Nucleosides and oligonucleotides containing 1,2,3-triazole residues with nucleobase tethers: Synthesis via the azide-alkyne 'click' reaction

Article abstract of DOI:10.1016/j.bmc.2008.08.026

A series of novel 1,2,3-triazole nucleosides linked to DNA nucleobases were prepared via copper(I)-catalyzed 1,3-dipolar cycloaddition of N-9 propargylpurines or N-1 propargylpyrimidines with the tolouyl protected 1-azido-2-deoxyribofuranose 2 followed by

Full text of DOI:10.1016/j.bmc.2008.08.026

Bioorganic & Medicinal Chemistry 16 (2008) 8427–8439
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Nucleosides and oligonucleotides containing 1,2,3-triazole residues with
nucleobase tethers: Synthesis via the azide-alkyne ‘click’ reaction
*
Padmaja Chittepu, Venkata Ramana Sirivolu, Frank Seela
Laboratory of Bioorganic Chemistry and Chemical Biology, Center for Nanotechnology, Heisenbergstrasse 11, 48149 Münster, Germany
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie, Universität Osnabrück, Barbarastrasse 7, 49069 Osnabrück, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel 1,2,3-triazole nucleosides linked to DNA nucleobases were prepared via copper(I)-
catalyzed 1,3-dipolar cycloaddition of N-9 propargylpurines or N-1 propargylpyrimidines with the tol-
ouyl protected 1-azido-2-deoxyribofuranose 2 followed by treatment with NaOMe/MeOH or aq NH3.
The antiviral activity of such compounds against selected RNA viruses is reported. The strongly fluo-
rescent 1,2,3-triazole compounds 16 and 17 were synthesized from propargylated uracil 1a and prop-
argylated adenine 1c with coumarin azide 15, and the fluorescence properties were studied. The
nucleosides 4 and 6 were incorporated into DNA using the phosphoramidite building blocks and
employed in solid-phase synthesis. Melting experiments demonstrated that such 1,2,3-triazole nucleo-
sides have a negative impact on the duplex stability when they are placed opposite to the canonical
bases as well as abasic sites. The nucleobases attached to the triazole ring cannot involve in the base
pair formation with the opposite bases because of the structural variations induced by the triazole
ring. The stacking of such nucleosides when positioned at the end of oligonucleotides retains the sta-
bility of DNA duplexes. The duplex structures were studied by molecular modelling which support the
results of melting experiments.
Received 16 June 2008
Revised 4 August 2008
Accepted 13 August 2008
Available online 15 August 2008
Keywords:
Huisgen–Meldal–Sharpless cycloaddition
Propargylated nucleobases
1,2,3-Triazole nucleosides
Oligonucleotides
Base pairing
Fluorescence
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
1,2,3-triazole nucleosides that are linked to DNA nucleobases as
recognition elements. They were prepared by the copper(I)-cata-
The copper(I)-catalyzed Huisgen–Sharpless–Meldal 1,3-dipolar
cycloaddition (‘click’ chemistry) between alkynes and azides
resulting in the formation of 1,4-disubstituted 1,2,3-triazoles has
gained significant importance because of its wide range of applica-
tions in various fields of drug discovery,1 bioconjugation,2,3 and
material or surface science.4–7 Five-membered triazole nucleosides
are of special interest because of their pronounced biological activ-
ities. Among them, a 1,2,4-triazole ring derivative ribavarin (viraz-
ole) and 1,2,3-triazole TSAO analogues are used for the treatment
of hepatitis C and HIV-1 virus.8–10
lyzed 1,3-dipolar cycloaddition reaction between a terminal C„C
bond of propargylated purine or pyrmidine bases with the azide
moiety of the sugar component 2 (Scheme 1). The antiviral activ-
ity of propargylated bases and 1,2,3-triazole nucleosides is eval-
uated. The 1,2,3-triazole nucleoside conjugates
4 and 6 are
incorporated into DNA by solid-phase synthesis and the duplex
stability was studied.
2. Results and discussion
Various 1,2,3-triazole acyclonucleosides were synthesized
from the propargylated nucleobases11 using the copper free
Huisgen 1,3-dipolar cycloaddition to evaluate their anti-HIV
activity.12 Recently, 2-ethynylfuro[2,3-b]pyrazines were coupled
with various sugars using microwave-assisted ‘click’ reaction to
form 1,2,3-triazole derivatives.13 Moreover, a related class of
modified nucleosides called ‘fleximers’ were developed by Seley
and co-workers. In this class of molecules the nucleobases were
‘split’ but still retained the key recognition sites of DNA bases.14
In this manuscript, we wish to report on the synthesis of novel
2.1. Synthesis of the monomers
The Huisgen 1,3-dipolar cycloaddition15 of alkynes and azides is
used for the synthesis of the nucleobase linked triazole nucleo-
sides. Previously 1,2,3-triazole acyclonucleosides were prepared
via 1,3-dipolar cycloaddition reaction using toluene as solvent at
elevated temperatures (110 °C) yielding a mixture of regioisom-
ers.12 Cu(I) dramatically enhances the rate of reaction and controls
the product formation at room temperature. Thus, the copper as-
sisted cycloaddition proceeds in a chemoselective way with the
formation of 1,4-disubstituted 1,2,3-triazoles as the sole
products.16,17
* Corresponding author. Tel.: +49 251 53406 500; fax: +49 251 53406 587.
E-mail address: Frank.Seela@uni-osnabrueck.de (F. Seela).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.08.026

8428
P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
Base
O
O
N3
N
TolO
TolO
N
"Click" reaction
N
Base
TolO
TolO
1a-e
2
3a-e
a = Uracil, b = Cytosine, c = Adenine, d = 6-Chloro-7-deazapurine,
e = 2-Amino-6-chloro-7-deazapurine
Base
Scheme 1. Copper(I)-catalyzed ‘click’ reaction between propargylated bases and the azido sugar 2.
We decided to explore the feasibility of the ‘click’ chemistry for
was treated with propargyl bromide to form 1c11,12 and d followed
by cycloaddition with the azido sugar 2 thereby forming the pro-
tected nucleosides 3c and d. Then, the protected nucleoside 3c or
d was converted (sodium methoxide or aqueous ammonia in an
autoclave at 90 °C) to produce nucleosides 6 or 9 (Scheme 3). The
etheno nucleoside 7 was prepared from the 1,2,3-triazole nucleo-
side 6, which was reacted with chloroacetaldehyde (pH 4.5–5) to
give the strongly fluorescent N6-etheno derivative 7. The reaction
was performed in a similar way as reported for the synthesis of
the etheno derivatives of adenine and cytosine.21
In addition to the above nucleosides we also demonstrated the
application of ‘click’ reaction in the 7-deazaguanine series using
the 2-amino-6-chloro-7-deazapurine (10)22 as starting material.
This was converted into the N9-propargylated base 1e by using
the same reaction conditions discussed above. Compound 1e
was ligated to the azido sugar 2 to yield the protected nucleoside
3e. The displacement of the 6-chloro group as well as the
deblocking of the toluoyl protecting groups of 3e was performed
with 0.2 M NaOMe/MeOH affording the nucleoside 11, while
deblocking of the protected nucleoside 3e with methanolic
ammonia (saturated at 0 °C at rt) gave compound 13. The 2,6-dia-
the construction of novel 1,2,3-triazole nucleosides with pyrimi-
dine and purine tethers connected via a flexible methylene linker.
At first, the pyrimidine 1,2,3-triazole nucleosides 4 and 5 were syn-
thesized. For this the pyrimidine nucleobases uracil and cytosine
were used as starting materials which were treated with propargyl
bromide in the presence of K2CO3 and DMF11,12 to give N-1 isomers
1a and b exclusively (71% and 75% yield). Next, the terminal C„C
bonds of propargylated nucleobases 1a and b were ligated to the
azide residue of the toluoyl protected 2-deoxyribose 218,19 using
the copper(I)-catalyzed 1,3-dipolar cycloaddition to afford the click
products 3a and b (Scheme 2). The reaction was performed in THF/
H2O/t-BuOH (3:1:1) in the presence of sodium ascorbate and cop-
per sulfate with a little excess of azide at room temperature lead-
ing to the product yields 80–85%. Finally, the sugar protecting
groups of the triazoyl nucleosides 3a and b were removed using
0.2 M NaOMe/MeOH to generate the nucleosides 4 and 5.
In a similar fashion 1,2,3-triazole nucleosides containing the
adenine and 7-deazapurine bases 1c and d at the 4-position of
the triazole moiety were prepared using the above reaction condi-
tions. For that purpose, adenine or 6-chloro-7-deazapurine20 (8)
Scheme 2. Reagents and conditions: (i) CuSO4, Na-ascorbate, THF/H2O/t-BuOH (3:1:1), rt; (ii) 0.2 M NaOMe/MeOH, rt.

P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
8429
NH2
N
NH2
N
N
N
N
N
N
N
N
N
NH2
N
N
N
N
N
N
N
(i)
(ii)
(iii)
2
N
N
N
N
N
N
N
N
N
O
O
O
TolO
HO
HO
1c
TolO
HO
HO
3c
6
7
NH2
N
Cl
N
Cl
N
N
N
N
N
N
Cl
N
(iv)
(i)
(v)
N
N
N
N
2
N
N
N
H
N
N
O
O
TolO
HO
8
1d
3d
9
TolO
HO
Scheme 3. Reagents and conditions: (i) CuSO4, Na-ascorbate, THF/H2O/t-BuOH (3:1:1), rt; (ii) 0.2 M NaOMe/MeOH, rt; (iii) ClCH2CHO, 1 M aq NaOAC buffer (iv) Propargyl
bromide, K2CO3, DMF, rt; (v) 28% NH4OH-dioxane (4:1), 90 °C.
mino nucleoside 12 was obtained from 3e when treated with aq
NH3 (90 °C); the guanosine analogue 14 became accessible from
compound 11 (Scheme 4).
The structure of all compounds was confirmed on the basis of
1H, 13C NMR spectra as well as by elemental analysis. The regiose-
lective formation of the N1/N9 of pyrimidines/purines toward
propargylation was determined on the basis of gated-decoupled
spectra (Table 1). The terminal alkynyl C-atom of the N1/N9 prop-
1
argylated bases 1a and c shows C, H couplings of JC,H = 252 Hz,
(2JC,H) = 51 Hz and the CH2 group of the propargyl side chain shows
a
coupling of (1JC,H) = 147 Hz (Table 2). The propargyl side
chain coupling constants are similar in both cases. The site of
alkylation was determined to be N1/N9 based on the 13C NMR
chemical shifts of related N1/N9 substituted uracil/adenine bases
Cl
OMe
N
N
N
N
N
N
NH2
N
NH2
Cl
Cl
N
(ii)
(i)
(iii)
N
N
N
N
2
N
N
NH2
N
N
N
N
NH2
H
O
O
TolO
HO
10
1e
TolO
HO
3e
11
(vi)
(iv)
(v)
NH2
N
Cl
O
N
NH
NH2
N
N
N
N
NH2
N
N
NH2
N
N
N
N
N
N
N
N
N
O
O
O
HO
HO
HO
HO
HO
HO
14
12
13
Scheme 4. Reagents and conditions: (i) propargyl bromide, K2CO3, DMF, rt; (ii) CuSO4, Na-ascorbate, THF/H2O/t-BuOH (3:1:1), rt; (iii) 0.2 M NaOMe/MeOH, rt; (iv) 28%
NH4OH-dioxane (4:1), 90 °C; (v) NH3/MeOH, rt; (vi) 2 N NaOH, reflux.

8430
P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
Table 1
13C NMR chemical shifts (d) of nucleobases measured in DMSO-d6 at 298 K
Compound
C(2)a
C(2)b
C(4)a
C(5)a
C(6)a
C(4)b
C(7)a
C(5)b
C(8)a
C(6)b
CH2
Triple bonds
C(7a)b
C(4a)b
C„CH
C„CH
1a
150.4
155.2
152.4
152.8
150.3c
150.2c
159.4
159.5
—
—
—
—
163.6
166.0
155.3
156.0
150.3c
150.6c
151.0
151.4
101.7
94.1
—
144.5
144.8
139.3
140.2
128.3
130.8
123.2
125.8
36.6
37.4
—
32.3
—
33.9
—
33.2
78.1
79.5
—
78.4
—
78.3
—
79.0
75.8
75.3
—
76.0
—
76.0
—
75.5
1b
Aded
1c
8
1d
10
1e
151.3
149.1
151.9
150.8c
154.7
153.2
117.6
118.5
116.6
116.8
108.7
108.6
—
98.8
99.1
98.8
99.3
a
Purine numbering.
Systematic numbering.
Tentative.
Adenine.
b
c
d
Table 2
Coupling constants JC,H (Hz) of nucleobases measured in DMSO-d6 at 298 K
C(5)a/C(7)b C(6)a/C(8)b
CH2
CH2AC„CH
(2JC,H
CH2AC„CH
(3JC,H
)
(1JC,H
)
(3JC,H
)
(1JC,H
)
(3JC,H
)
(1JC,H
)
(3JC,H
)
(2JC,H
)
)
(1JC,H
)
1a
1b
1c
1d
1e
175.9
172.4
—
181.9
179.5
2.3
3.7
—
7.3
7.3
182.1
179.7
212.6
190.9
189.7
4.1
3.9
3.5
3.9
3.9
146.7
145.5
146.2
146.3
146.9
3.6
3.7
3.6
3.4
3.5
50.6
50.5
50.9
50.8
50.6
8.8
8.9
9.1
9.1
8.9
252.7
252.0
252.9
252.9
252.4
4.0
4.0
4.0
4.1
4.0
a
Pyrimidine.
Purine.
b
or nucleosides.22–25 According to 1H NMR spectra of the ‘click’
products the terminal triple bonded proton signal (dH = 3.3 ppm)
of the alkynyl base disappeared and the newly formed triazole
was now observed at 8–9 ppm. The triazole ring formation can also
be identified from the 13C-spectra with the new signals of the ole-
finic C-atoms of the 1,2,3-triazole moiety at d(C5 = 122–124 ppm)
and d(C4 = 142–144 ppm). The large difference in the chemical
shifts (dC4–dC5 = 20 ppm) indicates the formation of 1,4-regioi-
somers (Table 3). An unambiguous assignment was performed on
nucleoside 4 on the basis of the 1H/13C heteronuclear long-range
correlations spectra (HMBC). Figure 1 displays the essential part
of the spectrum (complete spectrum not shown). The triazole C5
3
shows JC,H couplings with the anomeric proton C10-H as well as
with the methylene group of the linker. In contrary C4 does not
show any long-range coupling with C10-H confirming the forma-
tion of the 4-substituted triazole moiety.
2.2. Antiviral activity
Triazole nucleosides show pronounced biological activities. The
five-membered 1,2,4-triazole nucleoside ribavarin (virazole) was
the first synthetic nucleoside showing a broad spectrum of antivi-
Table 3
13C NMR chemical shifts (d) of modified nucleosides and their derivatives measured in DMSO-d6 at 298 K
C(2)a
C(2)b
C(4)a
C(5)a
C(6)a
C(4)b
C(7)a
C(5)b
C(8)a
C(6)b
Triazolec
C(10)
C(20)
C(30)
C(40)
C(50)
C(7a)b
C(4a)b
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
3a
4
3b
5
6
7
150.8
150.8
155.7
155.8
152.8
138.5
150.1e
150.2
159.4
159.5
159.7
157.6
152.8
150.8
151.9
151.6
150.8
152.6
—
—
—
—
—
—
—
—
163.8
163.8
166.1
166.2
156.0
141.1
150.4e
156.4
151.3
151.4
163.2
156.1
159.6
163.8
152.4
152.2
162.5
155.9
101.3
101.3
93.8
93.9
—
145.5
145.6
145.9
146.1
140.9
137.0
131.2
124.5
123.2
126.3
122.6
121.0
120.7
145.3
144.9
144.7
145.5
140.7
142.9
142.7
143.6
143.6
142.9
142.8
143.0
143.6
143.6
143.5
144.0
143.9
144.2
142.4
142.6
142.2
142.5
142.7
123.3
122.2
123.5
122.5
122.5
122.3
123.2
122.2
123.2
122.4
122.4
122.2
122.6
123.0
122.7
123.0
123.2
124.5
87.8
88.1e
87.8
88.1e
88.2e
88.1e
87.9
88.0
87.8
88.2e
88.2e
88.0e
88.4e
86.1e
88.4e
87.6e
—
74.6
70.5
74.7
70.7
70.6
70.4
74.6
70.5
74.6
70.6
70.7
70.5
70.8
70.4
70.6
70.4
—
82.3
88.3e
82.3
88.4e
88.4e
88.3e
82.3
88.3
82.3
88.4e
88.4e
88.3e
88.5e
87.5e
88.5e
86.1e
—
63.9
61.6
64.0
61.8
61.7
61.5
63.9
61.6
64.0
61.8
61.8
61.7
62.0
64.0
61.8
64.1
—
149.4
140.7
150.7e
148.9
153.3
153.4
153.9
151.5
150.5
—
118.6
122.6
116.8
102.2
108.6
122.4
98.5
100.0
102.1
—
—
3d
9
98.9
99.6
99.0
99.2
97.2
95.5
100.1
101.3
—
3e
13
11
12
14
20
22
23
16
17
150.3
150.2
—
125.2
125.1
—
—
101.3
—
—
—
149.3
118.6
—
—
—
—
a
b
c
Purine numbering.
Systematic numbering.
Triazole carbons.
Superimposed by the signal of DMSO-d6.
Tentative.
d
e

P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
8431
Figure 1. HMBC spectrum of 4 measured in DMSO-d6.
ral activities against many RNA and DNA viruses.26 This prompted
Table 5
Activity of 1,2,3-triazole nucleosides and propargyl bases against selected ssRNA
viruses in BHK cell lines
us to evaluate the antiviral activity of the related 1,2,3-triazole
nucleosides as well as propargylated bases. Among the tested
viruses, representatives of flavivirus which belong to the class of
positive-sense single-stranded RNA (ss RNA+) viruses were se-
lected. These are Hepacivirus (hepatitis C; HCV), yellow fever virus
(YFV), dengue virus type 2 (DENV-2), and West Nile virus (WNV).
Unfortunately, none of the compounds show selective antiviral
activity against the tested ssRNA+ viruses. First, regarding the
activity in the HCV replicon system a series of compounds were se-
lected for testing (Table 4). Their ability to inhibit the HCV replicon
Compound
YFVa
% Inhib.b
DENVa
WNVa
% Inhib.b
% Tox.c
% Inhib.b
% Tox.c
% Tox.c
3a
4
3b
5
3e
1a
1d
6
8.56
4.59
2.03
ꢀ1.14
ꢀ4.57
ꢀ1.98
ꢀ4.62
2.38
0.82
0.75
3.37
9.5
ꢀ2.84
ꢀ2.67
ꢀ3.67
0.24
ꢀ1.44
ꢀ4.57
3.81
ꢀ0.43
ꢀ2.44
ꢀ1.16
ꢀ2.95
2.42
3.37
9.5
ꢀ2.26
4.08
9.41
0.17
2.76
ꢀ9.83
7.85
ꢀ2.95
5.54
0.31
3.14
1.3
4.08
9.41
0.17
2.76
ꢀ9.83
7.85
ꢀ2.95
5.54
0.31
3.14
1.3
7.89
0.95
ꢀ6.17
ꢀ11.35
ꢀ0.25
8.36
4.39
8.76
ꢀ2.39
ꢀ4.6
ꢀ0.85
1.02
2.23
1.02
0.08
ꢀ19.45
ꢀ2.12
ꢀ11.67
ꢀ2.91
ꢀ1.39
4.48
replications at the test concentration of 15
HCV replicon assay (ELISA) as previously described.27 The cellular
toxicity was determined at the test concentration of 15 M of drug
lM was evaluated by an
7
22
3d
9
13
1c
1e
l
0.82
ꢀ1.05
in GS4.1 cells. For all compounds inhibition of the HCV replicon
replication is accompanied by cytotoxicity. Only the toluoyl pro-
tected 7-deazaadenine and 7-deazaguanine nucleosides (3d and
e) as well as its propargylated bases 1d and e develop less cytotox-
icity than other triazole compounds in the HCV replicon system.
Next, these compounds were tested against YFY, DENV, WNV mea-
sured by the inhibition of virus-induced cytopathogenicity in
10.98
13.33
ꢀ2.81
ꢀ3.21
3.43
8.55
2.49
5.71
ꢀ7.46
ꢀ3.4
3.8
ꢀ2.0
ꢀ1.77
3.8
ꢀ2.0
0.72
1.56
0.07
a
Flavivirus.
b
The observed inhibition against Flavivirus at the test concentration 15 lM of
drug in acutely infected BHK cell lines as determined by CPE (cytopathic effect)
assay.
c
The observed cellular toxicity at the test concentration of 15 lM of drug in BHK
cell lines.
Table 4
Activity of 1,2,3-triazole nucleosides and propargyl bases in the HCVa replicon system
Compound
HCV Replicon
Compound
HCV Replicon
% Inhib.b
% Tox.c
% Inhib.b
% Tox.c
acutely infected baby hamster kidney (BHK-21) cells. The data
are summarized in Table 5. However, in all the compounds the
antiviral activity is accompanied by cytotoxicity for the host cell
lines (BHK-21) within the same concentration range.
3a
4
3b
5
3e
1a
1d
6
11.32
0.04
ꢀ4.12
ꢀ5.59
ꢀ8.12
ꢀ5.59
6.49
7
6.07
9.02
49.25
ꢀ3.8
2.93
2.99
44.82
—
ꢀ2.24
ꢀ3.55
20.77
ꢀ3.79
ꢀ3.3
ꢀ10.4
1.35
22
3d
9
13
1c
1e
—
15.64
10.08
32.06
12.09
69.22
7.54
2.3. Fluorescence properties of 1,2,3-triazole coumarin
conjugates bearing nucleobase tethers
ꢀ8.85
1.84
ꢀ0.61
—
Earlier, the copper(I)-catalyzed ‘click’ reaction has been used to
introduce various reporter groups into DNA.28,29 Our laboratory
has reported on the fluorescence properties of four DNA octadiynyl
nucleosides attached with a coumarin dye.30,31 Coumarin dyes are
a
Hepacivirus.
b
The observed inhibition of HCV replicon replication at the test concentration of
15
lM of drug as determined by the HCV replicon (ELISA) assay.
c
The observed cellular toxicity at the test concentration of 15
lM of drug.

8432
P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
O
N
O
HN
HN
O
O
O
N
O
(i)
1a
N
N
OH
N
O
O
OH
16
N3
15
NH2
N
3-azido-7-hydroxycoumarin
N
N
N
(i)
O
O
NH2
N
N
N
N
OH
N
N
N
17
1c
Non-Fluorescent
Fluorescent
Scheme 5. Reagents and conditions: (i) CuSO4, Na-ascorbate, THF/H2O/t-BuOH (3:1:1), rt.
widely used because of their favourable photophysical properties.
They are utilized in the blue spectra region with strong emission
intensities and applied as nucleobase specific quenchers to deter-
mine structural dynamics of DNA.32 From the previous reports it
can be concluded that a dye attached to a nucleobase via a rigid lin-
ker (N3-propargylated 20-dU) enhances the fluorescence of nucleo-
sides compared to more flexible linker units (5-octadiynyl-20dU).33
In order to test this phenomenon at the nucleobase level we report
on the fluorescence properties of nucleobase-coumarin conjugates.
The 1,2,3-triazole ring formed from the terminal alkyne and azido
coumarin was found to generate fluorescence.34 In order to study
the fluorescence behavior of dye molecule when attached to ade-
nine and uracil bases via a short linker a series of compounds were
synthesized. First, the uracil base 1a containing a N1-terminal
alkynyl group was selectively conjugated to the azide residue of
coumarin azide 15 to form fluorescent 1,2,3-triazolyl nucleobase-
dye conjugate 16. Next, the terminal triple bond of adenine 1c
was conjugated with the non-fluorescent coumarin azide 15, lead-
ing to the formation of adduct 17 in 78% yield (Scheme 5). The
‘click’ functionalized compounds were identified by 1H, 13C NMR,
gated-decoupled spectra as well as by mass spectra. The 1H NMR
spectra of click products revealed the disappearance of two termi-
nal C„C hydrogens (singlet at 3.23 ppm); two new singlets ap-
peared at a range of d 8–9 ppm attributed to the hydrogens from
the newly formed triazole rings. The characteristic signals of the
olefinic carbon atoms of the newly formed 1,2,3-triazole moiety
were identified in the range of d(C) 123.2 and 142.5 ppm (Table
3). Moreover, the chemical shifts of the triazole ring carbons are
clearly evidenced by 1H, 13C, DEPT-135 NMR spectra. The DEPT-
135 NMR spectra show no signals for the quaternary carbons
(dC4) and an inverted signal for the triazole carbon (dC5) at around
125.4 ppm.
Previous reports have shown that the fluorescence of coumarin
derivatives is pH dependent, different absorption bands are ob-
served at different pH values due to the presence of neutral and
phenolate anionic species. Alkaline pH leads to the predominant
existence of phenolate anion.35 The fluorescence spectrum of ura-
cil-dye compound 16 shows an emission at 476 nm when excited
at 346 nm at pH 7.0, Tris–HCl buffer (Fig. 2). Similar emission
and excitation wavelengths were found for the adenine dye conju-
Figure 2. Fluorescence emission and excitation spectra of compounds 16 (a) and 17 (b) and 1:1 mixture of 16 + 17 (c) at an equimolar concentration (2 ꢁ 10-5 M) measured in
pH 7.0 and pH 8.5 Tris–HCl buffer.

P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
8433
gate 17 (Fig. 2b). Mixing an equal amount of A–U dye conjugates
(16 and 17) does not lead to any fluorescence quenching, thus base
pair formation seems not to occur. Similar results were observed
when the above experiment was performed in pH 8.5 Tris–HCl
buffer, but the excitation was shifted bathochromically to
394 nm with an identical emission (476 nm), which revealed the
existence of phenolate anionic species of coumarin. According to
previous reports the fluorescence properties of nucleobase-couma-
rin pairs are strongly influenced by the length of the side chain
between nucleobase and dye resulting in strong interactions
between the residues.36,33 From the above experiments we conclude
that 1,2,3-triazolyl dye conjugates 16 and 17 employing a rigid short
chain do not lead to significant fluorescence quenching.
which possess strong fluorescence properties.39 So, the photophys-
ical properties of the click product 7 were studied and compared
with N6-ethenoadenosine. Compound 7 shows an emission at
409 nm and an excitation observed at 292 nm with a stoke shift
of 118 nm (Fig. 4). A similar emission is observed for the related
N6-ethenoadenosine derivative.39 In order to understand the influ-
ence of the triazole ring on the fluorescence, the quantum yield of
the nucleoside 7 was determined under neutral conditions in
water. The quantum yield (U) of the 1,2,3-triazole etheno nucleo-
side 7 was found to be 0.51, which is almost similar to that of N6-
ethenoadenosine (0.52).39 From Figure 3A and B both etheno deriv-
atives show similar/comparable fluorescence.
In order to compare the fluorescence properties of the dye at-
tached to the nucleobases as well as nucleosides, compounds 16,
18, and 1933 were chosen. The signal intensity observed in the fluo-
rescence spectra of the dye conjugates is in the order of
(16 > 18 > 19) with the lowest fluorescence observed for the flexi-
ble linker side chain conjugate 19 (Fig. 3). The emission maximum
for 18 and 19 is found to be almost identical with a 476 nm emis-
sion when excited at 393 nm at pH 8.5. Compared to the nucleo-
side dye conjugates 18 and 19, the base conjugate 16 shows the
highest fluorescence due to the placement of dye at N1 position
2.4. Synthesis of phosphoramidites and properties of
oligonucleotides
Next, the influence of 1,2,3-triazolyl modified nucleosides 4 and
6 containing uracil and adenine nucleobases on the stability of
DNA was studied. For this various oligonucleotides were prepared.
The amino group of 6 was protected with the benzoyl residue using
the protocol of transient protection.40 Then, the pyrimidine nucle-
oside 4 and protected purine compound 22 were converted into
the DMTr-derivatives 20 and 23, and further transformed into
the phosphoramidite building blocks 21 and 24 under standard
conditions (Scheme 6). The structure of all compounds were char-
acterized by 1H, 13C (see Table 3), and 31P NMR spectroscopy as
well as by elemental analysis.
of the base moiety. Moreover, an increase of the
p-system by an
electronic coupling between the dye and the nucleobase via the
linker moiety can make a favorable contribution.
Next, similar experiments were performed with the etheno
nucleosides. The fluorescence properties of etheno nucleosides37
enable them to be used as structural and functional probes in
DNA and RNA.38 Our laboratory has reported on different 7-dea-
zapurine, 8-azapurine, and 2,8-dideazapurine etheno nucleosides,
Oligonucleotide synthesis was carried out on solid phase with
an ABI 392-08 synthesizer at a 1 lmol scale employing the synthe-
sized phosphoramidites 21 and 24 as well as standard building
blocks. The coupling yields were always higher than 95%. The
Figure 3. Fluorescence excitation and emission spectra of compounds 16, 18, and 19 at an equimolar concentration (2 ꢁ 10ꢀ5 M) measured in 0.1 M Tris–HCl buffer (pH 8.5).
Figure 4. Steady-state excitation and emission spectra of nucleoside 7 (A), and N6-ethenoadenosine (B) at an equimolar concentration (2.8 ꢁ 10-5 M) measured in water at
room temperature.

8434
P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
O
O
N
HN
HN
O
N
O
(i)
(ii)
N
N
4
N
N
N
N
O
O
DMTrO
DMTrO
HO
O
P
20
NCCH2CH2O N(i-Pr)2
21
NHBz
NHBz
NHBz
N
N
N
N
N
N
N
N
N
N
N
N
(iii)
(i)
(ii)
6
N
N
N
N
N
N
N
N
N
O
O
O
HO
DMTrO
DMTrO
HO
HO
O
P
22
23
NCCH2CH2O N(i-Pr)2
24
Scheme 6. Reagents and conditions: (i) DMTr-Cl, pyridine, rt; (ii) i-Pr2NP(Cl)OCH2CH2CN, i-Pr2EtN, CH2Cl2, 30 min, rt; (iii) Me3SiCl, benzoyl chloride, pyridine, 2 h, rt.
Table 7
synthesis of oligonucleotides was performed by employing the
DMT-on mode. After cleavage from the solid support, the oligo-
mers were deprotected in 25% aqueous ammonia solution for
14–16 h at 60 °C. The purification of the 50-dimethoxytritylated
oligomers was carried out by reversed-phase HPLC (see Section
4). The molecular masses of the oligonucleotides were determined
by Applied Biosystems Voyager DE PRO with 3-hydroxypicolinic
acid (3-HPA) as a matrix. The detected masses were close to the
calculated values (see Section 4, Table 7).
Molecular masses of oligonucleotides measured by MALDI-TOF mass spectrometry
Oligonucleotide
Mass (calcd)
Mass (found)
50-d(TTT TT4 TTT TTT) 27
50-d(AAA AAA 6AA AAA) 28
50-d(ATAT6T6T6TAT) 30
50-d(4T23) 33
50-d(A236) 34
50-d(TTT TTX TTT TTT) 35
50-d(AAA AAA XAA AAA) 36
3655.4
3777.6
3885.7
7305.8
7536.2
3464.3
3563.4
3655.0
3777.1
3886.4
7306.7
7536.3
3464.7
3563.5
Hybridization experiments were performed using the oligonu-
cleotide duplex 50-d(TTT TTT TTT TTT)-30 (25) and 30-d(AAA AAA
AAA AAA) (26) as a reference (see Table 6). One incorporation of
1,2,3-triazole compound 4 replacing dT in the standard oligomer
25 results in a Tm decrease of 12 °C when compared to the standard
duplex. A similar result was observed when dA was replaced by 6
in the standard sequence, but this decrease is less when compared
to that of 4. When both nucleosides 4 and 6 are placed opposite to
each other the Tm decreases by 27 °C. These results clearly indicate
that such 1,2,3-triazole nucleosides with nucleobase tethers when
placed in DNA cannot form base pairs because the methylene
linked nucleobases are flexible and not stacked. They do not show
any base pairing. These results prompted us to evaluate the effect
of single and multiple incorporations of 1,2,3-triazole nucleosides
in different sequence motifs. For that purpose a self-complemen-
tary sequence 29 was used which shows a Tm value of 36 °C.
Replacing three dA’s with 6 (30) results in a Tm of <10 °C. However,
when compounds 4 and 6 are located at the 50-end (33ꢂ32), 30-end
(34ꢂ31), or placed opposite to each other (33ꢂ34) their stability is
retained. These results imply that the terminal 1,2,3-triazole
nucleosides 4 and 6 develop stacking interaction when they are
positioned at the ends without disturbing the duplex structure.
Table 6
Tm values of oligonucleotide duplexes containing 1,2,3-triazole nucleosidesa
Duplex
Tm [°C]
Duplex
Tm [°C]
50-d(TTT TTT TTT TTT) 25
30-d(AAA AAA AAA AAA) 26
50-d(TTT TTT TTT TTT) 25
30-d(AAA AA6 AAA AAA) 28
50-[d(ATATATATATAT)]2 29
50-d(T24) 31
44
50-d(TTT TT4 TTT TTT) 27
30-d(AAA AAA AAA AAA) 26
50-d(TTT TT4 TTT TTT) 27
30-d(AAA AA6 AAA AAA) 28
50-[d(ATAT6T6T6TAT)]2 30
50-d(4T23) 33
32
35
17
36
67
<10
67
30-d(A24) 32
30-d(A24) 32
50-d(A236) 34
30-d(T24) 31
50-d(TTT TTX TTT TTT) 35
30-d(AAA AAA AAA AAA) 26
50-d(TTT TTT TTT TTT) 25
30-d(AAA AAX AAA AAA) 36
67
33
36
50-d(4T23) 33
30-d(6A23) 34
50-d(TTT TTX TTT TTT) 35
30-d(AAA AA6 AAA AAA) 28
50-d(TTT TT4 TTT TTT) 27
30-d(AAA AAX AAA AAA) 36
67
19
14
a
Measured at 260 nm in 1 M NaCl, 100 mM MgCl2 and 60 mM Na-cacodylate (pH
M single-strand concentration. X = abasic site.
7.0) with 5
l

P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
8435
Figure 5. Molecular models (A) of non-self complementary duplex 50-d(TTT TT4 TTT TTT)ꢂ30-d(AAA AAA AAA AAA) (27ꢂ26) (B) duplex 50-d(TTT TT4 TTT TTT)ꢂ30-d(AAA AA6
AAA AAA) (27ꢂ28) (C) duplex 50-d(TTT TTX TTT TTT)ꢂ30-d(AAA AA6 AAA AAA) (35ꢂ28). The models were constructed using Hyperchem 7.0/8.0 and energy minimized using the
MM+ calculations.
To give more space for the nucleoside conjugates 4 and 6 in the
DNA duplex, an abasic site was introduced into the standard se-
quence 25ꢂ26 opposite to them. A single incorporation of abasic
site replacing dT gives a Tm of 33 °C against dA and 36 °C against
dT. Similar values were observed for the nucleosides 4 (27ꢂ26)
and 6 (25ꢂ28) when they are placed at same position as that of aba-
sic site. When an abasic site is placed opposite to the 1,2,3-triazole
nucleosides, even a two times higher duplex destabilization was
observed. These results demonstrate that tethered 1,2,3-triazole
nucleosides behave like abasic sites. On the basis of this informa-
tion, the energy minimized molecular models of DNA duplexes
containing triazole nucleosides 4 and 6 were constructed using
MM+ force field by Hyperchem 7.0/8.0 program (Hypercube Inc.,
Gainesville, FL, USA, 2001). The duplexes 27ꢂ26, 27ꢂ28, and 35ꢂ28
were built as B-type DNA and are shown in Figure 4. According
to the models, the nucleobases of 4 and 6 are arranged perpendic-
ular to the triazole moiety and the methylene bridge forces the
base moiety apart, disrupting hydrogen bond formation with the
complementary bases leading to the destabilization (Figs. 5A–C).
The 1,2,3-triazole moiety cannot take part in stacking interactions
with adjacent bases even though they are well accommodated in
the double helix. The stacking of triazole moieties was recently
reported to be important for the thermal stabilization of
duplexes.41
behaving as an abasic site, with a destabilizing effect on the DNA
duplexes; when they are placed at the terminus of a duplex stabil-
ity is retained.
4. Experimental
4.1. General
4.1.1. Monomers
All chemicals were purchased from Acros, Aldrich, Sigma, or
Fluka (Sigma–Aldrich Chemie GmbH, Deisenhofen, Germany). Sol-
vents were of laboratory grade. Thin-layer chromatography (TLC):
aluminum sheets, silica gel 60 F254, 0.2 mm layer (VWR Interna-
tional, Darmstadt, Germany). Column flash chromatography (FC):
silica gel 60 (VWR International, Darmstadt, Germany) at 0.4 bar;
Sample collection with an UltroRac II fractions collector (LKB
Instruments, Sweden). UV spectra: U-3200 spectrometer (Hitachi,
Tokyo, Japan); kmax (e) in nm. NMR Spectra: Avance-250 or
Avance-300 spectrometers (Bruker, Karlsruhe, Germany), at
250.13 or 300.15 MHz for 1H and 13C; d in ppm relative to Me4Si
as internal standard or external 85% H3PO4 for 31P. The J values
in Hz. Elemental analyses were performed by Mikroanalytisches
Laboratorium Beller (Göttingen, Germany). Electron spray ioniza-
tion (ESI) MS for the nucleosides: Bruker-Daltonics-MicroTOF spec-
trometer with loop injection (Bremen, Germany).
3. Conclusions
4.1.2. Fluorescence measurements
Various alkynyl nucleobases of pyrimidines, purines, or 7-dea-
zapurines were synthesized and utilized as starting materials in
the ‘click’ reaction to attach azido residues. We accomplished a
convenient methodology for the synthesis of novel 1,2,3-triazole
nucleosides using the Cu(I)-catalyzed Huisgen–Sharpless–Meldal
alkyne-azide cycloaddition reaction. This reaction has an advan-
tage over high temperature cycloaddition, which resulted in the
formation of a mixture of regioisomers. The antiviral activity of
such 1,2,3-triazole compounds was studied. Moreover, alkynyl
bases were conjugated with azido coumarin yielding strongly fluo-
rescent 1,2,3-triazole conjugates. These rigid short chain 1,2,3-
triazolyl compounds are considered as useful fluorescent probes
because of the absence of interactions between the dye and nucle-
obase. The nucleosides 4 and 6 were converted into their phospho-
ramidite building blocks and incorporated into DNA using solid-
phase oligonucleotide synthesis. These nucleosides are almost
All measurements were done in double-distilled water at 20 °C.
Absorption spectra were measured with a Cary 100 Bio UV–visible
spectrophotometer. In order to avoid inner filter effects the sample
was not allowed to exceed 0.1 at the excitation wavelength using
standard quartz cuvettes with a pathlength of 1 cm. Fluorescence
spectra were recorded in the wavelength range between 320 and
600 nm using the Fluorescence Spectrophotometer F-2500 (Hit-
achi, Tokyo, Japan). For all calculations the water background
was subtracted from the sample. The fluorescence quantum yields
were determined using quinine sulfate in 0.1 N H2SO4 (fluores-
cence quantum yield 0.53)42 as a standard with the following
relation: Uf:sample
¼ Uf:standard ꢁ ðFsample=FstandardÞ ꢁ ðAstandard=AsampleÞ,
where Uf.sample is the unknown fluorescence quantum yield of
the fluorophore, F is the integrated fluorescence intensity; A is
the absorbance in 1 cm cuvettes and always not exceeds 0.1 at
and above the excitation wavelength.

8436
P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
4.1.3. Synthesis, purification, and characterization of
oligonucleotides
The oligonucleotide synthesis was performed on a DNA synthe-
sizer, model ABI 392-08 (Applied Biosystems, Weiterstadt, Ger-
zone yielded 4 as a colorless solid (220 mg, 86%). TLC: Rf (CH2Cl2/
MeOH, 9:1): 0.31. UV (MeOH): kmax 264 (10,300). 1H NMR
(DMSO-d6): 2.30–2.39 (m, 1H, H -C(20)); 2.54–2.64 (m, 1H, Hb-
a
C(20)); 3.46 (m, 2H, H-C(50)); 3.85 (m, 1H, H-C(40)); 4.36 (m, 1H,
H-C(30)); 4.89 (t, J = 5.3 Hz, 1H, OH-C(50)); 4.94 (s, 2H, CH2); 5.34
(d, J = 4.17 Hz, 1H, OH-C(30)); 5.57 (d, J = 7.7 Hz, 1H, H-C(5)); 6.34
(t, J = 6.1 Hz, 1H, H-C(10)); 7.73 (d, J = 7.8 Hz, 1H, H-C(6)); 8.25 (s,
many) at 1-lmol scale using the phosphoramidites 21 and 24
following the synthesis protocol for 30-cyanoethyl phosphorami-
dites (user’s manual for the 392 DNA sythesizer, Applied Biosys-
tems, Weiterstadt, Germany). The coupling efficiency was always
higher than 95%. After cleavage from the solid support, the oligo-
nucleotides were deprotected with 25% aqueous NH3 for 14–16 h
at 60 °C (during this process the amine protecting groups of the
adenine with tBPA (4-tert-butylphenoxy)acetyl protecting group
were removed. The purification of the 50-O-(dimethoxytrityl)oligo-
mers was carried out on reversed-phase HPLC (Merck-Hitachi-
HPLC: 250 ꢁ 4 mm RP-18 column with the following gradient sys-
tem [A, 0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5; B, MeCN]; gradi-
ent: 3 min 20% B in A, 12 min 20–50% B in A, and 25 min 20% B
in A with a flow rate of 1 mL/min. The purified ‘trityl-on’ oligonu-
cleotides were treated with 2.5% dichloroacetic acid in CH2Cl2 for
5 min at 0 °C to remove the 4,40-dimethoxytrityl residues. The det-
ritylated oligomers were purified again by reversed-phase HPLC
(gradient: 0–20 min 0–20% B in A, flow rate 1 mL/min). The oligo-
mers were desalted on a short column (RP-18, silica gel) and lyoph-
ilized on a Speed-Vac evaporator to yield colorless solids which
were frozen at ꢀ24 °C.
1H, C@CH); 11.31 (s, 1H, NH). Anal. Calcd for
C12H15N5O5
(309.11): C, 46.60; H, 4.89; N, 22.64. Found: C, 46.71; H, 4.83; N,
22.70.
4.1.6. 2-[(2-Deoxy-3,5-di-O-p-toluyl-b-D-erythro-
pentofuranosyl)-1,2,3-triazol-4-yl-methyl]cytosine (3b)
To a mixture of N-1-propargylcytosine 1b (0.4 g, 2.68 mmol)
and b-azido-2-deoxyribose 2 (0.83 g, 2.1 mmol) in THF/H2O/t-
BuOH, 3:1:1, (36 mL) was added sodium ascorbate (1.1 mL,
1.09 mmol) of freshly prepared 1 M solution in water, followed
by the addition of copper (II) sulfate pentahydrate 7.5% in water
(0.8 mL, 0.24 mmol). The heterogeneous reaction mixture was stir-
red for 6 h at rt and then evaporated and applied to FC (silica gel,
column 3 ꢁ 10 cm, CH2Cl2/MeOH 9:1), which gave compound 3b
(1.14 g, 78%) as a colorless foam. TLC: Rf (CH2Cl2/MeOH, 94:6):
0.34. UV (MeOH): kmax 240 (38,000), 270 (10,000). 1H NMR
(DMSO-d6): 2.37–2.39 (m, 6H, 2CH3); 2.75–2.85 (m, 1H, H -
a
C(20)); 3.09–3.20 (m, 1H, Hb-C(20)); 4.38–4.59 (m, 3H, H-C(50), H-
C(40)); 4.89 (s, 2H, CH2); 5.65 (d, J = 6.7 Hz, 1H, H-C(5)); 5.75 (m,
1H, H-C(30)); 6.59 (‘t’, J = 6.4 Hz, 1H, H-C(10)); 7.04 (br s, 2H,
NH2); 7.70 (d, J = 7.2 Hz, 1H, H-C(6)); 7.20–7.94 (m, 8H, arom. H);
8.25 (s, 1H, C@CH). Anal. Calcd for C28H28N6O6 (544.21): C, 61.76;
H, 5.18; N, 15.43. Found: C, 61.68; H, 5.26; N, 15.35.
Melting curves were measured with a Cary-1/3 UV/vis spectro-
photometer (Varian, Australia) equipped with a Cary thermoelec-
trical controller. The temperature was measured continuously in
the reference cell with a Pt-100 resistor, and the thermodynamic
data of duplex formation were calculated by the Meltwin 3.0 pro-
gram. Cary 100 Bio UV–vis spectrophotometer was used for the
UV-melting curves of oligonucleotides (Table 6) with a heating rate
of 1 °C per min.
4.1.7. 2-[(2-Deoxy-b-D-erythro-pentofuranosyl)-1,2,3-triazol-4-
yl-methyl]cytosine (5)
The molecular masses of the oligonucleotides were determined
in negative mode by Applied Biosystems Voyager DE PRO spec-
trometer with 3-hydroxypicolinic acid (3-HPA) as a matrix. The de-
tected masses were identical to the calculated values (Table 7).
Extinction coefficients e260 of the nucleosides: dA 15400 (H2O),
dT 8800 (H2O), 4 10,200 (MeOH), 6 15,000 (MeOH).
A solution of compound 3b (550 mg, 1.01 mmol) in 0.2 M
NaOMe/MeOH (40 mL) was stirred at rt overnight. The clear solu-
tion was evaporated, and the residue was applied to FC (silica
gel, column 4 ꢁ 16 cm, CH2Cl2/MeOH, 85:15). After evaporation,
the main zone yielded 5 as a colorless solid (270 mg, 87%). TLC:
Rf (CH2Cl2/MeOH, 85:15): 0.14. UV (MeOH): kmax 274 (8000). 1H
NMR (DMSO-d6): 2.30–2.38 (m, 1H, H -C(20)); 2.54–2.64 (m, 1H,
a
4.1.4. 2-[(2-Deoxy-3,5-di-O-p-toluyl-b-
pentofuranosyl)-1,2,3-triazol-4-yl- methyl]uracil (3a)
D
-erythro-
Hb-C(20)); 3.46 (m, 2H, H-C(50)); 3.85 (m, 1H, H-C(40)); 4.36 (m,
1H, H-C(30)); 4.88–4.91 (m, 3H, OH-C(50), CH2); 5.34 (d, J = 4.3 Hz,
1H, OH-C(30)); 5.68 (d, J = 7.2 Hz, 1H, H-C(5)); 6.35 (‘t’, J = 6.3 Hz,
1H, H-C(10)); 7.03 (br s, 2H, NH2); 7.69 (d, J = 7.2 Hz, 1H, H-C(6));
8.17 (s, 1H, C@CH). Anal. Calcd for C12H16N6O4 (308.12): C, 46.75;
H, 5.23; N, 27.26. Found: C, 46.78; H, 5.20; N, 27.09.
To a mixture of N-1-propargyluracil 1a (0.22 g, 1.46 mmol) and
b-azido-2-deoxyribose 2 (0.5 g,1.26 mmol) in THF/H2O/t-BuOH,
3:1:1, (16 mL) was added sodium ascorbate (0.68 mL, 0.67 mmol)
of a freshly prepared 1 M solution in water, followed by the addi-
tion of copper (II) sulfate pentahydrate 7.5% in water (0.54 mL,
0.162 mmol). The heterogeneous reaction mixture was stirred for
15 h at rt and then evaporated and applied to FC (silica gel, column
3 ꢁ 10 cm, CH2Cl2/MeOH, 98:2), which gave compound 3a (0.61 g,
76%) as a colorless foam. TLC: Rf (CH2Cl2/MeOH, 98:2): 0.25. UV
(MeOH): kmax 241 (34,000), 271 (11,000). 1H NMR (DMSO-d6):
4.1.8. 7-(2-Deoxy-b-
yl-methyl]adenine (6)
To a mixture of N-9-propargyladenine 1c (0.17 g, 1.01 mmol)
and b-azido-2-deoxyribose (0.5 g,1.26 mmol) in THF/H2O/t-
BuOH, 3:1:1, (16 mL) was added sodium ascorbate (680 L,
D-erythro-pentofuranosyl)-1,2,3-triazol-4-
2
l
2.36–2.39 (m, 6H, 2CH3); 2.77–2.87 (m, 1H, H -C(20)); 3.10–3.21
0.67 mmol) of freshly prepared 1 M solution in water, followed
a
(m, 1H, Hb-C(20)); 4.37–4.63 (m, 3H, H-C(50), H-C(40)); 4.94 (s, 2H,
CH2); 5.56 (d, J = 7.8 Hz, 1H, H-C(5)); 5.75 (m, 1H, H-C(30)); 6.59
(‘t’, J = 6.2 Hz, 1H, H-C(10)); 7.72 (d, J = 7.9 Hz, 1H, H-C(6)); 7.27–
7.99 (m, 8H, arom. H); 8.33 (s, 1H, C@CH); 11.33 (s, 1 H, NH). Anal.
Calcd for C28H27N5O7 (545.19): C, 61.64; H, 4.99; N, 12.84. Found:
C, 61.76; H, 5.10; N, 12.76.
by the addition of copper (II) sulfate pentahydrate 7.5% in water
(540 lL, 0.16 mmol). The heterogeneous reaction mixture was stir-
red for 48 h at rt, then evaporated and the product was used di-
rectly without further purification. A solution of above crude
compound in 0.2 M NaOMe/MeOH was stirred at rt overnight.
The clear solution was evaporated, and the residue was applied
onto FC (silica gel, column 4 ꢁ 16 cm, CH2Cl2/MeOH, 85:15). After
4.1.5. 2-[(2-Deoxy-b-
D-erythro-pentofuranosyl)-1,2,3-triazol-4-
evaporation, the main zone yielded 6 as a colorless solid
yl-methyl]uracil (4)
(220 mg, 67%). TLC: Rf (CH2Cl2/MeOH, 85:15): 0.12. UV (MeOH):
A solution of compound 3a (450 mg, 0.82 mmol) in 0.2 M
NaOMe/MeOH (30 mL) was stirred at rt overnight. The solution
was evaporated, and the residue was applied to FC (silica gel, col-
umn 4 ꢁ 16 cm, CH2Cl2/MeOH, 85:15). After evaporation, the main
kmax 261 (15,000). 1H NMR (DMSO-d6): 2.28–2.38 (m, 1H, H -
a
C(20)); 2.52–2.62 (m, 1H, Hb-C(20)); 3.45 (m, 2H, H-C(50)); 3.85
(m, 1H, H-C(40)); 4.35 (m, 1H, H-C(30)); 4.86 (t, J = 5.4 Hz, 1H, OH-
C(50)); 5.34 (d, J = 4.32 Hz, 1H, OH-C(30)); 5.43 (s, 2H, CH2); 6.32

P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
8437
(‘t’, J = 6.15 Hz, 1H, H-C(10)); 7.24 (br s, 2H, NH2); 8.14 and 8.19 (2s,
2H, H-C(2), H-C(8)); 8.29 (s, 1H, C@CH). Anal. Calcd for C13H16N8O3
(332.13): C, 46.98; H, 4.85; N, 33.72. Found: C, 46.79; H, 4.90; N,
33.80.
colorless solid (187 mg, 83%). TLC: Rf (CH2Cl2/MeOH 85:15): 0.2.
UV (MeOH): kmax 273 (8500). 1H NMR (DMSO-d6): 2.27–2.37 (m,
1H, H -C(20)); 2.52–2.60 (m, 1H, Hb-C(20)); 3.29–3.56 (m, 2H, H-
a
C(50)); 3.84 (m, 1H, H-C(40)); 4.35 (m, 1H, H-C(30)); 4.88 (t, 1H,
OH-C(50)); 5.39 (m, 3H, OH-C(30), H-CH2); 6.30 (‘t’, J = 6.25 Hz, 1H,
H-C(10)); 6.64 (d, 1H, J = 3.5 Hz, H-C(7)); 7.22 (d, J = 3.4 Hz, 1H,
H-C(8)); 7.36 (br s, 2H, NH2); 8.13 (s,1H, H-C(2)); 8.22 (s, 1H,
C@CH). Found: ESI-HR-MS: 332.14, C14H17N7O3; requires 331.14.
4.1.9. 7-[(2-Deoxy-b-D-erythro-pentofuranosyl)-imidazo[1,2-c]-
1,2,3-triazol-4-yl-methyl]adenine (7)
To a stirred solution of compound 6 (300 mg, 0.90 mmol) in
aqueous sodium acetate (1 M, pH 4.5–5.0, 33 mL) chloroacetalde-
hyde (50% aqueous solution, 5.6 mL) was added at 40–50 °C and
stirring was continued for 2 days. The reaction mixture was evap-
orated, and the residue was applied to FC (silica gel, column
4 ꢁ 16 cm, CH2Cl2/MeOH, 90:10) to give compound 7 as colorless
foam (0.25 g, 78%). TLC: Rf (CH2Cl2/MeOH, 85:15): 0.15. UV
(MeOH): kmax 229 (29,000), 275 (5200), 293 (3000), 266 (5100).
4.1.13. 2-Amino-4-chloro-7-prop-1-ynyl-7H-pyrrolo[2,3-
d]pyrimidine (1e)
2-Amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (10) (0.63 g,
3.75 mmol), potassium carbonate (0.51 g, 3.69 mmol) and propar-
gyl bromide (0.43 g, 3.61 mmol) were stirred in anhydrous DMF
(16 mL) at rt for 48 h. The solvent was evaporated under reduced
pressure and applied to FC (silica gel, column 4 ꢁ 16 cm, CH2Cl2/
MeOH, 98:2) to give 1e as a colorless solid (0.57 g, 74%). TLC: Rf
(CH2Cl2/MeOH 97:3): 0.50. UV (MeOH): kmax 261 (4000), 318
(5500). 1H NMR (DMSO-d6): 3.38 (t, 1H, J = 2.5 Hz, C„C–H); 4.87
(d, 1H, J = 2.5 Hz, CH2C„C); 6.34 (d, 1H, J = 3.8 Hz, H-C(7)); 6.76
(br s, 2H, NH2); 7.21 (d, J = 3.5 Hz, 1H, H-C(8)). Anal. Calcd for
C9H7ClN4 (206.04): C, 52.31; H, 3.41; N, 27.11. Found: C, 52.46;
H, 3.48; N, 26.95.
1H NMR (DMSO-d6): 2.28–2.37 (m, 1H, H -C(20)); 2.56–2.64 (m,
a
1H, Hb-C(20)); 3.42 (m, 2H, H-C(50)); 3.84 (m, 1H, H-C(40)); 4.35
(m, 1H, H-C(30)); 4.88 (t, 1H, OH-C(50)); 5.32 (d, 1H, OH-C(30));
5.61 (s, 2H, CH2); 6.31 (‘t’, J = 6.15 Hz, 1H, H-C(10)); 7.54 (s, 1H,
H-C(11)); 8.07 (s, 1H, H-C(10)); 8.32 (s, 1H, H-C(8)); 8.37 (s, 1H,
C@CH); 9.28 (s, 1H, H-C(2)). Anal. Calcd for C15H16N8O3 (356.13):
C, 50.56; H, 4.53; N, 31.45. Found: C, 50.52; H, 4.61; N, 31.30.
4.1.10. 4-Chloro-7-prop-1-ynyl-7H-pyrrolo[2,3-d]pyrimidine
(1d)
4.1.14. 2-Amino-4-chloro-7-[(2-deoxy-3,5-di-O-p-toluyl-b-
erythro-pentofuranosyl)-1,2,3-triazol-4-yl-methyl] 7H-
pyrrolo[2,3-d]pyrimidine (3e)
D-
4-Chloro-7H-pyrrolo[2,3-d]pyrimidine (8) (1.5 g, 9.8 mmol),
potassium carbonate (2.02 g, 14.66 mmol), and propargyl bromide
(1.74 g,14.7 mmol) were stirred in anhydrous DMF (27 mL) at rt for
24 h. The solvent was evaporated under reduced pressure and ap-
plied to FC (silica gel, column 4 ꢁ 16 cm, CH2Cl2/MeOH, 98:2). The
resulting solid was recrystallised from methanol to give 1d as col-
orless needles (1.33 g, 71%). TLC: Rf (CH2Cl2/MeOH, 98:2): 0.8. UV
(MeOH): kmax 271 (5000). 1H NMR (DMSO-d6): 3.47 (t, 1H,
J = 2.5 Hz, C„C–H); 5.17 (d, 1H, J = 2.5 Hz, CH2C„C); 6.70 (d, 1H,
J = 3.8 Hz, H-C(7)); 7.82 (d, J = 3.8 Hz, 1H, H-C(8)); 8.67 (s, 1H, H-
C(2)). Anal. Calcd for C9H6ClN3 (191.03): C, 56.41; H, 3.16; N,
21.93. Found: C, 56.36; H, 3.20; N, 22.01.
To a mixture of 1e (0.94 g, 4.55 mmol) and b-azido-2-deoxyri-
bose 2 (2.0 g, 5.05 mmol) in THF/H2O/t-BuOH, 3:1:1, (43 mL) was
added sodium ascorbate (2.6 mL, 2.58 mmol) of freshly prepared
1 M solution in water, followed by the addition of copper (II) sul-
fate pentahydrate 7.5% in water (2.0 mL, 0.6 mmol). The heteroge-
neous reaction mixture was stirred for 24 h at rt and then
evaporated and applied to FC (silica gel, column 3 ꢁ 10 cm, PE/
EtOAc, 1:1), gave compound 3e (2.32 g, 85%) as a colorless solid.
TLC: Rf (CH2Cl2/MeOH, 9:1): 0.33. UV (MeOH): kmax 317 (5700).
1H NMR (DMSO-d6): 2.36–2.38 (m, 6H, 2 CH3); 2.75–2.85 (m, 1H,
H -C(20)); 3.08–3.19 (m, 1H, Hb-C(20)); 4.38–4.61 (m, 3H, H-C(50),
a
4.1.11. 4-Chloro-7-[(2-deoxy-3,5-di-O-p-toluyl-b-
pentofuranosyl)-1,2,3-triazol-4-yl-methyl]-7H-pyrrolo[2,3-
d]pyrimidine (3d)
D
-erythro-
H-C(40)); 5.31 (s, 2H, CH2); 5.73 (m, 1H, H-C(30)); 6.30 (d, 1H,
J = 3.7 Hz, H-C(7)); 6.56 (‘t’, J = 6.5 Hz, 1H, H-C(10)); 6.70 (br s, 2H,
NH2); 7.15 (d, 1H, J = 3.5 Hz, H-C(8)); 7.25–7.93 (m, 8H, arom. H);
8.22 (s, 1H, C@CH). Anal. Calcd for C30H28ClN7O5 (601.18): C,
59.85; H, 4.69; N, 16.29. Found: C, 59.69; H, 4.79; N, 16.15.
To a mixture of 1d (0.21 g, 1.09 mmol) and b-azido-2-deoxyri-
bose 2 (0.5 g, 1.26 mmol in THF/H2O/t-BuOH, 3:1:1, (16 mL) was
added sodium ascorbate (0.63 mL, 0.62 mmol) of freshly prepared
1 M solution in water, followed by the addition of copper (II) sul-
fate pentahydrate 7.5% in water (0.5 mL, 0.15 mmol). The heteroge-
neous reaction mixture was stirred for 24 h at rt and then
evaporated and applied to FC (silica gel, column 3 ꢁ 10 cm, PE/
EtOAc, 1:1), which gave compound 3d (0.55 g, 85%) as a colorless
solid. TLC: Rf (PE/EtOAc, 1:1): 0.62. UV (MeOH): kmax 242
(32,000), 271 (7000). 1H NMR (DMSO-d6): 2.35–2.38 (m, 6H,
4.1.15. 2-Amino-4-chloro-7-[(2-deoxy-b-D-erythro-
pentofuranosyl)-1,2,3-triazol-4-yl-methyl]7H-pyrrolo[2,3-
d]pyrimidine (13)
A solution of compound 3e (0.9 g, 1.49 mmol) in NH3/MeOH
(saturated at 0 °C, 60 mL) was stirred overnight at rt. The clear
solution was evaporated, and the residue was applied to FC (silica
gel, column 4 ꢁ 16 cm, CH2Cl2/MeOH, 85:15). After evaporation,
the main zone yielded 13 as a colorless solid (470 mg, 86%). TLC:
2CH3); 2.79–2.83 (m, 1H, H -C(20)); 3.10–3.15 (m, 1H, Hb-C(20));
a
4.37–4.59 (m, 3H, H-C(50), H-C(40)); 5.57 (s, 2H, CH2); 5.73 (m,
1H, H-C(30)); 6.56 (‘t’, 1H, H-C(10)); 6.65 (d, 1H, J = 3.2 Hz, H-
C(7)); 7.22–7.92 (m, 8H, arom. H); 7.74 (d, 1H, H-C(8)); 8.32 (s,
1H, C@CH); 8.64 (s,1H, H-C(2)). Anal. Calcd for C30H27ClN6O5
(586.17): C, 61.38; H, 4.64; N, 14.32. Found: C, 61.40; H, 4.74; N,
14.26.
Rf (CH2Cl2/MeOH, 9:1): 0.35. UV (MeOH): kmax 260 (4000), 317
1
(5600). H NMR (DMSO-d6): 2.32–2.37 (m, 1H, H -C(20)); 2.55–
a
2.60 (m, 1H, Hb-C(20)); 3.37–3.49 (m, 2H, H-C(50); 3.81–3.85 (m,
1H, H-C(40)); 4.35 (m, H-C(30)); 4.85 (t, 1H, J = 5.5 Hz, OH-C(50));
5.30–5.38 (m, 3H, OH-C(30), H-CH2); 6.29–6.40 (m, 2H, H-C(7), H-
C(10)); 6.69 (br s, 2H, NH2); 7.18 (d, 1H, J = 3.75 Hz, H-C(8)); 8.19
(s, 1H, C@CH). Anal. Calcd for C14H16ClN7O3 (365.10): C, 45.97; H,
4.41; N, 26.81. Found: C, 46.03; H, 4.50; N, 26.70.
4.1.12. 4-Amino-7-[(2-deoxy-b-D-erythro-pentofuranosyl)-
1,2,3-triazol-4-yl-methyl]-7H-pyrrolo[2,3-d]pyrimidine (9)
A suspension of 3d (0.4 g, 0.68 mmol) in dioxane (40 mL) and
25% aq NH3 (50 mL) was introduced into an autoclave and stirred
at 90 °C for 24 h. The above reaction mixture was evaporated,
and the residue was applied to FC (silica gel, column 4 ꢁ 16 cm,
CH2Cl2/MeOH, 9:1). After evaporation, the main zone yielded 9 as
4.1.16. 2-Amino-7-[(2-deoxy-b-D-erythro-pentofuranosyl)-4-
methoxy-1,2,3-triazol-4-yl-methyl]-7H-pyrrolo[2,3-
d]pyrimidine (11)
Compound 3e (1.6 g, 2.65 mmol) was suspended in 0.2 N
NaOMe in MeOH (100 mL). The mixture was kept stirring at rt

8438
P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
for 15 h. The mixture was evaporated and applied to FC (silica gel,
column 2 ꢁ 10 cm), and eluted with CH2Cl2/MeOH (9:1). After
evaporation, the main zone yielded 11 as a colorless solid (0.8 g,
83%). TLC: Rf (CH2Cl2/MeOH, 97:3): 0.37. UV (MeOH): kmax 262
(‘t’, J = 5.3 Hz, 1H, H-C(10)); 6.82–7.31 (m, 13H, arom. H); 7.61
(d, J = 7.8 Hz, 1H, H-C(6)); 8.22 (s, 1H, C@CH); 11.30 (s, 1H,
NH). Anal. Calcd for C33H33N5O7 (611.24): C, 64.80; H, 5.44; N,
11.45. Found: C, 48.57; H, 5.04; N, 28.01.
(9000), 287 (8000). 1H NMR (DMSO-d6): 2.27–2.37 (m, 1H, H -
a
C(20)); 2.52–2.62 (m, 1H, Hb-C(20)); 3.39–3.51 (m, 2H, H-C(50);
3.81–3.99 (m, 3H, H-C(40), H-OMe); 4.35 (m, 1H, H-C(30)); 4.86 (t,
1H, J = 5.25 Hz, OH-C(50)); 5.25 (s, 2H, H-CH2); 5.33(d, 1H,
J = 4.37 Hz, OH-C(30)); 6.20–6.23 (m, 3H, H-C(7), NH2); 6.31 (‘t’,
1H, J = 6.25 Hz, H-C(10)); 6.91 (d, 1H, J = 3.50 Hz, H-C(8)); 8.15 (s,
1H, C@CH). Anal. Calcd for C15H19N7O4 (361.15): C, 49.86; H,
5.30; N, 27.13. Found: C, 49.92; H, 5.22; N, 27.10.
4.1.20. 2-[(2-Deoxy-5-O-(4,40-dimethoxytrityl)-b-
D-erythro-
pentofuranosy)-1,2,3-triazol-4-yl-methyl]uracil 30-(2-
Cyanoethyl diisopropylphosphoramidite) (21)
To a solution of 20 (100 mg, 0.16 mmol) in anhyd CH2Cl2 (4 mL)
were added (i-Pr)2EtN (0.05 lL, 0.28 mmol) and 2-cyanoethyl dii-
sopropylphosphoramidochloridite (0.08 mL, 0.35 mmol) while stir-
ring under Ar at rt stirring was continued for another 30 min, and
then CH2Cl2 (15 mL) was added. The soln. was then washed with
5% aq NaHCO3 soln. (15 mL), the aq layer extracted with CH2Cl2
(2ꢁ 30 mL), the combined org. layer dried over Na2SO4 and evapo-
rated, the resulting residue subjected to FC (silica gel, column
3 ꢁ 12 cm, pre wet with CH2Cl2/TE A, 98:2, v/v, until basic and
eluted with CH2Cl2/Me2CO/TEA, 96:4:1) to give compound 21 as
a colorless foam (100 mg, 75%). TLC: Rf (CH2Cl2/MeOH, 98:2):
0.63. 31P NMR (CDCl3): 150.41, 150.23.
4.1.17. [(2-Deoxy-b-D-erythro-pentofuranosyl)-1,2,3-triazol-4-
yl-methyl]7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (12)
A suspension of 3e (0.6 g, 1.0 mmol) in dioxane (30 mL) and 25%
aq NH3 (50 mL) was introduced into an autoclave and stirred at
120 °C for 24 h. The solution was evaporated and the residue was
applied to FC (silica gel, column 3 ꢁ 10 cm, CH2Cl2/MeOH, 20:1),
which gave compound 12 (238 mg, 69%) as a colorless solid. TLC:
Rf (CH2Cl2/MeOH, 9:1): 0.26. UV (MeOH): kmax 266 (7000), 286
1
(6000), H NMR (DMSO-d6): 2.27–2.61 (m, 2H, H -C(20), Hb-
4.1.21. 4-[(Benzoylamino)-7-(2-deoxy-b-D-erythro-
a
C(20)); 3.41–3.47 (m, 2H, H-C(50); 3.83 (m, 1H, H-C(40)); 4.35 (m,
1H, H-C(30)); 4.92 (t, 1H, J = 5.2 Hz, OH-C(50)); 5.19 (s, 2H, H-
CH2); 5.38 (d, 1H, OH-C(30)); 6.03 (br s, 2H, NH2); 6.31 (‘t’, 1H,
J = 6.47 Hz, H-C(10)); 6.43 (d, 1H, J = 3.5 Hz, H-C(7)); 6.80 (d, 1H,
J = 3.7 Hz, H-C(8)); 7.06 (br s, 2H, NH2); 8.16 (s, 1H, C@CH). Found:
ESI-HR-MS: 347.15, C14H18N8O3; requires 346.15.
pentofuranosyl)-1,2,3-triazol-4-ylmethyl]adenine (22)
Me3SiCl (1.2 mL, 9.41 mmol) was added to a solution of com-
pound 6 (300 mg, 0.9 mmol) in anhydrous pyridine (5.4 mL), and
was stirred at rt. After 30 min, the benzoyl chloride (4.5 mmol)
was introduced and the solution was kept at rt for another 2 h.
The mixture was cooled to 0 °C, diluted with H2O (3.6 mL), and stir-
red for 10 min. After the addition of 25% aq NH3 (3.6 mL), stirring
was continued for 2 h at rt. The solution was evaporated and the
residue was applied to FC (silica gel, column 3 ꢁ 10 cm, CH2Cl2/
MeOH, 9:1), which gave compound 22 (0.29 g, 74%) as a colorless
4.1.18. 2-Amino-7-[(2-deoxy-b-D-erythro-pentofuranosyl)-
1,2,3-triazol-4-yl-methyl]-3,7-dihydro-4H-pyrrolo[2,3-
d]pyrimidine-4-one (14)
Compound 11 (150 mg, 0.41 mmol) was dissolved in 2 N NaOH
(36 mL) and 1,4-dioxane (6 mL). The mixture was stirred under re-
flux for 6 h. After neutralization with 1 N HCl, The solution was
evaporated and the residue was applied to FC (silica gel, column
3 ꢁ 10 cm, CH2Cl2/MeOH 20:1), which gave compound 14
(80 mg, 55%) as a colorless solid. TLC: Rf (CH2Cl2/MeOH, 85:15):
0.4. UV (MeOH): kmax 262 (12,000). 1H NMR (DMSO-d6): 2.28–
solid. TLC: Rf (CH2Cl2/MeOH, 9:1): 0.24. UV (MeOH): kmax 263
1
(13,000). H NMR (DMSO-d6): 2.29–2.39 (m, 1H, H -C(20)); 2.53–
a
2.68 (m, 1H, Hb-C(20)); 3.47 (m, 2H, H-C(50)); 3.86 (m, 1H, H-
C(40)); 4.35 (m, 1H, H-C(30)); 4.87 (t, J = 5.4 Hz, OH-C(50)); 5.34 (d,
J = 4.32 Hz, 1H, OH-C(30)); 5.59 (s, 2H, CH2); 6.34 (‘t’, J = 6.2 Hz,
1H, H-C(10)); 7.43–8.05 (m, 7H, arom.H); 8.37 and 8.56 (2s, 2H,
H-C(2), H-C(8)); 8.74 (s, 1H, C@CH); 11.11 (s, NH). Anal. Calcd for
2.37 (m, 1H, H -C(20)); 2.53–2.66 (m, 1H, Hb-C(20)); 3.39–3.54
C20H20N8O4 (436.16): C, 55.04; H, 4.62; N, 25.68. Found: C, 55.09;
a
(m, 2H, H-C(50); 3.85 (m, 1H, H-C(40)); 4.35 (m, 1H, H-C(30)); 4.86
(t, 1H, J = 5.25 Hz, OH-C(50)); 5.18 (s, 2H, H-CH2); 5.34 (m, 1H,
OH-C(30)); 6.22–6.31 (m, 4H, NH2, H-C(10); H-C(7)); 6.72 (d, 1H,
J = 3.50 Hz, H-C(8)); 8.14 (s, 1H, C@CH); 10.31(s, 1H, NH) Anal.
Calcd for C14H17N7O4 (347.13): C, 48.41; H, 4.93; N, 28.23. Found:
C, 48.57; H, 5.04; N, 28.01.
H, 4.74; N, 25.49.
4.1.22. 4-[(Benzoylamino)-7-(2-deoxy-5-O-(4,40-
dimethoxytrityl)-b-D-erythro-pentofuranosyl)-1,2,3-triazol-4-
yl-methyl]adenine (23)
Compound 22 (200 mg, 0.45 mmol) was dried by repeated co-
evaporation with anhydrous pyridine (2ꢁ 3 mL) and dissolved in
anhydrous pyridine (6 mL). Then, 4,40-dimethoxytrityl chloride
(0.35 g, 1.03 mmol) was added at rt while stirring, and stirring
was continued for another 4 h. Thereupon, MeOH (2 mL) was
added and the stirring was continued for 10 min. The mixture
was poured into a 5% aq NaHCO3 soln (40 mL) and extracted
with CH2Cl2 (2ꢁ 40 mL). The combined organic layer was dried
(Na2SO4) and evaporated and the resulting residue subjected to
FC (silica gel, column 3 ꢁ 12 cm, pre wet with CH2Cl2/TEA,
98:2, v/v, until basic and eluted with CH2Cl2/MeOH/TEA,
4.1.19. 2-[(2-Deoxy-5-O-(4,40-dimethoxytrityl)-b-
D-erythro-
pentofuranosyl)-1,2,3-triazol-4-yl-methyl]uracil (20)
Compound 4 (100 mg, 0,32 mmol) was dried by repeated co-
evaporation with anhydrous pyridine (2 ꢁ 3 mL) and dissolved in
anhydrous pyridine (4 mL). Then, 4,40-dimethoxytrityl chloride
(217 mg, 0.64 mmol) was added at rt and stirring was continued
for another 4 h. Thereupon, MeOH (1 mL) was added and the
stirring was continued for 10 min. The mixture was poured into
a 5% aq NaHCO3 soln (30 mL) and extracted with CH2Cl2 (2ꢁ
30 mL). The combined organic layer was dried (Na2SO4) and
evaporated and the resulting residue subjected to FC (silica gel,
column 3 ꢁ 12 cm, pre wet with CH2Cl2/TEA, 98:2, v/v, until ba-
sic and eluted with CH2Cl2/MeOH/TEA, 96:4:1) to yield a color-
less foam 20 (160 mg, 81%). TLC: Rf (CH2Cl2/MeOH, 93:7): 0.46.
UV (MeOH): kmax 233 (22,000), 265 (11,000). 1H NMR (DMSO-
98:2:1) to yield
a colorless foam 23 (0.27 g, 80%). TLC: Rf
(CH2Cl2/MeOH 93:7): 0.45. UV (MeOH): kmax 273 (15,000), 231
(33,000). 1H NMR (DMSO-d6): 2.31–2.41 (m, 1H, H -C(20));
a
2.62–2.71 (m, 1H, Hb-C(20)); 3.06 (m, 2H, H-C(50)); 3.72 (s, 6H,
3MeO); 3.95 (m, 1H, H-C(40)); 4.39 (m, 1H, H-C(30)); 5.39 (d,
J = 4.8 Hz, 1H, OH-C(30)); 5.55 (s, 2H, CH2); 6.37 (t, J = 5.5 Hz,
1H, H-C(10)); 6.82–8.05 (m, 19H, arom. H); 8.32 and 8.49 (s,
2H, H-C(2), H-(8)); 8.69 (s, 1H, C@CH); 11.18 (s, 1H, NH). Anal.
Calcd for C41H38N8O6 (738.29): C, 66.65; H, 5.18; N, 15.17.
Found: C, 66.75; H, 5.30; N, 15.07.
d6): 2.32–2.42 (m, 1H, H -C(20)); 2.66–2.73 (m, 1H, Hb-C(20));
a
3.06 (m, 2H, H-C(50)); 3.72 (s, 6H, 3MeO); 3.95 (m, 1H, H-
C(40)); 4.41 (m, 1H, H-C(30)); 4.89 (s, 2H, CH2); 5.40 (d,
J = 4.8 Hz, 1H, OH-C(30)); 5.47 (d, J = 7.8 Hz, 1H, H-C(5)); 6.37

P. Chittepu et al. / Bioorg. Med. Chem. 16 (2008) 8427–8439
8439
4.1.23. 4-[(Benzoylamino)-7-(2-deoxy-5-O-(4,40-
support by Roche Diagnostics GmbH and ChemBiotech, Germany,
is gratefully acknowledged. Several antiviral evaluations were car-
ried out by Dr. Michel Liuggi (Idenix Senior Director Virology,
Laboratorio Cooperativo Idenix-Universita di Caliari, Italy). His
contribution is gratefully acknowledged. The work was supported
in part by Idenix Pharmaceuticals (F.S.).
dimethoxytrityl)-b-D-erythro-pentofuranosyl)-1,2,3-triazol-4-
yl-ethyl]adenine 30-(2-cyanoethyl diisopropylphosphoramidite)
(24)
To a solution of 23 (200 mg, 0.27 mmol) in anhydrous CH2Cl2
(6 mL) were added (i-Pr)2EtN (80 lL, 0.44 mmol) and 2-cyanoethyl
diisopropylphosphoramidochloridite (0.13 mL, 0.59 mmol) while
stirring under Ar at rt. Stirring was continued for another 30 min,
and then CH2Cl2 (20 mL) was added. The soln was then washed
with 5% aq NaHCO3 soln (20 mL), the aq layer extracted with
CH2Cl2 (2ꢁ 30 mL), the combined organic layer dried (Na2SO4)
and evaporated, the resulting residue subjected to FC (silica gel,
column 3 ꢁ 12 cm, pre wet with CH2Cl2/TEA, 98:2, v/v, until basic
and eluted with CH2Cl2/Me2CO/TEA, 96:4:1) to give compound
24 as a colorless foam (200 mg, 79%). TLC: Rf (CH2Cl2/MeOH,
93:7): 0.64. 31P NMR (CDCl3): 150.43, 150.23.
References and notes
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4.1.24. Preparation of the conjugate 16 from 1a and 15 by the
Cu(I)-catalyzed cycloaddition
To a solution of compound 1a (120 mg, 0.8 mmol) and 3-azido-
7-hydroxycoumarin (15; 160 mg, 0.79 mmol) in THF/H2O/t-BuOH,
10. Alvarez, R.; Velazquez, S.; San-Felix, A.; Aquaro, S.; De Clercq, E.; Perno, C.-F.;
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2:1:1, (8 mL), was added sodium ascorbate (330 lL, 0.32 mmol)
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tion of copper (II) sulfate pentahydrate 7.5% in water (0.26 mL,
0.08 mmol). The emulsion was stirred for 18 h at rt evaporated,
and applied to FC (silica gel, column 3 ꢁ 10 cm, CH2Cl2/MeOH,
95:5). From the main zone compound 16 (230 mg, 81%) was iso-
lated as a yellowish solid. TLC: Rf (CH2Cl2/MeOH, 95:5): 0.25. UV
(MeOH): kmax 258 (8200), 346 (12,100). 1H NMR (DMSO-d6): 5.04
(s, 2H, CH2); 5.62 (d, J = 7.8, 1H, H-C(5)); 6.83–6.92 (m, 2H, H-
arom.); 7.75 (d, 1H, J = 8.5, H-arom.); 7.83 (d, J = 7.9, 1H, H-C(6));
8.54 (s, 1H, triazole C@CH); 8.59 (s, 1H, H-arom.); 10.90 (s, 1H,
H-OH); 11.34 (s, 1H, NH). Found: ESI-HR-MS: 376.06 (M+Na)+,
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C16H11N5O5; requires 353.08.
4.1.25. Preparation of the conjugate 17 from 1c and 15 by the
Cu(I)-catalyzed cycloaddition
To a solution of compound 1c (100 mg, 0.58 mmol) and 3-azi-
do-7-hydroxycoumarin (15; 117 mg, 0.58 mmol) in THF/H2O/t-
BuOH, 2:1:1, (8 mL), was added sodium ascorbate (0.29 mL,
0.28 mmol) of a freshly prepared 1 M solution in water, followed
by the addition of copper (II) sulfate pentahydrate 7.5% in water
(230 lL, 0.07 mmol). The emulsion was stirred for 24 h at rt
evaporated, and applied to FC (silica gel, column 10 ꢁ 3 cm,
CH2Cl2/MeOH, 90:10). From the main zone compound 17
(170 mg, 78%) was isolated as
a yellowish solid. TLC: Rf
(CH2Cl2/MeOH 90:10): 0.34. UV (MeOH): kmax 258 (10,400),
346 (12,700). 1H NMR (DMSO-d6): 5.55 (s, 2H, CH2); 6.84–6.90
(m, 2H, H-arom.); 7.25 (s, 2H, NH2); 7.73 (d, 1H, J = 8.5, H-
arom.); 8.16 (s, 1H, H-C(8)), 8.26 (s, 1H, H-C(2)); 8.56 (s, 1H,
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Acknowledgments
We thank Dr. Simone Budow and Dr. Peter Leonard for reading
the manuscript and measurement of the NMR spectra. We also
thank Dr. Alexander Hepp for the HMBC spectrum of 4. Financial
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ˇ
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