Replacement of canonical DNA nucleobases by benzotriazole and 1,2,3-triazolo[4,5-d]pyrimidine: Synthesis, fluorescence, and ambiguous base pairing

Article abstract of DOI:10.1002/hlca.200590052

The syntheses and the fluorescence properties of 7H-3,6-dihydro-1,2,3- triazolo[4,5-d]pyrimidin-7-one 2′-deoxy-β-D-ribonucleosides (=2′-deoxy-8-azainosine) 3 (N³), 15 (N²), and 16 (N¹) as well as of 1,2,3-benzotriazole 2′-O-methyl-β- or -α-D-ribofuranosides 6 (N¹) and 24 (N¹) are described. Also the fluorescence properties of 1,2,3-benzotriazole 2′-deoxy-β-D-ribofuranosides 4 (N¹) and 5 (N²) are evaluated. From the nucleosides 3-6, the phosphoramidites 19, 26a, 26b, and 28 are prepared and employed in solid-phase oligonucleotide synthesis. In 12-mer DNA duplexes, compound 3 shows similar ambiguous base-pairing properties as 2′-deoxyinosine (1), while the nucleosides 4-6 show strong pairing with each other and discriminate very little the four canonical DNA constituents.

Full text of DOI:10.1002/hlca.200590052

Helvetica Chimica Acta ± Vol. 88 (2005)
751
Replacement of Canonical DNA Nucleobases by Benzotriazole
and 1,2,3-Triazolo[4,5-d]pyrimidine: Synthesis, Fluorescence,
and Ambiguous Base Pairing
by Frank Seela*, Anup M. Jawalekar, and Ingo Münster
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie, Universität Osnabrück,
Barbarastrasse 7, D-49069 Osnabrück, and Center for Nanotechnology (CeNTech), Gievenbecker Weg 11,
D-48149 Münster
(phone: ‡ 49-541-9692791; fax: ‡ 49-541-9692370; e-mail: Frank.Seela@uni-osnabrueck.de; www.seela.net)
The syntheses and the fluorescence properties of 7H-3,6-dihydro-1,2,3-triazolo[4,5-d]pyrimidin-7-one 2'-
deoxy-b-d-ribonucleosides (ˆ2'-deoxy-8-azainosine) 3 (N3), 15 (N2), and 16 (N1) as well as of 1,2,3-
benzotriazole 2'-O-methyl-b- or -a-d-ribofuranosides 6 (N1) and 24 (N1) are described. Also the fluorescence
properties of 1,2,3-benzotriazole 2'-deoxy-b-d-ribofuranosides 4 (N1) and 5 (N2) are evaluated. From the
nucleosides 3 ± 6, the phosphoramidites 19, 26a, 26b, and 28 are prepared and employed in solid-phase
oligonucleotide synthesis. In 12-mer DNA duplexes, compound 3 shows similar ambiguous base-pairing
properties as 2'-deoxyinosine (1), while the nucleosides 4 ± 6 show strong pairing with each other and
discriminate very little the four canonical DNA constituents.
Introduction. ± DNA-Duplex stability does not only depend on the horizontal
interactions of the bases (H-bonding), but also vertical interactions (base stacking) are
of utmost importance [1 ± 6]. These interactions are controlled by the properties of the
nucleobases (regular or modified ones) but also by the structure of the sugar± phos-
phate backbone (N or S sugar conformation, PNA, LNA) and the environmental
conditions such as counter ions and H2O molecules. Within the series of base-modified
oligonucleotides, a number of heterocyclic systems (hydrophilic or hydrophobic) were
synthesized and incorporated in DNA or RNA for various purposes [6 ± 13]. This led to
the development of the so-called universal or ambiguous nucleosides which are
categorized into two types: i) those forming base pairs by H-bonding and ii) those
stabilizing duplexes by stacking.
The 2'-deoxyinosine (1) is one of the most widely used universal nucleosides for
primers and probes in DNA technology. It forms H-bonds with all four natural
constituents of DNA; however, a wide range of Tm values is noticed [9][10]. Earlier, we
have studied the ambiguous base-pairing properties of the related 2'-deoxy-7-
deazainosine (2) in DNA duplexes [11]. The oligonucleotides incorporating 2 show
similar base-pairing properties to those of 2'-deoxyinosine (1), and 2 emerges also as a
universal nucleoside. As almost all of the universal nucleosides decrease the Tm values
of the oligonucleotide duplexes, we were looking for analogous compounds not
showing such drawbacks. Recently, it was reported that an additional N-atom in the 8-
position of purines leads to duplex stabilization compared to the parent purines [12 ±
15]. This prompted us to synthesize 2'-deoxy-8-azainosine (ˆ 7H-3,6-dihydro-1,2,3-
triazolo[4,5-d]pyrimidin-7-one 2'-deoxy-b-d-ribonucleoside; 3; systematic numbering
ꢀ 2005 Verlag Helvetica Chimica Acta AG, Zürich

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is used throughout the General Part; purine numbering is used exceptionally only when
data are compared with those of purine analogues), and to study its potential as
universal nucleoside. Likewise, related 1,2,3-benzotriazole nucleosides such as 4 and 5
as well as the 2'-methoxy derivative 6 were prepared. Compounds 4 ± 6 are not able to
form H-bonds but can develop stacking interactions through the benzene rings which
are replacing the pyrimidinone moieties of compound 3. Moreover, 1,2,3-benzotriazole
nucleosides (ˆ 8-azapurine nucleosides) are known to be fluorescent; therefore, their
fluorescence properties are of particular interest for studying the behavior in various
biological systems. This communication reports on the synthesis and the fluorescence
properties of the 3H-1,2,3-triazolo[4,5-d]pyrimidine (ˆ 8-azapurine) and benzotriazole
nucleosides 3 ± 6. Corresponding phosphoramidites were synthesized, and the base-
pairing properties of oligonucleotides containing these modified nucleosides were
investigated.
Results and Discussion. ± 1. Monomers. The synthesis of 3H-1,2,3-triazolo[4,5-
d]pyrimidine (8-azapurine) nucleosides encounters difficulties for the reason that
regioisomers as well as anomers can be formed upon glycosylation [16]. The synthesis
of our target nucleoside 3 was first described by Montgomery [16b]: The reaction of N-
(3H-1,2,3-triazolo[4,5-d]pyrimidin-7-yl)nonanamide with 3,5-bis-O-(4-chlorobenzoyl)-
2-deoxy-d-ribofuranosyl chloride in the presence of Et3N in refluxing benzene yielded
an anomeric mixture, which was separated and deacylated; compound 3 was then
obtained by deamination of the 3H-1,2,3-triazolo[4,5-d]pyrimidin-7-amine 3-(2'-deoxy-
b-d-ribofuranoside) (ˆ2'-deoxy-8-azaadenosine) [16]. However, this route resulted in
a low overall yield of 3. Our laboratory has reported the synthesis of 3H-1,2,3-
triazolo[4,5-d]pyrimidine-7-amine 3-(2'-deoxy-b-d-ribofuranoside) via the amination

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753
Scheme 1
i) 1m MeONa/MeOH (30 ml), r.t., 30 min. ii) 1m NaOH (30 ml), 508, 3 h.
of the corresponding methoxy nucleoside [17]. The glycosylation of 7-methoxy-3H-
1,2,3-triazolo[4,5-d]pyrimidine (7a) with 2'-deoxy-3,5-bis-O-(4-methylbenzoyl)-a-d-
erythro-pentofuranosyl chloride (8) was undertaken affording a mixture of the
regioisomeric glycosylation products 9a (N3), 10a (N2), and 11a (N1) in a ratio of
nearly 3 :4:3 [17] ( Scheme 1). As the methoxy nucleosides are susceptible for the
nucleophilic displacement by OHÀ ions, they are target molecules for the preparation
of 7H-3,6-dihydro-1,2,3-triazolo[4,5-d]pyrimidin-7-one (ˆ2'-deoxy-8-azainosine) nu-
cleosides 3, 15, and 16. We also tried to alter the ratio of regioisomers and opted for the
glycosylation of 7-isopropoxy-3H-1,2,3-triazolo[4,5-d]pyrimidine (7b) carrying a bulky
residue in the 7-position. For that, 7-isopropoxy-3H-1,2,3-triazolo[4,5-d]pyrimidine
(7b) was synthesized and glycosylated with pentofuranosyl chloride 8 to give a mixture
of the glycosylation products regioisomeric at N(3) (9b, 29%), N(2) (10b, 27%), and

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N(1) (11b, 31%). As the proportion was not significantly changed in favor of the N3
isomer 9b, the MeO derivatives were chosen for further manipulation [17].
Deprotection of compounds 9a, 10a, and 11a in 1m NaOMe/MeOH afforded 7-
methoxy nucleosides 12 ± 14, which, on further treatment with 1m NaOH, led to 1,2,3-
triazolo[4,5-d]pyrimidin-7-one 2'-deoxyribonucleosides (ˆ2'-deoxy-8-azainosine de-
rivatives) 3, 15, and 16, respectively (Scheme 1). The site of glycosylation was assigned
by 13C-NMR spectra. As the precursor methoxy compounds 9a ± 11a used in the
experiment were already assigned earlier [17], we followed the same assignment for 3,
15, and 16. The 13C-NMR spectra of the regioisomers 3, 15, and 16 show changes in the
chemical shifts of the bridgehead C-atoms C(3a) and C(7a). As compared to 3 (N3
isomer), a downfield shift of C(3a) (8 ppm) of 15 (N2) and a strong upfield shift of
C(7a) (11 ppm) for compound 16 (N1) confirms the site of glycosylation as N(3), N(2),
and N(1) for 3, 15 and 16, respectively. Compound 3 was also prepared enzymatically
from 9a with adenosine deaminase from calf intestinal mucosa.
Next, nucleoside 3 was converted into the 4,4'-dimethoxytrityl derivative 17 which
on further reaction with PCl3/N-methylmorpholine/1H-1,2,4-triazole afforded the 3'-
phosphonate 18. The phosphoramidite 19 was prepared from 17 with 2-cyanoethyl
diisopropylphosphoramidochloridite [13] (Scheme 2).
Scheme 2

Helvetica Chimica Acta ± Vol. 88 (2005)
755
Likewise, the benzotriazole nucleosides were prepared. We have already reported
the synthesis of the benzotriazole 2'-deoxyribonucleosides 4 and 5 by nucleobase-anion
glycosylation [18]. The glycosylation of 1H-1,2,3-benzotriazole (20) with 1,3,5-tri-O-
benzoyl-2-O-methyl-d-ribofuranose (21) was carried out in nitromethane with BF3 ´
Et2O as catalyst (Scheme 3). In this case, the N1-glycosylation product was the only
regioisomer formed, which is the result of the thermodynamic control of the reaction
(high temperature); unfortunately, an anomeric mixture of the N1-glycosylated
compounds 22 and 23 was obtained [19]. The O-benzoyl groups were removed with
methanolic ammonia to give the 1-(2-O-methyl-b/a-d-ribofuranosyl)-1H,2,3-benzo-
triazoles 6 and 24 in a 2 :1 ratio (Scheme 3). The site of glycosylation and the anomeric
configuration of 6 and 24 were assigned on the basis of both 1H-NMR and UV spectra.
Scheme 3
The UV spectra of 6 and 24 are strikingly similar. The 1H-NMR spectral comparison of 6 and 24 revealed
that HÀC(1') and HÀC(4') of the a-d-anomer 24 always resonate downfield from that of the b-d-anomer 6 [20]
[21]. The correct identity of the anomers was assigned by 1H-NMR NOE difference spectroscopy. Irradiation of
HÀC(1') of 6 resulted in an NOE at HÀC(4') of 1.8%, while that of 24 gave an NOE at HÀC(2') of 7.5% and at
HÀC(3') of 0.8%. Due to the similar environment of HÀC(1') and HÀC(4') in 6 and that of HÀC(1'),
HÀC(2'), and HÀC(3') in 24, they were assigned to be the b-d- and a-d-anomers, respectively.
Treatment of the nucleosides 4, 5, or 6 with 4,4'-dimethoxytrityl chloride
((MeO)2TrCl) yielded the (MeO)2Tr derivatives 25a, 25b, and 27 (Scheme 4), from
which the phosphoramidites 26a, 26b, and 28 were prepared with 2-cyanoethyl
diisopropylphosphoramidochloridite (Scheme 4).
All monomeric compounds were characterized by elemental analysis as well as by
1H-, 13C-, and 31P-NMR spectra (see Exper.Part and Tables 1 and 2). The 13C-NMR
signals were identified by gated-decoupled 13C-NMR spectra or heteronuclear
1H,13C-NMR correlation spectra.
2. Fluorescence Properties. The fluorescence of 5-amino-3,6-dihydro-7H-1,2,3-
triazolo[4,5-d]pyrimidin-7-one ribonucleoside (8-azaguanosine) and its 2'-deoxy de-

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Scheme 4
Table 1. 13C-NMR Chemical Shifts of 1,2,3-Triazolo[4,5-d]pyrimidinesa)b)
C(3a)
C(5)
160.1
156.5
156.1
156.5
155.3
148.8
147.0
C(7)
123.8
161.9
161.5
160.7
150.1
157.2
158.3
C(7a)
71.1
114.8
126.3
125.7
130.0
131.5
119.0
OCH
C(1')
C(2')
C(3')
C(4')
C(5')
7b
9b
10b
11b
3
153.4155.5
153.9
159.2
150.5
148.6
72.1
71.5
63.5
88.2
94.1
82.2
85.2
93.6
87.7
36.3
36.8
.335.474 85.6
74.0
73.9
82.2
82.9
71.7
88.5
88.8
88.7
63.5
63.4
38.2
70.6
70.5
70.6
61.9
62.0
61.8
c
15
16
156.3
153.2
)
)
c
a) Measured in (D6)DMSO. b) Systematic numbering. c) Superimposed by DMSO.
rivative at neutral pH increases by several orders when the pH of the solution is raised
to 11.0 [22][23]. It was, therefore, of interest to investigate the fluorescence properties
of compounds 3, 15, 16, and 4 ± 6 in neutral and alkaline medium (see the Fig. and
Table 3): all three 3,6-dihydro-7H-1,2,3-triazolo[4,5-d]pyrimidin-7-one 2'-deoxyribo-
nucleosides 3, 15, and 16 are fluorescent. The fluorescence of analogue 15 (N2) at
pH 7.0 is enhanced by a factor of 10 when the pH of the solution is raised to 11.0. Also,
the weak fluorescence of compounds 3 and 16 at neutral pH is increased by a factor of
5 ± 10 at pH 11.0 (see the Fig.). According to this, the significant fluorescence at
pH 11.0 must be due to the anionic species, which was also supported by pK values. The

Helvetica Chimica Acta ± Vol. 88 (2005)
757
Table 2. 13C-NMR Chemical Shifts of Benzotriazole Nucleosidesa)b)
C(3a)
C(4)
C(5)
C(6)
C(7)
C(7a)
4
5
145.5
143.6
145.5
144.8
145.3
145.1
145.5
145.4
145.4
119.2
118.2
119.2
118.3
119.5
119.0
119.3
118.9
119.3
124.3
127.0
124.3
127.0
124.7
124.0
124.5
124.1
124.5
127.6
127.0
127.5
127.5
128.2
127.4
127.9
127.3
127.9
111.1
118.2
111.0
112.9
110.8
111.8
111.2
112.9
113.0
132.4
143.6
132.6
143.6
132.8
132.9
132.4
132.7
132.8
25a
27
22
23
6
24
25b
C(1')
C(2')
C(3')
C(4')
C(5')
MeO
CˆO
c
4
5
86.2
93.1
85.1
92.8
87.6
87.6
88.486.469.2
88.8
87.6
)
)
)
)
70.6
70.7
70.2
70.2
71.9
71.481.5 .2
61.5
69.9
69.8
88.1
88.7
85.9
85.1
81.1
61.8
62.1
63.9
62.1
63.6
c
c
25a
27
22
23
6
54.9 (5')
54.9 (5')
58.6 (2')
')
c
80.0
80.2
165.1, 165.4
165.1, 165.5
64 59.2 (2
57.6 (2
61.1
63.458.0 (2
82.1
')
24
25b
85.6
85.2
81.7
82.6
58.6 (2')
'), 55.0 (5')
a) Measured in (D6) DMSO. b) Systematic numbering. c) Superimposed by DMSO.
pK values of protonation and deprotonation were determined UV-spectrophotometri-
cally in 0.1m sodium phosphate buffer (pH 7.0). Only one pKa was found for 3 (7.9) and
15 (8.5). Comparison of the pKa value of 3 with that of its counterparts 1 (8.9) and 2
(9.9) establishes that the extra N-atom (N(2)) in the 1,2,3-triazolo[4,5-d]pyrimidine
nucleosides increases their acidity. This indicates that the enhancement of fluorescence
at pH 11.0 is induced by the formation of anionic species. The 1H-1,2,3-benzotriazole 2-
(2'-deoxy-d-ribofuranoside) (5) exhibits a significant fluorescence (H2O, 10À5 m) with
an emission maximum at 368 nm (excitation at 281 nm). A quantum yield (Ff) of 0.1
was obtained for nucleoside 5, which is remarkably high compared to canonical
nucleosides. As expected, the fluorescence of 5 is pH-independent in the range of 7.0 to
11.0, so it is the neutral species which emits the light. The nucleosides 4 and 6 are
nonfluorescent.
From the above observations, it can be noted that the N2-glycosylated triazole
nucleosides are always fluorescent while the regioisomeric glycosylation products show
almost no fluorescence. In the case of the 3,6-dihydro-7H-1,2,3-triazolo[4,5-d]pyrimi-
din-7-one analogues, a large Stokes shift of ca. 80 nm is observed at pH 7.0 which even
increases at pH 11.0. In particular, compound 15 shows a maximal shift of 121 nm at
pH 11.0. A significant Stokes shift is also observed in case of the benzotriazole 2-(2'-
deoxy-d-ribofuranoside) 5 (94nm) which is pH-independent. Thus, nucleosides with
N(2) as glycosylation site exhibit the strongest fluorescence which becomes even
stronger in alkaline medium.
3. Synthesis and Base-Pairing Properties of Oligonucleotides. The oligonucleotide
synthesis was performed on the solid phase employing the phosphoramidites 19, 26a,
26b, and 28 as well as the phosphonate 18 (Tables 4 and 5). The coupling yield was

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Helvetica Chimica Acta ± Vol. 88 (2005)
Figure. Fluorescence spectra: a) 3 at pH 7.0 and 11.0 (excitation 274nm and emission 362 nm), b) 15 at pH 7.0
(excitation 272 nm and emission 340 nm) and pH 11.0 (excitation 290 nm and emission 411 nm), c) 16 at pH 7.0
(excitation 278 nm and emission 360 nm) and pH 11.0 (excitation 291 nm and emission 404 nm), and d) 5 at
pH 7.0 and 11.0 (excitation 281 nm and emission 368 nm).
Table 3. Spectral Properties of 1,2,3-Triazolo[4,5-d]pyrimidin-7-one Derivatives 3, 15, and 16 and of
Benzotriazole Nucleoside 5a)
pH
UV absorption
lmax [nm]
Fluorescence
lmax [nm]
Quantum yield
Ff
3
15
16
5
7.0
11.0
7.0
11.0
7.0
11.0
7.0
258
270
271
291
360
404
340
411
362
0.004
0.1
< 0.001
0.006
0.003
0.04
< 0.001
257
274362
274368
274368
11.0
0.1
a) UV Absorption and fluorescence spectra were measured in aqueous medium. The fluorescence quantum
yield (Ff) was determined by using quinine sulfate dihydrate in 0.1m HClO4 as standard (Ff ˆ 0.59).
always higher than 95%. Deprotection was performed with 25% aqueous NH3 solution
(608, 16 h), and the oligonucleotides were purified by reversed-phase HPLC. The
oligonucleotides 29 ± 46 were characterized by MALDI-TOF-MS, and the detected
masses were in good agreement with the calculated values (Table 6). The duplexes 5'-

Helvetica Chimica Acta ± Vol. 88 (2005)
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Table 4. Comparisons of Tm Values and Thermodynamic Data of Oligonucleotides Containing Compounds 1, 2,
or 3 Opposite to the Canonical Nucleosidesa)
Duplex
Tm DG8310
Duplex
Tm DG8310
[8] [kcal/mol]
[8] [kcal/mol]
5'-d(TACCTCAATACT)-3' (29)
3'-d(ATGGAGTTATGA)-5' (30)
5'-d(TACCTCAATACT)-3' (29)
3'-d(AT11AGTTATGA)-5' (31)
5'-d(TACCTCAATACT)-3' (29)
3'-d(AT22AGTTATGA)-5' (32)
5'-d(TACCTCAATACT)-3' (29)
3'-d(AT33AGTTATGA)-5' (33)
5'-d(TAAATCAATACT)-3' (34)
3'-d(AT11AGTTATGA)-5' (31)
5'-d(TAAATCAATACT)-3' (34)
3'-d(AT22AGTTATGA)-5' (32)
5'-d(TAAATCAATACT)-3' (34)
3'-d(AT33AGTTATGA)-5' (33)
4
6À 10.4
À 7.6
À 7.3
À 7.8
À 6.1
À 6.9
À 5.6
36
35
36
31
33
25
5'-d(TAGGTCAATACT)-3' (35) 22
3'-d(AT11AGTTATGA)-5' (31)
5'-d(TAGGTCAATACT)-3' (35) 20
3'-d(AT22AGTTATGA)-5' (32)
5'-d(TAGGTCAATACT)-3' (35) 24
3'-d(AT22AGTTATGA)-5' (33)
À 4.9
À 4.1
À 5.8
À 5.2
À 5.3
À 5.7
5'-d(TATTTCAATACT)-3' (36)
3'-d(AT11AGTTATGA)-5'
5'-d(TATTTCAATACT)-3' (36)
3'-d(AT22AGTTATGA)-5' (32)
5'-d(TATTTCAATACT)-3' (36)
3'-d(AT33AGTTATGA)-5' (33)
21
24
21
a) Measured at 260 nm in 100 mm NaCl, 10 mm MgCl2, and 10 mm Na-cacodylate (pH 7.0) with 5 mm single-
strand concentration.
Table 5. Tm Values and Thermodynamic Data of Oligonucleotides Containing 4, 5 or 6 Opposite to Regular
Nucleosidesa)
Duplex
Tm DG8310
Duplex
Tm DG8310
[8] [kcal/mol]
[8] [kcal/mol]
5'-d(TAGGTCAATACT)3' (35)
3'-d(ATCCAGTTATGA)-5' (37)
5'-d(TAGGTCAATACT)-3' (35)
3'-d(ATCC4GTTATGA)-5' (38)
5'-d(TAGGCCAATACT)-3' (39)
3'-d(ATCC4GTTATGA)-5' (38)
50
36
35
À 12.0
À 7.45
À 7.2
À 7.7
À 8.45
À 7.0
À 6.8
'-d(TAGGACAATACT)-3' (40) 37
3'-d(ATCC5GTTATGA)-5' (42)
5'-d(TAGGGCAATACT)-3' (41) 38
3'-d(ATCC5GTTATGA)-5' (42)
À 7.4
À 7.6
À 7.3
À 7.0
À 7.3
À 8.0
0À 8.4
À 8.1
À 8.0
5'-d(TAGGACAATACT)-3' (40) 37
3'-d(ATCC4GTTATGA)-5' (38)
5'-d(TAGGTCAATACT)-3' (35)
3'-d(ATCC6GTTATGA)-5' (43)
36
5'-d(TAGGGCAATACT)-3' (41)
3'-d(ATCC4GTTATGA)-5' (38)
5'-d(TAGGTCAATACT)-3' (35)
3'-d(ATCC5GTTATGA)-5' (42)
5'-d(TAGGCCAATACT)-3' (39)
3'-d(ATCC5GTTATGA)5-' (42)
5'-d(TAGG4CAATACT)-3' (44)
3'-d(ATCC4GTTATGA)-5' (38)
5'-d(TAGG5CAATACT)-3' (45)
3'-d(ATCC5GTTATGA)-5' (42)
5'-d(TAGG6CAATACT)-3' (46)
3'-d(ATCC6GTTATGA)-5' (43)
5'-d(TAGG4CAATACT)-3' (44)
3'-d(ATCC5GTTATGA)-5' (42)
4
1
'-d(TAGGCCAATACT)-3' (39) 35
3'-d(ATCC6GTTATGA)-5' (43)
5'-d(TAGGACAATACT)-3' (40) 36
3'-d(ATCC6GTTATGA)-5' (43)
5'-d(TAGGGCAATACT)-3' (41) 39
3'-d(ATCC6GTTATGA)-5' (43)
35
33
4
0 À 8.45
À 8.0
'-d(TAGG5CAATACT)-3' (45)
3'-d(ATCC4GTTATGA)-5' (38)
5'-d(TAGG6CAATACT)-3' (46)
3'-d(ATCC4GTTATGA)-5' (38)
5'-d(TAGG6CAATACT)-3' (46)
3'-d(ATCC5GTTATGA)-5' (42)
4
39
39
39
39
39
À 7.8
À 8.2
a) Measured at 260 nm in 1m NaCl, 100 mm MgCl2, and 60 mm Na-cacodylate (pH 7.0) with 5 mm single-strand
concentration.

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‡
Table 6. Molecular Masses ([M ‡ H] ) of Oligonucleotides Measured by MALDI-TOF-MS
‡
‡
Oligonucleotide
[M ‡ H] (calc.)
[M ‡ H] (found)
5'-d(A-G-T-A-T-T-G-A-3-3-T-A)-3' (33)
5'-d(A-G-T-A-T-T-G-4-C-C-T-A)-3' (38)
5'-d(A-G-T-A-T-T-G-5-C-C-T-A)-3' (42)
5'-d(A-G-T-A-T-T-G-6-C-C-T-A)-3' (43)
5'-d(T-A-G-G-4-C-A-A-T-A-C-T)-3' (44)
5'-d(T-A-G-G-5-C-A-A-T-A-C-T)-3' (45)
5'-d(T-A-G-G-6-C-A-A-T-A-C-T)-3' (46)
3694.50
3628.41
3628.41
3658.41
3638.41
3638.41
3668.41
3693.10
3628.46
3629.58
3659.60
3639.52
3638.59
3667.85
d(TACCTCAATACT)-3' ´ 3'-d(ATGGAGTTATGA)-5' (29 ´ 30) and 5'-d(TAGGT-
CAATACT)-3' ´ 3'-d(ATCCAGTTATGA)-5' (35 ´ 37) were used as a standard for the
evaluation of the strength of the base pairing (Tables 4 and 5).
The hybridization properties of oligonucleotides containing 7H-1,2,3-triazolo[4,5-
d]pyrimidin-7-one 2ꢁ-deoxyribonucleoside (2'-deoxy-8-azainosine) 3 were investigated
first and were compared to the corresponding oligomers containing either 2'-
deoxyinosine (1) and its 7-deazapurine analogue 2. For that, two consecutive
incorporations of 3 were done replacing the dG residue of the sequence 5'-
d(AGTATTGAGGTA) ( 30). The resulting oligomer 33 was hybridized with the
complementary strands containing four canonical bases opposite to 3 (Table 4). From
Table 4, it is apparent that the 3,6-dihydro-7H-1,2,3-triazolo[4,5-d]pyrimidin-7-one 2'-
deoxyribonucleoside 3 located opposite to dC, dG, dA, and dT behaved similar to that
of 2'-deoxyinosine (1) and 2'-deoxy-7-deazainosine (2). Surprisingly, we observed a
rather low stability of duplexes when compound 3 was located opposite to dA (34 ´ 33).
The above observations indicate that the base-pairing modes for compound 3 against
dC, dG, dA, and dT were almost the same as those of compounds 1 and 2, and support
the various base-pair motifs suggested earlier [11]. Although compound 3 did not
increase duplex stability as compared to compounds 1 and 2, its fluorescence properties,
in particular the strong change going from the neutral to the alkaline medium, is of
interest.
Next, the hybridization properties of the benzotriazole nucleosides 4 ± 6 were
investigated. For that, they were incorporated in oligonucleotides replacing the dA
residue of 5'-d(AGTATTGACCTA)-3' (37), and for the self-pairing, the dT moiety of
5'-d(TAGGTCAATACT)-3' (35). The pairing properties of 4 ± 6 opposite the
canonical nucleosides and opposite of each other were investigated (Table 5). For
duplexes containing 4, 5, or 6 opposite to canonical nucleosides, a maximum
stabilization of 2 ± 58 was observed opposite to dA or dG as compared to that opposite
to dC or dT. The present results indicate the preference of hydrophobic bases for the
purines over the pyrimidine bases at stacking, which were also observed for other
compounds by Leonard et al. [24]. The duplexes containing the nucleosides 4, 5, or 6
preferred to pair with themselves than with the natural nucleosides.
Despite of the different glycosylation sites, the nucleosides 4 and 5 paired among
each other and as well as with 6, without any discrimination. Even though, the presence
of a 2'-O-methyl group in 6 did not destabilize the duplexes. As there is no possibility of
H-bonding, the interstrand interaction in which the benzotriazole rings stack upon each
other is the main driving force for the observed stability [25][26]. Interstrand stacking

Helvetica Chimica Acta ± Vol. 88 (2005)
761
can be controlled by the surface area of the nucleobases, the hydrophobicity of the
residues, and their molecular polarizability. Thus, the higher molecular polarizability of
guanine and adenine (14.06 and 13.72 Æ 0.5 10À24, resp.) over thymine and cytosine
(11.77 and 10.82 Æ 0.5 10À24, resp.) contributes favorably to their enhanced interactions
[27][28]. Likewise, an increased hydrophobicity and surface area of self-paired
benzotriazole nucleobase (molecular polarizability, 13.76 Æ 0.5 10À24) give a ca. 28
stabilization over dG ´ dA. This implies that the natural DNA bases are not particularly
effective for an interstrand stacking. Nevertheless, it is surprising that the regioisomeric
benzotriazole nucleosides 4 and 5, when paired with each other, resulted in the same
degree of duplex stabilization. It seems that the DNA backbone is flexible enough to
accommodate these changes.
We thank Dr. Helmut Rosemeyer for the NMR measurements, Dr. Hong Li for the measurement of
fluorescence spectra, Mrs. Elisabeth Michalek for oligonucleotide synthesis, Mr. Khalil I.Shaikh for MALDI-
TOF spectra, and Mrs. Eva-Maria Becker for her help in determining pKa values. We also appreciate a generous
gift of 1,3,5-tri-O-benzoyl-2-O-methyl-a-d-ribofuranose by Dr. Xiaomei Zhang. Financial support by the
ꢂGraduiertenkollegꢁ (Nr. 612) of the Deutsche Forschungsgemeinschaft is gratefully acknowledged.
Experimental Part
General. All chemicals were purchased from Aldrich, Sigma, or Fluka (Sigma-Aldrich Chemie GmbH,
Deisenhofen, Germany). Solvents were of laboratory grade. TLC: aluminium sheets, silica gel 60F254, 0.2-mm
layer (VWR, Germany). Column flash chromatography (FC): silica gel 60 (Merck, Germany) at 0.4bar; sample
collection with an UltroRac-II fraction collector (LKB Instruments, Sweden). Solvent systems for TLC and FC:
CH2Cl2/MeOH 98 :2 (A), CH2Cl2/MeOH 95 :5 (B), CH2Cl2/MeOH 9 :1 (C), CH2Cl2/MeOH 8 :2 (D),
petroleum ether/acetone 8 :2 (E), petroleum ether/AcOEt 7:3 (F), petroleum ether/AcOEt 8 :2 (G),
petroleum ether/AcOEt 5 :1 (H), CH2Cl2/acetone 98 :2 (I), CH2Cl2/acetone 95 :5 (J). UV Spectra: U-3200
spectrometer (Hitachi, Tokyo, Japan); lmax (e) in nm. NMR Spectra: Avance-250 or AMX-500 spectrometers
(Bruker, Karlsruhe, Germany), at 250.13 MHz for 1H and 13C; d in ppm rel. to Me4Si as internal standard, J
values in Hz. Elemental analyses were performed by Mikroanalytisches Laboratorium Beller (Göttingen,
Germany).
Synthesis, Purification, and Characterization of the Oligonucleotides 29 ± 46. The oligonucleotide synthesis
was performed on a DNA synthesizer, model 392 (Applied Biosystems, Weiterstadt, Germany). Melting curves
were measured with a Cary 1/3 UV/VIS spectrophotometer (Varian, Australia) equipped with a Cary
thermoelectric controller. The temp. 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 program. Oligonucleotide
analysis was carried out by reversed-phase HPLC (Merck-Hitachi HPLC; 250 Â 4mm RP-18 column; gradients
of 0.1m (Et3NH)OAc (pH 7.0)/MeCN 95 :5 (K) and MeCN (L); gradient a: 15 min 0 ± 50% L in K, flow rate
1 ml/min; gradient b: 25 min 0 ± 20% L in K, 25 ± 35 min 20 ± 20% L in K, flow rate 1 ml/min. MALDI-TOF-MS:
Biflex-III spectrometer (Bruker Saxonia, Leipzig, Germany).
7-Isopropoxy-3H-1,2,3-triazolo[4,5-d]pyrimidine (7b). A suspension of 6-chloropyrimidine-4,5-diamine
(1.45 g, 10 mmol) in 1,4-dioxane (75 ml) was treated with pentyl nitrite (2.1 ml, 16 mmol) at 80 ± 908 for 30 min.
The mixture was cooled in an ice bath and concentrated to 1/3 of its volume. Then 1m Na in iPrOH (10 ml) was
added, and stirring was continued at 808 for 30 min. The mixture was evaporated, the residue dissolved in H2O
(20 ml), and the soln. treated with charcoal. After filtration, the aq. soln. was acidified to pH 5 with AcOH, and
the precipitating crude product was removed by filtration. The solid was then dissolved in MeOH, adsorbed on
silica gel, and subjected to FC (column 20 Â 5 cm, C): 7b (985 mg, 55%). Colorless solid. TLC (C): Rf 0.33. UV
(MeOH): 258 (8712). 1H-NMR ((D6)DMSO): 1.45 (d, J ˆ 6.1, Me2CHO); 5.66 (q, J ˆ 6.1, Me2CHO); 8.68
(s, HÀC(5)). Anal. calc. for C7H9N5O (179.18): C 46.92, H 5.06, N 39.09; found: C 47.14, H 5.11, N 38.83.
Glycosylation of 7b with 2-Deoxy-3,5-bis-O-(4-methylbenzoyl)-a-d-erythro-pentofuranosyl Chloride (8).
The suspension of 7b (1.98 g, 11 mmol) in MeCN (70 ml) was treated with NaH (97%; 280 mg, 11.3 mmol), and
the mixture was stirred for 15 min at r.t. Then 8 (4.27 g, 10.98 mmol) was added portionwise within 30 min, and
stirring was continued for 30 min. The mixture was filtered through Celite and the solvent evaporated. The

762
Helvetica Chimica Acta ± Vol. 88 (2005)
resulting oil was subjected to FC (column 20 Â 5 cm, D) and separated in three main zones. The faster migrating
zone was always the N3 isomer, zone II contained the N2 isomer, and the slower migrating zone was the N1
isomer.
3-[2-Deoxy-3,5-bis-O-(4-methylbenzoyl)-b-d-erythro-pentofuranosyl]-7-isopropoxy-3H-1,2,3-triazolo[4,5-d]-
pyrimidine (9b): Colorless foam (1.69 g, 29%). TLC (F): Rf 0.25. UV (MeOH): 242 (31869). 1H-NMR
((D6)DMSO): 1.45, 1.50 (2d, J ˆ 6.0, Me2CHO); 2.44, 2.39 (2s, 2 Me); 3.03 (m, HbÀC(2')); 3.57 (m, HaÀC(2'));
4.38, 4.52 (2m, 2 HÀC(5')); 4.75 (m, HÀC(4')); 5.64( m, Me2CHO); 5.95 (m, HÀC(3')); 6.99 (t, J ˆ 5.0,
HÀC(1')); 7.34, 7.86 (2m, arom. H); 8.77 (s, HÀC(5)). Anal. calc. for C28H29N5O6 (531.56): C 63.27, H 5.50; N
13.18; found: C 63.49, H 5.51, N 13.08.
2-[2-Deoxy-3,5-bis-O-(4-methylbenzoyl)-b-d-erythro-pentofuranosyl]-7-isopropoxy-2H-1,2,3-triazolo[4,5-d]-
pyrimidine (10b): Colorless foam (1.58 g, 27%). TLC (F): Rf 0.18. UV (MeOH): 241 (34878), 270 (11958).
1H-NMR ((D6)DMSO): 1.45, 1.46 (2d, J ˆ 6.0, Me2CHO); 2.43, 2.37 (2s, 2 Me); 3.03 (m, HbÀC(2')); 3.47
(m, HaÀC(2')); 4.49, 4.61 (2m, 2 HÀC(5')); 4.77 (m, HÀC(4')); 5.65 (m, Me2CHO); 5.95 (m, HÀC(3')); 6.90
(dd, J ˆ 3.8, 6.8, HÀC(1')); 7.34, 7.86 (2m, arom. H); 8.77 (s, HÀC(5)). Anal. calc. for C28H29N5O6 (531.56): C
63.27, H 5.50; N 13.18; found: C 63.20, H 5.70, N 13.05.
1-[2-Deoxy-3,5-bis-O-(4-methylbenzoyl)-b-d-erythro-pentofuranosyl]-7-isopropoxy-1H-1,2,3-triazolo[4,5-d]-
pyrimidine (11b): Colorless foam (1.81 g, 31%). TLC (F): Rf 0.09, UV (MeOH): 240 (34383), 271 (8383).
1H-NMR ((D6)DMSO): 1.45, 1.50 (2d, J ˆ 6.0, Me2CHO); 2.44, 2.39 (2s, 2 Me); 3.03 (m, HbÀC(2')); 3.57
(m, HaÀC(2')); 4.38, 4.52 (2m, 2 HÀC(5')); 4.76 (m, HÀC(4')); 5.64( m, Me2CHO); 5.95 (m, HÀC(3')); 7.00
(t, J ˆ 5.0, HÀC(1')); 7.36, 7.86 (2m, arom. H); 8.77 (s, HÀC(5)). Anal. calc. for C28H29N5O6 (531.56): C 63.27, H
5.50; N 13.18; found: C 63.35, H 5.61, N 13.15.
Deprotection of Compounds 9a ± 11a. A soln. of 9a, 10a, or 11a (1.26 g, 2.5 mmol) in 1m NaOMe/MeOH
(30 ml) was stirred at r.t. for 30 min. The mixture was adsorbed on silica gel and subjected to FC (column 35 Â
5 cm, B).
3-(2-Deoxy-b-d-erythro-pentofuranosyl)-7-methoxy-3H-1,2,3-triazolo[4,5-d]pyrimidine (12): Colorless solid
(494 mg, 74%). TLC (B): Rf 0.10. 1H-NMR ((D6)DMSO): 2.51 (m, HbÀC(2')); 3.09 (m, HaÀC(2')); 3.41, 3.57
(2m, 2 HÀC(5')); 3.92 (m, HÀC(4')); 4.23 (s, MeO); 4.59 (m, HÀC(3')); 4.72 (t, J ˆ 5.7, OHÀC(5')); 5.41
(d, J ˆ 4.6, OHÀC(3')); 6.73 (t, J ˆ 6.2, HÀC(1')); 8.80 (s, HÀC(5)). Anal. calc. for C10H13N5O4 (267.24): C
44.94, H 4.90, N 26.21; found: C 44.82, H 4.92, N 26.04.
2-(2-Deoxy-b-d-erythro-pentofuranosyl)-7-methoxy-2H-1,2,3-triazolo[4,5-d]pyrimidine (13): Colorless solid
(450 mg, 67%). TLC (B): Rf 0.05. 1H-NMR ((D6)DMSO): 2.55 (m, HbÀC(2')); 2.91 (m, HaÀC(2')); 3.45, 3.58
(2m, 2 HÀC(5')); 3.97 (m, HÀC(4')); 4.20 (s, MeO); 4.56 (m, HÀC(3')); 4.71 (t, J ˆ 5.4, OHÀC(5')); 5.41
(d, J ˆ 4.8, OHÀC(3')); 6.64( dd, J ˆ 4.0, 6.8, HÀC(1')); 8.77 (s, HÀC(5)). Anal. calc. for C10H13N5O4 (267.24):
C 44.94, H 4.90, N 26.21; found: C 45.05, H 4.82, N 26.09.
1-(2-Deoxy-b-d-erythro-pentofuranosyl)-7-methoxy-1H-1,2,3-triazolo[4,5-d]pyrimidine (14): Colorless solid
(449 mg, 67%). TLC (C): Rf 0.22. 1H-NMR ((D6)DMSO): 2.49 (m, HbÀC(2')); 3.11 (m, HaÀC(2')); 3.33, 3.49
(2m, 2 HÀC(5')); 3.92 (m, HÀC(4')); 4.22 (s, MeO); 4.56 (m, HÀC(3')); 4.65 (t, J ˆ 5.6, OHÀC(5')); 5.42
(d, J ˆ 4.4, OHÀC(3')); 6.64( t, J ˆ 5.8, HÀC(1')); 8.76 (s, HÀC(5)). Anal. calc. for C10H13N5O4 (267.24): C
44.94, H 4.90, N 26.21; found: C 45.07, H 4.98, N 26.10.
8-Aza-2'-deoxyinosines 3, 15, and 16. A soln. of compound 12, 13, or 14 (668 mg, 2.5 mmol) in 1n NaOH
(30 ml) was stirred for 3 h at 508. The mixture was cooled to r.t. and neutralized with AcOH. The soln. was
diluted with H2O (250 ml) and subjected to Amberlit XAD-2 (20 ± 50 mesh) (column 5 Â 35 cm). The column
was washed with H2O (300 ml) and eluted with H2O/iPrOH 9 :1. Evaporation and repetitive co-evaporation
with acetone afforded the isomeric 8-aza-2'-deoxyinosines 3, 15, and 16.
3-(2-Deoxy-b-d-erythro-pentofuranosyl)-3,6-dihydro-7H-1,2,3-triazolo[4,5-d]pyrimidin-7-one (3): Color-
less solid (443 mg, 70%). TLC (D): Rf 0.33. UV (MeOH): 256 (8200). 1H-NMR ((D6)DMSO): 2.44
(m, HbÀC(2')); 2.98 (m, HaÀC(2')); 3.38, 3.55 (2m, 2 HÀC(5')); 3.90 (m, HÀC(4')); 4.54 (m, HÀC(3')); 4.78
(br. s, OHÀC(5')); 5.43 (br. s, OHÀC(3')); 6.56 (dd, J ˆ 6.1, 6.0, HÀC(1')); 8.30 (s, HÀC(5)); 12.80 (br. s, NH).
Anal. calc. for C9H11N5O4 (253.21): C 42.69, H 4.38, N 27.66; found: C 42.82, H 4.56, N 27.39.
2-(2-Deoxy-b-d-erythro-pentofuranosyl)-2,6-dihydro-7H-1,2,3-triazolo[4,5-d]pyrimidin-7-one (15): Color-
less solid (405 mg, 64%). TLC (D): Rf 0.33. UV (MeOH): 271 (9400). 1H-NMR ((D6)DMSO): 2.46
(m, HbÀC(2')); 2.83 (m, HaÀC(2')); 3.39, 3.55 (2m, 2 HÀC(5')); 3.91 (m, HÀC(4')); 4.52 (m, HÀC(3')); 4.80
(br. s, OHÀC(5')); 5.45 (br. s, OHÀC(3')); 6.49 (dd, J ˆ 4.2, 6.7, HÀC(1')); 8.16 (s, HÀC(5)); 12.40 (br. s, NH).
Anal. calc. for C9H11N5O4 (253.21): C 42.69, H 4.38, N 27.66; found: C 42.82, H 4.56, N 27.39.
1-(2-Deoxy-b-d-erythro-pentofuranosyl)-1,6-dihydro-7H-1,2,3-triazolo[4,5-d]pyrimidin-7-one (16): Color-
less solid (267 mg, 42%). TLC (D): Rf 0.33. UV (MeOH): 255 (7000). 1H-NMR ((D6)DMSO): 2.45

Helvetica Chimica Acta ± Vol. 88 (2005)
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(m, HbÀC(2')); 2.99 (m, HaÀC(2')); 3.38, 3.52 (2m, 2 HÀC(5')); 3.90 (m, HÀC(4')); 4.51 (m, HÀC(3')); 4.75
(s, OHÀC(5')); 5.43 (s, OHÀC(3')); 6.46 (dd, J ˆ 5.8, 6.0, HÀC(1')); 8.13 (s, HÀC(5)). Anal. calc. for
C9H11N5O4 (253.21): C 42.69, H 4.38, N 27.66; found: C 42.82, H 4.56, N 27.39.
Enzymatic Synthesis of 3 from 9a. The 7-methoxy compound 9a (1.34g, 2.66 mmol) was disolved in H 2O
(500 ml) and treated with adenosine-deaminase ADA (from calf intestine; soln. in glycerol, 50 ml). After stirring
for 16 h at 308, the soln. was evaporated followed by co-evaporation with acetone. The crude product was
purified by FC (silica gel, column 3 Â 10 cm, C): 3 (529 mg, 79%). Colorless solid. Data: identical with that
described above.
3-{5-O-[Bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-b-d-erythro-pentofuranosyl}-3,6-dihydro-7H-1,2,3-
triazolo[4,5-d]pyrimidin-7-one (17). A suspension of 3 (771 mg, 3.05 mmol) in anh. pyridine (3 ml) was stirred
with (MeO)2TrCl (1.23 g, 3.63 mmol) for 1 h. The reaction was quenched by the addition of AcOEt (20 ml),
followed by brine (10 ml). The mixture was extracted twice with AcOEt (20 ml, each), the combined org. layer
washed with H2O (10 ml), dried (MgSO4), and evaporated and the residue purified by FC (column 6 Â 20 cm,
gradient A ! B): 17 (901 mg, 53%). Colorless foam. TLC (E): Rf 0.17. 1H-NMR ((D6)DMSO): 2.50
(m, HbÀC(2')); 2.98 ± 3.32 (m, HaÀC(2'), 2 HÀC(5')); 3.72, 3.73 (2s, 2 MeO); 4.04 (m, HÀC(4')); 4.62
(m, HÀC(3')); 5.44 (s, OHÀC(3')); 6.63 (dd, J ˆ 3.6, 6.9, HÀC(1')); 8.28 (s, HÀC(5)); 6.73 ± 7.28 (m, 13 arom.
H); 12.79 (br. s, NH). Anal. calc. for C30H29N5O6 (555.58): C 64.85, H 5.26, N 12.61; found: C 64.76, H 5.25, N
12.51.
3-{5-O-[Bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-b-d-erythro-pentofuranosyl}-3,6-dihydro-7H-1,2,3-
triazolo[4,5-d]pyrimidin-7-one 3'-(2-Cyanoethyl diisopropylphosphoramidite) (19). To a soln. of 17 (580 mg,
1.04mmol) and iPr2EtN (0.27 ml, 1.57 mmol) in anh. THF (15 ml), 2-cyanoethyl diisopropylphosphoramido-
chloridite (0.35 ml, 1.55 mmol) was added dropwise. After stirring at r.t. for 70 min, the reaction was quenched
by the addition of a 5% aq. NaHCO3 soln. (5 ml). The aq. layer was extracted with CH2Cl2 (2 Â 10 ml), the
combined org. layer washed with brine (10 ml), dried (MgSO4), and evaporated, and the resulting oil subjected
to FC (column 9 Â 3 cm, C): 19 (224mg, 28%). Colorless foam. TLC ( E): Rf 0.50. 31P-NMR (CDCl3): 150.1,
150.2.
3-{5-O-[Bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-b-d-erythro-pentofuranosyl}-3,6-dihydro-7H-1,2,3-
triazolo[4,5-d]pyrimidin-7-one 3'-(Triethylammonium Phosphonate) (18). To a soln. of PCl3 (0.22 ml,
2.52 mmol) and 4-methylmorpholine (2.75 ml, 25.0 mmol) in CH2Cl2 (10 ml), 1H-1,2,4-triazole (553 mg,
8.0 mmol) was added. The soln. was cooled to 08, then 17 (278 mg, 0.50 mmol) in CH2Cl2 (15 ml) was added
dropwise at r.t., and stirring was continued for 30 min. The mixture was poured into 1m (Et3NH)HCO3 (ˆ TBK;
pH 8.0, 25 ml), the aq. layer extracted with CH2Cl2 (3 Â 20 ml), the combined org. layer dried (Na2SO4) and
evaporated, and the residue subjected to FC (column 15 Â 3 cm, D). Evaporation of the main zone afforded a
colorless oil, which was dissolved in CH2Cl2. The soln. was extracted with aq. 0.1m TBK (10 Â 20 ml), dried, and
evaporated: 18 (250 mg, 70%). Colorless foam. TLC (D): Rf 0.15. 31P-NMR ((D6)DMSO): 1.12 (dd, J ˆ 5.87,
9.1).
Glycosylation of 1H-1,2,3-Benzotriazole (20) with 1,3,5-tri-O-Benzoyl-2-O-methyl-a-d-ribofuranose (21).
A soln. of 21 (2.74g, 5.75 mmol) and 1 H-1,2,3-benzotriazole (20; 850 mg, 7.14mmol) in dry nitromethane
(160 ml) was brought to reflux for 15 min. Then BF3 ´ Et2O (1.32 ml, 10.7 mmol) was added, and the mixture was
refluxed for further 15 min, cooled, and evaporated. The resulting oil was dissolved in CH2Cl2 (50 ml), the soln.
washed with 5% NaHCO3 soln. (20 ml), dried (Na2SO4), and evaporated, and the residue purified by FC
(column 20 Â 3 cm, I): 22 (1.1 g, 40%; less polar) and 23 (500 mg, 18%; more polar).
1-(3,5-Di-O-benzoyl-2-O-methyl-b-d-ribofuranosyl)-1H-1,2,3-benzotriazole (22): Colorless foam. TLC (I):
Rf 0.5. UV (MeOH): 231 (25500), 275 (5200), 282 (5100). 1H-NMR ((D6)DMSO): 3.39 (s, MeOÀC(2')); 4.51
(m, 2 HÀC(5')); 4.82 (m, HÀC(4')); 5.17 (m, HÀC(2')); 6.0 (m, HÀC(3')); 6.82 (d, J ˆ 4.11, HÀC(1')); 7.43 ±
8.13 (m, 14arom. H). Anal. calc. for C 26H23N3O6 (473.48): C 65.95, H 4.90, N 8.87; found: C 65.99, H 4.96, N 8.78.
1-(3,5-Di-O-benzoyl-2-O-methyl-a-d-ribofuranosyl)-1H-1,2,3-benzotriazole (23): Colorless foam. TLC (I):
Rf 0.42. UV (MeOH): 232 (26300), 275 (5500), 282 (5400). 1H-NMR ((D6)DMSO): 3.07 (s, MeOÀC(2'));
4.67 ± 4.75 (m, HÀC(4'), 2 HÀC(5')); 5.25 (m, HÀC(2')); 5.84( m, HÀC(3')); 7.19 (d, J ˆ 5.41, HÀC(1')); 7.46 ±
8.10 (m, 14arom. H). Anal. calc. for C 26H23N3O6 (473.48): C 65.95, H 4.90, N 8.87; found: C 66.05, H 5.01, N 8.82.
1-(2-O-Methyl-b-d-ribofuranosyl)-1H-1,2,3-benzotriazole (6). Compound 22 (600 mg, 1.27 mmol) in
methanolic ammonia (60 ml) was stirred overnight in a sealed bottle. The mixture was evaporated and the
residue subjected to FC (column 12 Â 3 cm, C): 6 (310 mg, 92%). Colorless oil. TLC (C): Rf 0.5. UV (MeOH):
255 (6200), 282 (3500). 1H-NMR ((D6)DMSO): 3.33 (s, MeOÀC(2')); 3.59 (m, 2 HÀC(5')); 4.05 (m, HÀC(4'));
4.46 (m, HÀC(2')); 4.56 (m, HÀC(3')); 4.97 (t, J ˆ 4.8, OHÀC(5')); 5.36 (d, J ˆ 4.4, OHÀC(3')); 6.46 (d, J ˆ

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Helvetica Chimica Acta ± Vol. 88 (2005)
5.1, HÀC(1')); 7.42 ± 7.56 (2ꢁtꢁ, HÀC(5), HÀC(6)); 8.07, 8.10 (d, HÀC(4), HÀC(7)). Anal. calc. for C12H15N3O4
(265.27): C 54.33, H 5.70, N 15.84; found: C 54.38, H 5.75, N 15.73.
1-(2-O-Methyl-a-d-ribofuranosyl)-1H-1,2,3-benzotriazole (24). As described for 6, 23 (300 mg, 0.63 mmol)
in methanolic ammonia (30 ml) gave 24 (139 mg, 83%). Colorless oil. TLC (C): Rf 0.5. UV (MeOH): 256
(6200), 282 (3800). 1H-NMR ((D6)DMSO): 2.99 (s, MeOÀC(2')); 3.50, 3.65 (2m, 2 HÀC(5')); 4.22
(m, HÀC(4')); 4.31 ± 4.37 (m, HÀC(2'), HÀC(3')); 4.97 (t, J ˆ 5.6, OHÀC(5')); 5.36 (d, J ˆ 6.6, OHÀC(3'));
6.87 (d, J ˆ 4.7, HÀC(1')); 7.37 ± 7.56 (2ꢁtꢁ, HÀC(5), HÀC(6)); 7.94, 8.8.06 (2d, HÀC(4), HÀC(7)). Anal. calc.
for C12H15N3O4 (265.27): C 54.33, H 5.70, N 15.84; found: C 54.56, H 5.91, N 15.92.
1-{5-O-[Bis(4-methoxyphenyl)phenylmethyl]-2-O-methyl-b-d-ribofuranosyl}-1H-1,2,3-benzotriazole
(25b). Compound 6 (200 mg, 0.75 mmol) was dried by repeated co-evaporation with anh. pyridine and
suspended in dry pyridine (3 ml), to which (MeO)2TrCl (320 mg, 0.94mmol) was added. The mixture was
stirred for 24h, quenched by the addition of 5% aq. NaHCO 3 soln. (10 ml), and extracted with CH2Cl2 (3 Â
10 ml). The combined org. layer was dried (Na2SO4) and evaporated and the residue subjected to FC (column
12 Â 3 cm, J): 25b (300 mg, 70%). Colorless foam. TLC (J): Rf 0.45. UV (MeOH): 235 (23700), 276 (6600).
1H-NMR ((D6)DMSO): 2.94, 3.15 (2m, 2 HÀC(5')) 3.47 (s, MeOÀ(2')); 4.16 (m, HÀC(4')); 4.62 ± 4.66
(m, HÀC(2'), HÀC(3')); 5.36 (d, J ˆ 4.4, OHÀC(3')); 6.58 (d, HÀC(1')); 6.67 ± 7.20 (m, 13 arom. H); 7.42,
7.55 (2't', HÀC(5), HÀC(6)); 8.00, 8.11 (d, HÀC(4), HÀC(7)). Anal. calc. for C33H33N3O6 (567.63): C 69.83, H
5.86, N 7.40; found: C 69.94, H 5.97, N 7.39.
1-{5-O-[Bis(4-methoxyphenyl)phenylmethyl]-2-O-methyl-b-d-ribofuranosyl}-1H-1,2,3-benzotriazole 3'-(2-
i
Cyanoethyl diisopropylphosphoramidite) (26b). To a soln. of 25b (100 mg, 0.18 mmol) and Pr2EtN (60 ml,
0.34mmol) in anh. CH 2Cl2 (2 ml), 2-cyanoethyl diisopropylphosphoramidochloridite (65 ml, 0.29 mmol) was
added at r.t. After stirring for 25 min, the mixture was diluted with CH2Cl2 (10 ml) and quenched by adding a 5%
NaHCO3 soln. (20 ml). Then, the aq. layer was extracted with CH2Cl2 (3 Â 15 ml), the combined org. layer dried
(Na2SO4) and evaporated, and the resulting oil subjected to FC (column 10 Â 3 cm, I): 26b (95 mg, 70%).
Colorless foam. TLC (I): Rf 0.5. 31P-NMR (CDCl3): 151.8, 152.2.
1-{5-O-[Bis(4-methoxyphenyl)phenylmethyl)-2-deoxy-b-d-erythro-pentofuranosyl}-1H-1,2,3-benzotriazole
(25a). Compound 4 (250 mg, 1.06 mmol) was dried by repeated co-evaporation with anh. pyridine and
suspended in dry pyridine (2 ml). The soln. was stirred in the presence of N,N-dimethylpyridin-4-amine (8 mg,
0.07 mmol) and (MeO)2TrCl (398 mg, 1.18 mmol) for 5 h [13]. The mixture was diluted with 5% aq. NaHCO3
soln. (10 ml) and extracted with CH2Cl2 (3 Â 10 ml). The combined org. layer was dried (Na2SO4) and
evaporated and the residue subjected to FC (column 12 Â 3 cm, B). Colorless foam (400 mg, 70%). TLC (B): Rf
0.44. UV (MeOH): 236 (23300), 275 (6900). 1H-NMR ((D6) DMSO): 2.49 (m, HaÀC(2')); 3.05 (m, HbÀC(2'));
3.02, 3.46 (m, 2 HÀC(5')); 3.67, 3.68 (2s, 2 MeO)); 4.05 (m, HÀC(4')); 4.62 (m, HÀC(3')); 5.49 (d, OHÀC(3'));
6.83 (m, HÀC(1')); 6.61 ± 7.20 (m, arom. H); 7.48, 7.59 (2ꢂtꢁ, HÀC(5), HÀC(6)); 8.01, 8.1 (2d, HÀC(4),
HÀC(7)). Anal. calc. for C32H31N3O5 (537.61): C 71.49, H 5.81, N 7.82; found: C 71.42, H 5.80, N 7.84.
2-{5-O-[Bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-b-d-erythro-pentofuranosyl}-2H-1,2,3-benzotriazole
(27). As described for 25a, with 5 (250 mg, 1.06 mmol), N,N-dimethylpyridin-4-amine (8 mg, 0.07 mmol),
(MeO)2TrCl (398 mg, 1.18 mmol), and dry pyridine (2 ml) for 5 h: 27 (383 mg, 67%). Colorless foam. TLC (B):
Rf 0.38. UV (MeOH): 234(19600), 279 (12500). 1H-NMR ((D6)DMSO): 2.51 (m, HaÀC(2')); 2.96
(m, HbÀC(2')); 3.07, 3.46 (m, 2 HÀC(5')); 3.65, 3.68 (2s, 2 MeO); 4.06 (m, HÀC(4')); 4.70 (m, HÀC(3'));
5.48 (d, J ˆ 5.1, OHÀC(3')); 6.75 (m, HÀC(1')); 6.66 ± 7.27 (m, arom. H); 7.45, 7.47 (2ꢁtꢁ, HÀC(5), HÀC(6));
7.91, 7.94(2 d, HÀC(4), HÀC(7)). Anal. calc. for C32H31N3O5 (537.61): C 71.49, H 5.81, N 7.82; found: C 71.58, H
5.75, N 7.84.
1-{5-O-[Bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-b-d-erythro-pentofuranosy}-1H-1,2,3-benzotriazole
3'-(2-Cyanoethyl diisopropylphosphoramidite) (26a). As described for 26b, with 25a (150 mg, 0.28 mmol),
iPr2EtN (92 ml, 0.53 mmol), 2-cyanoethyl diisopropylphosphoramidochloridite (92 ml, 0.41 mmol), and anh.
CH2Cl2 (2 ml) at r.t. for 30 min: 26a (180 mg, 87%). Colorless foam. TLC (I): Rf 0.41. 31P-NMR (CDCl3): 150.1,
150.4.
2-{5-O-[Bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-b-d-erythro-pentofuranosyl}-2H-1,2,3-benzotriazole
3'-(2-Cyanoethyl diisopropylphosphoramidite) (28). As described for 26a, with 27 (150 mg, 0.28 mmol), iPr2EtN
(92 ml, 0.53 mmol), and 2-cyanoethyl diisopropylphosphoramidochloridite (92 ml, 0.41 mmol): 28 (170 mg,
83%). Colorless foam. TLC (I): Rf. 0.41. 31P-NMR (CDCl3): 150.0, 150.2.

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Received October 25, 2004