Oligonucleotides containing pyrazolo[3,4-d]pyrimidines: 8-aza-7-deazaadenines with bulky substituents in the 2- or 7-position

Article abstract of DOI:10.1080/15257770500265745

□ The synthesis of the 2′-deoxyadenosine analogues 1b, 2b, and 3c modified at the 7- and/or 2-position is described. The effect of 7-chloro and 2-methylthio groups on the duplex stability is evaluated. For that, the nucleosides 1b, 2b, and 3c were convert

Full text of DOI:10.1080/15257770500265745

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Oligonucleotides Containing Pyrazolo[3,4-
d]Pyrimidines: 8-Aza-7-deazaadenines With Bulky
Substituents in the 2- or 7-Position
Frank Seela a b , Anup M. Jawalekar a b , Lijuan Sun a b & Peter Leonard a b
a Laboratorium für Organische und Bioorganische Chemie , Institut für Chemie, Universität
Osnabr ück , Osnabrück, Germany
b Laboratorium für Organische und Bioorganische Chemie , Center for Nanotechnology
(CeNTech) , Münster, Germany
Published online: 16 Aug 2006.
To cite this article: Frank Seela , Anup M. Jawalekar , Lijuan Sun & Peter Leonard (2005) Oligonucleotides Containing
Pyrazolo[3,4-d]Pyrimidines: 8-Aza-7-deazaadenines With Bulky Substituents in the 2- or 7-Position, Nucleosides, Nucleotides
and Nucleic Acids, 24:10-12, 1485-1505, DOI: 10.1080/15257770500265745
To link to this article: http://dx.doi.org/10.1080/15257770500265745
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Nucleosides, Nucleotides, and Nucleic Acids, 24:1485–1505, 2005
c
Copyright ꢀ Taylor & Francis Group, LLC
ISSN: 1525-7770 print/1532-2335 online
DOI: 10.1080/15257770500265745
OLIGONUCLEOTIDES CONTAINING PYRAZOLO[3,4-d]PYRIMIDINES:
8-Aza-7-deazaadenines With Bulky Substituents in the 2- or
7-Position
ᮀ
Frank Seela, Anup M. Jawalekar, Lijuan Sun, and Peter Leonard
Labora-
torium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie, Universita¨t Osna-
bru¨ck, Osnabru¨ck, Germany, and Center for Nanotechnology (CeNTech), Mu¨nster, Germany
ᮀ
The synthesis of the 2 -deoxyadenosine analogues 1b, 2b, and 3c modified at the 7- and/or
2-position is described. The effect of 7-chloro and 2-methylthio groups on the duplex stability
is evaluated. For that, the nucleosides 1b, 2b, and 3c were converted to the corresponding
phosphoramidites 15, 19, and 22, which were employed in the solid-phase oligonucleotide synthesis.
In oligonucleotide duplexes, compound 1b forms stable base pairs with dT, of which the separated
1b-dT base pairs contribute stronger than that of the consecutive base pairs. Compound 2b shows
universal base pairing properties while its N8 isomer 3c forms duplexes with lower stability.
Keywords Nucleosides; Oligonucleotides; Pyrazolo[3,4-d]pyrimidines; Bulky susbtitu-
ents; Base pairing; Duplex stability
INTRODUCTION
8-Aza-7-deaza-2ꢁ-deoxyadenosine (1a)[1] (purine numbering is used
throughout the discussion section, Figure 1) is an ideal substitute of 2ꢁ-
deoxyadenosine within duplex DNA. The multiple incorporations of 1a-dT
in place of dA-dT do not cause a significant change of the Tm values.[2,3] Ex-
tensive studies have been performed modifying the 8-aza-7-deaza-2ꢁ-deoxy-
adenosine (1a) at the 7-position with bulky substituents. It was shown that
the 7-substituents of moderate size stabilize the duplex DNA significantly.[3–6]
In memory to my good friend the late Professor Dr. John A. Montgomery.
Received 19 January 2005; accepted 30 March 2005.
We thank Dr. Helmut Rosemeyer for the NMR measurements, Mrs. Elisabeth Michalek for
oligonucleotide synthesis, Mrs. Monika Dubiel for her help during the preparation of the manuscript,
and Mr. Khalil Shaikh for MALDI-TOF spectra. Financial support by the Roche Diagnostic GmbH,
Penzberg, is gratefully acknowledged.
Address correspondence to F. Seela, Organische und Bioorganische Chemie, Institut fu¨r Chemie,
Universita¨t Osnabru¨ck, Barbarastrasse 7, D-49069 Osnabru¨ck, Germany. Fax: +49 541 969 2370; E-mail:
frank.seela@uni-osnabrueck.de
1485

1486
F. Seela et al.
FIGURE 1 8-Aza-7-deazapurine-2 -deoxyribonucleosides.
This has been demonstrated on halogenated and alkynylated nucleosides.
Up to now the bromo, iodo, propynyl, and hexynyl compounds 1c-1f have
been studied and their bulky side chains were found to be accommodated
well in the major groove of B-DNA.[3–6] We are further extending the se-
ries of 7-substituted compounds with the 7-chloro nucleoside 1b. Different
from the 7-substituted nucleosides, 2-substituted purine or purine like nu-
cleobases do not show such favorable properties. 2-Substituted purine nu-
cleosides with halogeno substituents have already been prepared by Mont-
gomery and by others.[7–12] Unlike these nucleosides only a few pyrazolo
pyrimidine nucleosides containing halogens in the 2-position have been
synthesized.[13,14] As 2-substituents are located in the minor groove of B-
DNA, their dimension is limited by the narrow size of this groove. Recently,
the effect of 2-chloro substituent was studied by incorporating the 2-chloro-
8-aza-7-deazaadenine nucleoside 2a in duplex DNA.[14] The 2-chloro group
causes a steric clash with the 2-oxo group of dT resulting in duplex desta-
bilization. Nature uses even bulkier 2-methylthio groups to modify adenine
residues in RNA[15–17]—a phenomenon which has recently been studied on
the oligonucleotide level.[15] We became interested in such a modification
on 8-aza-7-deazapurine nucleosides (2b and 3c). Contrary to purines, an 8-
aza-7-deazapurine cannot form Hoogsteen base pairs and will be devoid base
pairing within duplex DNA but will allow base stacking. This manuscript re-
ports on the incorporation and the base pairing of the 8-aza-7-deazaadenine
nucleosides 1b, 2b, and 3c containing oligonucleotide duplexes. For that
the phosphoramidites 15, 19, and 22 are prepared which are employed in
solid-phase synthesis.

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines
1487
RESULTS AND DISCUSSION
Synthesis and Properties of Monomers
For the synthesis of the target nucleosides 1b, 2b, and 3c, 8-aza-7-chloro-
7-deaza-6-methoxypurine (4) and 8-aza-2-methylthio-7-deaza-6-isopropoxy-
purine (8) served as precursors. Treatment of 8-aza-7-deaza-6-methoxypu-
rine[1,18] with N-chlorosuccinimide (NCS) in DMF gave 4, while the reaction
of 8-aza-2-methylthio-7-deaza-6-chloropurine[19] with 1M NaOiPr yielded 8.
Glycosylation of compound 4 under nucleobase anion conditions (KOH/
TDA-1) with 2-deoxy-3,5-di-O-(p-toluoyl)-β-D-erythro-pentofuranosyl chloride
(5)[20,21] afforded compound 6 as major product. Likewise, the glycosylation
of 8 with 5 gave the regioisomeric N9- and N8-glycosylated compounds 9 and
10 in nearly 2:1 proportion.[14] The latter were deprotected with 0.1 or 0.4
M Na in corresponding alcohols to yield the nucleosides 7, 11, and 12 as
colorless compounds.[4] Subsequently, the 6-alkoxy groups were displaced
by an amino group in NH3/MeOH in a steel bomb (90◦C) furnishing the
nucleosides 1b, 2b, and 3c (Schemes 1 and 2). As 2ꢁ-deoxyribonucleosides
are sensitive to acidic depurination, the stability of the N-glycosylic bonds
of 1b, 2b, and 3c were evaluated UV-spectrophotometrically in 2 NHCl at
40◦C. Compound 2b (t1/2 5.8 h) was much more stable on its N-glycosylic
bond than its regioisomer 3c (t1/2 3.8 h) and 8-aza-7-chloro-7-deaza-2ꢁ-deoxy-
adenosine 1b (t1/2 2.3 h).
Next, the protection of the 6-amino group of the nucleosides 1b, 2b, and
3c was performed. For that, the nucleosides 1b and 2b were treated with
dimethylformamide dimethylacetal at r.t. or 50◦C yielding the (dimethyl-
amino)methylidene compounds 13 and 16 (Schemes 3 and 4).[4] The
nucleoside 3c was treated with isobutyryl chloride affording the protected
compound 20.[22] The stability of the protecting groups was determined by
SCHEME 1

1488
F. Seela et al.
SCHEME 2
UV spectrophotometrically in 25% ammonia at r.t. While the formamidine
derivative 13 showed a half life τ = 1.6 min, at 230 nm, the compound
16 was rather labile to ammonia treatment. So acetamidine protection was
employed for the amino function of 2b which was therefore treated with
dimethylacetamide dimethyl acetal in methanol at 50◦C to yield compound
SCHEME 3

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines
1489
SCHEME 4
17 (τ = 21.5 min, at 320, in 25% ammonia).[23] For compound 20, the
isobutyryl group was found to be the most suitable protecting group (τ =
128 min, at 330 nm). The 5ꢁ-OH groups of the nucleosides 13, 17, and 20
were protected with the 4,4ꢁ-dimethoxytrityl residue → 14, 18, and 21 under
standard conditions and the 3ꢁ-OH groups were phosphitylated to give the
phosporamidites 15, 19, and 22.[14] (Schemes 3–5).
1
All compounds were characterized by H-, 13C-, or 31P-NMR spectra
(Table 1 and Experimental), as well as by elemental analysis or mass spectra.
The 13C-NMR signals were assigned by gated-decoupled 13C-NMR or het-
eronuclear [1H, 13C]-NMR correlation spectra. Nucleoside 1b was assigned
as N9 isomer according to the published literature.[4] The assignment of the
glycosylation position of 2b and 3c, was performed on the basis of chemical
shift analyses. In 13C-NMR, the carbon signal next to the glycosylation site is
shifted by 8 ppm upfield when the glycosylation position changes from N9
to N8 (Table 1). This is also the case for the corresponding derivatives 9-
12. The introduction of the acetamidine protecting group (17) has a strong
influence on the 13C-NMR chemical shifts of the heterocyclic system. NOE
data were used to confirm the anomeric configuration as well as the glyco-
sylation position of the nucleoside 3c. Irradiation of 3c on H-(7) gave NOEs

1490
F. Seela et al.
SCHEME 5
at H-(1ꢁ) (8.8%), H-(2ꢁ) (1.5%), and H-(3ꢁ) (1.8%). Due to the spatial re-
lationships of the H-atoms, the glycosylic bond of compound 3c is β-D, and
the glycosylation site is N8.
Oligonucleotides
Synthesis. Oligonucleotide synthesis was performed applying the phos-
phoramidites 15, 19 and 22 in solid-phase oligonucleotide chemistry in an
automatized DNA synthesizer. The coupling yields were always higher than
95%. The oligonucleotides were deprotected in 25% NH3 solution and pu-
rified by RP-18 HPLC. The homogeneity of the oligonucleotides was estab-
lished by reversed-phase HPLC. The modified oligonucleotides were char-
acterized by MALDI-TOF mass spectroscopy, and the detected masses were
found in good agreement with the calculated values (Table 2).
Oligonucleotide Duplex Stability. Among the 8-aza-7-deaza-2ꢁ-deoxy-
adenosines, the 7-substituted Br, I, propynyl and hexynyl nucleosides (1c-1f)
have shown a positive effect on the duplex stability.[3–6] In this regard, the
contribution of a 7-chloro substituent (1b) is unknown. For that purpose, a
series of oligonucleotides were synthesized replacing dA at the various posi-
tions by 1b in the duplex 5ꢁ-d(TAGGTCAATACT)•3ꢁ-d(ATCCAGTTATGA)
(23•24) (Table 3).
A systematic evaluation of the duplexes with an increasing number of 1b
reveals the importance of different positions of incorporation to the ther-
mal stability (Table 3). Thus, a single incorporation of 1b at the central
position (→ 25) stabilizes the dA-dT base pair by 2◦C, while a consecutive
incorporation (→ 26) gives a stabilization of 1.5◦C per modification. In an-
other sequence (→ 27), two incorporations of 1b at different positions led

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines
1491
TABLE 1 13C-NMR Chemical Shifts of Pyrazolo[3,4-d]pyrimidine Nucleosidesa
Compoundb
C(3)
C(3a)
C(4)
C(6)
C(7a)
MeS
OCH3/2CH3
4
6
7
1b
8
9
10
11
2b
12
3c
13
14
16
17
18
20
21
130.9
132.3
131.6
131.7
131.9
132.9
125.5
133.5
133.3
124.4
125.7
133.4
133.4
133.9
133.8
133.7
129.8
129.7
98.9
100.3
100.1
97.4
162.9
163.1
163.0
157.2
168.3
169.5
168.5
169.1
168.9
168.1
169.6
161.9
161.9
168.5
168.5
168.5
167.7
167.5
156.1
156.8
156.6
157.7
161.5
161.5
163.0
161.5
157.0
163.0
161.1
156.4
156.4
161.3
160.8
160.8
161.0
161.1
156.3
155.5
155.4
154.5
156.8
155.7
161.5
155.6
154.3
161.3
159.2
155.1
155.1
155.2
154.9
154.8
153.7
153.7
54.7
54.6
99.0
13.7
13.7
13.7
13.7
13.3
13.6
14.0
21.6
21.5
21.5
21.5
100.2
100.5
100.1
98.2
100.2
100.4
104.0
104.0
105.4
105.7
105.7
100.6
100.9
21.5
54.8
13.7
13.6
13.6
13.3
13.4
54.9
19.1
19.1
C(1 )
C(2 )
C(3 )
C(4)
C(5)
6
7
1b
9
84.3
84.2
83.7
81.3
90.3
83.9
83.5
90.6
91.2
83.7
83.6
83.7
83.7
83.6
90.6
90.1
35.2
37.8
37.7
35.2
36.7
37.7
78.4
70.6
70.7
74.6
74.4
70.9
71.1
70.3
71.7
70.7
70.4
71.1
71.1
70.8
70.7
70.2
87.6
87.7
87.6
84.2
82.2
87.6
87.4
88.3
89.2
87.6
85.1
87.5
87.5
85.2
88.5
86.1
63.6
62.0
62.2
63.9
63.8
62.3
62.4
61.6
63.1
62.2
64.0
62.5
62.5
64.4
62.1
63.8
10
11
2b
12
3c
13
14
16
17
18
20
21
37.7
c
c
37.7
38.0
37.7
37.7
38.0
c
c
aMeasured in DMSO-d6.
bSystematic numbering.
cSuperimposed by DMSO-d6.
to a stronger stabilization (3◦C per modification). A combination of consec-
utive and two separated incorporations in a same sequence (→ 28) reflect
to the stability and contribute 1.75◦ per modification. From the above ob-
servation, it can be concluded that the incorporation of 1b stabilizes the du-
plexes significantly and the separated base pairs contribute stronger than
that of the consecutive ones. The increase in the duplex stability can be
attributed to the stacking of the nearest neighbors and the flexibility ob-
tained to the 7-Cl substituent so as to accommodate well in the major groove.

1492
F. Seela et al.
TABLE 2 Molecular Masses Determined by MALDI-TOF Mass Spectroscopy
Oligonucleotides
[M H ] (calc.)
[M H ] (found)
5 -d(TAGGTC1bATACT) (25)
5 -d(TAGGTC1b1bTACT) (26)
3 -d(ATCC1bGTT1bTGA) (27)
5 -d(T1bGGTC1b1bT1bCT) (28)
5 -d(TAGGTCA1aTACT) (36)
5 -d(TAGGTCA2bTACT) (41)
5 -d(TAGGTCA3cTACT) (45)
3680
3714
3714
3783
3645
3691
3691
3679
3714
3713
3783
3645
3691
3692
Next, the self-complementary oligonucleotides containing alternating
bases or base tracts were studied. Earlier investigations on oligonucleotides
with modified bases showed that self-complementary duplexes formed by al-
ternating 5ꢁ-d(A-T)n are extraordinarily sensitive to base modifications. It re-
sults from the particular structure of these oligomers.[24] For that reason, the
self-complementary oligomer 5ꢁ-d(1b-dT)6 was evaluated. When compared
to 5ꢁ-d(1c-dT)6, 5ꢁ-d(1d-dT)6, 5ꢁ-d(1e-dT)6, and 5ꢁ-d(1f-dT)6 duplexes[4–6]
(Table 4), the Tm values of the alternating duplexes differ significantly from
each other. From Table 4, it is apparent that the oligonucleotides with alter-
nating 7-substituted 1b-1f show a stabilization of 15–30◦ over unsubstituted
1a. Thermal stability increases from 1a-1e with the exception of 1f. We at-
tribute the increase in Tm values from 1a-1e to the bulkiness of the 7-sub-
stituted groups (Table 5).[25] Being most bulky, the 7-hexynyl substituted
compound 1f (Table 5) does not lead to a duplex stabilization compared to
TABLE 3 Tm Values and Thermodynamic Data of Oligonucleotides Containing Compd. 1ba
Tm
∆Tm ( ) per
∆H
∆S
∆G
310
Duplexes
( C)
modification
(kcal/mol) (cal/mol K) (kcal/mol)
5 -d(TAGGTCAATACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTC1bATACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTC1b1bTACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTCAATACT)
3 -d(ATCC1bGTT1bTGA)
5 -d(TAG GTC1bATACT)
3 -d(ATCC1bGTT1bTGA)
5 -d(TAGGTC1b1bTACT)
3 -d(ATCC1bGTT1bTGA)
23
24
25
24
26
24
23
27
25
27
26
27
47
49
50
53
54
56
54
58
−84
−93
−89
−90
−95
−92
−92
−95
−236
−264
−250
−252
−265
−253
−256
−261
−10.6
−11.2
−11.5
−12.1
−12.7
−12.9
−12.4
−13.9
+2.0
+1.5
+3.0
+2.3
+2.25
+1.75
+1.8
5 -d(T1bGGTC1b1bT1bCT) 28
3 -d(ATCCAGTTATGA) 24
5 -d(T1bGGTC1b1bT1bCT) 28
3 -d(ATCC1bGTT1bTGA) 27
aMeasured in 100 mM NaCl, 10 mM MgCl2, and 10 mM Na-cacodylate (pH 7.0) with 5 µM single-
strand concentration.

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines
1493
TABLE 4 Tm Values and Thermodynamic Data of Self-complimentary Oligonucleotides Containing
the Nucleosides 1a-fa
Tm
∆Tm ( ) per
∆H
∆S
∆G
310
Duplexes
( C)
modification
(kcal/mol)
(cal/mol K)
(kcal/mol)
5 -d[(A-T)6]2-3
5 -d[(1a-T)6]2-3
5 -d[(1b-T)6]2-3
5 -d[(1c-T)6]2-3
5 -d[(1d-T)6]2-3
5 -d[(1e-T)6]2-3
5 -d[(1f-T)6]2-3
29•29
30•30
31•31
32•32
33•33
34•34
35•35
33
36
51
52
56
66
56
−45
—
−125
—
−6.3
—
0.25
1.5
1.6
1.9
2.75
1.9
−56
−59
−61
−91
−61
−152
−157
−163
−246
−163
−9.3
−9.7
−10.5
−14.5
−10.6
aMeasured in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with 5 µM single-strand
concentration.
the 7-propynyl residue 1e. This suggests that the major groove can accom-
modate the bulky 7-substituents up to a certain size, thereafter it does reflect
to the stability. Thus, the size of the 7-substituted groups plays an important
role to the stabilization of the duplexes. Moreover, all the halogenated com-
pounds increase the duplex stability compared to 1a.
In contrary to the 7-substituted compounds 1a-1f, 2-substituted com-
pounds influence the base pairing property as they are located in the core
of the helix. Earlier, the contribution of the 2-chloro group of 2-chloro-8-
aza-7-deaza-2ꢁ-deoxyadenosine (2a) has been evaluated incorporating it in
a duplex 23•24.[14] The 2-chloro substituent causes steric clash with the 2-
oxo group of dT as well as with the 2-amino group of dG and results to the
destabilization. Besides that, it shows the stacking effect opposite to dC and
dA. Recent studies on the RNA duplexes containing 2-methylthio adenosine
and its derivatives have shown that 2-methylthio groups enhance the stack-
ing interactions with adjacent base pairs.[15] Therefore, it was anticipated
that the base pairing strength opposite to dC and dA can be maximized
with the large size 2-MeS group (Table 5), which could lead to universal
base pairing properties. For that, a single incorporation of 2-methylthio-8-
TABLE 5 The Molar Volume (Size) of Nucleobases of 1a-f and 2a,
2b, or 3c
Molar volume
(cm3)
Van der Waals radii
(Å)
Substituent
H (1a)
7-Cl (1b) or 2-Cl (2a)
7-Br (1c)
83.8
95.7
3.0
3.0
3.0
3.0
5.0
5.0
5.0
1.20 (H)
1.80 (Cl)
1.95 (Br)
2.15 (I)
—
100.0
105.8
118.3
167.4
116.2
7-I (1d)
7-Propynyl (1e)
7-Hexynyl (1f)
2-MeS (2b or 3c)
—
—

1494
F. Seela et al.
TABLE 6 Tm Values and Thermodynamic Data of Oligonucleotides Incorporating the Nucleosides
1a, 2a, and 2b Opposite to the Canonical Nucleosides
Tm
∆Tm
∆H
(kcal/mol)
∆S
(cal/mol K)
∆G
310
(kcal/mol)
Duplexes
( C)a
( C)a
5 -d(TAGGTCAATACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTCA1aTACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTCA1aTACT)
3 -d(ATCCAGTCATGA)
5 -d(TAGGTCA1aTACT)
3 -d(ATCCAGTAATGA)
5 -d(TAGGTCA1aTACT)
3 -d(ATCCAGTGATGA)
5 -d(TAGGTCA2aTACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTCA2aTACT)
3 -d(ATCCAGTCATGA)
5 -d(TAGGTCA2aTACT)
3 -d(ATCCAGTAATGA)
5 -d(TAGGTCA2aTACT)
3 -d(ATCCAGTGATGA)
5 -d(TAGGTCA2bTACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTCA2bTACT)
3 -d(ATCCAGTCATGA)
5 -d(TAGGTCA2bTACT)
3 -d(ATCCAGTAATGA)
5 -d(TAGGTCA2bTACT)
3 -d(ATCCAGTGATGA)
5 -d(TAGGTCASTACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTCASTACT)
3 -d(ATCCAGTCATGA)
5 -d(TAGGTCASTACT)
3 -d(ATCCAGTAATGA)
5 -d(TAGGTCASTACT)
3 -d(ATCCAGTGATGA)
23
24
36
25
36
37
36
38
36
39
40
24
40
37
40
38
40
39
41
24
41
37
41
38
41
39
42
24
42
37
42
38
42
39
50
51
36
38
44
44
31
38
38
41
37
40
39
33
34
34
33
−90
−73
−64
−64
−70
−76
−58
−71
−60
−73
−67
−69
−73
−47
−52
−51
−48
−252
−204
−181
−181
−196
−213
−166
−203
−167
−206
−192
−196
−209
−129
−146
−141
−131
−12.0
−10.2
−7.5
−8.0
−9.0
−9.5
−6.7
−8.1
−7.9
−8.7
−7.6
−8.2
−8.0
−7.0
−7.1
−7.2
−7.0
+1
−15
−13
−6
−6
−19
−12
−12
−9
−13
−10
−11
−17
−16
−16
−17
aMeasured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with 5 µM
single-strand concentration.
aza-7-deaza-2ꢁ-deoxyadenosine 2b or 3c was made replacing dA residue in
the sequence 23, leading to oligonucleotides 41 and 45 (Tables 6 and 7).
The data for the 2-chloro compound 2a used for the comparison is taken
from the previously published article.[14]
According to the Tm values shown in Table 6, the 2-methylthio nucleo-
side 2b behaves almost identically towards all the DNA constituents (dA, dG,
dT, and dC) and shows universal base pairing. Tm values are generally 10–
12◦C lower than that of parent duplex 23•24. As compared to the 2-chloro
nucleoside 2a, a degree of stabilization of 2b against dC is 6◦C higher and

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines
1495
TABLE 7 Tm Values and Thermodynamic Data of Oligonucleotides Incorporating the Nucleosides
3a, 3b, and 3c Opposite to the Canonical Nucleosides
Tm
∆Tm
∆H
(kcal/mol)
∆S
(cal/mol K)
∆G
310
(kcal/mol)
Duplexes
( C)a
( C)a
5 -d(TAGGTCAATACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTCA3aTACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTCA3aTACT)
3 -d(ATCCAGTCATGA)
5 -d(TAGGTCA3aTACT)
3 -d(ATCCAGTAATGA)
5 -d(TAGGTCA3aTACT)
3 -d(ATCCAGTGATGA)
5 -d(TAGGTCA3bTACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTCA3bTACT)
3 -d(ATCCAGTCATGA)
5 -d(TAGGTCA3bTACT)
3 -d(ATCCAGTAATGA)
5 -d(TAGGTCA3bTACT)
3 -d(ATCCAGTGATGA)
5 -d(TAGGTCA3cTACT)
3 -d(ATCCAGTTATGA)
5 -d(TAGGTCA3cTACT)
3 -d(ATCCAGTCATGA)
5 -d(TAGGTCA3cTACT)
3 -d(ATCCAGTAATGA)
5 -d(TAGGTCA3cTACT)
3 -d(ATCCAGTGATGA)
23
24
43
24
43
37
43
38
43
39
44
24
44
37
44
38
44
39
45
24
45
37
45
38
45
39
50
43
44
44
46
40
46
46
41
39
47
46
39
−90
−74
−79
−72
−75
−76
−83
−81
−77
−53
−77
−77
−50
−252
−210
−226
−202
−209
−216
−233
−227
−221
−147
−218
−219
−134
−12.0
−9.1
−9.3
−9.4
−9.8
−8.4
−10.3
−10.1
−8.8
−8.0
−9.7
−9.4
−8.0
−7
−6
−6
−4
−10
−4
−4
−9
−11
−3
−4
−11
aMeasured at 260 nm in 1 M NaCl, 100 mM MgCl2, and 60 mM Na-cacodylate (pH 7.0) with 5 µM
single-strand concentration.
that against dA is 2◦C higher. When 2b is incorporated against dC, it be-
haves like 1a, and against dA it gives a 2◦C stabilization over that of 1a. This
is a direct result of a contribution of the 2-methylthio group to the stability
by enhanced stacking interactions. When there is face-face base pairing, the
2-MeS group should accommodate in the minor grove and could result in
labile duplexes. Based on the earlier reports of the purine polynucleotides:
the 2-methylthio group of purine polynucleotides alters the Watson-Crick
base pairs due to the steric repulsion with the 2-oxo group of heterocyclic
rings and stabilizes the duplex by Hoogsteen base pairing. But this is not
the case in 2b as it cannot form the Hoogsteen base pairs.[17] Therefore,
we believe that when compound 2b pairs opposite to canonical bases, the
base moiety might turn around protruding the 2-MeS group into the major
groove which results in a stabilization by simple stacking interaction. This is
also supported by the abasic residue S showing Tm values of ∼33◦C against
the canonicals. Thus, the presence of a 2-methylthio group of 2b harmonizes

1496
F. Seela et al.
the base pairing and stabilizes the DNA duplexes by stacking interactions re-
sulting in universal base pairing properties.
However, this is not the case with the unusually linked nucleoside 3c
(Table 7). Compound 3c does not show universal base pairing properties of
the nucleoside 3a and forms two sets of duplexes showing similar stabilities.
It gives maximum stabilization when located opposite dA and dC than those
opposite dG and dT. The effect of the 2-MeS group of the N8 isomer 3c is
exactly similar to that of the 2-chloro group of 3b. The base pair motifs
of 3b are already suggested by He et al.[14] According to that, the 2-MeS
group of the nucleoside 3c must be located in the major groove of DNA
helices. In case of 3c-dC or 3c-dA base pairs, the 2-MeS substituent is well
accommodated in the major groove and has enough steric freedom during
its interaction with the substituents of the cognate base. This may result to
the maximum stabilization opposite to dC or dA. By contrast, when 3c is
situated opposite to dT or dG, the 2-MeS group clashes with the oxo groups
of dT or dG resulting in 6–7◦C lower Tm values than that of 3c-dC or 3c-dA
base pairs. Note that all the duplexes are more stable than those containing
the abasic residue S (Table 6).
EXPERIMENTAL
General
All chemicals were purchased from Aldrich, Acros, Sigma, or Fluka
(Sigma-Aldrich Chemie GmbH, Deisenhofen, Germany). Solvents were of
laboratory grade.Thin layer chromatography (TLC) silica gel 60 F254 (0.2
mm) (VWR, Damstadt, Germany) and flash chromatography (FC) with 0.4
bar on silica gel 60 H (VWR, Darmstadt, Germany). NMR Spectra were
measured on an Avance-DPX-250 spectrometer (Bruker, Germany), 250.13
1
MHz for H, 62.89MHz for 13C, 101.256Hz for 31P, δ values are in ppm
related to internal SiMe4 (1H, 13C) or external H3PO4. Elemental analyses
were performed by Mikroanalytisches Labor Beller (Go¨ttingen, Germany).
3-Chloro-4-methoxy-1H-pyrazolo[3,4-d]pyrimidine (4). N-Chlorosuc-
cinimide (5.0 g, 37.45 mmol) was added to a solution of 4-methoxy-1H-
pyrazolo[3,4-d]pyrimidine[1,18] (2.81 g, 18.72 mmol) in anh. DMF (50 mL).
After stirring for 32 h at 50◦C, the solvent was removed in vacuo. The
residue was applied to FC (column 50 × 3.5 cm). Elution was performed
with CH2Cl2/MeOH, 98:2 (300 mL) followed by 95:5 (300 mL) affording
compound 4 as a white solid (2.4 g, 69%). TLC (CH2Cl2/MeOH 9:1): Rf
1
0.57; UV (MeOH) λmax 246 (ε 6600); H-NMR (DMSO-d6) δ 4.10 (s, 3 H,
OCH3), 8.56 (s, 1 H, H-6), 14.20 (s, 1 H, NH); Anal. Calc. for C6H5N4OCl
(184.58): C 39.04, H 2.73, N30.35, Cl 19.21, found: C 39.34, H 2.86, N30.65,
Cl 19.34.

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines
1497
3-Chloro-1-[2-deoxy-3,5-di-O-(p-toluoyl)-β-D-erythro-pentofuranosyl]-4-
methoxy-1H-pyrazolo[3,4-d]pyrimidine (6). To a suspension of compound
4 (370 mg, 2.01 mmol) in MeCN(50 mL), KOH (85%, 214 mg, 3.81 mmol)
and TDA-1 (tris[2-(2-methoxyethoxy)ethyl]amine, 35 µL, 0.11 mmol) were
added. After stirring at r.t. for 10 min, 2-deoxy-3, 5-di-O-p-toluoyl-α-D-erythro-
pentofuranosyl chloride (5)[20,21] (1.00 g, 2.57 mmol) were added, and
stirring was continued for another 30 min.[4] Insoluble material was filtered
off, the solvent evaporated, and the residue subjected to FC (column 40 ×
2.5 cm). Elution was performed with petroleum ether/ethyl acetate, 3:1
(300 mL), 2:1 (300 mL), and 1:1 (300 mL). Compound 6 was crystallized
from petroleum ether/ethyl acetate yielding colorless needles (344 mg,
32%). TLC (petroleum ether/ethyl acetate 2:1): Rf 0.6; UV (MeOH) λmax
241 (ε 23,500); 1H-NMR (DMSO-d6) δ 2.37, 2.40 (2s, 6 H, 2 CH3), 2.78 (m,
1 H, Hα-2ꢁ), 3.18 (m, 1 H, Hβ-2ꢁ), 4.13 (s, 3 H, OCH3), 4.47 (m, 2 H, H-
5ꢁ), 4.56 (m, 1 H, H-4ꢁ); 5.81 (m, 1 H, H-3ꢁ), 6.82 (t, 1 H, J = 6.20, H-1ꢁ),
7.30–7.96 (m, 8 H, arom), 8.68 (s, 1 H, H-6); Anal. Calc. for C27H25N4O6Cl
(536.96): C 60.39, H 4.69, N10.43; found: C 60.42, H 4.78, N10.39.
3-Chloro-1-(2-deoxy-β-D-erythro-pentofuranosyl)-4-methoxy-1H-pyrazo-
lo[3,4-d]pyrimidine (7). Compound 6 (343.9 mg, 0.64 mmol) was stirred
for 4 h in 0.4 M NaOMe/MeOH (150 mL). The solution was evaporated
to dryness, and the residue was subjected to FC (column 40 × 2.5 cm,
CH2Cl2/MeOH 9:1). Compound 7 (131 mg, 68%) was obtained as a white
solid. TLC (CH2Cl2/MeOH 9:1): Rf 0.6; UV (MeOH) λmax 271 (ε 7400),
1
246 (ε 9100); H-NMR (DMSO-d6) δ 2.31 (m, 1 H, Hα-2ꢁ), 2.80 (m, 1 H,
Hβ-2ꢁ), 3.48 (m, 2 H, H-5ꢁ), 3.82 (m, 1 H, H-4ꢁ), 4.12 (s, 3 H, OCH3), 4.43
(m, 1 H, H-3ꢁ), 4.71 (t, 1 H, J = 5.77, OH-5ꢁ), 5.33 (d, 1 H, J = 4.69, OH-3ꢁ),
6.62 (t, 1 H, J = 6.67, H-1ꢁ), 8.67 (s, 1 H, H-6); Anal. Calc. for C11H13N4O4Cl
(300.70): C 43.94, H 4.36, N18.63; found: C 44.04, H 4.26, N18.56.
4-Amino-3-chloro-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1H-pyrazolo
[3,4-d]pyrimidine (1b). Compound 7 (435.9 mg, 1.45 mmol) was sus-
pended in sat. NH3/MeOH (0◦C) solution (200 mL) and the reaction
mixture were stirred at 90◦C for 4 h in a steel bomb. After cooling the solvent
was removed in vacuo, and the residue was applied to FC (column 40 × 2.5
cm, CH2Cl2/MeOH 95:5). Compound 1b (256 mg, 62%) was obtained as
a white solid. TLC (CH2Cl2/MeOH 9:1): Rf 0.3; UV (MeOH) λmax 228 (ε
1
7400), 260 (ε 7500), 280 (ε 10,300); H-NMR (DMSO-d6) δ 2.24 (m, 1 H,
Hα-2ꢁ), 2.75 (m,1 H, Hβ-2ꢁ), 3.47 (m, 2 H, H-5ꢁ), 3.78 (m, 1 H, H-4ꢁ), 4.39
(m, 1 H, H-3ꢁ), 4.76 (t, 1 H, J = 5.55, OH-5ꢁ), 5.29 (d, 1 H, J = 4.36, OH-3ꢁ),
6.51 (t, 1 H, J = 6.30, H-1ꢁ), 7.18, 8.07 (br, 2 H, NH2), 8.23 (s, 1 H, H-6);
Anal. Calc. for C10H12N5O3Cl (285.69): C 42.04, H 4.23, N24.51; found: C
42.20, H 4.16, N24.39.

1498
F. Seela et al.
3-Chloro-1-(2-deoxy-β-D-erythro-pentofuranosyl)-4-{[(dimethylamino)
methylidene]amino}-1H -pyrazolo[3,4 -d]pyrimidine (13). A solution of
compound 1b (200 mg, 0.7 mmol) in MeOH (20 mL) was treated with N,N-
dimethylformamide dimethyl acetal (1.62 mL, 12.1 mmol) for 20 min at
r.t. After evaporation, the residue was applied to FC (column 12 × 3.0 cm,
CH2Cl2/MeOH 95:5). Compound 13 was isolated as foam (226 mg, 95%).
1
TLC (CH2Cl2/MeOH 9:1): Rf 0.45; UV (MeOH) λmax 320 (ε 32,000); H-
NMR (DMSO-d6) δ 2.27 (m, 1 H, Hα-2ꢁ), 2.76 (m,1 H, Hβ-2ꢁ), 3.19, 3.24
(2s, 6 H, Me2N), 3.46 (m, 2 H, H-5ꢁ), 3.81 (m, 1 H, H-4ꢁ), 4.41 (m, 1 H,
H-3ꢁ), 4.76 (t, 1 H, J = 5.66, OH-5ꢁ), 5.29 (d, 1 H, J = 4.50, OH-3ꢁ), 6.56 (t,
1 H, J = 6.39, H-1ꢁ), 8.45 (s, 1 H, H-6), 8.95 (s, 1 H, N=CH); Anal. Calc. for
C13H17N6O3Cl (340.77): C 45.82, H 5.03, N24.66; found: C 46.13, H 5.10,
N24.12.
3-Chloro-1-(2-deoxy-5-O-(4,4 -dimethoxytriphenylmethyl)-β-D-erythro-
pentofuranosyl)- 4 -{[(dimethylamino)methylidene]amino}-1H -pyrazolo
[3,4-d]pyrimidine (14). To a solution of compound 13 (180.0 mg, 0.53
mmol) in dry pyridine (1.5 mL) was added 4,4ꢁ-dimethoxytriphenylmethyl
chloride (217.0 mg, 0.64 mmol). After stirring at r.t. for 2 h, the mixture
was poured into ice-cold 3% aq. NaHCO3 soln. (5 mL), the aq. layer was
extracted with CH2Cl2 (2 × 50 mL), the combined organic layers were dried
(Na2SO4), filtered and evaporated. The residue was applied to FC (column
12 × 3 cm, CH2Cl2/MeOH 95:5). Compound 14 was isolated as colorless
foam (291 mg, 86%). TLC (CH2Cl2/MeOH 9:1): Rf 0.5; UV (MeOH) λmax
233 (ε 28,700), 320 (ε 29,500); 1H-NMR (DMSO-d6) δ 2.31 (m, 1 H, Hα-2ꢁ),
2.76 (m,1 H, Hβ-2ꢁ), 3.18, 3.23 (2s, 6 H, Me2N), 3.07 (m, 2 H, H-5ꢁ), 3.68
(2s, 6 H, 2 MeO), 3.92 (m, 1 H, H-4ꢁ), 4.53 (m, 1 H, H-3ꢁ), 5.35 (d, 1 H, J =
4.85, OH-3ꢁ), 6.59 (2d, 1 H, J = 4.13, H-1ꢁ), 6.71–7.32 (m, 13 H, arom), 8.49
(s, 1 H, H-6), 8.95 (s, 1 H, N=CH); Anal. Calc. for C34H35N6O5Cl (643.13):
C 63.50, H 5.49, N13.07; found: C 63.47, H 5.38, N12.89.
3-Chloro-1-(2-deoxy-5-O-(4,4 -dimethoxytriphenylmethyl)-β-D-erythro-
pentofuranosyl)- 4 -{[(dimethylamino)methylidene]amino}-1H -pyrazolo
[3,4-d]pyrimidine 3 -O-[(2-Cyanoethyl) N,N-Diisopropylphosphoramidite]
(15). To a solution of compound 14 (237.8 mg, 0.37 mmol) in dry
CH2Cl2 (20 mL) anh. N,N-diisopropylethylamine (0.118 mL, 0.68 mmol)
and chloro(2-cyanoethoxy) (N,N-diisopropylamino)phosphine (0.118 mL,
0.53 mmol) were added under Ar. After stirring for 30 min, the reaction
mixture was diluted with CH2Cl2 (20 mL), and 5% aq. NaHCO3 (10 mL)
was added. The mixture was extracted with CH2Cl2 (2 × 10 mL), the
organic layers were dried (Na2SO4), filtered, and evaporated. The residue
was applied to FC (column 12 × 2 cm, CH2Cl2/acetone, 92:8). Compound

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines
1499
15 was isolated as foam (187 mg, 60%). TLC (CH2Cl2/acetone 9:1): Rf 0.6,
0.7. 31P-NMR (CDCl3) δ 149.64, 149.53.
6-Methylthio-4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidine (8). A solu-
tion of 6-methylthio-4-chloro-1H-pyrazolo[3,4-d]pyrimidine[19] (5.0 g, 24.91
mmol) in 1 M sodium isopropoxide in isopropanol (200 mL) was refluxed
for 2 h. Upon cooling NaCl was precipitated with Et2O and was filtered
off. The filtrate was neutralized with acetic acid and evaporated to dry-
ness. The residue was dissolved in 200 mL of methanol. An addition of
ice-cold water precipitated out 8 as pale yellow needles (4.10 g, 73%). TLC
(CH2Cl2/MeOH 95:5): Rf 0.48. UV (MeOH) λmax 278 (ε 12,300), 236 (ε
1
18,600); H-NMR (DMSO-d6) δ 1.36, 1.39 (2s, 6 H, 2 CH3), 2.52 (s, 3 H,
CH3S), 5.54 (m, 1 H, CH), 8.04 (s, 1 H, H-3), 13.72 (br s, 1 H, NH); Anal.
Calc. for C9H12N4OS (224.28): C 48.20, H 5.39, N24.98; found: C 48.18, H
5.25, N24.81.
1-[2-Deoxy-3,5-di-O-(p-toluoyl)-β-D-erythro-pentofuranosyl]-6-methyl-
thio-4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidine (9). A solution of 8 (500
mg, 2.23 mmol) and KOH (500 mg, 8.91 mmol) was stirred in MeCNfor
15 min and followed by the addition of TDA-1 (81 µL, 0.25 mmol). After
15 min 1-chloro-2-deoxy-3,5-di-O-toluoyl-α-D-erythro-pentofuranose (5)[20,21]
(1.04 g, 2.68 mmol) was added in portions. The reaction mixture was stirred
for another 15 min and filtered. The filtrate was concentrated and applied
to FC (column 15 × 3 cm, CH2Cl2/MeOH 98:2). The fast migrating zone
furnished 9 as colorless foam (688 mg, 54%). TLC (CH2Cl2/MeOH 98:2):
Rf 0.6. UV (MeOH) λmax 279 (ε 15,700), 238 (ε 49,300); 1H-NMR (DMSO-
d6) δ 1.39 (br s, 6 H, 2 CH3), 2.39 (br s, 6 H, 2 CH3O), 2.58 (s, 3 H, CH3S),
2.79 (m, 1 H, Hα-2ꢁ), 3.37 (m, Hβ-2ꢁ, D2O), 4.40–4.52 (m, 3 H, H-4ꢁ, H-5ꢁ),
5.53 (m, 1 H, CH), 5.86 (m, 1 H, H-3ꢁ), 6.76 (m, 1 H, H-1ꢁ), 7.33–7.98 (m,
8 H, arom), 8.19 (s, 1 H, H-3); Anal. Calc. for C30H32N4O6S (576.66): C
62.48, H 5.59, N9.72; found: C 62.69, H 5.47, N9.58.
2-[2-Deoxy-3,5-di-O-(p-toluoyl)-β-D-erythro-pentofuranosyl]-6-methyl-
thio-4-isopropoxy-2H-pyrazolo[3,4-d]pyrimidine (10). The slow migrating
zone afforded 10 as colorless foam (375 mg, 29%). TLC (CH2Cl2/MeOH
1
98:2): Rf 0.5. UV (MeOH) λmax 282 (ε 11,700), 240 (ε 51,100): H-NMR
(DMSO-d6) δ 1.34, 1.36 (2s, 6 H, (CH3)2CH), 2.35, 2.40 (2s, 6 H, 2 CH3O),
2.54 (s, 3 H, CH3S), 2.79 (m, 1 H, Hα-2ꢁ), 3.18 (m, 1 H, Hβ-2ꢁ), 4.43–4.64
(m, 3 H, H-4ꢁ, H-5ꢁ), 5.49 (m, 1 H, OCH), 5.87 (m, 1 H, H-3ꢁ), 6.50 (t, 1 H,
J = 5.43, H-1ꢁ), 7.22–7.95 (m, 8 H, arom), 8.70 (s, 1 H, H-3); Anal. Calc.
for C30H32N4O6S (576.66): C 62.48, H 5.59, N9.72; found: C 62.68, H 5.56,
N9.78.

1500
F. Seela et al.
1-[2-Deoxy-β-D-erythro-pentofuranosyl]-6-methylthio-4-isopropoxy-1H-
pyrazolo[3,4-d]pyrimidine (11). A suspension of 9 (3 g, 5.20 mmol) in
200 mL of 0.1 M NaOiPr was stirred for 15 min. The clear solution was
evaporated and applied to FC (column 12 × 3 cm). Stepwise elution with 2%
MeOH in CH2Cl2, followed by 10% MeOH in CH2Cl2, gave 11 as a colorless
solid (1.5 g, 85%). TLC (CH2Cl2/MeOH 95:5): Rf 0.25. UV (MeOH) λmax
1
279 (ε 14,500), 238 (ε 18,800); H-NMR (DMSO-d6) δ 1.37, 1.39 (2s, 6 H,
2 CH3), 2.58 (s, 3 H, CH3S), 2.28 (m, 1 H, Hα-2ꢁ), 2.82 (m, 1 H, Hβ-2ꢁ),
3.49 (m, 2 H, H-5ꢁ), 3.80 (m, 1 H, H-4ꢁ), 4.45 (m, 1 H, H-3ꢁ), 4.71 (m, 1 H,
OH-5ꢁ), 5.30 (m, 1 H, OH-3ꢁ), 5.56 (m, 1 H, CH), 6.61 (t, 1 H, J = 6.36,
H-1ꢁ), 8.15 (s, 1 H, H-3); Anal. Calc. for C14H20N4O4S (340.40): C 49.40, H
5.92, N16.46; found: C 49.68, H 5.83, N16.29.
2-[2-Deoxy-β-D-erythro-pentofuranosyl]-6-methylthio-4-isopropoxy-2H-
pyrazolo[3,4-d]pyrimidine (12). As described for 11, compound 10 (3 g,
5.20 mmol) gave 12 as a colorless solid (1.6 g, 90%). TLC (CH2Cl2/MeOH
9:1): Rf 0.48. UV (MeOH) λmax 282 (ε 10,800), 240 (ε 19,700), 227 (ε
1
17,500); H-NMR (DMSO-d6) δ 1.35, 1.38 (2s, 6 H, (CH3)2CH), 2.51 (s,
3 H, CH3S), 2.34 (m, 1 H, Hα-2ꢁ), 2.62 (m, 1 H, Hβ-2ꢁ), 3.61 (m, 2 H, H-5ꢁ),
3.89 (m, 1 H, H-4ꢁ), 4.40 (m, 1 H, H-3ꢁ), 4.95 (m, 1 H, OH-5ꢁ), 5.35 (m,
1 H, OH-3ꢁ), 5.52 (m, 1 H, (CH3)2CH), 6.27 (t, 1 H, J = 6.11, H-1ꢁ), 8.71 (s,
1 H, H-3); Anal. Calc. for C14H20N4O4S (340.40): C 49.40, H 5.92, N16.46;
found: C 49.37, H 5.80, N16.41.
4-Amino-1-[2-deoxy-β-D-erythro-pentofuranosyl]-6-methylthio-1H-pyra-
zolo[3,4-d]pyrimidine (2b). A suspension of 11 (200 mg, 0.59 mmol) in
methanol, saturated with dry ammonia (100 mL), was stirred in a glass sealed
bottle for 5 days at r.t. The clear solution was adsorbed on silica gel and
applied to FC (column 15 × 3 cm, CH2Cl2/MeOH 9:1). Compound 2b was
isolated as a colorless solid (160 mg, 92%). TLC (CH2Cl2/MeOH 9:1): Rf
1
0.48. UV (MeOH) λmax 278 (ε 14,600), 242 (ε 28,800); H-NMR (DMSO-
d6) δ 2.48 (s, 3 H, CH3S), 2.25 (m, 1 H, Hα-2ꢁ), 2.81 (m, 1 H, Hβ-2ꢁ), 3.51
(m, 2 H, H-5ꢁ), 3.79 (m, 1 H, H-4ꢁ), 4.42 (m, 1 H, H-3ꢁ), 4.73 (t, 1 H, J =
5.75, OH-5ꢁ), 5.27 (d, 1 H, J = 4.47, OH-3ꢁ), 6.50 (t, 1 H, J = 6.5, H-1ꢁ),
7.71, 7.88 (m, 2 H, NH2), 8.05 (s, 1 H, H-3); Anal. Calc. for C11H15N5O3S
(297.33): C 44.43, H 5.08, N23.55; found: C 44.48, H 5.03, N23.46.
4-Amino-2-[2-deoxy-β-D-erythro-pentofuranosyl]-6-methylthio-2H-pyra-
zolo[3,4-d]pyrimidine (3c). As described for 2b, compound 3c was obtained
from 12 (200 mg, 0.59 mmol) as a colorless solid (160 mg, 92%). TLC
(CH2Cl2/MeOH 95:5): Rf 0.5. UV (MeOH) λmax 278 (ε 11,500), 240 (ε
21,500);1H-NMR (DMSO-d6) δ 2.51 (s, 3 H, CH3S), 2.32 (m, 1 H, Hα-2ꢁ),
2.62 (m, 1 H, Hβ-2ꢁ), 3.43–3.57 (m, 2 H, H-5ꢁ), 3.89 (m, 1 H, H-4ꢁ), 4.37 (m,

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines
1501
1 H, H-3ꢁ), 4.87 (m, 1 H, OH-5ꢁ), 5.31 (d, 1 H, J = 4.31, OH-3ꢁ), 6.26 (t,
1 H, J = 6.14, H-1ꢁ), 7.69 (m, 2 H, NH2), 8.42 (s, 1 H, H-3); Anal. Calc. for
C11H15N5O3S (297.33): C 44.43, H 5.08, N23.55; found: C 44.55, H 5.02,
N23.46.
1-[2-Deoxy-β-D -erythro -pentofuranosyl]- 4{[(dimethylamino)methyli-
dene]amino}-6-methylthio-1H-pyrazolo[3,4-d]pyrimidine (16). A solution
of compound 2b (250 mg, 0.84 mmol) in MeOH was stirred with N,N-
dimethylformamide dimethylacetal (1.4 g) at 50◦C for 6 h. The solution was
evaporated and applied to FC (column 20 × 3 cm, CH2Cl2/MeOH 95:5) to
give compound 16 as colorless solid (250 mg, 84.4%). TLC (CH2Cl2/MeOH
95:5): Rf 0.4. UV (MeOH) λmax 315 (ε 17,400), 292 (ε 18,300), 272 (ε
1
16,400), 245 (ε 18,700); H-NMR (DMSO-d6) δ 2.55 (s, 3 H, CH3S), 2.28
(m, 1 H, Hα-2ꢁ), 2.85 (m, 1 H, Hβ-2ꢁ), 3.15, 3.23 (2s, 6 H, 2 CH3), 3.38–
3.54 (m, 2 H, H-5ꢁ), 3.82 (m, 1 H, H-4ꢁ), 4.44 (m, 1 H, H-3ꢁ), 4.74 (t, 1 H,
J = 5.72, OH-5ꢁ), 5.30 (d, 1 H, J = 4.44, OH-3ꢁ), 6.56 (t, 1 H, J = 6.43,
H-1ꢁ), 8.06 (s, 1 H, N=CH), 8.86 (s, 1 H, H-3); Anal. Calc. for C14H20N6O3S
(352.41): C 47.71, H 5.72, N23.85; found: C 47.41, H 5.80, N23.67.
1-[2-Deoxy-β-D-erythro-pentofuranosyl]-4{[(dimethylamino)ethylidene]
amino}-6-methylthio-1H -pyrazolo[3,4 -d]pyrimidine (17). A solution of
compound 2b (150 mg, 0.50 mmol) in MeOH was stirred with N,N-di-
methylacetamide dimethylacetal (1 mL) at 40◦C for 16 h. The solution was
evaporated and applied to FC (column 20 × 3 cm, CH2Cl2/acetone 8:2) to
give compound 17 as colorless solid (170 mg, 92%). TLC (CH2Cl2/acetone
8:2): Rf 0.38. UV (MeOH) λmax 219 (ε 13,800), 269 (ε 12,400), 246 (ε
1
20,300); H-NMR (DMSO-d6) δ 2.26 (s, 3 H, CH3), 2.51 (s, 3 H, CH3S),
2.27 (m, 1 H, Hα-2ꢁ), 2.85 (m, 1 H, Hβ-2ꢁ), 3.15, 3.23 (2s, 6 H, 2 CH3), 3.38–
3.54 (m, 2 H, H-5ꢁ), 3.82 (m, 1 H, H-4ꢁ), 4.44 (m, 1 H, H-3ꢁ), 4.71 (t, 1 H,
J = 5.70, OH-5ꢁ), 5.27 (d, 1 H, J = 4.41, OH-3ꢁ), 6.56 (t, 1 H, J = 6.43,
H-1ꢁ), 7.96 (s, 1 H, H-3); Anal. Calc. for C15H22N6O3S (366.44): C 49.17, H
6.05, N22.93; found: C 49.26, H 6.10, N22.78.
1-[2-Deoxy-5-O-(4,4 -dimethoxytriphenylmethyl)-β-D-erythro-pentofura-
nosyl]- 4{[(dimethylamino)ethylidene]amino} - 6 - methylthio-1H -pyrazolo
[3,4-d]pyrimidine (18). Compound 17 (125 mg, 0.34 mmol) in 2 mL of
pyridine was stirred with (MeO)2TrCl (150 mg, 0.44 mmol) at r.t. for 2.5 h.
The reaction was quenched by the addition of 5% NaHCO3 solution (10
mL) and extracted with dichloromethane (3 × 15 mL). The organic layer
was dried over Na2SO4 and evaporated. The resulting residue was subjected
to FC (column 15 × 3 cm, CH2Cl2/acetone 9:1) to give compound 18 as
colorless foam (192 mg, 84%). TLC (CH2Cl2/acetone 9:1): Rf 0.55. UV
1
(MeOH) λmax 282 (ε 14,300), 273 (ε 14,600), 237 (ε 33,600); H-NMR

1502
F. Seela et al.
(DMSO-d6) δ 2.22 (s, 3 H, CH3), 2.51 (s, 3 H, CH3S), 2.32 (m, 1 H, Hα-
2ꢁ), 2.84 (m, 1 H, Hβ-2ꢁ), 3.15, 3.23 (2s, 6 H, 2 CH3), 3.02–3.15 (m, 2 H,
H-5ꢁ), 3.71, 3.72 (2s, 6 H, 2 CH3O), 3.93 (m, 1 H, H-4ꢁ), 4.56 (m, 1 H, H-
3ꢁ), 5.31 (d, 1 H, J = 4.83, OH-3ꢁ), 6.58 (m, 1 H, H-1ꢁ), 7.90 (s, 1 H, H-3),
6.74–7.32 (m, 13 H, arom); Anal. Calc. for C36H40N6O5S (668.81): C 64.65,
H 6.03, N12.57; found: C 65.04, H 6.04.
1-[2-Deoxy-5-O-(4,4 -dimethoxytriphenylmethyl)-β-D-erythro-pentofura-
nosyl]-4{[(dimethylamino)ethylidene]amino}-6-methylthio-1H-pyrazolo[3,
4-d]pyrimidine 3 -[(2-Cyanoethyl)-N,N-diisopropyl-phosphoramidite] (19).
To a soln. of compd. 18 (90 mg, 0.14 mmol) and (iPr)2EtN(30 µL, 0.17
mmol) in anh. CH2Cl2 (2 mL), 2-cyanoethyl diisopropylphosphoramido-
chloridite (50 µL, 0.29 mmol) was added at r.t.. After stirring for 20 min,
the mixture was diluted with CH2Cl2 (10 mL) and quenched by adding a 5%
aq. NaHCO3 soln. (20 mL). Then, the aq. layer was extracted with CH2Cl2
(3 × 15 mL), the combined org. layer dried over Na2SO4 and evaporated.
The resulting oil was applied to FC (column 10 × 3 cm, CH2Cl2/acetone
95:5) affording colorless foam (85 mg, 73%). TLC (CH2Cl2/acetone 95:5):
Rf 0.52, 0.48. 31P-NMR (CDCl3) δ 149.9, 149.6.
2-[2-Deoxy-β-D-erythro-[pentofuranosyl]-4{[isobutyryl]amino}-6-methyl-
thio-2H-pyrazolo[3,4-d]pyrimidine (20). Compound 3c (200 mg, 0.67
mmol) was repeatedly evaporated with pyridine and suspended in pyridine
(2 mL). TMSCl (1.15 mL, 9.06 mmol) was added and stirred for 30 min,
followed by the addition of isobutyryl chloride (0.14 mL, 1.34 mmol) and
stirring was continued for 2 h at r.t. The reaction mixture was cooled in an
ice-bath, 1 mL H2O was added. After 5 min, 1 mL 25% aq. NH3-soln. was
added, and the solution stirred for 15 min more. The solution evaporated
to dryness, dissolved in 10 mL H2O, and extracted with CH2Cl2 (3 × 15
mL).The organic layer was dried over Na2SO4, evaporated and applied to
FC (column 15 × 3 cm, CH2Cl2/MeOH 95:5). Compound 20 was obtained
as colorless foam (150 mg 60.69%). TLC (CH2Cl2/MeOH 95:5): Rf 0.46.
UV (MeOH) λmax 297 (ε 10,800), 245 (ε 16,400), 216 (ε 14,000); 1H-NMR
(DMSO-d6) δ 1.13, 1.14 (2s, 6 H, 2 CH3), 2.52 (s, 3 H, CH3S), 2.34 (m, 1 H,
Hα-2ꢁ), 2.67 (m, 1 H, Hβ-2ꢁ), 2.89 (m, 1 H, CH(CH3)2), 3.48–3.61 (m, 2 H,
H-5ꢁ), 3.92 (m, 1 H, H-4ꢁ), 4.45 (m, 1 H, H-3ꢁ), 4.86 (t, 1 H, J = 5.55, OH-
5ꢁ), 5.32 (d, 1 H, J = 4.4, OH-3ꢁ), 6.41 (t, 1 H, J = 5.8, H-1ꢁ), 8.91 (s, 1 H,
H-3), 11.2 (s, 1 H, NH); Anal. Calc. for C15H21N5O4S (367.42): C 49.03, H
5.76, N19.06; found: C 48.96, H 5.65, N18.89.
2-[2-Deoxy-5-O-(4,4 -dimethoxytriphenylmethyl)-β-D-erythro-pentofu-
ranosyl]-4{[isobutyryl]amino}-6-methylthio-2H-pyrazolo[3,4-d]pyrimidine
(21). To a stirred solution of 20 (100 mg, 0.27 mmol ) in anhydrous

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines
1503
pyridine (2 mL) was added 4-dimethylaminopyridine (25 mg, 0.20 mmol),
followed by (MeO)2TrCl (125 mg, 0.37 mmol ). The reaction was worked
up as described for 18. Compound 21 was obtained as colorless foam (107
mg, 59%). TLC (CH2Cl2/acetone 9:1): Rf 0.62. UV (MeOH) λmax 275 (ε
12,200), 236 (ε 30,300); 1H-NMR (DMSO-d6) δ 1.13, 1.14 (2s, 6 H, 2 CH3),
2.53 (s, 3 H, CH3S), 2.34 (m, 1 H, Hα-2ꢁ), 2.83 (m, 1 H, Hβ-2ꢁ), 2.86 (m, 1 H,
CH(CH3)2), 3.10–3.31 (m, 2 H, H-5ꢁ), 3.67, 3.69 (2s, 6 H, 2 CH3O), 3.98
(m, 1 H, H-4ꢁ), 4.50 (m, 1 H, H-3ꢁ), 5.36 (d, 1 H, J = 5.0, OH-3ꢁ), 6.45 (m,
1 H, H-1ꢁ), 8.94 (s, 1 H, H-3), 11.21 (s, 1 H, NH), 6.70-7.29 (m, 13 H, arom);
Anal. Calc. for C36H39N5O6S (669.79): C 64.56, H 5.87, N10.46; found: C
64.60, H 5.99, N10.40.
2-[2-Deoxy-5-O-(4,4 -dimethoxytriphenylmethyl)-β-D-erythro-pentofura-
nosyl]-4{[isobutyryl]amino}-6-methylthio-2H-pyrazolo[3,4-d]pyrimidine 3 -
[(2-Cyanoethyl)-N,N-diisopropyl-phosphoramidite] (22). As described for
19, with 21 (70 mg, 0.11 mmol), (iPr)2EtN(25 µL, 0.145 mmol) and 2-
cyanoethyl diisopropylphosphoramidochloridite (50 µL, 0.22 mmol): 22 (70
mg, 77%). Colorless foam. TLC (CH2Cl2/acetone 95:5): Rf 0.60, 0.58. 31P-
NMR (CDCl3) δ 150.1, 150.4.
Oligonucleotides
The synthesis was performed with DNA solid phase synthesizer, Model
392 (Applied Biosystems, Weiterstadt, Germany) with “trityl-on” mode. The
oligonucleotides were purified and desalted by HPLC with RP-18 (5 µm, 4 ×
250 mm and 4 × 125 mm) (LiChrospher®, Merck, Germany), lyophilized
with SpeedVac centrifuge (Savant Instruments, Farmingdale, New York).
MALDI-TOF spectra were measured with Biflex III spectrometer (Bruker
Saxonia, Leipzig, Germany). Melting curves were measured with a Cary
1/1E UV/VIS spectrophotometer (Varian, Australia) equipped with a ther-
moelectrical controller; the actual temperature was measured in the refer-
ence cell with a PT-100 resistor. UV-spectra were measured on U-3200 spec-
trophotometer (Hitachi, Japan). The oligonucleotides were cleaved from
the solid support by 25% aq. NH3-soln., and purified by HPLC with the
gradient: 3 min 20% B in A, 12 min 20–40% B in A with a flow rate of
1.0 mL/min (A, 0.1 M (Et3NH) OAc (pH 7.0)/MeCN 95:5; B, MeCN).
They were detritylated with 2.5% dichloroacetic acid in dichloromethane,
lyophilized, and coevaporated with methanol. The residue was dissolved
in bidistilled water and purified by HPLC with the gradient 20 min 0–
20% B in A with a flow rate of 1 mL/min. The purified oligonucleo-
tides were desalted with water and eluted with water/methanol (3:2). The
oligonucleotides were characterized by MALDI-TOF mass spectrometry
(Table 2).

1504
CONCLUSION
F. Seela et al.
The 8-aza-7-deazaadenine 2ꢁ-deoxyribonucleosides 1b with a chloro
residue at the 7-position strengthen the dA-dT base pair stability, as it was
shown for related oligonucleotides containing bromine or iodine at that
position. On the other hand, 2-chloro or 2-methylthio groups have an
unfavorable influence on the duplex stability, leading to harmonization of
base pair stability when 2b is located opposite to dC and dA. Thus, this
nucleoside can be considered as a universal nucleoside. A universal base
pairing was also reported for the N8-glycosylated nucleoside. However, the
ambiguous base pairing of 3c is not so striking. It forms two sets of duplex
stability showing a maximum stabilization against dA or dC than that of dG
or dT.
REFERENCES
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pyrazolo[3,4-d]pyrimidine via phase-transfer glycosylation. Helv. Chim. Acta 1985, 68, 563–570.
2. Seela, F.; Kaiser, K. 8-Aza-7-deazaadenine N8- and N9-(β-D-2 -deoxyribofuranosides): Building
blocks for automated DNA synthesis and properties of oligodeoxyribonucleotides. Helv. Chim.
Acta 1988, 71, 1813–1823.
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influence of 7-substituted 8-aza-7-deazaadenines on the duplex structure and stability. J. Chem.
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5. He, J.; Seela, F. Propynyl groups in duplex DNA: Stability of base pairs incorporating 7-substituted
8-aza-7-deazapurines or 5-substituted pyrimidines. Nucleic Acids Res. 2002, 30, 5485–5496.
6. Seela, F.; He, Y.; He, J.; Becher, G.; Kro¨schel, R.; Zulauf, M.; Leonard, P. Base-modified
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adenosine: Facile enzymatic preparation and growth inhibitory effects on human cell lines.
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deoxyribonucleosides. Helv. Chim. Acta 1994, 77, 194–202.
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of nucleosides with nitrogen-8 as a glycosylation position. Org. Biomol. Chem. 2003, 1, 1873–1883.

Oligonucleotides Containing Pyrazolo[3,4-d]pyrimidines
1505
15. Kierzek, E.; Kierzek, R. The thermodynamic stability of RNA duplexes and hairpins containing
N6-alkyladenosines and 2-methylthio-N6-alkyladenosines. Nucleic Acids Res. 2003, 31, 4472–4480.
16. Esberg, B.; Bjo¨rk, G.R. The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthio-
adenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the
tRNA. J. Bacteriol. 1995, 177, 1967–1975.
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allopurinol and its methyl derivatives by a carbon-13 NMR spectroscopic method. Khim.
Geterotsikl. Soedin. 1979, 11, 1543–1546.
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d]pyrimidines. J. Am. Chem. Soc. 1956, 78, 784–790.
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erythro-pentofuranosyl chloride. Synth. Commun. 1997, 27, 3505–3511.
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nucleoside forming specific hydrogen bonds with the four canonical DNA constituents. Nucleic
Acids Res. 2000, 28, 3224–3232.
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25. Molar volume values were calculated using the program ChemSketch (version 4.55, provided by
Advanced Chemistry Developments Inc., Toronto, Canada; http:/www.acdlabs.com).