Synthesis and hybridization properties of 2′-O-(tetrazol-5-yl)ethyl-modified oligonucleotides

Article abstract of DOI:10.1016/j.tet.2008.02.075

2′-O-(1H-Tetrazol-5-yl)ethyladenosine was synthesized using 2′-O-cyanoethyladenosine derivative as a key intermediate. The 2′-O-(1H-tetrazol-5-yl)ethyl modifications exhibited intriguing properties such as the change in the structure of the tetrazole residue between a protonated and a deprotonated form. The T_m experiments of various oligodeoxynucleotides having a 2′-O-(1H-tetrazol-5-yl)ethyl-modified adenosine showed reduced hybridization affinity in comparison to the unmodified oligonucleotides toward their complementary oligodeoxynucleotides. The mechanism of the reduced hybridization affinity was discussed on the basis of the structure and the physicochemical properties of the tetrazole moiety.

Full text of DOI:10.1016/j.tet.2008.02.075

Available online at www.sciencedirect.com
Tetrahedron 64 (2008) 4370e4376
www.elsevier.com/locate/tet
Synthesis and hybridization properties of 20-O-(tetrazol-5-yl)ethyl-
modified oligonucleotides
Hisao Saneyoshi a,b, Keigo Tamaki a,b, Akihiro Ohkubo a,b, Kohji Seio a,b, Mitsuo Sekine a,b,
*
a Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midoriku, Yokohama 226-8501, Japan
b CREST, JST (Japan Science and Technology Agency), Nagatsuta, Midoriku, Yokohama 226-8501, Japan
Received 19 October 2007; received in revised form 22 February 2008; accepted 22 February 2008
Available online 4 March 2008
Abstract
20-O-(1H-Tetrazol-5-yl)ethyladenosine was synthesized using 20-O-cyanoethyladenosine derivative as a key intermediate. The 20-O-(1H-
tetrazol-5-yl)ethyl modifications exhibited intriguing properties such as the change in the structure of the tetrazole residue between a protonated
and a deprotonated form. The Tm experiments of various oligodeoxynucleotides having a 20-O-(1H-tetrazol-5-yl)ethyl-modified adenosine
showed reduced hybridization affinity in comparison to the unmodified oligonucleotides toward their complementary oligodeoxynucleotides.
The mechanism of the reduced hybridization affinity was discussed on the basis of the structure and the physicochemical properties of the
tetrazole moiety.
Ó 2008 Published by Elsevier Ltd.
Keywords: 20-O-Modified oligonucleotide; Cyanoethyl; (1H-Tetrazol-5-yl)ethyl; pH-dependent hybridization
1. Introduction
interaction site for the hybridization affinity. There are numer-
ous studies that utilized the high versatility of tetrazole.7e10 For
example, in nucleic acid chemistry, tetrazoles have been used as
a proton donor/nucleophile for activating the phosphoramidite
derivatives,11 a component of 30-N-tetrazolylthymidine (AZT
analogue),12 water-soluble inter-nucleotidic bond mimics,13
and as a N-tetrazolyl nucleobase for a metal-mediated base
pair.14 On the other hand, because of the acidity of the 5-carbon
substituted tetrazole function (pKa¼5.5e5.6),15 the tetrazole
moiety is in the monoanionic form at the physiological condi-
tions. This character could affect the hybridization property
of oligonucleotides containing tetrazole residues (Fig. 1).
In this paper, we report the synthesis of oligonucleotides
containing 20-O-[(1H-tetrazol-5-yl)ethyl]adenosine (At) and
Chemically modified oligonucleotides have been utilized as
indispensable materials for DNA chip technology,1 gene ther-
apy,2 gene regulation,3 and recent nanotechnology,4 because
of their hybridization affinity for target DNA or RNA mole-
cules. Modification of their carbohydrate residues at the 20-
position has been studied as the principal strategy for improving
hybridization affinity, nuclease resistance, and conjugation with
functional molecules in nucleic acids.5 We have recently re-
ported that 20-O-cyanoethyl RNAs have superior properties
compared to simple 20-O-alkylated RNAs with respect to their
hybridization ability and nuclease resistance.6 It is well known
that the cyano group can be converted to other functional groups
by a variety of chemical reactions. Therefore, 20-O-cyanoethyl-
ated ribonucleosides may be useful precursors for the synthesis
of further functionalized 20-O-modified ribonucleosides.
OH-
H+
H
N
R
R
N
N
N
N
N
N
In this study, we focused on the conversion of the cyanoethyl
group into the (1H-tetrazol-5-yl)ethyl group that could be a new
N
R = oligonucleotide
* Corresponding author. Tel.: þ81 45 924 5706; fax: þ81 45 924 5772.
E-mail address: msekine@bio.titech.ac.jp (M. Sekine).
Figure 1. Interconversion between a neutral and a dissociated species of the
tetrazole moiety.
0040-4020/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.tet.2008.02.075

H. Saneyoshi et al. / Tetrahedron 64 (2008) 4370e4376
4371
20-O-[(1-methyl-1H-tetrazol-5-yl)ethyl]adenosine (Amt) as
a reference material and their hybridization affinity for the com-
plementary DNA oligomers. In addition, 20-O-[(1H-tetrazol-5-
yl)ethyl]adenosine 30-phosphoramidite building block and its
methylated derivative were synthesized via 20-O-cyanoethyl-
adenosinepreviouslyreportedbyus astheprecursorsforthesyn-
thesis of the corresponding 30-phosphoramidite building blocks
and were successfully incorporated into oligonucleotides.
the S-type (C20-endo) conformation caused by the introduction
of the tetrazolylethyl group.
To incorporate 3 into oligonucleotides, a suitable protecting
group of the tetrazole moiety was required because the unpro-
tected tetrazole moiety would accelerate the decomposition of
the phosphoramidite unit. Protecting groups such as trityl,
tetrahydropyranyl, benzyl, and cyanoethyl have been used
for the NH function of the tetrazole residue.7b We chose the
cyanoethyl group as the protecting group because it could be
removed under the same conditions as those prescribed for
the removal of base and phosphate.
2. Results and discussion
Treatment of 3 with phenoxyacetyl chloride in pyridine
produced an 80% yield of the N-protected derivative 6. In
a previous paper, we reported a new method for the cyanoeth-
ylation of the hydroxyl group using Cs2CO3 in t-BuOH.6
Therefore, this method was initially used for the cyanoethyl-
ation of the tetrazole moiety of 1. However, cyanoethylation
at the base moiety occurred simultaneously. The basicity of
Cs2CO3 seemed to be responsible for this result, because
Cs2CO3 could abstract tetrazolic proton and the relatively
acidic proton of the amide group on the base moiety too.
Therefore, we used NaHCO3 as a less basic reagent to avoid
the side reaction. However, cyanoethylation occurred neither
at the tetrazole moiety nor the base moiety. In this case, the
NaHCO3 as a base was too weak to activate tetrazole moiety
at room temperature. Finally, we found that the reaction at
80 ꢀC in t-BuOH produced a mixture of N-cyanoethylated
regioisomers 7a and 7b. From the mixture, 7a and 7b were
isolated with yields of 12 and 23%, respectively, and the cya-
noethylated site was determined using HMBC spectra to con-
firm the structures shown in Scheme 1. Next, to observe the
effect of the acidic proton of the tetrazole moiety on hybridiza-
tion affinity, we synthesized the adenosine derivatives 8a and
2.1. Synthesis of 20-O-[(1H-tetrazol-5-yl)ethyl]adenosine
derivatives and protected adenosine 30-phosphoramidite
derivatives
Conversion of the nitrile group of the 20-O-cyanoethylated
adenosine derivative (1)16 to a tetrazole ring was studied. The
combined use of sodium azide and ammonium chloride,17
which have been widely used for tetrazole ring formation,
resulted in the formation of complex mixtures. These reaction
media are slightly acidic; therefore, an increase in the temper-
ature of the reaction led to considerable loss of the 1,1,3,3-
tetraisopropyldisiloxane-1,3-diyl (TIPDS) group. However,
the use of trimethylsilylazide (TMSN3) in the presence of
a catalytic amount of Bu2SnO18 produced an 89% yield of
the desired product 2 without decomposition.
The treatment of the resulting product 2 with Et3N$3HF
produced a 67% yield of 20-O-[(1H-tetrazol-5-yl)ethyl]adeno-
sine (3). The sugar conformation of 3 was analyzed by H
NMR and compared with that of 20-O-methyladenosine (4)
and 20-O-(2-cyanoethyl)adenosine (5),6 as shown in Table S1.
The analyses revealed a slight increase in the population of
1
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
N
N
TMSN3
Et3N-3HF
Et3N
THF
N
O
O
O
nBu2SnO
O
HO
Si
O
Si
O
O
At
Toluene
H
N
H
N
O
O
Si
O
O
Si
CN
HO
O
N
N
N
N
N
N
3:67%
1
2:89%
Phenoxyacetyl chloride
pyridine
O
O
O
O
O
O
NH
NH
NH
N
N
N
N
N
N
N
N
N
N
N
N
or
MeI
CN
O
O
O
O
O
O
NaHCO3
Si
O
Si
O
Si
O
t-BuOH or DMF
R
N
H
N
O
O
O
O
Si
Si
O
O
N
Si
N
R
N
N
N
N
N
N
N
N
6:80%
7b (R = Ce):23%
8b (R = Me):21%
7a (R = Ce):12%
8a (R = Me):12%
Scheme 1. Synthesis of 20-O-[(1H-tetrazol-5-yl)ethyl]adenosine derivatives and its N-alkylated derivatives.

4372
H. Saneyoshi et al. / Tetrahedron 64 (2008) 4370e4376
Table 1
Modified oligodeoxynucleotides synthesized using 11 and 14
8b containing the N-methylated tetrazoyl group. Methylation
of 6 with methyl iodide in DMF in the presence of NaHCO3
produced a 12% yield of the desired compound 8a. In this
case, a 21% yield of the alkylated regioisomer 8b was also ob-
tained. The alkylated sites were determined from the HMBC
spectra.
The 20-O-(2-cyanoethyl-1H-tetrazol-5-yl)adenosine 30-phos-
phoramidite derivative 11 was synthesized from compound 7b,
as shown in Scheme 2. Treatment of 7b with Et3N$3HF
produced a 99% yield of the 30,50-O-free derivative 9. The usual
tritylation of 9 produced a 73% yield of the 50-O-protected
derivative 10. Phosphitylation of the resulting product 10 with
(i-Pr2N)2POCH2CH2CN produced a 71% yield of 11. Similarly,
the 20-O-(1-methyl-1H-tetrazol-5-yl)adenosine 30-phosphor-
amidite derivative 14 was synthesized from the compound 8a
via compounds 12 and 13, as shown in Scheme 2.
Oligonucleotide
MALDI-TOF MASS
Calcd.
Found
a
15
16
17
18
19
20
a
50-d(CGTAGAtCTATGCTAT)-30
4677.8
4691.9
5014.0
5070.0
5014.0
5070.0
4679.1
4695.2
5015.7
5066.2
5018.8
5075.1
b
50-d(CGTAGAmtCTATGCTAT)-30
a
50-d(CGTAtGAtCTAtTGCTAtT)-30
b
50-d(CGTAmtGAmtCTAmtTGCTAmtT)-30
a
50-d(CGTGCAtAtAtAtTGTCTT)-30
b
50-d(CGTGCAmtAmtAmtAmtTGTCTT)-30
Deprotection conditions, NH4OH, 55 ꢀC, 5 h.
b
Deprotection conditions, NH4OH, rt, 8 h. At: 20-O-[(1H-tetrazol-5-yl)-
ethyl]adenosine, Amt: 20-O-[(1-methyl-1H-tetrazol-5-yl)ethyl]adenosine.
to evaluate the effect of the attachment of the 20-substituent
and the anionic modification. The Tm data are described in
Table 2. As a results, the introduction of At (15) and Amt
(16) reduced the hybridization affinities toward 22 in compar-
ison with unmodified 21 by 2.9 and 2.0 ꢀC, respectively. In ad-
dition, the stability of the 15/22 duplex was proved to be
0.9 ꢀC lower than the 16/22 duplex, probably because the
deprotonation of the tetrazole ring of At generated an addi-
tional negative charge and caused anioneanion repulsion
with internucleotide phosphates.
2.2. Synthesis and properties of modified oligodeoxy-
nucleotides
Next, to observe the effects of the tetrazole-type modifica-
tions on duplex stability when methylated, 20-O-tetrazolyl-5-
ylethyl-modified oligodeoxynucleotides 15, 17, and 19, and
20-O-methyltetrazol-5-ylethyl-modified 16, 18, and 20 with
either one or four 20-O-modified adenosine residues were syn-
thesized by solid-phase synthesis, as summarized in Table 1.
We noticed that the cyanoethyl groups on the tetrazole moie-
ties in the oligonucleotides were not completely deprotected
under NH4OH at room temperature for 8 h (conditions b) in
the case of 15, 17, and 19 according to the anion-exchange
HPLC analysis. Therefore, the deprotection reactions were
carried out in NH4OH at 55 ꢀC for 5 h (conditions a) for these
oligonucleotides. The modified oligomers 15 and 16 have one-
point modification with At and Amt, respectively, at the
central position. Oligomers 17 and 18 have four modified
nucleosides at discontinuous positions. Oligomers 19 and 21
have a consecutive sequence of four modified nucleosides.
Hybridization affinities of unmodified 50-d(CGTAGAC-
TATGCTAT)-30 (21), 15, and 16 toward the complementary
strand 30-d(GCATCTGATACGATA)-50 (22) were measured
Table 2
Effects of the 20-substituents on the duplex stabilitiesa
Oligonucleotide
Tm (ꢀC)
DTm (ꢀC)
21
15
16
a
50-d(CGTAGACTATGCTAT)-30
50-d(CGTAGAtCTATGCTAT)-30
50-d(CGTAGAmtCTATGCTAT)-30
48.5
45.6
46.5
d
ꢁ2.9
ꢁ2.0
Conditions: 2.0 mM of 21, 15, or 16 and 2.0 mM of 22 in 10 mM sodium
phosphate buffer (pH 7.0), 0.1 M NaCl, 0.1 mM EDTA.
The 20-O-cationic modified oligonucleotides are known to
stabilize duplexes due to the neutralization of phosphate an-
ions in the case of a discontinuous sequence.19 However, a con-
secutive sequence of modified nucleosides retains or decreases
the hybridization affinity due to the cationecation charge re-
pulsions.19 Therefore, we next examined the anionic repul-
sions between dissociated tetrazole moieties and phosphate
anions in duplexes with four At or Amt modifications using
O
O
O
O
O
NH
O
NH
NH
N
N
N
N
N
(i-Pr2N)2POCH2CH2CN
Diisopropylamine
N
N
N
N
N
N
Et3N-3HF
Et3N
N
DMTrO
1 H-tetrazole
CH2Cl2, rt
HO
DMTrCl
pyridine, rt
O
O
O
DMTrO
7b or 8a
O
THF, rt
O
P
O
HO
O
R
HO
O
R
CN
R
N
9: 99% from 7b
12: 98% from 8a
10: 73% from 9
13: 72% from 12
11: 71% from 10
14: 55% from 13
CH3
N
N
N
CN
N
N
R =
and
for 12, 13, 14
for 9, 10, 11
N
N
N
Scheme 2. Synthesis of protected adenosine 30-phosphoramidite derivatives.

H. Saneyoshi et al. / Tetrahedron 64 (2008) 4370e4376
4373
the duplexes 17/22, 18/22, 19/23{30-d(GCACGTTTTACA-
GAA)-50}, and 20/23.
phosphoramidite derivative 11. The 20-O-(1H-tetrazol-5-yl)-
ethyl modification provided a new property different from that
of 20-O-cyanoethylated oligonucleotides. The oligonucleotides
incorporating At having a 1H-tetrazol-5-yl group showed re-
duced hybridization affinity toward the complementary strands
probably due to the anionic charges of the tetrazole moiety as
clarified by the comparative Tm analyses by use of the oligonu-
cleotides incorporating Amt having a 1-methyl-1H-tetrazol-5-
yl group. Although the methylation of the acidic NH group
slightly improved the hybridization properties, the Tm values
of the duplexes modified with Amt were still much lower than
thoseof theunmodified duplexes. Therefore, notonlytheanionic
charges of the tetrazolide moiety but also the 20-O-(1H-tetrazol-
5-yl)ethyl skeleton itself seemed to contribute to the duplex
instability. Although such duplex destabilize, this ionizable
modification would make it possible to interact with a metale
cation, proton and/or protonated amine for the regulation of
thehybridizationaffinities. Itshouldbenoted, however, although
thepKa of 1H-tetrazole(5.5e5.6)supported the deprotonation of
thetetrazole moietyofAtintheoligonucleotides, thestructureof
the tetrazole moiety in the oligonucleotides has not yet been de-
termined directly. Detailed physicochemical experiments such
as NMR and UV titration of the oligonucleotides are necessary
to clarify this point. The studies on the structures and the appli-
cability of the oligonucleotides modified with the (1H-tetrazol-
5-yl)ethyl are underway and will be reported elsewhere.
As shown in Table 3, the introduction of the four At modifi-
cations into 17 and 19 significantly decreased the hybridization
affinity as compared to their neutral counterparts 18 and 20 by
3.3 and 5.0 ꢀC, respectively. As clarified by the comparison of
the two DTm values, the consecutively introduced At residues in
19 decreased the hybridization affinity more than the non-
consecutively modified 17. It should be noted that although
the Tm of the 18/22 duplex became higher than that of the
anionic 17/22 duplex, it was still much lower than that of the
unmodified 21/22 duplex. Therefore, it seemed that not only
the anionic charge of the At residue but also the steric and
conformational effects of At and Amt residues contributed to
the duplex destabilization. A similar trend was observed for
the 19/23 and 20/23 duplexes in comparison with the unmodi-
fied duplex of 50-d(CGTGCAAAATGTCTT)-30 (24)/23, which
gave the Tm of 55.7 ꢀC.
Table 3
The sequence dependency of the duplex stabilitiesa
Oligonucleotide
Tm (ꢀC)
DTm (ꢀC)
17
18
19
20
a
50-d(CGTAtGAtCTAtTGCTAtT)-30
34.3
37.6
43.6
48.6
d
þ3.3b
50-d(CGTAmtGAmtCTAmtTGCTAmtT)-30
50-d(CGTGCAtAtAtAtTGTCTT)-30
50-d(CGTGCAmtAmtAmtAmtTGTCTT)-30
d
þ5.0c
Conditions: 2.0 mM 17, 18, 19, or 20 and 2 mM of 22 in 10 mM sodium
phosphate buffer (pH 7.0), 0.1 M NaCl, 0.1 mM EDTA.
b
DTm¼(Tm of 18/22 duplex)ꢁ(Tm of 17/22 duplex).
c
DTm¼(Tm of 20/23 duplex)ꢁ(Tm of 19/23 duplex).
4. Experimental section
Finally, the thermodynamic parameters of the duplexes 17/
22 and 18/22 were also estimated (Table 4). The DH0 values of
17/22 and 18/22 duplexes were ꢁ81.5 and ꢁ86.8 kcal/mol,
respectively. These data suggest that the less negative enthalpy
value of 17 compared to 18 was the main reason for the lower
hybridization affinity.
4.1. General remarks
1H NMR spectra were recorded at 500 MHz and the chemi-
cal shifts were measured from the solvent peak as an internal
standard, and 13C NMR spectra were recorded at 500 MHz and
the chemical shifts were measured from the solvent peak used
as an internal standard and dioxane (D2O) used as an external
standard. 31P NMR spectra were recorded at 109 MHz and the
chemical shifts were measured from 85% H3PO4 as an external
standard. Pyridine was distilled twice from p-toluenesulfonyl-
chloride and CaH2 after being refluxed for several hours and
Table 4
Thermodynamic parameters of the duplexes containing At and Amta
Duplex
DH0 (kcal/mol)
DS0 (cal/K mol)
DG037 (kcal/mol)
17/22
18/22
a
ꢁ81.5
ꢁ86.8
ꢁ237
ꢁ252
ꢁ8.0
ꢁ8.7
˚
stored over molecular sieves 4 A. TLC was performed on
Conditions: 10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl,
0.1 mM EDTA and 60, 50, 30, 25, 15, 10, 5, 3, 2, 1.5, 1.0, 0.6, and 0.5 mM
duplex, respectively. Eq: 1/Tm¼DS/DHþR ln(Ct/4)/DH.
Merck Kieselgel 60 F254 precoated glass plates. Column chro-
matography was performed with silica gel C-200, C-300
(Wako Co., Ltd.), 60 N (Kanto Chemical, Co., Inc.), and NH
(Fuji Silysia Chemical Ltd.), and a minipump for a goldfish
bowl was conveniently used to attain sufficient pressure for
rapid chromatographic separation. Recycling preparative
HPLC was performed by use of an apparatus from Japan
Analytical Industry Co., Ltd. Anion-exchange HPLC was
performed using the following systems. System A: anion-
exchange HPLC was performed on a Waters Aliance system
with a Waters 3D UV detector and a Gen-Pak FAX column
(Waters, 4.6ꢂ100 mm). A linear gradient (0e60%) starting
from 25 mM sodium phosphate buffer (pH 6.0) and applying
25 mM sodium phosphate buffer (pH 6.0) containing 1 M
NaCl (pH 6.0) was used at a flow rate of 1 mL/min for
3. Conclusion
In this paper, a new class of artificially 20-modified ribonu-
cleosides were synthesized using a 20-O-cyanoethylated adeno-
sine derivative (1) as a precursor. Chemical conversion of the
nitrile function of the cyanoethyl group into the 1H-tetrazol-
5-yl group was carried out using trimethylsilylazide in the
presence of a catalytic amount of dibutyltin oxide without
losing the 30,50-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)
group. The NH function of the tetrazole moiety was pro-
tected with the cyanoethyl group by a Michael reaction.
Selective N-protection was achieved for preparing the

4374
H. Saneyoshi et al. / Tetrahedron 64 (2008) 4370e4376
45 min at 50 ꢀC. High resolution ESI mass spectrometry was
performed by use of a Mariner (PerSeptive Biosystems, Inc.).
166.8. HRMS calcd for C33H49N9O7Si2 (MþHþ) 740.3366,
found 740.3330.
4.1.1. 30,50-O-(1,1,3,3-Tetraisopropyldisiloxane-1,3-diyl)-
20-O-[2-(tetrazol-5-yl)ethyl]adenosine (2)
4.1.4. 20-O-[(1-N-Cyanoethyltetrazol-5-yl)ethyl]-6-N-
phenoxyacetyl-30,50-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)adenosine (7a) and its regioisomer (7b)
Compound 1 (23.3 g, 41.4 mmol) was dissolved in anhy-
drous toluene (200 mL). To the solution were added trimethyl-
silyilazide (27.5 mL, 207 mmol) and n-Bu2SnO (2.06 g,
8.28 mmol). After being stirred at 110 ꢀC for 23 h, the mixture
was evaporated in vacuo. The residue was chromatographed on
a column of silica gel with hexane/CHCl3/MeOH (1:0:0/1:1:0/
Compound 6 (0.5 g, 0.676 mmol) was dissolved in t-BuOH
(3.4 mL). To the solution were added acrylonitrile (221 ml,
3.4 mmol) and NaHCO3 (57 mg, 0.680 mmol). After being
stirred at 80 ꢀC for 23 h, the reaction mixture was evaporated
in vacuo. The residue was diluted with ethyl acetate and washed
with brine. The organic phase was dried over Na2SO4 and
filtrated. The solution was evaporated in vacuo. The residue
was chromatographed on a column of silica gel with CHCl3/
MeOH (99.7:0.3, v/v) to give compounds 7a (66 mg, 12%)
and 7b (125 mg, 23%) was eluted with CHCl3/MeOH
1
0:1:0/0:98:2, v/v/v) to give compound 2 (22.4 g, 89%). H
NMR (CDCl3, 500 MHz) d 0.94e1.13 (28H, m), 3.36e3.40
(2H, m), 4.06e4.10 (2H, m), 4.20 (1H, d, J¼8.6 Hz), 4.24
(1H, d, J¼4.6 Hz), 4.30 (1H, d, J¼13.6 Hz), 4.38e4.41 (1H,
m), 4.67e4.70 (1H, m), 5.92 (2H, br), 6.09 (1H, s), 8.17 (1H,
s), 8.39 (1H, s); 13C NMR (CDCl3) d 12.3, 12.9, 13.1, 13.6,
16.9, 17.2, 17.4, 17.5, 24.4, 59.4, 68.1, 69.2, 81.8, 82.6, 88.4,
120.5, 138.0, 148.5, 153.3, 153.8, 155.8. HRMS calcd for
C25H43N9O5Si2 (MþHþ) 606.2999, found 606.3051.
1
(99.6:0.4, v/v). H NMR of 7a (CDCl3, 500 MHz) d 0.92e
1.10 (28H, m), 3.10e3.14 (2H, m), 3.32e3.37 (2H, m), 3.97
(2H, d, J¼11.0 Hz), 4.18e4.22 (2H, m), 4.25e4.30 (2H, m),
4.65e4.67 (2H, m), 4.71e4.80 (2H, m), 4.86 (2H, br), 6.00
(1H, s), 7.03e7.06 (3H, m), 7.32e7.35 (2H, m), 8.27 (1H, s),
8.74 (1H, s), 9.42 (1H, br); 13C NMR of 7a (CDCl3) d 12.5,
12.9, 13.0, 13.4, 16.9, 17.1, 17.1, 17.2, 17.4, 17.4, 17.5, 18.9,
24.8, 42.9, 59.7, 68.2, 69.3, 69.6, 81.8, 83.1, 88.3, 115.1,
116.1, 122.6, 123.4, 130.0, 141.5, 148.5, 150.8, 152.7, 153.8,
157.1, 166.8. HRMS of 7a calcd for C36H52N10O7Si2
(MþHþ) 793.3632, found 793.3548.
4.1.2. 20-O-[2-(Tetrazol-5-yl)ethyl]adenosine (3)
Compound 2 (151 mg, 0.25 mmol) was dissolved in anhy-
drous THF (2.5 mL). To the solution were added triethylamine
trihydrogen fluoride (141 ml, 0.88 mmol) and triethylamine
(62 ml, 0.45 mmol). After being stirred at room temperature
for 1 h, the mixture was evaporated in vacuo. The residue was
chromatographed on a column of silica gel with hexane/ethyl
acetate (3:1/1:1, v/v) and passed through Dowex (Hþ) to give
compound 3 (61 mg, 67%). 1H NMR (D2O, 500 MHz)
d 3.13e3.17 (2H, m), 3.82e3.90 (3H, m), 4.17e4.22 (1H,
m), 4.28e4.30 (1H, m), 4.47e4.54 (1H, m), 4.43e4.54 (1H,
m), 4.73e4.85 (1H, m), 5.93 (1H, d, J¼7.1 Hz), 8.17 (1H, s),
8.19 (1H, s); 13C NMR (D2O) d 24.5, 62.2, 68.0, 69.7, 82.4,
86.9, 87.5, 119.5, 141.2, 148.5, 152.0, 155.4, 155.8. HRMS
calcd for C13H17N9O4 (MþHþ) 364.1476, found 364.1483.
1H NMR of 7b (CDCl3, 500 MHz) d 0.98e1.10 (28H, m),
3.09e3.11 (2H, t, J¼6.8 Hz), 3.23e3.35 (2H, m), 3.98e4.02
(1H, m), 4.09e4.20 (3H, m), 4.32e4.33 (1H, d, J¼4.6 Hz),
4.39e4.43 (1H, m), 4.77 (1H, q, J¼4.6 Hz), 4.83e4.86 (4H,
m), 6.00 (1H, s), 7.05e7.08 (3H, m), 7.33e7.37 (2H, m),
8.28 (1H, s), 8.75 (1H, s), 9.41 (1H, br); 13C NMR of 7b
(CDCl3) d 12.7, 12.9, 13.0, 13.5, 17.0, 17.1, 17.2, 17.3,
17.4, 17.5, 18.3, 26.9, 48.0, 59.9, 68.2, 69.1, 69.8, 81.5,
82.4, 88.9, 115.1, 115.6, 122.5, 123.4, 129.9, 142.2, 148.4,
151.0, 152.6, 157.1, 165.0, 166.8. HRMS of 7b calcd for
C36H52N10O7Si2 (MþHþ) 793.3632, found 793.3656.
4.1.3. 6-N-Phenoxyacetyl-30,50-O-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)-20-O-[2-(tetrazol-5-yl)ethyl]adenosine (6)
Compound 3 (4.85 g, 8 mmol) was co-evaporated once with
anhydrous pyridine and dissolved in anhydrous pyridine
(40 mL). To the solution was added phenoxyacetyl chloride
(1.22 mL, 8.8 mmol). After being stirred at room temperature
for 4 h, the reaction mixture was quenched with H2O and evap-
orated invacuo. The residuewas diluted with CHCl3 and washed
with brine and aq NaHCO3. The organic phase was dried over
Na2SO4 and filtrated. The residue was chromatographed on
a column of silica gel with hexane/CHCl3/MeOH (50:50/
0:100:0/95:5, v/v) to give compound 6 (4.73 g, 80%). 1H
NMR (CDCl3, 500 MHz) d 3.34e3.40 (1H, m), 3.43e3.47
(1H, m), 3.98e4.03 (1H, m), 4.07e4.10 (1H, m), 4.25e4.27
(1H, m), 4.31e4.33 (2H, m), 4.51e4.54 (1H, m), 4.77 (1H,
dd, J¼4.6, 4.9 Hz), 4.85 (2H, br), 6.18 (1H, s), 7.04 (3H, m),
7.34e7.38 (2H, m), 8.36 (1H, s), 8.79 (1H, s), 9.47 (1H, br);
13C NMR (CDCl3) d 12.1, 12.9, 13.0, 13.5, 16.8, 17.1, 17.3,
17.4, 17.5, 24.6, 59.2, 68.1, 68.2, 68.9, 81.9, 83.1, 88.1, 115.1,
122.6, 123.5, 130.0, 141.1, 148.6, 150.7, 152.8, 153.8, 157.1,
4.1.5. 20-O-[(1-N-Methyltetrazol-5-yl)ethyl]-6-N-phenoxy-
acetyl-30,50-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-
adenosine (8a) and its regioisomer (8b)
Compound 6 (0.5 g, 0.676 mmol) was dissolved in anhy-
drous DMF (3.4 mL). To the solution were added NaHCO3
(57 mg, 0.680 mmol) and methyl iodide (210 ml, 3.4 mmol).
After being stirred at room temperature for 19 h, the mixture
was evaporated in vacuo. The residue was diluted with ethyl
acetate and washed with H2O three times. The organic layer
was dried over Na2SO4, and filtered. The solution was evapo-
rated in vacuo. The residue was chromatographed on a column
of silica gel with CHCl3 to give 8b (107 mg, 21%). Compound
8a (59 mg, 12%) was eluted with CHCl3/MeOH (99.6:0.4, v/v).
1H NMR of 8a (CDCl3, 500 MHz) d 0.92e1.11 (28H, m),
3.22e3.31 (2H, m), 3.96e4.00 (2H, m), 4.12 (3H, s), 4.18e
4.19 (1H, m), 4.21 (1H, s), 4.25e4.27 (2H, m), 4.64e4.67
(1H, m), 4.86 (2H, s), 6.00 (1H, s), 7.03e7.06 (3H, m),
7.32e7.36 (2H, m), 8.28 (1H, s), 8.74 (1H, s), 9.41 (1H, br);

H. Saneyoshi et al. / Tetrahedron 64 (2008) 4370e4376
4375
13C NMR of 8a (CDCl3) d 12.5, 12.9, 13.0, 13.4, 16.9, 17.1,
17.2, 17.4, 17.5, 24.7, 34.0, 59.7, 68.3, 69.2, 69.5, 81.8, 83.0,
88.3, 115.1, 122.6, 123.4, 130.0, 141.5, 148.5, 150.8, 152.7,
153.5, 157.1, 166.8. HRMS of 8a calcd for C34H52N9O7Si2
(MþNaþ) 776.3348, found 776.3300.
organic layer was dried over MgSO4 and filtered. The solution
was evaporated in vacuo. The residue was chromatographed on
a column of silica gel with hexane/ethyl acetate (1:1/0:1, v/v)
containing 0.5% triethylamine to give compound 10 as white
1
foam (5.15 g, 73%). H NMR (CDCl3, 500 MHz) d 3.10 (2H,
1H NMR of 8b (CDCl3, 500 MHz) d 0.97e1.14 (28H, m),
3.22e3.31 (2H, m), 4.00e4.03 (1H, m), 4.11e4.22 (3H, m),
4.27 (3H, s), 4.33e4.34 (1H, m), 4.37e4.39 (1H, m), 4.75e
4.81 (1H, m), 4.89 (2H, s), 6.02 (1H, s), 7.04e7.08 (3H, m),
7.32e7.37 (2H, m), 8.31 (1H, s), 8.75 (1H, s), 9.58 (1H, br);
13C NMR of 8b (CDCl3) d 12.7, 12.8, 13.0, 16.9, 17.0, 17.1,
17.2, 17.3, 17.5, 26.8, 39.2, 59.8, 68.2, 69.3, 69.7, 81.5,
82.3, 88.9, 115.0, 122.4, 123.3, 129.8, 142.1, 148.4, 150.9,
152.5, 157.1, 164.2, 166.9. HRMS of 8b calcd for
C34H52N9O7Si2 (MþNaþ) 776.3348, found 776.3493.
m), 3.20e3.21 (1H, m), 3.24 (1H, dd, J¼4.2, 5.1 Hz), 3.41e
3.44 (1H, m), 3.51e3.54 (1H, m), 3.78 (6H, s), 4.11e4.14
(1H, m), 4.16e4.19 (2H, m), 4.26 (1H, dd, J¼4.2, 3.7 Hz),
4.54 (1H, q, J¼4.9 Hz), 4.75 (1H, t, J¼4.6 Hz), 4.85e4.88
(4H, m), 6.16 (1H, d, J¼4.6 Hz), 6.80e6.83 (4H, m), 7.05e
7.08 (3H, m), 7.20e7.38 (9H, m), 7.43e7.45 (2H, m), 8.22
(1H, s), 8.71 (1H, s), 9.41 (1H, br); 13C NMR (CDCl3)
d 18.2, 26.3, 48.2, 55.3, 63.2, 67.5, 68.2, 69.9, 81.9, 84.4,
86.7, 87.0, 113.3, 115.1, 115.5, 122.6, 123.5, 127.1, 128.0,
128.3, 130.0, 130.2, 135.7, 142.4, 144.6, 148.4, 151.7, 152.6,
157.1, 158.7, 165.0, 166.7. HRMS calcd for C45H44N10O8
(MþNaþ) 875.3236, found 875.3234.
4.1.6. 20-O-[(2-N-Cyanoethyltetrazol-5-yl)ethyl)]-6-N-
phenoxyacetyladenosine (9)
Compound 7a (5.02 g, 6.35 mmol) was dissolved in anhy-
drous THF (32 mL). To the solution were added Et3N$3HF
(3.6 mL, 22.2 mmol) and triethylamine (1.6 mL, 11.4 mmol).
After being stirred at room temperature for 3 h, the mixture
was evaporated in vacuo. The residue was chromatographed
on a column of silica gel with hexane/CHCl3/MeOH (100:0:0/
4.1.9. 50-O-(4,40-Dimethoxyltrityl)-20-O-[(1-N-methyl-
tetrazol-5-yl)ethyl]-6-N-phenoxyacetyladenosine (13)
1H NMR (CDCl3, 500 MHz) d 2.95e3.00 (1H, m), 3.14e
3.16 (1H, m), 3.42 (1H, dd, J¼4.2, 6.6 Hz), 3.51 (1H, dd,
J¼3.2, 7.6 Hz), 3.78 (6H, m), 4.00 (3H, s), 4.15e4.17 (1H,
m), 4.28 (1H, t, J¼3.9 Hz), 4.30 (1H, d, J¼8.8 Hz), 4.65
(1H, q, J¼4.6 Hz), 4.78e4.80 (1H, m), 4.85 (2H, br), 5.20
(1H, d, J¼5.1 Hz), 6.14 (1H, J¼5.1 Hz), 6.81e6.83 (4H, m),
7.05e7.08 (3H, m), 7.20e7.29 (3H, m), 7.32e7.38 (6H, m),
7.43e7.45 (2H, m), 8.24 (1H, s), 8.71 (1H, s), 9.41 (1H, br);
13C NMR (CDCl3) d 24.3, 33.5, 55.4, 63.3, 65.7, 68.2, 70.1,
82.0, 84.5, 86.8, 87.1, 113.3, 115.1, 122.6, 123.5, 127.1,
128.1, 128.3, 130.0, 130.2, 135.8, 142.5, 144.7, 148.4,
151.8, 152.6, 153.2, 157.1, 158.7, 166.7. HRMS calcd for
C43H43N9O8 (MþNaþ) 836.3127, found 836.3192.
1
0:98:2, v/v/v) to give compound 9 (3.46 g, 99%). H NMR
(CDCl3, 500 MHz) d 3.07e3.22 (4H, m), 3.78e3.87 (2H, m),
3.99e4.05 (2H, m), 4.41 (1H, s), 4.57 (1H, br), 4.69e4.70
(1H, d, J¼4.2 Hz), 4.86e4.91 (5H, m), 5.92e5.94 (1H, d,
J¼7.6 Hz), 6.01 (1H, d, J¼10.5 Hz), 7.05e7.09 (3H, m),
7.34e7.38 (2H, m), 8.02 (1H, s), 8.80 (1H, s), 9.46 (1H, br).
13C NMR (CDCl3) d 18.3, 26.2, 48.3, 63.5, 66.9, 68.2, 70.7,
80.2, 81.6, 88.5, 89.5, 115.1, 115.5, 122.7, 124.7, 130.1,
143.9, 149.3, 150.8, 152.2, 157.1, 165.1, 166.7. HRMS calcd
for C24H26N10O6 (MþHþ) 551.2110, found 551.2127.
4.1.10. 20-O-[(2-N-Cyanoethyltetrazol-5-yl)ethyl]-50-O-
(4,40-dimethoxyltrityl)-6-N-phenoxyacetyladenosine
4.1.7. 20-O-[(1-N-Methyltetrazol-5-yl)ethyl]-6-N-phenoxy-
acetyladenosine (12)
30-(2-cyanoethyl N,N-diisopropylphosphoramidite) (11)
Compound 10 (341 mg, 0.4 mmol) was co-evaporated four
times with anhydrous toluene and once with anhydrous
CH2Cl2, and dissolved in anhydrous CH2Cl2 (2 mL) under argon
atmosphere. Tothesolutionwereadded2-cyanoethylN,N,N0,N0-
tetraisopropylphosphorodiamidite (152 ml, 0.48 mmol) and
diisopropylammonium tetrazolide (27 mg, 0.16 mmol). And
further 2-cyanoethyl N,N,N0,N0-tetraisopropylphosphorodiami-
dite (62 ml, 0.20 mmol) and diisopropylammonium tetrazolide
(20 mg, 0.12 mmol) were added. After being stirred at room
temperature for 6 h, the mixture was diluted with ethylacetate.
The solution was washed with brine and aq NaHCO3. The or-
ganic layer was dried over Na2SO4 and filtered. The solution
was evaporated in vacuo. The residue was purified by use of re-
cycling preparative HPLC eluted with ethyl acetate to give com-
1H NMR (CDCl3, 500 MHz) d 2.89e2.93 (1H, m), 3.04e
3.10 (1H, m), 3.80 (1H, m), 3.88e3.91 (1H, m), 3.96e4.03
(4H, m), 4.12e4.17 (1H, m), 4.41 (1H, s), 4.78 (1H, d,
J¼4.2 Hz), 4.84e4.88 (3H, m), 5.67 (1H, br), 5.94 (1H, d,
J¼7.8 Hz), 6.01 (1H, br), 7.03e7.06 (3H, m), 7.32e7.36
(2H, m), 8.07 (1H, s), 8.78 (1H, s), 9.57 (1H, br); 13C NMR
(CDCl3) d 24.1, 33.5, 63.4, 64.9, 68.2, 70.3, 81.5, 88.5,
89.2, 115.0, 122.6, 124.6, 130.0, 144.0, 149.2, 150.8, 152.1,
153.3, 157.1, 166.9. HRMS calcd for C22H25N9O6 (MþNaþ)
534.1820, found 534.1859.
4.1.8. 20-O-[(2-N-Cyanoethyltetrazol-5-yl)ethyl]-50-O-
(4,40-dimethoxyltrityl)-6-N-phenoxyacetyladenosine (10)
Compound 9 (3.46 g, 6.28 mmol) was co-evaporated four
times with anhydrous pyridine and dissolved in anhydrous pyr-
idine (31 mL). To the solution was added 4,4-dimethoxytrityl
chloride (2.55 g, 7.54 mmol). The mixture was stirred at
room temperature for 165 min and quenched with H2O. The
mixture was evaporated in vacuo and diluted with ethyl acetate.
The solution was washed with brine and aq NaHCO3. The
1
pound 11 as white foam (298 mg, 71%). H NMR (CDCl3,
500 MHz) d 1.10e1.31 (12H, m), 2.40e2.42 (1H, m), 2.65e
2.68 (1H, m), 3.05e3.08 (2H, m), 3.12e3.22 (2H, m), 3.35e
3.39 (1H, m), 3.53e3.73 (4H, m), 3.81 (6H, m), 3.84e4.01
(2H, m), 4.13e4.22 (1H, m), 4.36e4.43 (1H, m), 4.60e4.67
(1H, m), 4.78e5.00 (1H, m), 6.12e6.13 (1H, m), 6.82e6.85

4376
H. Saneyoshi et al. / Tetrahedron 64 (2008) 4370e4376
(4H, m), 7.08e7.11 (3H, m), 7.22e7.47 (11H, m), 8.18e8.22
(1H, m), 8.69e8.72 (1H, m), 9.40 (1H, br); 13C NMR (CDCl3)
d 18.1, 20.2, 20.3, 20.5, 20.6, 24.7, 24.8, 26.3, 43.2, 43.3, 43.4,
43.5, 48.0, 55.3, 57.9, 58.1, 58.8, 58.9, 62.8, 63.1, 67.9, 68.2,
68.4, 70.9, 71.0, 71.4, 71.5, 80.2, 80.5, 84.0, 84.2, 86.7, 86.8,
87.0, 87.1, 113.3, 115.4, 115.7, 115.8, 117.5, 117.9, 122.5,
123.6, 127.1, 128.0, 128.3, 128.4, 130.0, 130.2, 135.7, 135.8,
142.7, 142.9, 144.5, 144.6, 148.4, 152.0, 152.5, 152.6, 157.2,
158.7, 164.4, 164.5, 166.8. 31P NMR (CDCl3) d 151.1, 151.8.
HRMS calcd for C54H61N12O9P (MþHþ) 1053.4538, found
1052.4427.
Supplementary data
The 1H, 13C, and 31P NMR data of all new products are pro-
vided. Supplementary data associated with this article can be
found in the online version, at doi:10.1016/j.tet.2008.02.075.
References and notes
1. (a) Raddatz, S.; Mueller-Ibeler, J.; Kluge, J.; Wass, L.; Burdinski, G.;
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Havens, J. R.; Onofrey, T. J.; Wang, D.; Schweitzer, M. Nucleic Acids
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2005, 337, 332e337.
4.1.11. 50-O-(4,40-Dimethoxyltrityl)-20-O-[(1-N-methyl-
tetrazol-5-yl)ethyl]-6-N-phenoxyacetyladenosine 30-2-cyano-
ethyl N,N-diisopropylphosphoramidite (14)
2. (a) Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D. W. Y. Nat.
Chem. Biol. 2006, 12, 711e719; (b) Davis, S.; Lollo, B.; Freier, S.;
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Hovig, E.; Sioud, M. J. Mol. Biol. 2007, 90e108.
1H NMR (CDCl3, 500 MHz) d 1.03e1.16 (12H, m), 2.38e
2.40 (1H, m), 2.61e2.66 (1H, m), 3.12e3.20 (2H, m),
3.33e3.37 (1H, m), 3.47e3.66 (5H, m), 3.78e3.79 (6H, m),
3.99e4.00 (3H, m), 4.01e4.32 (2H, m), 4.34 (1H, m), 4.55e
4.70 (2H, m), 4.86 (2H, br), 6.13e6.17 (1H, m), 6.80e6.83
(4H, m), 7.05e7.08 (3H, m), 7.21e7.47 (11H, m), 8.21e8.27
(1H, m), 8.71e8.73 (1H, m), 9.40 (1H, br); 13C NMR (CDCl3)
d 7.21, 20.3, 20.4, 20.5, 24.4, 24.5, 24.6, 24.7, 24.8, 33.9,
43.2, 43.3, 43.4, 55.3, 55.4, 58.0, 58.1, 58.2, 58.3, 62.5, 63.0,
68.2, 68.4, 70.9, 71.0, 71.2, 71.3, 81.8, 83.8, 83.9, 86.8, 86.9,
87.1, 87.2, 113.3, 115.1, 117.5, 118.0, 122.5, 123.3, 127.1,
128.0, 128.3, 130.0, 130.2, 130.3, 135.5, 135.6, 142.0, 144.4,
144.5, 148.5, 151.6, 151.7, 152.6, 152.7, 153.1, 153.2, 157.2,
158.7, 158.8, 166.8. 31P NMR (CDCl3) d 150.8, 151.2. HRMS
calcd for C52H60N11O9P (MþHþ)1014.4385, found 1014.4324.
4. (a) Wengel, J. Org. Biomol. Chem. 2004, 2, 277e280; (b) Shu, W.; Liu,
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Docherty, P. H.; Rosair, G. M.; Quenardelle, R.; Kraft, A. Org. Lett.
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4.2. Oligonucleotide synthesis
8. (a) Cobb, A. J. A.; Longbottom, D. A.; Shaw, D. M.; Ley, S. V. Chem.
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Oligodeoxynucleotides were synthesized on an Applied Bi-
osystems 392 oligonucleotide synthesizer in 1 mmol, using pac
phosphoramidite building blocks. A 0.1 M solution of each 20-
O-tetrazol-5-ylethyl adenosine phosphoramidite was used and
the time for coupling was set to be 15 min. 5-Benzylthio-1H-
tetrazole (0.25 M) was used as the reaction activator. Release
of full modified oligonucleotide and deprotection of protecting
group at phosphate, base, and tetazole-5-yl was carried out
by use of NH4OH at room temperature for 8 h or 55 ꢀC for
5 h. The crude oligonucleotides were purified with standard
DMTr-on protocol by using C-18 cartridge and further purified
by anion-exchange HPLC.
´
10. Tominey, A.; Andrew, D.; Oliphant, L.; Rosair, G. M.; Dupre, J.; Kraft, A.
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Acknowledgements
2005, 11, 6246e6253.
15. Murlowska, K.; Sadlej-Sosnowska, N. J. Phys. Chem. A 2005, 109, 5590e
5595.
This work was supported by a Grant from CREST of JST
(Japan Science and Technology Agency) and a Grant-in-Aid
for Scientific Research from the Ministry of Education, Cul-
ture, Sports, Science, and Technology, Japan. This work was
also supported by the COE21 project. This study was also
supported by Industrial Technology Research Grant Program
in 2005 from New Energy and Industrial Technology Develop-
ment Organization (NEDO) of Japan.
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