(coumarin-7-yloxymethylene)-1,2,3-triazol-1-yl]-5-methyluridine
(16d)
It was obtained as white solid (yield = 80%). M. P. 180◦C–184◦C. IR
(KBr) νmax: 3404, 2924, 1760, 1742, 1696, 1466, 1389, 1289, 1149, 1073, and
849 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 1.79 (s, 3H, C-5 CH3), 3.58–3.61
(m, 1H, C-5ꢁHa), 3.73–3.76 (m, 1H, C-5ꢁHb), 4.49 (m, 1H, C-4ꢁH), 4.65 (m,
1H, C-3ꢁH), 5.27 (m, 3H, C-4ꢁꢁ CH2 and C-2ꢁH), 5.46 (brs, 1H, OH), 5.97
(d, 1H, J = 3.64 Hz, C-1ꢁH), 6.08 (brs, 1H, OH), 6.29 (d, 1H, J = 9.52 Hz,
C-3ꢁꢁꢁH), 7.02 (d, 1H, J = 8.04 Hz, C-6ꢁꢁꢁH), 7.64 (d, 1H, J = 8.08 Hz, C-8ꢁꢁꢁH),
7.87 (s, 1H, C-6H), 7.99 (d, 1H, J = 9.52 Hz, C-5ꢁꢁꢁH), 8.35 (s, 1H, C-5ꢁꢁH),
11.34 (s, 1H, NH); 13C NMR (100.6 MHz, DMSO-d6): δ 12.98, 60.66, 60.80,
61.57, 72.58, 81,34, 88.35, 101.22, 109.56, 112.46, 113.92, 123.87, 126.26,
126.37, 136.23, 141.52, 146.48, 150.70, 153.06, 160.13, 160.79, and 163.73;
HR-ESI-TOF-MS: m/z 484.1424 ([M+H]+), calcd. for [C22H21N5O8+H]+
484.1300.
3ꢀ-Deoxy-3ꢀ-[4-(4-methylcoumarin-7-yloxymethylene)-1,2,3-triazol-1-yl]-5-
methyluridine (16e)
It was obtained as white solid (yield = 82%). M. P. 234◦C–238◦C. IR
(KBr) νmax: 3419, 2928, 1745, 1715, 1713, 1693, 1477, 1290, 1137, 1013, and
837 cm−1; 1H NMR (400 MHz, DMSO-d6): δ 1.79 (s, 3H, C-5 CH3), 3.57–3.62
(m, 1H, C-5ꢁHa), 3.72–3.76 (m, 1H, C-5ꢁHb), 4.48 (q, 1H, J = 5.12 Hz, C-
4ꢁH), 4.64 (t, 1H, J = 5.84 Hz, C-3ꢁH), 5.22–5.27 (m, 3H, C-4ꢁꢁ CH2 and
C-2ꢁH), 5.38 (t, 1H, J = 4.40 Hz, OH), 5.97 (d, 1H, J = 5.12 Hz, C-1ꢁH),
6.02 (d, 1H, J = 5.12 Hz, C-3ꢁꢁꢁH), 6.21 (s, 1H, OH), 7.04 (dd, 1H, J = 2.16
and 8.76 Hz, C-6ꢁꢁꢁH), 7.16 (d, 1H, J = 2.20 Hz, C-8ꢁꢁꢁH), 7.68 (d, 1H, J =
8.80 Hz, C-5ꢁꢁꢁH), 7.85 (s, 1H, C-6H), 8.29–8.33 (m, 2H, C-5ꢁꢁH and C-4ꢁꢁꢁH),
11.37 (s, 1H, NH); 13C NMR (100.6 MHz, DMSO-d6): δ 12.27, 60.76, 60.99,
61.69, 72.52, 81.43, 88.23, 101.54, 109.62, 112.61, 112.69, 112.90, 126.42,
129.57, 136.30, 141.38, 144.36, 150.75, 155.35, 160.33, 161.22, and 163.78;
HR-ESI-TOF-MS: m/z 498.1580 ([M+H]+), calcd. for [C23H23N5O8+H]+
498.1581.
3ꢀ-Deoxy-3ꢀ-[4-(3-ethyl-4-methylcoumarin-7-yloxymethylene)-1,2,3-triazol-1-yl]-
5-methyluridine (16f)
It was obtained as white solid (yield = 79%). M. P. 240◦C–243◦C. IR (KBr)
νmax: 3397, 2930, 1749, 1684, 1465, 1387, 1257, 1156, 1071, and 780 cm−1; 1H
NMR (400 MHz, DMSO-d6): δ 0.95 (t, 3H, J = 7.68 Hz, -CH2CH3), 1.71 (s,

Chemoenzymatic Synthesis of 3ꢀ-Triazolylnucleosides
657
3H, C-5 CH3), 2.29 (s, 3H, C-4ꢁꢁꢁCH3), 2.46 (q, 2H, J = 7.32 Hz, -CH2CH3),
3.51 (dd, 1H, J = 3.64 and 12.44 Hz, C-5ꢁHa), 3.66 (dd, 1H, J = 3.68 and
11.72 Hz, C-5ꢁHb), 4.41 (q, 1H, J = 5.16 Hz, C-4ꢁH), 4.56 (t, 1H, J = 5.84 Hz,
C-3ꢁH), 5.14–5.19 (m, 3H, C-4ꢁꢁ CH2 and C-2ꢁH), 5.30 (t, 1H, J = 5.16 Hz,
OH), 5.89 (d, 1H, J = 5.16 Hz, C-1ꢁH), 5.93 (d, 1H, J = 5.12 Hz, OH), 6.93
(dd, 1H, J = 2.92 and 8.80 Hz, C-6ꢁꢁꢁH), 7.04 (d, 1H, J = 2.20 Hz, C-8ꢁꢁꢁH), 7.60
(d, 1H, J = 8.80 Hz, C-5ꢁꢁꢁH), 7.77 (s, 1H, C-6H), 8.25 (s, 1H, C-5ꢁꢁH), 11.29
(s, 1H, NH); 13C NMR (100.6 MHz, DMSO-d6): δ 12.27, 12.98, 14.36, 60.80,
61.57, 72.58, 81.34, 88.35, 101.22, 109.56, 112.46, 113.92, 123.87, 126.26,
126.37, 136.23, 141.52, 146.48, 150.70, 153.06, 160.13, 160.79, and 163.74;
HR-ESI-TOF-MS: m/z 526.1893 ([M+H]+), calcd. for [C25H27N5O8+H]+
526.1891.
3ꢀ-Deoxy-3ꢀ-[4-(coumarin-4-yloxymethylene)-1,2,3-triazol-1-yl]-
5-methyluridine (16g)
It was obtained as white solid (yield = 76%). M. P. 206◦C–210◦C. IR (KBr)
νmax: 3370, 2926, 1760, 1742, 1690, 1376, 1240, 1107, and 767 cm−1; 1H NMR
(400 MHz, DMSO-d6): δ 1.79 (s, 3H, C-5 CH3), 3.74–3.78 (m, 3H, C-5ꢁHa,
C-5ꢁHb and OH), 4.49–4.52 (m, 2H, C-4ꢁH and C-3ꢁH), 4.67–4.68 (m, 1H, C-
2ꢁH), 5.27 (t, 1H, J = 6.60 Hz, OH), 5.43 (s, 2H, C-4ꢁꢁ CH2), 5.98 (d, 1H, J =
5.12 Hz, C-1ꢁH), 6.17 (s, 1H, C-3ꢁꢁꢁH), 7.33 (t, 1H, J = 7.32 Hz, C-6ꢁꢁꢁH), 7.39
(d, 1H, J = 8.08 Hz, C-8ꢁꢁꢁH), 7.64 (t, 1H, J = 7.32 Hz, C-7ꢁꢁꢁH), 7.73 (d, 1H,
J = 8.04 Hz, C-5ꢁꢁꢁH), 7.86 (s, 1H, C-6H), 8.43 (s, 1H, C-5ꢁꢁH), 11.38 (s, 1H,
NH); 13C NMR (100.6 MHz, DMSO-d6): δ 12.33, 60.13, 61.77, 62.88, 72.65,
81.42, 88.37, 91.43, 109.68, 115.10, 116.56, 122.97, 124.33, 126.71, 132.93,
136.31, 140.57, 150.79, 152.84, 161.69, 163.84, and 164.48; HR-ESI-TOF-MS:
m/z 484.1424 ([M+H]+), calcd. for [C22H21N5O8+H]+ 484.1475.
3ꢀ-Deoxy-3ꢀ-[4-(naphthyl-2-yloxymethylene)-1,2,3-triazol-1-yl]-
5-methyluridine (16h)
It was obtained as white solid (yield = 85%). M. P. 139◦C–142◦C. IR (KBr)
νmax: 3407, 2927, 1756, 1686, 1459, 1396, 1238, 1098, and 772 cm−1; 1H NMR
(400 MHz, DMSO-d6): δ 1.82 (s, 3H, C-5 CH3), 3.64 (dd, 1H, J = 2.96 and
12.44 Hz, C-5ꢁHa), 3.80 (dd, 1H, J = 3.32 and 11.00 Hz, C-5ꢁHb), 4.54 (q,
1H, J = 5.12 Hz, C-4ꢁH), 4.72 (t, 1H, J = 6.00 Hz, C-3ꢁH), 5.28 (t, 1H, J =
6.60 Hz, C-2ꢁH), 5.38 (s, 2H, C-4ꢁꢁ CH2), 5.42 (t, 1H, J = 5.16 Hz, OH), 6.02
(d, 1H, J = 4.40 Hz, C-1ꢁH), 6.05 (d, 1H, J = 5.16 Hz, OH), 7.22 (d, 1H, J
= 7.32 Hz, C-1ꢁꢁꢁH), 7.44–7.55 (m, 4H, C-8ꢁꢁꢁH, C-5ꢁꢁꢁH, C-6ꢁꢁꢁH, and C-7ꢁꢁꢁH),
7.87–7.90 (m, 2H, C-6H and C-3ꢁꢁꢁH), 8.15 (d, 1H, J = 8.04 Hz, C-4ꢁꢁꢁH),
8.43 (s, 1H, C-5ꢁꢁH), 11.41 (s, 1H, NH); 13C NMR (100.6 MHz, DMSO-d6): δ
12.29, 60.65, 60.75, 61.76, 72.69, 81.33, 88.41, 105.78, 109.54, 120.34, 121.59,
124.88, 125.35, 125.88, 126.20, 126.50, 127.50, 134.06, 136.25, 142.34, 150.71,
153.58, and 164.76; HR-ESI-TOF-MS: m/z 466.1682 ([M+H]+), calcd. for
[C23H23N5O6+H]+ 466.1501.

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3ꢀ-Deoxy-3ꢀ-[4-(naphthyl-1-yloxymethylene)-1,2,3-triazol-1-yl]-
5-methyluridine (16i)
It was obtained as white solid (yield = 84%). M. P. 218◦C–220◦C. IR (KBr)
1
νmax: cm−1; H NMR (400 MHz, DMSO-d6): δ 1.80 (s, 3H, C-5 CH3), 3.61
(dd, 1H, J = 2.96 and 12.44 Hz, C-5ꢁHa), 3.76 (dd, 1H, J = 2.92 and 8.08 Hz,
C-5ꢁHb), 4.50 (q, 1H, J = 5.84 Hz, C-4ꢁH), 4.67 (t, 1H, J = 5.84 Hz, C-3ꢁH),
5.23–5.27 (m, 3H, C-2ꢁH, and C-4ꢁꢁ CH2), 5.40 (t, 1H, J = 5.16 Hz, OH), 5.99
(d, 1H, J = 4.40 Hz, C-1ꢁH), 6.03 (d, 1H, J = 5.12 Hz, OH), 7.20 (dd, 1H, J
= 2.92 and 9.52 Hz, C-2ꢁꢁꢁH), 7.35 (t, 1H, J = 8.08 Hz, C-3ꢁꢁꢁH), 7.47 (t, 1H,
J = 7.32 Hz, C-6ꢁꢁꢁH), 7.53 (d, 1H, J = 2.20 Hz, C-7ꢁꢁꢁH), 7.82–7.85 (m, 3H,
C-4ꢁꢁꢁH, C-5ꢁꢁꢁH, and C-8ꢁꢁꢁH), 7.87 (s, 1H, C-6H), 8.35 (s, 1H, C-5ꢁꢁH), 11.39
(s, 1H, NH); 13C NMR (100.6 MHz, DMSO-d6): δ 12.28, 60.67, 60.78, 61.14,
72.60, 81.38, 88.36, 107.09, 109.56, 118.68, 123.73, 126.08, 126.47, 126.78,
127.55, 128.61, 129.39, 134.23, 136.25, 142.05, 150.71, 156.02, and 163.75;
HR-ESI-TOF-MS: m/z 466.1680 ([M+H]+), calcd. for [C23H23N5O6+H]+
466.1501.
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