Synthesis of readily accessible triazole-linked dimer deoxynucleoside phosphoramidite for solid-phase oligonucleotide synthesis

Article abstract of DOI:10.1055/s-0030-1258243

A concise preparation of triazole-linked deoxynucleoside phosphoramidite for its direct use in solid-phase oligonucleo tide synthesis is reported. This dimer has successfully been utilized in solid-phase synthesis to make the 10- and 12-mer oligomers. Georg Thieme Verlag Stuttgart New York.

Full text of DOI:10.1055/s-0030-1258243

3710
PAPER
Synthesis of Readily Accessible Triazole-Linked Dimer Deoxynucleoside
Phosphoramidite for Solid-Phase Oligonucleotide Synthesis
Synthesis of
T
r
riazole-L
i
inked
v
D
ime
r
Deox
a
r
P
hosph
i
oramidite Chandrasekhar,*a Pabbaraja Srihari,a Chidalla Nagesh,a Nayani Kiranmai,a Narayana Nagesh,b
M. Mohammed Idris*b
a
Organic Chemistry Division – I, Indian Institute of Chemical Technology, Hyderabad 500 007, India
Fax +91(40)27160512; E-mail: srivaric@iict.res.in
Centre for Cellular and Molecular Biology, Hyderabad 500 007, India
b
E-mail: idris@ccmb.res.in.
Received 25 June 2010; revised 18 July 2010
triazole moieties throughout the chain makes it a limiting
factor for further exploration. To overcome this, we initi-
ated synthesis of a dimer nucleoside phosphoramidite
with a triazole linkage that can be utilized directly for oli-
gonucleotide synthesis on a solid support and substituted/
linked up at the requisite site(s) in the chain. In continua-
tion to our research focused on click chemistry,15 we here-
in wish to report the synthesis of a dimer nucleoside
phosphoramidite 1, wherein the two monomer nucleoside
units are interlinked through a triazole moiety rather than
the regular phosphate ester linkage 1¢ (Figure 2).
Abstract: A concise preparation of triazole-linked deoxynucleo-
side phosphoramidite for its direct use in solid-phase oligonucleo-
tide synthesis is reported. This dimer has successfully been utilized
in solid-phase synthesis to make the 10- and 12-mer oligomers.
Key words: nucleotides, triazole, click, oligomer, solid-phase syn-
thesis
Modified nucleosides/nucleotides continue to attract sig-
nificant focus in medical applications for gene silencing
such as, antisense, antigene, and RNA interference.1 As
naturally occurring oligonucleotides suffer from degrada-
tion due to endo- and exonucleases, modifications have
become necessary to enhance the stability and also im-
prove pharmacokinetic properties from a therapeutic
point of view. Since the introduction of antisense agents,
diverse analogues have been developed that include,
modifications in the phosphate backbone (see Figure 1);
phosphate analogues such as phosphorothioate A,
phosphoramidate, methyl phosphonate,2 and nucleotide
borano phosphates B;3 complete replacement of the phos-
phodiester linkage by more stable functionalities like
amide C,4 amine5 and heterocycles D, E,6 F;7 modifica-
tions in nucleoside bases;8 derivatization/substitution of
the sugar moiety9 and complete replacement of furano
phosphate moiety to result in PNA;10 and replacement of
the sugar moiety with morpholine etc.11 In parallel, the lat-
est, innovative reactions such as the click reaction, have
been well utilized to derive modified nucleosides.12 Our
own interest in the click reaction has encouraged us to uti-
lize it for the synthesis of new scaffolds from Baylis-
Hillman adducts to result in triazoles13 and tetrazoles14
that displayed potency when screened against TNF-a in-
hibition studies towards anti-arthritis.
O
O
O
O
O
T
O
T
T
O
O
O
S
NH
P
P
O
–
O–
O
O
BH3
O
O
O
T
T
T
O
O
O
C
A
B
H
O
O
O
O
T
O
T
T
N
N
N
N
N
N
N
N
N
O
T
O
T
O
9
T
O
O
HO
D
F
E
Recently, Isobe et al. have accomplished the synthesis of
a 10-mer TLDNA oligomer chain with click chemistry
wherein the phosphate group has been completely re-
placed with by a triazole.7 This new analogue was found
to form a stable double strand with the complementary
strand of natural DNA. The linkage of sugars through the
Figure 1 Some modified thymidine dinucleotides
Even though the replacement of the phosphodiester moi-
ety with heterocycles, such as imidazole and tetrazoles, is
known,6 replacement with the triazole moiety has not
been well explored.12,16 We started to design a new dinu-
cleoside phosphoramidite 1 with the sugar part of the nu-
cleotide unaltered. Retrosynthetic analysis for 1 revealed
two key fragments, azide 2 (AZT) and acetylene 3. Com-
SYNTHESIS 2010, No. 21, pp 3710–3714
x
x.
x
x
.
2
0
1
0
Advanced online publication: 07.09.2010
DOI: 10.1055/s-0030-1258243; Art ID: Z16210SS
© Georg Thieme Verlag Stuttgart · New York

PAPER
Synthesis of Triazole-Linked Dimer Deoxynucleoside Phosphoramidite
3711
odinane and was subjected to Corey–Fuchs protocol18 to
give dibromomethylene 7. Compound 7 was treated with
freshly generated ethylmagnesium bromide to give the
monomer 3 with a terminal acetylene moiety.19 Com-
pound 3 was subjected to cycloaddition with commercial-
ly available AZT 2 in the presence of copper metal in
ethanol at reflux temperature to give the nucleoside dimer
8 in 75% yield.15a Our next goal was to protect the free pri-
mary alcohol (5¢-OH) with 4,4¢-dimethoxytrityl chloride
and convert the protected hydroxy group at the 3¢-position
to a phosphoramidite to obtain the readily accessible
dimer nucleoside phosphoramidite. Thus, we proceeded
with the protection of free 5¢-OH group in 8 as the corre-
sponding dimethoxytrityl ether 9 with 4,4¢-dimethoxytri-
tyl chloride in the presence of pyridine and 4-
(dimethylamino)pyridine. Tetrabutylammonium fluoride
mediated deprotection of silyl ether 9 afforded 10 which
was converted into the target phosphoramidite 1 using 2-
cyanoethyl diisopropylchlorophosphoramidite in the
presence of N,N-diisopropylethylamine in anhydrous
dichloromethane at room temperature.16f Thus, we have
accomplished the synthesis of the target dimer deoxynu-
cleoside phosphoramidite 1 (Scheme 2).
T
T
O
O
O
O–
O
DMTrO
P
N
O
N
N
ODMTr
O
O
T
N
O
O
T
O
P
O
P
CN
N
CN
1
1'
Figure 2 Thymidine dimers
HO
O
O
O
T
T
T
+
1 (TT*)
HO
HO
4 thymidine
TBSO
N3
3
2 AZT
T = thymine
Scheme 1 Retrosynthesis for modified dimer nucleoside phosph-
oramidite 1
The synthesized dinucleoside 1 was a suitably protected
dimer that can be utilized directly for solid-phase oligonu-
cleotide synthesis. We have successfully demonstrated
the utilization of compound 1 for the synthesis of two oli-
gomers of length 10-mer poly mix GC(TT*)AG(TT*)TA
and 12-mer (poly T, TT(TT*)TT(TT*)TTTT which were
synthesized in the oligo synthesizer (Scheme 3). These
oligos were characterized by MALDI analysis with m/z
pound 3 was easily accessible from commercially avail-
able thymidine 4 in five steps (Scheme 1).
For the synthesis, we started with thymidine 4 which was
protected as its bis(silyl ether) 5 at the 3¢- and 5¢-positions.
Selective primary silyl deprotection was achieved with
10-camphorsulfonic acid17 to give the free primary alco-
hol 6. The alcohol 6 was oxidized with Dess–Martin peri-
TBSCl
imidazole
HO
HO
TBSO
CSA
O
1) DMP, CH2Cl2, r.t.
T
T
O
O
T
CH2Cl2, r.t.
99%
MeOH–CH2Cl2 (1:1)
2) CBr4, Ph3P, Et3N
CH2Cl2, 0 °C to r.t.
82%
0 °C
69%
TBSO
O
HO
TBSO
4
5
6
T
Br
Br
O
T
AZT, Cu, EtOH
T
DMTrCl, pyridine
EtMgBr, THF
O
sat. CuSO4 (cat.)
reflux,10 h
75%
DMAP, CH2Cl2
0 °C to r.t., 26 h
80%
–20 °C to r.t.
70%
N
OH
N
TBSO
TBSO
N
7
3
O
T
8
T
TBSO
T
T
O
O
O
DMTrO
DMTrO
DMTrO
N
N
TBAF, THF
NC(CH2)2OPClN(i-Pr)2
N
N
N
N
0 °C to r.t,. 1 h
99%
DIPEA, CH2Cl2
r.t., 2 h
N
O
N
T
N
O
O
T
49%
T
O
O
TBSO
HO
P
N
CN
9
10
O
NH
O
1
T =
N
modified dimer (TT*)
Scheme 2 Synthesis of modified dinucleotide 1 (triazole-linked dimer)
Synthesis 2010, No. 21, 3710–3714 © Thieme Stuttgart · New York

3712
S. Chandrasekhar et al.
PAPER
quenched with H2O (20 mL) and extracted with CH2Cl2 (3 × 20
mL). The combined organic layers were washed with brine (30
mL), dried (anhyd Na2SO4), and concentrated in vacuo. The residue
was subjected to column chromatography (silica gel, EtOAc–hex-
ane, 1:5) to yield 5 (5.8 g, 99%) as a white solid; mp 141–142 °C;
Rf = 0.80 (EtOAc–hexane, 1:1).
1H NMR (300 MHz, CDCl3): d = 8.66 (s, 1 H), 7.4 (d, J = 0.9 Hz, 1
H), 6.25 (dd, J = 7.3, 6.0 Hz, 1 H), 4.43–4.50 (m, 1 H), 3.72–3.98
(m, 3 H), 2.24 (ddd, J = 13.1, 5.89, 3.03 Hz, 1 H), 1.98 (ddd,
J = 13.1, 7.5, 6.5 Hz, 1 H), 1.91 (d, J = 0.9 Hz, 3 H), 0.94 (s, 9 H),
0.91 (s, 9 H), 0.11 (d, J = 0.9 Hz, 6 H), 0.08 (d, J = 3.4 Hz, 6 H).
2975.697 for 10-mer poly mix, GC(TT*)AG(TT*)TA and
m/z 3530.752 for 12-mer poly T, TT(TT*)TT(TT*)TTTT.
GC(TT*)AG(TT*)TA
poly-mix-oligomer
DNA synthesizer
1
TT(TT*)TT(TT*)TTTT
poly-T-oligomer
Scheme 3 Application of modified dimer in oligomer synthesis
13C NMR (75 MHz, CDCl3): d = 163.5, 150.1, 135.4, 110.7, 87.8,
84.8, 72.2, 62.9, 41.3, 25.9, 25.7, 18.3, 17.9, 12.5, –4.6, –4.8, –5.3.
In conclusion, a triazole-linked modified dinucleoside
phosphoramidite synthesis and its incorporation into
HRMS: m/z calcd [M + Na]+ for C22H42N2O5NaSi2: 493.2516;
long-chain oligonucleotides on solid phase were demon- found: 493.2529.
strated. Further investigations on the properties of this
1-[(2R,4S,5R)-4-(tert-Butyldimethylsilyloxy)-5-(hydroxymeth-
yl)tetrahydrofuran-2-yl]-5-methylpyrimidine-2,4(1H,3H)-di-
one (6)
dimer nucleoside phosphoramidite and its application in
long-chain oligomer synthesis and also the synthesis of
other dinucleotide combinations are in progress.
CSA (0.812 g, 3.49 mmol) was added portionwise to a stirred soln
of 5 (2.78 g, 5.91 mmol) in CH2Cl2–MeOH (1:1, 20 mL) at 0 °C un-
der N2 and the mixture was stirred at 0 °C for 16 h. The mixture was
quenched with aq NaHCO3 (20 mL) at 0 °C and stirred for 30 min.
The solvents were evaporated in vacuo and the aqueous layer was
extracted with CH2Cl2 (3 × 20 mL). The combined organic layers
were washed with brine (20 mL), dried (anhyd Na2SO4), and con-
centrated in vacuo. The residue was subjected to column chroma-
tography (silica gel, EtOAc–hexane, 2:3) to yield 6 (1.45 g, 69%) as
a white solid; mp 89–91 °C; Rf = 0.50 (EtOAc).
1H NMR (300 MHz, CDCl3): d = 9.80 (br s, 1 H), 7.36 (s, 1 H), 6.07
(t, J = 6.6 Hz, 1 H), 4.45–4.54 (m, 1 H), 3.82–3.94 (m, 2 H), 3.64–
3.76 (m, 1 H), 3.10 (br s, 1 H), 2.27–2.42 (m, 1 H), 2.21–2.25 (m, 1
H), 1.88 (s, 3 H), 0.90 (s, 9 H), 0.09 (s, 6 H).
All the reagents employed were obtained commercially from M/s.
Aldrich and used without further purifications unless otherwise stat-
ed. For anhydrous reactions, solvents were dried by literature pro-
cedures; removal of solvent was performed under reduced pressure
using a rotary evaporator. All reactions requiring anhydrous condi-
tions were carried out in oven-dried glassware under N2. All reac-
tions and fractions from column chromatography were monitored
by TLC using plates with a UV fluorescent indicator (normal silica
gel, Merck 60 F254). 1H NMR spectra were recorded at 300 or 400
MHz [relative to the solvent residual signal in CDCl3 (d = 7.26) or
to TMS], and 13C NMR spectra were recorded at 75 MHz/100 MHz
at r.t. [relative to the solvent signal CDCl3 (d = 77.00) unless other-
wise noted]. One or more of the following methods were used for
visualization: UV absorption by fluorescence quenching; I2 stain-
ing; phosphomolybdic acid/Ce(SO4)2/H2SO4/H2O (10 g:1.25 g:12
mL:238 mL) spray; anisaldehyde spray (EtOH–AcOH–H2SO4–
anisaldehyde; 200 mL:2 mL:6.35 mL:3 mL). Column chromatogra-
phy was performed using 60–120 mesh silica gel.
13C NMR (75 MHz, CDCl3): d = 164.2, 150.6, 137.2, 111.1, 87.7,
86.9, 71.7, 62.1, 40.7, 25.9, 18.1, 12.6, –4.5, –4.6.
HRMS: m/z calcd [M]+ for C16H28N2O5Si: 356.1776; found:
356.1767.
1-[(2R,4S,5R)-4-(tert-Butyldimethylsilyloxy)-5-(2,2-dibromovi-
nyl)tetrahydrofuran-2-yl]-5-methylpyrimidine-2,4(1H,3H)-di-
one (7)
Oligonucleotide Synthesis
The solid phase oligonucleotide synthesis was performed on an
ABI-394 DNA synthesizer (USA) following standard procedures.
Detritylation was achieved using 2–3% trichloroacetic acid in
CH2Cl2. The incoming base was first allowed to react with weak
acid tetrazole to form an active tetrazolyl phosphoramidite interme-
diate which reacts with the 5¢-hydroxy group of the recipient. Unre-
acted free hydroxy groups were capped by Ac2O and N-
methylimidazole in MeCN. Stabilization of the phosphate linkage
between two bases was achieved by treating the resin-supported oli-
gomer with I2 in THF–H2O. After the completion of the chain syn-
thesis, the mixture was incubated at 55 °C for 16 h to cleave the
resin as well as remove the protection groups. The modified base
phosphoramidite as well as DMTr+ on oligonucleotides were puri-
fied by passing through the DMTr+ Glen-Pak Cartridges (Glen Re-
search Inc, USA) and they were characterized by mass spectroscopy
and gel electrophoresis.
To a stirred soln of 6 (1.38 g, 3.87 mmol) in CH2Cl2 (30 mL) was
added Dess–Martin periodinane (1.78 g, 4.21 mmol) and the mix-
ture was stirred at r.t. for 30 min. The reaction was quenched with
sat. aq Na2S2O3 (10 mL) at 0 °C. The mixture was diluted with
CH2Cl2 (10 mL) and the aqueous layer was extracted with CH2Cl2
(2 × 10 mL). The combined organic layers were washed with sat. aq
NaHCO3 (10 mL) and brine (10 mL), dried (anhyd Na2SO4), and
concentrated in vacuo to give crude aldehyde (1.30 g) that was uti-
lized directly in the next reaction without further purification. To
the stirred soln of Ph3P (3.7 g, 14.12 mmol) in CH2Cl2 (10 mL) at 0
°C under N2 was added CBr4 (2.34 g, 7.06 mmol) dissolved in
CH2Cl2 (10 mL) slowly and the reaction was stirred at this temper-
ature for 30 min and then warmed to r.t. The mixture was cooled to
0 °C and Et3N (1.07 g, 10.59 mmol) was added and the mixture was
allowed to warm to r.t. for 15 min. The mixture was recooled to 0
°C and to this was added the above aldehyde (1.38 g, 3.53 mmol)
dissolved in CH2Cl2 (10 mL). When the reaction was complete, the
mixture was quenched with H2O (30 mL) and stirred for 30 min, the
organic layer was separated and the aqueous layer was extracted
with CH2Cl2 (3 × 20 mL). The combined organic layers were
washed with brine (20 mL), dried (anhyd Na2SO4), and concentrat-
ed in vacuo. The residue was subjected to column chromatography
(silica gel, EtOAc–hexane, 1:4) to yield 7 (1.62 g, 82%) as a light
brown solid; mp 149–151 °C; Rf = 0.50 (EtOAc–hexane, 4:6).
1-{(2R,4S,5R)-4-(tert-Butyldimethylsilyloxy)-5-[(tert-butyldi-
methylsilyloxy)methyl]tetrahydrofuran-2-yl}-5-methylpyrimi-
dine-2,4(1H,3H)-dione (5)
To the soln of thymidine 4 (3.0 g, 12.38 mmol) in CH2Cl2 (20 mL)
at 0 °C under N2 was added imidazole (2.52 g, 37.15 mmol) and the
mixture was stirred for 20 min. TBSCl (4.65 g, 30.96 mmol) in
CH2Cl2 (10 mL) was added dropwise and the mixture was stirred at
r.t. for 1 h. After completion of the reaction, the mixture was
Synthesis 2010, No. 21, 3710–3714 © Thieme Stuttgart · New York

PAPER
Synthesis of Triazole-Linked Dimer Deoxynucleoside Phosphoramidite
3713
1H NMR (300 MHz, CDCl3): d = 8.44 (br s, 1 H), 7.06 (d, J = 1.1
Hz, 1 H), 6.50 (d, J = 8.5 Hz, 1 H), 6.01 (t, J = 6.6 Hz, 1 H), 4.52
(dd, J = 8.5, 3.9 Hz, 1 H), 4.29–4.36 (m, 1 H), 2.19–2.40 (m, 2 H),
1.95 (d, J = 1.1 Hz, 3 H), 0.91 (s, 9 H), 0.12 (s, 3 H), 0.10 (s, 3 H).
13C NMR (75 MHz, CDCl3): d = 163.9, 150.4, 136.1, 135.5, 111.4,
87.1, 86.7, 75.5, 73.4, 40.4, 25.8, 18.1, 12.8, –4.4, –4.6.
1-[(2R,4S,5S)-5-{[Bis(4-methoxyphenyl)(phenyl)meth-
oxy]methyl}-4-(4-{(2R,3S,5R)-3-(tert-butyldimethylsilyloxy)-5-
[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]tetrahy-
drofuran-2-yl}-1H-1,2,3-triazol-1-yl)tetrahydrofuran-2-yl]-5-
methylpyrimidine-2,4(1H,3H)-dione (9)
Nucleoside 8 (0.500 g, 0.809 mmol) was co-evaporated with dry py-
ridine (2 × 10 mL) and dried under vacuum for 30 min. To a soln of
dried 8 (0.500 g, 0.809 mmol) in dry pyridine (40 mL) was added
DMAP (0.217 g, 1.78 mmol) and 4,4¢-dimethoxytrityl chloride
(0.329 g, 0.971 mmol) at r.t. under argon. The mixture was stirred
at r.t. for 20 h, at which time TLC indicated that the reaction was not
complete (only 20% conversion). To the mixture was added addi-
tional DMAP (0.217 g, 1.78 mmol) and 4,4¢-dimethoxytrityl chlo-
ride (0.329 g, 0.971 mmol). The mixture was stirred at r.t. until the
reaction was complete (~6 h). The solvent was removed, and the
residue was purified by chromatography (silica gel, MeOH–CHCl3,
2:98) to give 9 (0.591g, 80%) as a light yellow foam; mp 162–163
°C; Rf = 0.45 (MeOH–CHCl3, 1:9).
1H NMR (300 MHz, CDCl3): d = 8.94 (br s, 1 H), 8.75 (br s, 1 H),
7.69 (d, J = 1.1Hz, 1 H), 7.66 (d, J = 0.7 Hz, 1 H), 7.45 (s, 1 H,),
7.37–7.43 (m, 2 H), 7.24–7.35 (m, 7 H), 6.84 (dd, J = 8.8, 9.0 Hz, 4
H), 6.42–6.53 (m, 2 H), 5.32–5.41 (m, 1 H), 4.88 (d, J = 2.8 Hz, 1
H), 4.63–4.69 (m, 1 H), 4.39–4.46 (m, 1 H), 3.79 (s, 6 H), 3.70 (dd,
J = 11.4, 10.9 Hz, 1 H), 3.35 (dd, J = 10.9, 10.7 Hz, 1 H), 3.03–3.13
(m, 1 H), 2.58–2.82 (m, 2 H), 2.31–2.41 (m, 1 H), 1.87 (s, 3 H), 1.60
(s, 3 H), 0.87 (s, 9 H), 0.07 (s, 6 H).
HRMS: m/z calcd [M + Na]+ for C17H26Br2N2O4NaSi: 530.9897;
found: 530.9926.
1-[(2R,4S,5R)-4-(tert-Butyldimethylsilyloxy)-5-ethynyltetrahy-
drofuran-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dione (3)
EtBr (3.04 g, 27.93 mmol) was added to a suspension of Mg (0.50
g, 18.62 mmol) in anhyd THF (20 mL) dropwise. After generation
of EtMgBr, the mixture was cooled to –20 °C and to this was added
compound 7 (0.95 g, 1.86 mmol) in THF (5 mL). After consumption
of the starting material, the mixture was quenched with sat. aq
NH4Cl (10 mL) at 0 °C, the mixture was diluted with H2O (10 mL),
and the aqueous layer was extracted with EtOAc (2 × 20 mL). The
combined organic layers were washed with brine (10 mL), dried
(anhyd Na2SO4), and concentrated in vacuo. The residue was sub-
jected to column chromatography (silica gel, EtOAc–hexane, 1:4)
to yield 3 (0.45 g, 70%) as a white solid; mp 158–160 °C; Rf = 0.50
(EtOAc–hexane, 1:1).
1H NMR (400 MHz, CDCl3): d = 8.98 (br s, 1 H), 7.52 (s, 1 H), 6.42
(dd, J = 5.8, 8.1 Hz, 1 H), 4.58 (s, 1 H), 4.51 (d, J = 3.66, 4.3 Hz, 1
H), 2.75 (d, J = 1.5 Hz, 1 H), 2.43 (ddd, J = 4.4, 5.8, 13.1 Hz, 1 H),
2.16 (ddd, J = 4.4, 8.1, 13.1 Hz, 1 H), 1.94 (s, 3 H), 1.91 (s, 9 H),
0.12 (s, 6 H).
13C NMR (100 MHz, CDCl3): d = 163.7, 150.3, 135.5, 111.1, 86.8,
80.5, 77.6, 76.9, 40.5, 25.6, 17.9, 12.7, –4.8, –4.9.
13C NMR (75 MHz, CDCl3): d = 163.3, 158.8, 150.4, 150.1, 146.2,
143.9, 136.6, 135.3, 134.9, 134.8, 130.0, 128.1, 127.9, 127.3, 122.2,
113.3, 111.6, 111.2, 87.1, 86.2, 85.2, 83.6, 80.6, 77.1, 76.1, 62.0,
59.7, 55.2, 40.0, 38.3, 25.6, 17.9, 12.5, 12.0, 0.9. –4.8, –4.8.
HRMS: m/z calcd [M + H]+ for C48H58N7O10Si: 920.4009; found:
920.4003.
HRMS: m/z calcd [M + Na]+ for C17H26N2O4NaSi: 373.1556;
found: 373.1559.
1-[(2R,4S,5S)-5-{[Bis(4-methoxyphenyl)(phenyl)meth-
oxy]methyl}-4-(4-{(2R,3S,5R)-3-hydroxy-5-[5-methyl-2,4-di-
oxo-3,4-dihydropyrimidin-1(2H)-yl]tetrahydrofuran-2-yl}-1H-
1,2,3-triazol-1-yl)tetrahydrofuran-2-yl]-5-methylpyrimidine-
2,4(1H,3H)-dione (10)
A 1 M soln of TBAF (0.11 g, 0.43 mmol) was added to a stirred soln
of 9 (0.40 g, 0.43 mmol) in THF (5 mL) at 0 °C under N2 and the
mixture was stirred for 1 h. The reaction was quenched with sat.
NaHCO3 and stirred for 10 min. The aqueous layer was extracted
with EtOAc (3 × 5 mL), the combined organic layers were washed
with brine (5 mL), dried (anhyd Na2SO4), and concentrated in vac-
uo. The residue was subjected to column chromatography (silica
gel, MeOH–CHCl3, 5:95) to yield 7 (0.35 g, 99%) as a light foamy
white solid; mp 123–125 °C; Rf = 0.40 (MeOH–CHCl3, 1:9).
1H NMR (300 MHz, CDCl3): d = 8.22 (s, 1 H), 7.68 (s, 1 H), 7.62
(s, 2 H), 7.19–7.43 (m, 9 H), 6.82 (d, J = 7.7 Hz, 4 H), 6.42–6.58
(m, 2 H), 5.32–5.45 (m, 1 H), 5.04 (d, J = 5.0 Hz, 1 H), 4.68–4.81
(m, 1 H), 4.38–4.46 (m, 1 H), 3.77 (s, 6 H), 3.60–3.72 (m, 1 H),
3.32–3.43 (m, 1 H), 2.92–2.99 (m, 1 H), 2.59–2.83 (m, 2 H), 2.42–
2.53 (m, 1 H), 1.83 (s, 3 H), 1.55 (s, 3 H).
13C NMR (75 MHz, CDCl3): d = 163.6, 163.4, 158.8, 150.5, 150.3,
146.2, 144.0, 136.7, 135.3, 135.0, 134.8, 130.0, 128.1, 127.9, 127.3,
122.3, 113.3, 111.7, 111.3, 87.2, 86.0, 85.3, 83.7, 80.1, 75.2, 62.5,
60.2, 55.2, 39.3, 38.3, 12.5, 12.0.
1-[(2R,4S,5R)-4-(tert-Butyldimethylsilyloxy)-5-(1-{(2S,3S,5R)-
2-(hydroxymethyl)-5-[5-methyl-2,4-dioxo-3,4-dihydropyrimi-
din-1(2H)-yl]tetrahydrofuran-3-yl}-1H-1,2,3-triazol-4-yl)tet-
rahydrofuran-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dione (8)
Cu(0) (20 mg) was added to a stirred soln of AZT 2 (0.431 g, 1.61
mmol) and 3 (0.565 g, 1.61 mmol) in EtOH (20 mL). Sat. aq CuSO4
(0.5 mL) was added to the mixture and it was refluxed for 12 h. Af-
ter the completion of the reaction, the mixture was filtered through
Celite and washed with CHCl3, and the filtrate was concentrated in
vacuo. The residue was subjected to column chromatography (silica
gel, MeOH–CHCl3, 2:98) to yield 8 (0.74 g, 75%) as a white solid;
mp 139–141 °C; Rf = 0.30 (EtOAc).
1H NMR (300 MHz, CDCl3): d = 9.25 (s, 1 H), 9.21 (s, 1 H), 7.89
(s, 1 H), 7.59 (d, J = 1.1 Hz, 1 H), 7.49 (d, J = 0.95 Hz, 1 H), 6.28
(t, J = 6.8 Hz, 1 H), 6.22 (t, J = 6.2 Hz, 1 H), 5.42–5.51 (m, 1 H),
4.98 (d, J = 3.2 Hz, 1 H), 4.70–4.77 (m, 1 H), 4.42–4.48 (m, 1 H),
3.99–4.08 (m, 1 H), 3.74–3.85 (m, 1 H), 3.41–3.50 (m, 1 H), 2.91–
3.08 (m, 2 H), 2.64–2.72 (m, 1 H), 2.36 (ddd, J = 3.7, 6.2, 13.4 Hz,
1 H), 1.94 (d, J = 0.75 Hz, 3 H), 1.91 (d, J = 0.75 Hz, 3 H), 0.86 (s,
9 H), 0.03 (s, 3 H), 0.01 (s, 3 H).
13C NMR (75 MHz, CDCl3): d = 164.0, 150.8, 150.5, 146.1, 137.7,
137.6, 123.2, 111.1, 88.3, 87.5, 85.3, 81.0, 76.1, 61.1, 58.9, 39.8,
37.6, 29.6, 25.6, 17.9, 12.5, 12.4, –4.8.
HRMS: m/z calcd [M]+ for C27H39N7O8Si: 617.2629; found:
617.2638.
HRMS: m/z calcd [M + Na]+ for C42H43N7O10Na: 828.2970; found:
828.2969.
Synthesis 2010, No. 21, 3710–3714 © Thieme Stuttgart · New York

3714
S. Chandrasekhar et al.
PAPER
(2R,3S,5R)-2-({1-[(2S,3S,5R)-2-{[Bis(4-methoxyphenyl)(phe-
nyl)methoxy]methyl}-5-[5-methyl-2,4-dioxo-3,4-dihydropyrim-
idin-1(2H)-yl]tetrahydrofuran-3-yl}-1H-1,2,3-triazol-4-yl)-5-
[5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl]tetrahy-
drofuran-3-yl 2-Cyanoethyl diisopropylphosphoramidite (1)
To a soln of alcohol 10 (0.1 g, 0.12 mmol) in anhyd CH2Cl2 (8 mL)
at r.t. was added DIPEA (0.8 mL, 0.49 mmol) and 2-cyanoethyl di-
isopropylchlorophosphoramidite (0.053 g, 0.22 mmol) in CH2Cl2
(0.5 mL) under N2. After completion of the reaction (2 h), the mix-
ture was cooled to 0 °C and diluted with H2O (4 mL) and CH2Cl2 (8
mL). The organic layer was separated and washed with sat. aq
NaHCO3 (10 mL). The aqueous layer was extracted with CH2Cl2
(2 × 8 mL). The combined organic layers were dried (anhyd
Na2SO4), filtered, and concentrated in vacuo. The residue was puri-
fied via flash chromatography (silica gel 100–200 mesh, CHCl3–
MeOH–Et3N, 99:0.5:0.5) to obtain 1 (0.06 g, 49%) as a creamy
powder; Rf = 0.50 (MeOH–CHCl3, 1:9).
1H NMR (300 MHz, CDCl3): d = 7.81 (d, J = 0.9 Hz, 1 H), 7.73 (d,
J = 0.9 Hz, 1 H), 7.69 (br s, 1 H), 7.63–7.67 (m, 2 H), 7.48 (br s, 2
H), 7.36–7.42 (m, 4 H), 7.22–7.35 (m, 12 H), 6.80–6.89 (m, 8 H),
6.47–6.59 (m, 4 H), 5.28–5.40 (m, 2 H), 5.21 (br s, 1 H), 5.12 (br s,
1 H), 4.68–4.82 (m, 2 H), 4.41–4.54 (m, 2 H), 3.51–3.94 (m, 18 H),
3.30–3.39 (m, 2 H), 2.94–3.09 (m, 4 H), 2.45–2.77 (m, 8 H), 2.64 (t,
J = 6.4 Hz, 4 H), 1.86 (s, 6 H), 1.56 (d, J = 5.8 Hz, 6 H), 1.24–1.10
(m, 24 H).
(4) (a) Nina, M.; Fonne-Pfister, R.; Beaudegnies, R.; Chekatt,
H.; Jung, P. M. J.; Murphy-Kessabi, F.; de Mesmaeker, A.;
Wendeborn, S. J. Am. Chem. Soc. 2005, 127, 6027.
(b) de Mesmaeker, A.; Lebreton, J.; Waldner, A.; Fritsch,
V.; Wolf, R. M. Bioorg. Med. Chem. Lett. 1994, 4, 873.
(5) (a) Viswanadham, G.; Petersen, G. V.; Wengel, J. Bioorg.
Med. Chem. Lett. 1996, 6, 987. (b) Petersen, G. V.; Wengel,
J. Tetrahedron 1995, 51, 2145.
(6) (a) Matt, P. V.; Lochmann, T.; Altmann, K.-H. Bioorg. Med.
Chem. Lett. 1997, 7, 1549. (b) Matt, P. V.; Altmann, K.-H.
Bioorg. Med. Chem. Lett. 1997, 7, 1553. (c) Filichev, V. V.;
Malin, A. A.; Ostrovskii, V. A.; Pedersen, E. B. Helv. Chim.
Acta 2002, 85, 2847.
(7) Isobe, H.; Fujino, T.; Yamazaki, N.; Guillot-Nieckowski,
M.; Nakamura, E. Org. Lett. 2008, 10, 3729.
(8) For few recent publications, see: (a) Koissi, N.; Lonnberg,
H. Nucleosides, Nucleotides, Nucleic Acids 2007, 26, 1203.
(b) Krueger, A. T.; Lu, H.; Lee, A. H. F.; Kool, E. T. Acc.
Chem. Res. 2007, 40, 141. (c) Liu, H.; Gao, J.; Kool, E. T.
J. Am. Chem. Soc. 2005, 127, 1396. (d) Kool, E. T. Acc.
Chem. Res. 2002, 35, 936.
(9) For few recent sugar modified nucleosides, see:
(a) Stauffiger, A.; Leumann, C. J. Eur. J. Org. Chem. 2009,
1153. (b) Prakash, T. P.; Bhat, B. Curr. Top. Med. Chem.
2007, 7, 641. (c) Gagneron, J.; Gosselin, G.; Mathe, C. Eur.
J. Org. Chem. 2006, 4891. (d) Prakash, T. P.; Kawasaki, A.
M.; Lesnik, E. A.; Owens, S. R.; Manoharan, M. Org. Lett.
2003, 5, 403.
(10) (a) Nielsen, P. E. Pure Appl. Chem. 1998, 70, 105.
(b) Fujii, M.; Yoshida, K.; Hidaka, J.; Ohtsu, T. Bioorg.
Med. Chem. Lett. 1997, 7, 637.
(11) (a) Zhang, N.; Tan, C.; Cai, P.; Zhang, P.; Zhao, Y.; Jiang,
Y. Bioorg. Med. Chem. 2009, 17, 2441. (b) Summerton, J.;
Weller, D. Nucleosides Nucleotides 1997, 16, 889.
(12) For a recent review, see: Amblard, F.; Cho, J. H.; Schinazi,
R. F. Chem. Rev. 2009, 109, 4207; and references cited
therein.
13C NMR (75 MHz, CDCl3): d = 163.4, 163.3, 158.8, 150.3, 150.0,
146.1, 144.0, 136.6, 136.5, 135.3, 130.0, 128.1, 128.0, 127.3, 122.7,
122.2, 113.3, 111.5, 111.3, 87.2, 86.0, 85.8, 85.2, 85.0, 83.6, 83.5,
79.4, 79.1, 62.3, 62.2, 60.0, 55.2, 43.4, 43.2, 38.5, 38.4, 24.6, 24.5,
24.4, 24.4, 22.9, 12.6, 12.0.
31P NMR (161.91 MHz, CDCl3): d = 150.8, 149.1.
HRMS: m/z calcd [M + Na]+ for C51H60N9O11PNa: 1028.4042;
found: 1028.4048.
Acknowledgment
(13) Chandrasekhar, S.; Basu, D.; Rambabu, Ch. Tetrahedron
Ch. Nagesh thanks CSIR, New Delhi and N. Kiranmai thanks UGC,
New Delhi for the award of research fellowship. The authors thank
Dr. B. Jagadeesh for NMR analysis of the modified dimer nucleo-
side and Dr. Ganesh Kumar for mass analysis of the oligomers.
Lett. 2006, 47, 3059.
(14) Srihari, P.; Dutta, P.; Srinivasa Rao, R.; Yadav, J. S.;
Chandrasekhar, S.; Thombare, P.; Mohapatra, J.; Chatterjee,
A.; Jain, M. R. Bioorg. Med. Chem. 2009, 19, 5569.
(15) For other contributions on click chemistry from our group,
see: (a) Chandrasekhar, S.; Lohitha Rao, Ch.; Nagesh, Ch.;
Raji Reddy, Ch.; Sridhar, B. Tetrahedron Lett. 2007, 48,
5869. (b) Chandrasekhar, S.; Tiwari, B.; Parida, B. B.;
Raji Reddy, Ch. Tetrahedron: Asymmetry 2008, 19, 495.
(c) Chandrasekhar, S.; Seenaiah, M.; Lohitha Rao, Ch.;
Raji Reddy, Ch. Tetrahedron 2008, 64, 11325.
(16) (a) Lazrek, H. B.; Rochdi, A.; Engels, J. W. Nucleosides
Nucleotides 1999, 18, 1257. For recent papers with
triazoles, see: (b) Nuzzi, A.; Massi, A.; Dondoni, A. QSAR
Comb. Sci. 2007, 26, 1191. (c) Lucas, R.; Neto, V.;
Hadj Bouazza, A.; Zerrouki, R.; Granet, R.;
References
(1) (a) Koropatnick, J.; Berg, R. W.; Jason, T. L. H. Recent
Development in Gene Therapy, 1st ed.; Transworld
Research Network: Trivandrum, 2007, 151–180. (b) Haas,
J.; Engels, J. W. Tetrahedron Lett. 2007, 48, 8891.
(c) Buchini, S.; Leumann, C. J. Curr. Opin. Chem. Biol.
2003, 7, 717. (d) Kurreck, J. Eur. J. Biochem. 2003, 270,
1628.
(2) (a) For more recent papers on these derivatives see: Olesiak,
M.; Stec, W. J.; Okruszek, A. Org. Biomol. Chem. 2009, 7,
2162; and references cited therein. (b) Kanaori, K.; Tamura,
Y.; Wada, T.; Nishi, M.; Kanehara, H.; Morii, T.; Tajima,
K.; Makino, K. Biochemistry 1999, 38, 16058.
Krausz Champavier, Y. Tetrahedron Lett. 2008, 49, 1004.
(d) Lucas, R.; Zerrouki, R.; Granet, R.; Champavier, Y. K.
Tetrahedron 2008, 64, 5467. (e) Isobe, H.; Fujino, T.;
Yamazaki, N. Tetrahedron Lett. 2009, 50, 4101; and
references cited therein. (f) El-Sagheer, A. H.; Brown, T.
J. Am. Chem. Soc. 2009, 131, 3958.
(c) De Mesmaeker, A.; Altmann, K. H.; Waldner, A.;
Wendeborn, S. Curr. Opin. Struct. Biol. 1995, 5, 343.
(d) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 543.
(e) Brill, W. K.-D.; Nielsen, J.; Carruthers, M. H.
Tetrahedron Lett. 1988, 29, 5517.
(17) Chandrasekhar, S.; Rambabu, Ch.; Syamprasad Reddy, A.
Org. Lett. 2008, 10, 4355.
(18) (a) Corey, E. J.; Fuchs, P. L. Tetrahedron Lett. 1972, 13,
3769. (b) Takahashi, S.; Nakata, T. J. Org. Chem. 2002, 67,
5739.
(19) Initially, the reaction was tried with n-BuLi and the yield
was found to be poor as the reaction ended with an
intractable mixture of products.
(3) (a) Li, P.; Sergueeva, Z. A.; Dobrikov, M.; Shaw, B. R.
Chem. Rev. 2007, 107, 4746. (b) Higashida, R.; Oka, N.;
Kawanaka, T.; Wada, T. Chem. Commun. 2009, 2466. (c)
For other derivatives including boronates, see: Han, Q.;
Sarafianos, S. G.; Arnold, E.; Parniak, M. A.; Gaffney, B. L.;
Jones, R. A. Tetrahedron 2009, 65, 7915.
Synthesis 2010, No. 21, 3710–3714 © Thieme Stuttgart · New York