Base pairing of 8-Aza-7-deazapurine-2,6-diamine linked via the N(8)-position to the DNA backbone: Universal base-pairing properties and formation of highly stable duplexes when alternating with dT

Article abstract of DOI:10.1002/1522-2675(200205)85:5<1340::AID-HLCA1340>3.0.CO;2-H

The unusually N⁸-glycosylated pyrazolo[3,4-d]pyrimidine-4,6-diamine 2′-deoxyribonucleoside (3) was synthesized and converted to the phosphoramidite 11. Oligonucleotides were prepared by solid-phase synthesis, and the base pairing of compound 3

Full text of DOI:10.1002/1522-2675(200205)85:5<1340::AID-HLCA1340>3.0.CO;2-H

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Helvetica Chimica Acta Vol. 85 (2002)
Base Pairing of 8-Aza-7-deazapurine-2,6-diamine Linked via the
N(8)-Position to the DNA Backbone: Universal Base-Pairing Properties and
Formation of Highly Stable Duplexes when Alternating with dT
by Junlin He and Frank Seela*
Laboratorium f¸r Organische und Bioorganische Chemie, Institut f¸r Chemie, Universit‰t Osnabr¸ck,
Barbarastr. 7, D-49069 Osnabr¸ck
The unusually N8-glycosylated pyrazolo[3,4-d]pyrimidine-4,6-diamine 2'-deoxyribonucleoside (3) was
synthesized and converted to the phosphoramidite 11. Oligonucleotides were prepared by solid-phase synthesis,
and the base pairing of compound 3 was studied. In non-self-complementary duplexes containing compound 3
located opposite to the four canonical DNA constituents, strong base pairs are formed that show
ambiguous pairing properties. The self-complementary duplex d(3-T)6 (34 ¥ 34) is significantly more stable
than d(A-T)6.
Introduction. Earlier, it was shown that N8-glycosylated 8-aza-7-deazaadenine
(ˆ pyrazolo[3,4-d]pyrimidin-4-amine) 2'-deoxyribonucleoside (1) [1 3] acts as a
universal nucleoside when positioned opposite any of the four canonical DNA
constituents [4]. It forms an unusually stable base pair with thymidine when it replaces
2'-deoxyadenosine within d(A-T)6 (DTm ˆ 1.38/1 ¥ dT) (Fig. 1) [2]. Its N9-linked isomer
4 does not have such a property [5] (purine numbering is used throughout the Results
and Discussion section). Recently, the base-pairing capability of the related C8-linked
7-deazaguanosine (2) was reported [6]. It shows a base recognition pattern similar to
that of 2'-deoxyisoguanosine but one different from the regularly glycosylated 2'-
deoxyguanosine. Strong base pairs between compound 2 and 2'-deoxy-5-methylisocy-
tidine are observed in DNA duplexes with antiparallel chain orientation. To the
contrary, compound 2 forms base pairs of similar stability with dC in parallel DNA.
From model building, it was anticipated that compound 3 would exhibit pairing
properties similar to those of nucleoside 1. However, due to the presence of the
additional 2-amino group, a stronger base pair with dT was expected. We now report
the synthesis of a phosphoramidite building block 11, prepared from nucleoside 3, and
its base-pairing properties in self-complementary and non-self-complementary oligo-
nucleotide duplexes. The Tm data obtained from hybridization experiments will be
compared with those of compound 1 [5]. Moreover, the potential of this compound as a
universal nucleoside is studied. Ambiguous base pairing is not found for the N9-
glycosylated nucleosides 5 and 6 [7][8].
Results and Discussion. 1. Monomers. The sugar-protected compound 7 [9] was
used as the starting material. It was detoluoylated to yield nucleoside 8. Next, the
isopropoxy group of 8 was replaced by an amino group (25% aqueous NH3 solution,
608, 4d) to give nucleoside 3. For oligonucleotide solid-phase synthesis, the amino

Helvetica Chimica Acta Vol. 85 (2002)
1341
Fig. 1. N8- and N9-Glycosylated nucleobases
groups of 3 were protected. It has been reported that the reactivities of the two amino
groups of 5 and 6 are different, which complicated the synthesis of the oligonucleotide
building blocks [10]. Various protecting groups have been studied. As the isobutyryl
residue was already successfully used for the protection of compound 5, the same
residues were employed in the case of 3 [10 12]. The protocol of transient protection
was employed [13] (Scheme 1). The two isobutyrylation products isolated are
compound 9a carrying three isobutyryl residues (42% yield) and compound 9b
carrying one isobutyryl residue at each amino group (20%). The monoprotected 9c was
not obtained. The triisobutyrylated 9a was chosen for further experiments. Syntheses of
the (MeO)2Tr derivative 10 and of the phosphoramidite 11 were performed by standard
protocols (Scheme 1) [14].
The position of glycosylation as well as the anomeric configuration of compound 3
were assigned unambiguously by 13C-NMR chemical shifts as well as by NOE
difference spectra (Scheme 2). According to our definition [4], the orientation of the
base on N8-glycosylated nucleosides is −anti× when the distance between HÀC(1') and
HÀC(7) is minimal and −syn× when this distance is maximal (Scheme 2,b). This is
different to the definition of N9-linked nucleosides, in which the orientation of the base
is −syn× when the distance between HÀC(1') and HÀC(8) is minimal and −syn× when
this distance is maximal. As an NOE effect between HÀC(7) and HÀC(1') was
observed, the nucleoside 3 adopts a 55% −anti× population, which is similar to that of
nucleoside 1 (66% −anti×) [4].

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Helvetica Chimica Acta Vol. 85 (2002)
Scheme 1
i) 0.1m MeONa/MeOH, 458, 1 h. ii) 25% aq. NH3 soln., 608, 4 d. iii) (MeO)2TrCl, pyridine, r.t., 4 h.
iv) 2-Cyanoethyl diisopropylphosphoramidochloridite, CH2Cl2, r.t., 30 min.
Irradiation of HÀC(1') of 3 results in a strong NOE on HÀC(7) (6.3%) besides weaker NOEs on HÀC(2')
(4.8%) and HÀC(4') (1%) (Scheme 2,a). On the other hand, irradiation of HÀC(7) gives an NOE on HÀC(1')
(6.3%) (Scheme 2,b). The NOEs on HÀC(2') and HÀC(4') established the b-d configuration, while the NOE on
HÀC(7) indicated that the sugar moiety is connected via N(8). The −syn×/−anti× population of the nucleoside 3
was calculated by means of a calibration graph published earlier for regularly linked nucleosides [15].

Helvetica Chimica Acta Vol. 85 (2002)
1343
Scheme 2. NOE Data of the −anti× States of Nucleoside 3
3
The sugar conformation was studied by means of the vicinal J(H,H) coupling
constants of the 1H-NMR spectra measured in D2O applying the PSEUROT program
[16]. The S-conformer populations of all pyrazolo[3,4-d]pyrimidine nucleosides 1 and
3 5 (56 63%) are lower compared to that of 2'-deoxyadenosine (72%) [17]. For
nucleoside 3, the S-population is somewhat higher than for nucleoside 1 (61 vs. 56%).
The conformation about the C(4')ÀC(5') bond (g(‡)g > gt > g(À)g) of nucleoside 3
shows no preference for either the ‡ sc (43%) or the À sc (40%) rotamer population,
which is analogous to the results with other pyrazolo[3,4-d]pyrimidine nucleosides [4].
To study the stability of the protecting groups, the deprotection of compounds 9a
and 9b was monitored by UV spectroscopy in 25% aqueous NH3 solution at 408 (9a at
328 nm; 9b at 250 nm; at these two wavelengths, greatest absorbance changes during
hydrolysis were observed and thus used for monitoring). As the stepwise deprotection
was difficult to follow UV-spectrophotometrically, the reaction products were
identified by HPLC (Fig. 2,a for 9a, and Fig. 2,b for 9b). In both cases (after 50 min
for 9a and 150 min for 9b), the deprotection resulted in the formation of the
unprotected nucleoside 3 and a monoprotected derivative 9c. The latter was not
assigned unambiguously. Figs. 2,c and d, show the HPLC profiles of the hydrolysis
products together with their starting materials 9a and 9b. From these observations, it is
apparent that an isobutyryl residue protecting the 6-amino group is much more labile
than that protecting the 2-amino function [10][11]. As indicated by HPLC, all
protecting groups are completely removed under the conditions of oligonucleotide
deprotection (608, 25% aqueous NH3 solution, overnight). The stability of nucleoside 3
was also studied in 1n HCl at room temperature. When the reaction was followed by
HPLC for 1 h (for conditions, see Fig. 2), no significant decomposition was observed.
All compounds were characterized by 1H-and 13C-NMR spectra. The assignments
of the 13C-NMR resonances were made on the basis of 1H,13C coupling constants, which
were determined from gated-decoupled spectra (Tables 1 and 2). The 13C-NMR
chemical shifts of C(2), C(4), and C(6) of compound 3 are tentatively assigned. The
13C-NMR chemical shift of C(7), being almost identical to that of compound 1,
confirmed N(8) as the glycosylation site, in agreement with the NOE-difference
spectra. Next, the position of the isobutyryl residues of the triisobutyrylated derivative
9a was determined. For this purpose, the 2J and 3J 1H,13C coupling constants of

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Helvetica Chimica Acta Vol. 85 (2002)
Fig. 2. HPLC Profile of the deprotection a) of nucleoside 9a (25% aq. NH3 soln., 408, 50 min) and b) of
nucleoside 9b (25% aq. NH3 soln., 408, 150 min), c) of the mixture of 9a and its deprotection products, and d) of
the mixture of 9b and its hydrolysis products. The HPLC was performed on an RP-18 column (20 Â 1 cm) with
buffer B (see Exper. Part) and was monitored at 260 nm; flow rate 0.7 ml/min.
Table 1. 13C-NMR Chemical Shifts of Pyrazolo[3,4-d]pyrimidine Nucleosidesa)
C(2)b) C(4)b) C(5)b) C(6)b) C(7)b) C(1') C(2') C(3') C(4') C(5') CˆO, Me, CH
C(6)c) C(7a)c) C(3a)c) C(4)c) C(3)c)
d
1 [1] 156.7
3
4 [1] 156.2
5 [4] 156.9
159.6
101.4
97.6
100.6
95.5
159.5
124.0
90.5
90.1
87.7
87.4
90.1
91.0
90.6
90.7
)
70.7
71.0
71.2
71.3
70.5
70.5
70.7
70.4
88.4
88.3
84.2
83.3
88.2
88.7
88.6
86.5
62.1
62.5
62.6
62.7
61.9
61.9
62.1
64.3
162.7e) 162.3e)
159.6e) 124.3
40.4
38.1
38.0
153.8
158.3
164.1
161.4
161.3
161.4
158.2
162.7
163.2
156.4
155.2
156.5
133.2
133.3
123.7
130.1
129.1
130.4
d
8
161.5
155.6
154.8
155.6
97.8
)
)
)
21.7, 68.4
176.9, 179.6
175.3, 176.8
177.0, 179.5
d
9a
9b
10
101.6
100.6
101.8
d
40.5
a) Measured in (D6)DMSO. b) Purine numbering. c) Systematic numbering. d) Superimposed by DMSO.
e) Tentative.
Table 2. 13C,1H-Coupling Constants [Hz] of Pyrazolo[3,4-d]pyrimidine-4,6-diamine Nucleosidesa)
Coupling constant
1
3
9a
C(2)b) (C(6)c))
C(4)b) (C(7a)c))
C(7)b) (C(3)c))
1J(C(2), HÀC(2))
3J(C(4), HÀC(7))
1J(C(7), HÀC(7))
3J(C(7), HÀC(1'))
2J(C(5), HÀC(7))
3J(C(5), HÀN(6))
1J(C(1'), HÀC(1'))
2J(C(1'), HÀC(2'))
1J(C(1'), HÀC(3))
1J(C(4'), HÀC(4'))
1J(C(3'), HÀC(3'))
1J(C(5'), HÀC(5'))
194.6
7.4
194.1
1.6
7.1
4.6
167.5
7.3
3.3
146.9
147.6
139.6
7.1
191.4
1.6
7.7
3.9
165.4
6.9
2.6
146.9
147.6
139.9
7.3
203.4
1.4
6.9
4.4
C(5)b) (C(3a)c))
C(1')
169.3
C(4')
C(3')
C(5')
146.9
151.0
138.9
a) Measured in D2O at 303 K, b) Purine numbering. c) Systematic numbering.

Helvetica Chimica Acta Vol. 85 (2002)
1345
compounds 1, 3, and 9a (Fig. 3) were compared (Table 2). Nucleoside 3 shows a dt for
C(5), due to couplings with HÀC(7) and the two protons of the 6-amino group.
Analogous couplings were also observed for nucleoside 1. In compound 9a, the signal of
C(5) was a dd, due to couplings with one proton of the amide moiety and HÀC(7). This
indicates that only one isobutyryl group is attached to the 6-amino function of 9a, while
the 2-amino group carries two isobutyryl residues.
Fig. 3. 13C,1H-Coupling paths of nucleosides 1, 3, and 9a
2. Oligonucleotides. 2.1. Synthesis. The oligonucleotides 12 38 (Tables 3 7) were
prepared by solid-phase synthesis by means of phosphoramidite chemistry. The
coupling efficiency was always higher than 95%. Deprotection of the oligomers was
conducted in 25% aqueous ammonia at 608. Reversed-phase HPLC was applied for the
purification (see Exper. Part). The composition of the oligomers was verified after
tandem hydrolysis with snake-venom phosphodiesterase/alkaline phosphatase by
HPLC analysis as described. The composition of oligonucleotides 15 (Fig. 4,a) and
38 (Fig. 4,b) was analyzed by reversed-phase HPLC after enzymatic digestion; the
relative ratios of all nucleosides were in agreement with that calculated for the
oligonucleotides. The retention time of the canonical nucleosides and compound 5
(Fig. 4,c) were used as reference. The chromatographic mobility of the N8-glycosylated
nucleoside 3 is greater than that of the N9-compound 5 (Fig. 4,c). MALDI-TOF Mass
spectrometry indicated that the masses of the oligomers were in agreement with the
calculated values (see Exper. Part).
2.2. Base-Pairing Properties. To study the base-pairing properties of nucleoside 3,
non-self-complementary and self-complementary oligonucleotides were prepared that
were derived from the duplexes 5'-d(TAG GTC AAT ACT) ¥ 3'-d(ATC CAG TTA
TGA) (12 ¥ 13), [5'-d(A-T)6]2 (31 ¥ 31), [5'-d(A)6-d(T)6] (35 ¥ 35), or [5'-d(T)6-d(A)6]2
(36 ¥ 36). The dA residues were partly or fully replaced by the N8-nucleoside 3, or by the
nucleosides 1 or 4 for comparison.
2.2.1. Stability of Non-Self-Complementary Duplexes. At first, the nucleoside 3 was
introduced into the non-self-complementary duplex 12 ¥ 13 opposite to dT. According
to Table 3, the Tm decrease is moderate when two modified residues are positioned in
the flanking region (see 15 ¥ 17) and stronger when they are located in the center of the
duplex (see 14 ¥ 15 or 15 ¥ 16) phenomena well-known from other modified duplexes
[10]. A particularly strong decrease is observed when the central dA-residues are

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Helvetica Chimica Acta Vol. 85 (2002)
Fig. 4. HPLC Profile of the enzymatic hydrolysis a) of oligonucleotide 15 and b) of oligonucleotide 38 by snake-
venom phosphodiesterase followed by alkaline phosphatase in 0.1m Tris ¥ HCl buffer (pH 8.3); c) HPLC profile
of nucleosides 3 and 5. Conditions, see Exper. Part.
Table 3. Tm Values and Thermodynamic Data of Antiparallel-Strand Oligonucleotide Duplexes Containing the
Nucleoside 3 Located Opposite dTa)
Tm [8]
DH8 [kcal/mol] DS8 [cal/mol K] DG8310 [kcal/mol]
5'-d(TAG GTC AAT ACT)-3' (12) [11]
3'-d(ATC CAG TTA TGA)-5' (13)
50 (47)
À 90
À 72
À 81
À 79
À 60
À 252
À 204
À 234
À 225
À 174
À 12.0
À 8.1
À 8.4
À 9.6
À 6.2
5'-d(TAG GTC A3T ACT)-3' (14)
3'-d(ATC C3G TTA TGA)-5' (15)
39
40
45
30
5'-d(TAG GTC 33T ACT)-3' (16)
3'-d(ATC C3G TTA TGA)-5' (15)
5'-d(T3G GTC AAT 3CT)-3' (17)
3'-d(ATC C3G TTA TGA)-5' (15)
5'-d(TAG GTC A3T ACT)-3' (14)
3'-d(ATC C3G TT3 TGA)-5' (18)
a) Measured at 260 nm in 1m NaCl, 100 mm MgCl2, and 60 mm Na cacodylate (pH 7.0) with 10 mm
oligonucleotide concentration. Data in parentheses were measured in 100 mm NaCl, 10 mm MgCl2, and
10 mm Na cacodylate (pH 7.0) with 10 mm oligonucleotide concentration. The standard errors of thermody-
namic data for DH8 and DS8 obtained from the curve fitting are within 15%.
replaced in the oligomer 13 (see 14 ¥ 18). This phenomenon is a structural feature of the
sequence and has also been observed in other cases. When duplexes containing
nucleoside 3 are compared with those having the abasic residue dS [4] (dS ˆ 1,2-
dideoxyribose; Table 4), the stabilization of the base is obvious. The effects of the
diamino compound 3 are in the range of the −adenine nucleoside× 1. However,
compound 3 gives higher DH values than compound 1. An increasing number of 3-
residues leads to a decrease of the Tm value.
The CD spectra of these duplexes are similar to those of the unmodified compounds
(Fig. 5,a). These findings indicate that the change of the glycosylation position can
meet the spatial demand for the base pairing within an appropriate distance to form H-

Helvetica Chimica Acta Vol. 85 (2002)
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Table 4. Comparison of the Influence of Nucleosides 1, 3, and dS on the Duplex Stabilitya)b)c)
Tm [8]
DH8 [kcal/mol]
À 82
DS8 [cal/mol K]
À 233
DG8310 [kcal/mol]
À 9.6
5'-d(TAG GTC A3T ACT)-3' (14)
3'-d(ATC CAG TTA TGA)-5' (13)
44 (43)
5'-d(TAG GTC AST ACT)3' (19)
3'-d(ATC CAG TTA TGA)-5' (13)
33
À 47
À 88
À 77
À 61
À 91
À 79
À 88
À 80
À 63
À 57
À 62
À 48
À 68
À 45
À 129
À 249
À 217
À 174
À 261
À 224
À 248
À 224
À 177
À 158
À 173
À 122
À 197
À 126
À 7.0
À 10.1
À 9.8
À 6.9
À 10.2
À 9.5
À 11.1
À 10.5
À 7.9
À 7.8
À 7.9
À 6.8
À 6.5
À 6.2
5'-d(TAG GTC AAT ACT)-3' (12)
3'-d(ATC C3G TTA TGA)-5' (15)
46
5'-d(TAG GTC AAT ACT)-3' (12)
3'-d(ATC C1G TTA TGA)-5' (20)
46 (42)
33
5'-d(TAG GTC AAT ACT)-3' (12)
3'-d(ATC CSG TTA TGA)-5' (21)
5'-d(TAG GTC 33T ACT)-3' (16)
3'-d(ATC CAG TTA TGA)-5' (13)
45
5'-d(TAG GTC 11T ACT)-3' (22)
3'-d(ATC CAG TTA TGA)-5' (13)
44
5'-d(T3G GTC AAT 3CT)-3' (17)
3'-d(ATC CAG TTA TGA)-5' (13)
49
5'-d(T1G GTC AAT 1CT)-3' (23)
3'-d(ATC CAG TTA TGA)-5' (13)
48
5'-d(TAG GTC AAT ACT)-3' (12)
3'-d(ATC C3G TT3 TGA)-5' (18)
39
5'-d(TAG GTC AAT ACT)-3' (12)
3'-d(ATC C1G TT1 TGA)-5' (24)
38
5'-d(T3G GTC AAT 3CT)-3' (17)
3'-d(ATC C3G TT3 TGA)-5' (18)
39
5'-d(T1G GTC AAT 1CT)-3' (23)
3'-d(ATC C1G TT1 TGA)-5' (24)
33
5'-d(TAG GTC 33T ACT)-3' (16)
3'-d(ATC C3G TT3 TGA)-5' (18)
32
5'-d(TAG GTC 11T ACT)-3' (22)
3'-d(ATC C1G TT1 TGA)-5' (24)
27
a) See Table 3. b) dS ˆ 1,2-dideoxyribose. c) The thermodynamic data of duplexes containing nucleoside 1 and
dS were from [4].
bonds and base-stacking interactions. However, an additional 2-amino group has
almost no effect on the duplex stability. Obviously, tridentate base pairs are not formed.
Nucleoside 1 can pair with the four natural nucleosides without much penalty of
stability [4]. It is acting as an universal nucleoside, behaving differently to its N9-
glycosylated counterpart 4. To study those properties, the base pairing between 3 and
the four canonical nucleosides were investigated in non-self-complementary duplexes
(Tables 5 and 6). Nucleoside 1 and the abasic residue dS were also incorporated into
the same positions to allow a comparison of data. When nucleoside 3 is located opposite
to dC, dA, or dG (Tables 5 and 6), similar effects on the Tm values are observed as
found for 1. Apparently, the additional 2-amino group contributes very little to the

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Helvetica Chimica Acta Vol. 85 (2002)
Table 5. Tm Values and Thermodynamic Data of Antiparallel-Strand Oligonucleotides 15 and 20 Containing the
Nucleosides 1 and 3 Located Opposite dT or Other Canonical Nucleosidesa)b)c)
Tm [8]
DH8 [kcal/mol]
À 90
DS8 [cal/mol K]
À 252
DG8310 [kcal/mol]
À 12.0
5'-d(TAG GTC AAT ACT)- 3' (12)
3'-d(ATC CAG TTA TGA)-5' (13)
50
5'-d(TAG GTC AAT ACT)- 3' (12)
3'-d(ATC C1G TTA TGA)-5' (20)
46
46
33
46
42
35
45
43
35
44
43
40
À 76
À 88
À 61
À 80
À 85
À 65
À 77
À 79
À 76
À 73
À 77
À 71
À 212
À 249
À 174
À 225
À 244
À 187
À 217
À 226
À 222
À 205
À 217
À 202
À 9.9
À 10.1
À 6.9
À 9.9
À 9.1
À 7.4
À 9.7
9.1
5'-d(TAG GTC AAT ACT)- 3' (12)
3'-d(ATC C3G TTA TGA)-5' (15)
5'-d(TAG GTC AAT ACT)- 3' (12)
3'-d(ATC CSG TTA TGA)-5' (21)
5'-d(TAG GCC AAT ACT)- 3' (25)
3'-d(ATC C1G TTA TGA)-5' (20)
5'-d(TAG GCC AAT ACT)- 3' (25)
3'-d(ATC C3G TTA TGA)-5' (15)
5'-d(TAG GCC AAT ACT)- 3' (25)
3'-d(ATC CSG TTA TGA)-5' (21)
5'-d(TAG GAC AAT ACT)- 3' (26)
3'-d(ATC C1G TTA TGA)-5' (20)
5'-d(TAG GAC AAT ACT)- 3' (26)
3'-d(ATC C3G TTA TGA)-5' (15)
5'-d(TAG GAC AAT ACT)- 3' (26)
3'-d(ATC CSG TTA TGA)-5' (21)
À 7.2
À 9.4
À 9.4
À 8.6
5'-d(TAG GGC AAT ACT)- 3' (27)
3'-d(ATC C1G TTA TGA)-5' (20)
5'-d(TAG GGC AAT ACT)- 3' (27)
3'-d(ATC C3G TTA TGA)-5' (15)
5'-d(TAG GGC AAT ACT)- 3' (27)
3'-d(ATC CSG TTA TGA)-5' (21)
a) See Table 3. b)c) See Table 4.
Fig. 5. CD Spectra of helixes formed a) by non-self-complementary duplexes containing 3 and b) by the
alternating duplex 34 ¥ 34

Helvetica Chimica Acta Vol. 85 (2002)
1349
Table 6. Tm Values and Thermodynamic Data of Antiparallel-Strand Oligonucleotides 16 and 22 Containing the
Nucleosides 1, 3, or the Abasic Residue dS Located Opposite the Canonical Nucleosidesa)b)c)
Tm [8]
DH8 [kcal/mol]
À 79
DS8 [cal/mol K]
À 224
DG8310 [kcal/mol]
À 9.5
5'-d(TAG GTC 11T ACT)-3' (22)
3'-d(ATC CAG TTA TGA)-5' (13)
44
5'-d(TAG GTC 33T ACT)-3' (16)
3'-d(ATC CAG TTA TGA)-5' (13)
45
33
42
43
34
42
43
34
39
42
33
À 91
À 47
À 79
À 85
À 52
À 78
À 86
À 51
À 67
À 79
À 48
À 261
À 129
À 225
À 243
À 146
À 223
À 248
À 141
À 190
À 227
À 131
À 10.2
À 7.0
À 9.0
À 9.2
À 7.1
À 9.1
À 9.2
À 7.2
À 8.1
À 8.8
À 7.0
5'-d(TAG GTC AST ACT)-3' (19)
3'-d(ATC CAG TTA TGA)-5' (13)
5'-d(TAG GTC 11T ACT)-3' (22)
3'-d(ATC CAG TCA TGA)-5' (28)
5'-d(TAG GTC 33T ACT)-3' (16)
3'-d(ATC CAG TCA TGA)-5' (28)
5'-d(TAG GTC AST ACT)-3' (19)
3'-d(ATC CAG TCA TGA)-5' (28)
5'-d(TAG GTC 11T ACT)-3' (22)
3'-d(ATC CAG TAA TGA)-5' (29)
5'-d(TAG GTC 33T ACT)-3' (16)
3'-d(ATC CAG TAA TGA)-5' (29)
5'-d(TAG GTC AST ACT)-3' (19)
3'-d(ATC CAG TAA TGA)-5' (29)
5'-d(TAG GTC 11T ACT)-3' (22)
3'-d(ATC CAG TGA TGA)-5' (30)
5'-d(TAG GTC 33T ACT)-3' (16)
3'-d(ATC CAG TGA TGA)-5' (30)
5'-d(TAG GTC AST ACT)-3' (19)
3'-d(ATC CAG TGA TGA)-5' (30)
a) See Table 3. b)c) See Table 4.
Table 7. Tm Values and Thermodynamic Data of Alternating Self-Complementary Oligonucleotides Containing
the Nucleosides 1, 3, and 4 Opposite dTa)
Tm [8]
DH8 [kcal/mol] DS8 [cal/mol K] DG8310 [kcal/mol]
[5'-d( ATATA TATATAT )-3 ']2 (31 ¥ 31) [2] 33(26)
À 49.5
À 63
À 74
À 139.3
À 180
À 207
À 6.3
À 7.2
À 9.6
[5'-d(4T4T4 T4T4T4T)-3']2 (32 ¥ 32) [2]
[5'-d(1T1 T1T 1T1 T1T)-3']2 (33 ¥ 33) [4] 49(39)
[5'-d(3T3 T3T 3T3 T3T)-3']2 (34 ¥ 34) 58(55)
36
À 71.9
À 191.9
À 11.3
[5'-d(AAA AAA TTT TTT)-3']2 (35 ¥ 35) 46(40)
[5'-d(TTT TTT AAA AAA)-3']2 (36 ¥ 36) 33(29)
[5'-d(333 333 TTT TTT)-3']2 (37 ¥ 37)
[5'-d(TTT TTT 333 333)-3']2 (38 ¥ 38)
42(48), 19(14)
29(32)
39(44)
5'-d(333 333 TTT TTT)-3' (37)
5'-d(TTT TTT 333 333)-3' (38)
a) See Table 3.

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Helvetica Chimica Acta Vol. 85 (2002)
base-pair stability. In the case of two consecutive incorporations, compounds 1 and 3
lead to almost the same Tm values. But duplexes containing compound 1 or 3 give much
higher Tm values than those incorporating the abasic residue dS. This means that
interactive forces exist between 1 or 3 and the four canonical residues, such as H-
bonding and/or base stacking. The CD spectra (Fig. 6) show that the mismatched base
pairs do not disturb the duplex structure significantly. Thus, compound 3 has the
potential of a universal nucleoside as it is found for nucleoside 1.
Fig. 6. CD Spectra of duplexes containing nucleoside 3 opposite the four canonical nucleosides measured in 1m
NaCl, 100 mm MgCl2, and 10 mm Na cacodylate (pH 7.0)
2.2.2. Duplex Stability of Alternating Self-complementary Oligonucleotides. Earlier,
it was found that the oligomer d(1-T)6 (33 ¥ 33) forms a more stable duplex than the
parent duplex d(4-T)6 (32 ¥ 32) incorporating the regularly N9-glycosylated nucleoside
[4]. Thus, it is expected that the replacement of dA by the diamino compound 3
increases the duplex stability further as a third H-bond might be formed as shown in
base pair VI (5-dT; Fig. 7). According to Table 7 the duplex 34 ¥ 34 has a Tm 98 higher
than that of 33 ¥ 33 and 258 higher than that of the unmodified 31 ¥ 31. This corresponds
to a stabilization of each 3-dT pair by 0.758 compared to that of the 1-dT pair and by
2.18 compared to that of dA-dT. The duplex 32 ¥ 32 (base pair V; Fig. 7) containing the
regularly N9-linked pyrazolo[3,4-d]pyrimidine nucleoside 4 is only slightly more stable
than the parent duplex 31 ¥ 31. The CD spectrum of 34 ¥ 34 is very similar to that of 33 ¥
33 both of them having a negative lobe around 308 nm, while the control duplex has a
negative lobe around 245 nm [2][18]. This indicates that the incorporation of N8-
glycosylated nucleoside residues into duplexes with alternating dA* ¥ dT results in an
autonomous DNA. Apparently, the additional 2-amino group of the nucleoside 3 can
form H-bonds in this particular duplex, and a tridendate base pair is formed.
The chain orientation of the alternating duplexes incorporating either compound 1
or 3 (33 ¥ 33 or 34 ¥ 34) (Table 7) can be antiparallel (I and III) or parallel (II and IV)
(Fig. 7). However, the antiparallel duplex d(A-T)6 (31 ¥ 31) is more stable than the
parallel one [19]. This might not be the case for the duplexes 33 ¥ 33 and 34 ¥ 34, which
can form an autonomous DNA structure with possibly much better stacked nucleobase
residues. To clarify this problem, the block oligomers d(3)6-(T)6 (37 ¥ 37) and d(T)6-(3)6
(38 ¥ 38) as well as control duplexes (see 35 ¥ 35 and 36 ¥ 36) were synthesized (Table 7).

Helvetica Chimica Acta Vol. 85 (2002)
1351
Only in the antiparallel case, stable duplexes are expected, while in a parallel
arrangement, rather labile species should be formed (maximally six instead of twelve
base pairs). In the parallel case, a stable duplex is expected only when 37 is hybridized
with 38. The melting curves show unexpected profiles (Fig. 8,a). The duplex 38 ¥ 38
gives a monophasic melting profile, while the melting profile of 37 ¥ 37 is biphasic. Both
modified duplexes show a higher Tm value under low salt buffer concentration than
under high salt concentration conditions (see Table 3), a phenomenon difficult to
understand. Also the CD spectra show significant changes (Fig. 8,b). The CD spectrum
of 38 ¥ 38 looks exactly like that of the control duplex 36 ¥ 36 (not shown), while the
spectra of 37 ¥ 37 and 37 ¥ 38 are different from that of 38 ¥ 38. From these experiments,
no definite conclusion about chain orientation can be drawn. However, it seems more
likely that the base pair 3-dT is of the Watson-Crick type with antiparallel chain
orientation for the alternating and the block oligonucleotides. Otherwise, the rather
high Tm values of 37 ¥ 37 and 38 ¥ 38 cannot be explained.
Fig. 7. Base-pair motifs formed between nucleosides 1 5 and dT forming Watson-Crick or reverse Watson-Crick
base pairs. aps ˆ antiparallel strand, ps ˆ parallel strand.

1352
Helvetica Chimica Acta Vol. 85 (2002)
Fig. 8. a) Melting profile of helices formed by the self-complementary block sequences 37 and 38 (conditions, see
Exper. Part). b) CD spectra of duplexes 37 ¥ 37, 38 ¥ 38, and 37 ¥ 38.
Conclusions.
The N8-glycosylated nucleoside 3 shows similar base pairing
behavior as compound 1 when it replaces dA residues in dA ¥ dT base pairs. It shows
ambiguous base pairing with the four canonical DNA constituents. The 2-amino group
of 3 does not increase the base-pair stability. Thus, nucleoside 3 has the potential to be
an universal nucleoside when it replaces only a few of the canonical nucleosides. The
situation changes when the nucleoside 3 residue alternates with dT. In this case, the 12-
mer duplex incorporating compound 3 is significantly more stable than that containing
compound 1 or dA. Here, the additional 2-amino group of 3 has a great impact on the
stability of the duplex structure. From these observations, it is concluded that only in
the latter case does the 2-amino group participate in H-bonding with the 2-oxo group of dT.
We thank Dr. H. Rosemeyer for helpful discussions and Mr. Yang He for the NMR spectra. We also appreciate
the syntheses of the oligonucleotides and their characterization by MALDI-TOF spectra by E. Feiling. Financial
support by the Deutsche Forschungsgemeinschaft and the Roche Diagnostics GmbH is gratefully acknowledged.
Experimental Part
General. See [10]. Monomers. Thin-layer chromatography (TLC): aluminium sheets, silica gel 60 F254
(0.2 mm, Merck, Germany). Flash chromatography (FC): 0.4 bar; silica gel 60 H (Merck, Darmstadt,
Germany). Solvent systems for TLC and FC: CH2Cl2/MeOH 10 :1 (A), CH2Cl2/MeOH 9 :1 (B), CH2Cl2/
MeOH 95 :5 (C), CH2Cl2/acetone 10 :1 (D), and CH2Cl2/acetone 9 :1 (E). M.p.: B¸chi-SMP-20 apparatus
(B¸chi, Switzerland); uncorrected. NMR Spectra: Avance-DPX-250 and AMX-500 spectrometers (Bruker,
Germany); d values in ppm rel. to internal SiMe4 (1H, 13C) or external H3PO4 (85%), J in Hz. Microanalyses
were performed by the Mikroanalytisches Labor Beller (Gˆttingen, Germany).
Oligonucleotides. Oligonucleotide synthesis: ABI-392-DNA synthesizer (Applied Biosystems, Weiterstadt,
Germany) in the −trityl-on× mode. Melting curves: Cary-1- or Cary-1E-UV/VIS spectrophotometers (Varian,
Australia) equipped with a Cary thermoelectrical controller; the temp. was measured continuously in the
reference cell with a Pt-100 resistor, and the thermodynamic data of duplex formation were calculated with the
program Meltwin 3.0 [20]. UV Spectra: 150-20 spectrometer (Hitachi, Japan); lmax(e) in nm. CD Spectra: Jasco-
600 (Jasco, Japan) spectropolarimeter with thermostatically (Lauda RCS-6 bath) controlled 1-cm cuvettes. The
enzymatic hydrolysis of the oligomers was performed as described below [3], and the following extinction
coefficients e260 were used: dA 15400, dG 11700, dT 8800, and dC 7300. Snake-venom phosphodiesterase
(EC 3.1.15.1, Crotallus adamanteus) and alkaline phosphatase (EC 3.1.3.1, E. coli) were generous gifts from
Roche Diagnostics GmbH, Germany. MALDI-TOF-MS: Biflex-III spectrometer (Bruker Saxonia-Analytik
GmbH, Leipzig, Germany); results in Table 8.

Helvetica Chimica Acta Vol. 85 (2002)
1353
‡
Table 8. Molecular Masses (MH ) of Oligonucleotides Measured by MALDI-TOF Mass Spectrometry
‡
‡
MH (calc.)
MH (found)
5'-d(TAG GTC A3T ACT)-3' (14)
5'-d(AGT ATT G3C CTA)-3' (15)
5'-d(TAG GTC 33T ACT)-3' (16)
5'-d(T3G GTC AAT 3CT)-3' (17)
5'-d(AGT 3TT G3C CTA)-3' (18)
5'-d(3T3 T3T 3T3 T3T)-3' (34)
5'-d(333 333 TTT TTT)-3' (37)
5'-d(TTT TTT 333 333)-3' (38)
3659
3659
3673
3673
3673
3732
3732
3732
3660
3662
3675
3677
3676
3732
3733
3733
The synthesis was carried out on a 1-mmol scale with the monomer 11 and the 3'-phosphoramidites of
[(MeO)2Tr]ib2Gd, [(MeO)2Tr]bz6Ad, [(MeO)2Tr]bz4Cd, and [(MeO)2Tr]Td. After the −trityl-on× oligonucleo-
tides were cleaved from the solid support, they were deprotected in 25% aq. NH3 soln. for 12 15 h at 608. Then,
the purification was performed by reversed-phase HPLC (250 Â 4 mm, RP-18 column, Merck, Germany;
gradient (A, 0.1m (Et3NH)OAc (pH 7.0)/MeCN 95 :5; B, MeCN): 3 min 20% B in A, 12 min 20 40% B in A,
flow rate 1.0 ml/min). The purified −trityl-on× oligonucleotides were treated with 2.5% CHCl2COOH/CH2Cl2 for
5 min at r.t. to remove the 4,4'-dimethoxytrityl residues. The detritylated oligomers were purified again by
reversed-phase HPLC (gradient: 20 min 0 20% B in A, flow rate 1 ml/min). The oligomers were desalted on a
short column (RP-18, silica gel): colorless solids, which were stored at À248.
The composition of oligonucleotides was established as follows: The oligonucleotides were dissolved in 0.1m
Tris ¥ HCl buffer (pH 8.3, 200 ml), and treated with snake-venom phosphodiesterase (3 ml) at 378 for 45 min, and
subsequently with alkaline phosphatase (3 ml) at 378 for another 30 min. The mixtures were analyzed by HPLC
(RP-18, at 260 nm, gradient A, 0.7 ml/min) to give the ratio of nucleosides incorporated in the oligonucleotide.
The composition analysis of oligonucleotides 15 and 38 is shown in Fig. 4.
2-(2-Deoxy-b-d-erythro-pentofuranosyl)-4-isopropoxy-2H-pyrazolo[3,4-d]pyrimidin-6-amine (8). The
protected nucleoside 2-[2-deoxy-3,5-di-O-(p-toluoyl)-b-d-erythro-pentofuranosyl]-4-isopropoxy-2H-pyrazo-
i
lo[3,4-d]pyrimidin-6-amine (7) [9] (7.5 g, 13.8 mmol) was suspended in 0.1m PrONa/iPrOH (400 ml), stirred,
and heated gently (458) for 1 h (TLC monitoring). The mixture was neutralized with AcOH and evaporated.
The residue was dissolved in MeOH (ca. 50 ml). The soln. was adsorbed on silica gel (20 g); this material was
loaded on top of a silica gel column (7 cm  30 cm) and submitted to FC (6 cm  30 cm, A). The main zone
furnished 8 (3.0 g, 71%). Colorless solid. Rf (B) 0.38. 1H-NMR ((D6DMSO): 1.34 (m, Me2CH); 2.30
(m, 1 HÀC(2')); 2.61 (m, 1 HÀC(2')); 3.58 (m, 2 HÀC(5')); 3.87 (m, HÀC(4')); 4.39 (m, HÀC(3')); 5.04
(m, OHÀC(5')); 5.28 (m, OHÀC(3')); 5.47 (m, Me2CH); 6.15 (t, Jˆ 6.0, HÀC(1')); 6.39 (s, NH); 8.41 (s, HÀC(7)).
2-(2-Deoxy-b-d-erythro-pentofuranosyl)-2H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (3). A suspension of
8 (3.1 g, 10 mmol) in 25% aq. NH3 soln. was heated in an autoclave at 608 for 4 d under stirring. The mixture was
concentrated to a small volume, MeOH (100 ml) was added, and the soln. was adsorbed on silica gel (10 g). FC
(silica gel, 6 Â 20 cm, A) gave 3 (2.3 g, 86%). White solid. Rf (B) 0.48. UV (MeOH): 225 (25000), 264 (9200),
295 (7600). 1H-NMR ((D6)DMSO): 2.30 (m, 1 HÀC(2')); 2.58 (m, 1 HÀC(2')); 3.53 (m, 2 HÀC(5')); 3.88
(m, HÀC(4')); 4.40 (m, HÀC(3')); 5.09 (m, OHÀC(5')); 5.30 (m, OHÀC(3')); 5.79 (s, NH2); 6.15 (t, J ˆ 5.88,
HÀC(1')); 7.21 (br., NH2), 8.23 (s, HÀC(7)). Anal. calc. for C10H14N6O3 (266.26): C 45.11, H 5.30, N 31.56;
found: C 45.21, H 5.23, N 31.19.
Isobutyrylation of 3. Compd. 3 (0.9 g, 3.38 mmol) was co-evaporated with anh. pyridine (3 times) and then
dissolved in anh. pyridine (15 ml). Me3SiCl (2.16 ml, 17.0 mmol) was added to the soln. while stirring for 15 min
at r.t. Then, isobutyric anhydride (3.56 ml, 21.5 mmol) was added, and stirring was continued for 3 h. The
mixture was cooled in an ice-bath and diluted with cold H2O (6 ml). After 5 min, aq. 25% NH3 soln. (6 ml) was
added and the soln. stirred for 30 min. The mixture was evaporated and co-evaporated with toluene (3 times).
The residue was purified by FC (A): fast migrating 9a (0.57 g, 42%) and slow migrating 9b (0.27 g, 20%).
2-(2-Deoxy-b-d-erythro-pentofuranosyl)-N4,N6,N6-triisobutyryl-2H-pyrazolo[3,4-d]pyrimidine-4,6-diamine
1
(9a): Colorless amorphous solid. Rf (B) 0.38. UV (MeOH): 276 (10850), 302 (7600). H-NMR ((D6)DMSO):
1.12 (m, Me2CH); 2.42 (m, 1 HÀC(2')); 2.64 (m, 1 HÀC(2')); 2.92 (m, Me2CH); 3.51, 3.61 (m, 2 HÀC(5')); 3.93
(m, HÀC(4')); 4.45 (m, HÀC(3')); 4.87 (t, J ˆ 5.35, OHÀC(5')); 5.36 (d, J ˆ 4.35, OHÀC(3')); 6.50 (t, J ˆ 5.63,
HÀC(1')); 9.12 (s, HÀC(7)); 11.47 (s, NH); 7.27 7.94 (m, arom. H); 8.44 (s, HÀC(7)). Anal. calc. for
C22H32N6O6 (476.53): C 55.45, H 6.77, N 17.64; found: C 55.41, H 6.51, N 17.11.

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Helvetica Chimica Acta Vol. 85 (2002)
2-(2-Deoxy-b-d-erythro-pentofuranosyl)-N4,N6-diisobutyryl-2H-pyrazolo[3,4-d]pyrimidine-4,6-diamine
(9b): Rf (B) 0.32. UV (MeOH): 239 (41600), 285 (10900). 1H-NMR ((D6)DMSO): 1.13 (m, Me2CH); 2.39
(m, 1 HÀC(2')); 2.61 (m, 1 HÀC(2')); 3.00 (m, Me2CH); 3.50, 3.60 (m, 2 HÀC(5')); 3.91 (m, HÀC(4')); 4.44
(m, HÀC(3')); 4.92 (t, J ˆ 5.50, OHÀC(5')); 5.33 (d, J ˆ 4.40, OHÀC(3')); 6.41 (t, J ˆ 5.65, HÀC(1')); 8.87
(s, HÀC(7)); 10.08, 11.00 (s, NH). Anal. calc. for C18H26N6O5 (406.44): C 53.19, H 6.45, N 20.68; found: C 53.76,
H 6.55, N 20.03.
2-[2-Deoxy-5-O-(4,4'-dimethoxytrityl)-b-d-pentofuranosyl]-N4,N6,N6-triisobutyryl-2H-pyrazolo[3,4-d]pyri-
midine-4,6-diamine (10). Compound 9a (0.55 g, 1.15 mmol) was co-evaporated with anh. pyridine (3 times) and
dissolved in pyridine (2.5 ml). (MeO)2TrCl (0.6 g, 1.76 mmol) was added, and the mixture was stirred at r.t. for
3 h. The reaction was quenched by the addition of MeOH and the mixture evaporated and co-evaporated with
toluene. FC (6 cm  30 cm, A) furnished 10 (0.26 g, 29%). Colorless foam. Rf (C) 0.3. UV (MeOH): 275
(13000), 305 (7600). 1H-NMR ((D6)DMSO): 1.12 (m, 3 Me2CH); 2.46 (m, 1 HÀC(2')); 2.72 (m, Me2CH); 2.91
(m, 1 HÀC(2')); 3.08 (m, 2 HÀC(5')); 3.65, 3.68 (s, 2 MeO); 4.05 (m, HÀC(4')); 4.53 (m, HÀC(3')); 5.40
(d, J ˆ 5.0, OHÀC(3')); 6.60 (m, HÀC(1')); 6.69 7.79 (m, arom. H); 9.17 (s, HÀC(7)); 11.50 (s, NH). Anal.
calc. for C43H50N6O8 (778.89): C 66.31, H 6.42, N 10.79; found: C 66.48, H 6.53, N 10.59.
2-[2-Deoxy-5-O-(4,4'-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-N4,N6,N6-triisobutyryl-2H-pyrazo-
lo[3,4-d]pyrimidine-4,6-diamine 3'-(2-Cyanoethyl Diisopropylphosphoramidite) (11). To a soln. of 10 (0.6 g,
0.77 mmol) in anh. CH2Cl2 (30 ml) under Ar, (iPr)2EtN (0.27 ml, 1.55 mmol) and 2-cyanoethyl diisopropyl-
phosphoramidochloridite (260 ml, 1.14 mmol) were added, and the mixture was stirred at r.t. for 30 min. The
reaction was monitored by TLC. The mixture was diluted with CH2Cl2 and the soln. washed with 5% aq.
NaHCO3 soln. and brine. The org. phase was dried (Na2SO4) and evaporated and the product separated by FC
(2.5 cm  6 cm, D): 11 (0.5 g, 66%). Colorless foam. Rf (E) 0.50, 0.59. 1H-NMR ((D6)DMSO): 1.10
(m, Me2CH); 2.44 (m, 1 HÀC(2')); 2.64 (m, Me2CH); 2.99 (m, 1 HÀC(2'), 2 HÀC(5')); 3.36 (m, CH2CH2);
3.78 (s, 2 MeO); 4.32 (m, HÀC(4')); 4.74 (m, HÀC(3')); 6.35 (m, HÀC(1')); 6.74 7.35 (m, arom. H); 8.28
(s, HÀC(7)); 9.15 (s, NH). 31P-NMR (CDCl3), 150.22, 150.50.
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Received November 19, 2001

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