Major-groove-halogenated DNA: The effects of bromo and iodo substituents replacing H-C(7) of 8-Aza-7-deazapurine-2,6-diamine or H-C(5) of uracil residues

Article abstract of DOI:10.1002/1522-2675(20010516)84:5<1048::AID-HLCA1048>3.0.CO;2-9

Oligonucleotides containing halogenated 'purine' and pyrimidine bases were synthesized. Bromo and iodo substituents were introduced at the 7-position of 8-aza-7-deazapurine-2,6-diamine (see 2b,c) or at the 5-position of uracil residues (see 3b,c). Phospho

Full text of DOI:10.1002/1522-2675(20010516)84:5<1048::AID-HLCA1048>3.0.CO;2-9

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Helvetica Chimica Acta ± Vol. 84 (2001)
Major-Groove-Halogenated DNA: The Effects of Bromo and Iodo
Substituents Replacing H C(7) of 8-Aza-7-deazapurine-2,6-diamine or
H C(5) of Uracil Residues
by Georg Becher, Junlin He, and Frank Seela*
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie, Universität Osnabrück,
Barbarastrasse 7, D-49069 Osnabrück (fax: ‡49-(0)541-969-2370; e-mail: Frank.Seela@uni-osnabrueck.de)
Oligonucleotides containing halogenated ꢀpurineꢁ and pyrimidine bases were synthesized. Bromo and iodo
substituents were introduced at the 7-position of 8-aza-7-deazapurine-2,6-diamine (see 2b,c) or at the 5-position
of uracil residues (see 3b,c). Phosphoramidites were synthesized after protection of 2b with the isobutyryl
residue and of 2c with the benzoyl group. Duplexes containing the residues 2b or 2c gave always higher Tm
values than those of the nonmodified counterparts containing 2'-deoxyadenosine, the purine-2,6-diamine
2'-deoxyribonucleoside (1), or 2a at the same positions. Six 2b residues replacing dA in the duplex
5'-d(TAGGTCAATACT)-3' (11) ´ 5'-d(AGTATTGACCTA)-3' (12) raised the Tm value from 48 to 758 (4.58 per
modification (Table 3)). Contrary to this, incorporation of the 5-halogenated 2'-deoxyuridines 3b or 3c into
oligonucleotide duplexes showed very little influence on the thermal stability, regardless of which ꢀpurineꢁ
nucleoside was located opposite to them (Tables 4 and 5). The positive effects on the thermal stability of
duplexes observed in DNA were also found in DNA ´ RNA hybrids or in DNA with parallel chain orientation
(Tables 8 and 9, resp.).
Introduction. ± The thermal stability of oligonucleotide duplexes increases when
7-substituted 8-aza-7-deazapurines (ˆ pyrazolo[3,4-d]pyrimidines) replace purines
[1][2]. This effect is particularly strong when the heterocyclic base is an 8-aza-7-
deazaguanine and carries a halogeno substituent at the 7-position [1][3][4]. Recent
studies on the substitution of purine-2,6-diamine by the 7-bromo derivative of 8-aza-7-
deazapurine-2,6-diamine have shown that this modified purine derivative forms as
stable base a pair with thymine as guanine does with cytosine [5]. In contrast, the base
pair of the structurally related purine-2,6-diamine 2'-deoxyribonucleoside (1; n2Ad)
with thymidine is rather weak when introduced into duplex DNA [5][6].

Helvetica Chimica Acta ± Vol. 84 (2001)
1049
This investigation compares the effects of halogeno substituents such as the bromo
and iodo atom introduced at the 7-position of 8-aza-7-deazapurine-2,6-diamine 2'-
deoxyribonucleosides (see 2b,c) and at the 5-position of 2'-deoxyuridine (see 3b,c)
(purine numbering is used throughout the General Part; see 1). These nucleosides are
incorporated into various positions of duplex DNA, and the number of incorporations
is increased stepwise. As the halogeno substituents are directed into the major groove,
in both the ꢀpurineꢁ- and pyrimidine-nucleoside series, it is of interest to quantify the
effects. Furthermore, the effect of multiple incorporations is investigated, as it is known
from other duplex-stabilizing nucleosides that the stabilizing effects level off with an
increasing number of modified nucleoside incorporations [7][8]. Oligonucleotide
duplexes incorporating 7-halogenated 7-deazapurines (ˆ pyrrolo[2,3-d]pyrimidines)
show differences in the series of 7-iodo and 7-bromo compounds [9] that result from
the spatial requirements of the halogeno substituent (van der Waals radii: Brˆ 1.85 Š;
I ˆ 1.98 Š [10]) and/or from the more hydrophobic character of the iodo substituents
compared to the bromo residues. Thus, the 7-iodo derivative 2c of the 8-aza-7-
deazapurine-2,6-diamine nucleoside is synthesized and converted to a phosphorami-
dite, and a number of oligonucleotides are synthesized containing this 7-iodinated 8-
aza-7-deazapurine-2,6-diamine nucleoside 2c. A comparison of the thermal stability of
these duplexes illustrates the striking differences in duplex stabilization among
halogeno-substituted pyrimidine or 8-aza-7-deazapurine residues located in a similar
environment of the major groove of B-DNA.
Results and Discussion. ± 1. Synthesis and Properties of Monomers. The 7-iodinated
8-aza-7-deazapurine-2,6-diamine 2'-deoxyribonucleoside 2c was prepared from the
6-isopropoxy compound 4 [11] upon treatment with 25% aqueous NH3 solution
at 708 for 4 days in an autoclave. The nucleosides 2a,b [11] as well as the 2'-deoxy-
uridine derivatives 3a ± c have been described elsewhere [12]. The halogeno
substituents change the mobility of the nucleosides on a reversed-phase HPLC
column, with the iodinated 2c and 3c as the slowest migrating compounds (Fig. 1,a
and b). The retention times refer to the hydrophobic character of the nucleosides; the
data were used later for the composition analysis of the base-modified oligonucleo-
tides.
The nucleobases of the modified nucleosides 2a ± c and 3a ± c influence the N $ S
pseudorotational equilibrium of the sugar moiety. This affects the conformation of the
1
sugar-phosphate backbone of DNA. For this reason, the H-NMR spectra of the
pyrazolo[3,4-d]pyrimidine nucleoside 2c as well as of the pyrimidine nucleosides 3a ± c
3
were measured in D2O, and J(H,H) NMR coupling constants were determined. The
conformational analysis was performed on the basis of these coupling constants with
the program PSEUROT [13]. According to Table 1, the 8-aza-7-deazapurin-2,6-
diamine nucleoside 2a shows a higher N-conformer population than the corresponding
purine nucleoside 1. The conformation around the C(4') C(5') bond indicates that the
8-aza-7-deazapurine-2,6-diamine nucleoside 2c, as the 8-aza-7-deaza-2'-deoxyguano-
sines [14], prefers the gt-( sc)-rotamer population, while, for the regular purine
nucleosides, the gg‡-(‡sc)- or the gg -(ap) conformation is predominant [15]. The
conformation of the sugar moieties of the pyrimidine nucleosides 3a ± c is also
influenced by the substitution at the 5-position (Table 1). The main change is observed

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Helvetica Chimica Acta ± Vol. 84 (2001)
Fig. 1. HPLC Profiles of a) the nucleosides 1 and 2a ± c and b) the nucleosides 3a ± c and dT. c) d) HPLC Profiles
of the enzymatic-hydrolysis products of the oligonucleotides 21 and 28, respectively, obtained by digestion with
snake-venom phosphodiesterase followed by alkaline phosphatase in 0.1m Tris ´ HCl buffer (pH 8.3) at 378. The
nucleoside mixtures were analyzed by reversed-phase HPLC at 260 nm on a RP-18 column (200 Â 10 mm).
Gradient for a): 0 ± 50 min B in A (0% ± 100%), for b) and d): 0 ± 30 min 100% A; for c) 0 ± 20 min 100% A, 20 ±
50 min B in A (0% ! 100%), 0.7 ml/min (A ˆ buffer (see Exper. Part), B ˆ MeCN).
Table 1. 3J(H,H) Coupling Constants of the Sugar Moieties and N/S-Conformer Populations of the 2'-
Deoxyribonucleosides 1 ± 3 at 303 Ka)
3J(H,H)/Hz
J(1',2') J(1',2'') J(2',3') J(2'',3') J(3',4') J(4',5') J(4',5'') %N %S gg
Conformation
‡
gt
gg
1
2a
2c
dT [16]
3a
7.30
6.60
6.60
±
6.70
6.45
6.50
6.10
6.80
6.70
±
6.60
6.50
6.50
7.00
6.90
6.85
±
6.50
6.50
6.50
3.10
3.70
3.90
±
4.30
4.70
4.50
3.40
3.60
3.85
±
4.10
4.50
4.40
3.20
4.00
4.30
±
3.60
3.40
3.40
4.30
5.80
5.90
±
5.20
4.70
4.80
31
37
38
36
30
34
33
69
63
62
64
70
66
67
62
25 13
42 22
43 26
36
31
±
47
55
54
±
±
35 18
30 15
31 15
3b
3c
a) Solvent D2O; r.m.s. <0.4 Hz; j DJmax j <0.4 Hz.
between 3a without a 5-substituent and the derivatives dTor 3b,c with a Me or halogeno
substituent.
As the reactivities of the amino groups of the nucleoside 2b are rather different
from those of 1, various residues were studied for the base protection. Earlier,
nucleoside 2b was protected with the N,N-dibutylformamidine (dnb) residue. Now, the
isobutyryl (ibu) group was introduced by the protocol of transient protection (2b ! 5a)
[17] (Scheme). As a by-product, the monoprotected nucleoside 6 was isolated (22%).
The formation of mono-acylated compounds has been observed in the case of other
purine-2,6-diamine nucleosides [18]. When performing the alkaline hydrolysis of
compounds 5a or 6 in 25% aqueous NH3 solution at 408, a fast deprotection was
observed. The half-life for 5a and 6 were determined and found to be 4.5 and 20.5 min,
respectively, indicating that this protecting group is appropriate for the solid-phase
oligonucleotide synthesis. For the protection of the iodo nucleoside 2c, the transient

Helvetica Chimica Acta ± Vol. 84 (2001)
1051
Scheme
i) 5a: 2b, Me3SiCl, pyridine, isobutyric anhydride, r.t., 4 h; 5b: 2c, Me3SiCl, pyridine, benzoyl chloride, r.t., 24 h.
ii) (MeO)2TrCl, pyridine, r.t., 4 h. iii) 2-Cyanoethyl diisopropylphosphoramidochloridite, CH2Cl2 (for 8b in
THF), r.t., 30 min.
protection protocol was used as in 2b, but in this case, a benzoyl group was chosen
(2c ! 5b). The time for complete deprotection of 5b (25% aqueous NH3 solution, 408,
HPLC monitoring at 260 nm) was 450 min. A half-life was not determined because of
the stepwise nature of the reaction.

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Helvetica Chimica Acta ± Vol. 84 (2001)
The base-protected nucleosides 5a,b as well as the pyrimidine nucleosides 3a ± c
were converted to the (MeO)2Tr derivatives 7a,b, and 9a ± c under standard reaction
conditions [19]. Phosphitylation of the (MeO)2Tr derivatives 7a,b was performed in
CH2Cl2 or THF in the presence of 2-cyanoethyl diisopropylphosphoramidochloridite
to furnish the phosphoramidites 8a,b (Scheme); the pyrimidine building blocks 10a ± c
were prepared from 9a ± c in CH2Cl2 [19]. The phosphoramidites 8a,b and 10a ± c were
then employed in solid-phase oligonucleotide synthesis. All compounds were
1
characterized by H-, 13C-, and 31P-NMR spectra and by elemental analysis (Table 2
and Exper. Part). Table 2 summarizes the 13C-NMR data of 8-aza-7-deazapurine as well
Table 2. 13C-NMR Chemical Shifts of Pyrazolo[3,4-d]pyrimidine-4,6-diamine 2'-Deoxyribonucleosidesa)
C(2)b) C(4)b) C(5)b) C(6)b)d) C(7)c) CˆO/ˆCH CˆO/ˆCH C(1') C(2') C(3') C(4') C(5')
C(6)c) C(7)c) C(38)c) C(4)c)
C(3)c)
2b 157.6
2c 157.0
3a 163.2
3b 159.1
3c 160.4
5a 155.8
5b 149.6
157.4
157.6
150.5
149.7
150.0
153.8
152.1
156.5
154.9
152.1
158.0
159.1
160.5
94.5
91.2
101.8
95.6
162.7
162.2
140.6
140.6
145.0
156.2
153.4
157.5
157.5
153.2
144.7
144.7
144.2
119.2
98.3
83.0 37.5 70.9 87.3 62.4
83.1 37.6 70.9 87.3 62.4
84.2 38.5 70.5 87.4 61.3
84.7 38.4 69.9 87.5 60.7
e
69.9
84.6
)
69.2 87.4 60.7
104.7
105.7
97.4
121.4 175.2
96.3 169.4
119.1 175.3
117.2 174.7
176.5
177.5
83.6 37.5 70.7 87.7 62.2
84.4 38.7 71.6 88.6 63.1
83.3 37.5 70.8 87.5 62.2
6
156.0
7a 155.6
7b 149.5
9a 163.0
9b 158.0
9c 158.0
98.5
175.2
177.5
83.5 37.6 72.2 85.4 63.6
e
105.8
101.5
96.0
96.2
±
84.8
84.1
)
)
71.6 86.7 65.3
69.9 85.3 63.4
e
84.9 38.4 70.3 85.7 63.6
84.8 38.4 70.4 85.8 63.7
69.8
a) Measured in (D6)DMSO at 303 K. b) Purine numbering. c) Systematic numbering. d) Tentative. e) Super-
imposed by (D6)DMSO.
as those of pyrimidine nucleosides. The assignment was made according to gated-
decoupled spectra. The NMR data of 3a ± c were included, as a literature search gave
little information on that matter.
2. Oligonucleotides. 2.1. Synthesis and Characterization. Automated solid-phase
synthesis of the oligonucleotides (11 ± 37, Tables 3 ± 9) was performed with the
phosphoramidites 8a,b and 10a ± c as well as the standard building blocks. The
syntheses followed the standard protocol [20], and the coupling yields were always
higher than 97%. Oligonucleotides containing halogenated dU residues require the use
of the [4-(tert-butyl)phenoxy]acetyl (tac) group for the protection of the canonical
phosphoramidites [21]. In these cases, the deprotection was performed with conc.
ammonia at room temperature, while in all other cases, the deprotection was carried
out at 608. The oligonucleotides were detritylated and purified on purification
cartridges [22] or by reversed-phase HPLC (conditions for purification, see Exper.
Part). The homogeneity of the compounds was established by ion-exchange chroma-
tography (see Exper. Part). The composition of the oligonucleotides was determined by
tandem hydrolysis with snake-venom phosphodiesterase and alkaline phosphatase
followed by reversed-phase (RP-18) HPLC as described [23]. Typical examples are

Helvetica Chimica Acta ± Vol. 84 (2001)
1053
shown in Fig. 1,c and d. The newly incorporated iodonucleosides 2c or 3c migrate
slower than the canonical DNA constituents. The oligonucleotides were also
characterized by MALDI-TOF mass spectra. The detected masses were in good
agreement with the calculated values (see Exper. Part, Table 10).
2.2. Base-Pair Stabilities of the Oligonucleotide Duplexes. Previously, the 7-bromo
nucleoside 2b was found to stabilize DNA duplexes strongly, while the non-
halogenated compound 2a contributes very little to the duplex stability [1][5]. The
contribution of the purine-2,6-diamine nucleoside 1 to the duplex stability is even lower
[6][24]. Thus, DNA duplexes containing 1 ´ thymidine base pairs are only slightly more
stable than those with dA ´ dT pairs. As it was not known whether the stabilizing effect
of the bromo nucleoside 2b would increase continuously with an increasing number of
incorporations or would level off with multiple incorporations ± as it is reported for
other modified nucleosides [7] ± a series of oligonucleotides were synthesized that
contain the halogenated compound 2b in a consecutive manner or in distant position.
The modified residues were incorporated in one or both strands of a double-stranded
DNA. The total number of incorporations was increased in duplexes from 1 to 6. The
non-halogenated duplexes containing compound 2a were prepared for comparison.
Apart from the incorporation of the bromo nucleoside 2b, the iodo compound 2c was
also studied. For all experiments, the non-self-complementary duplex 5'-d(TAGGT-
CAATACT)-3' (11) ´ 5'-d(AGTATTGACCTA)-3' (12) was chosen.
The nonhalogenated nucleoside 2a increases the Tm value of the standard duplex
11 ´ 12 by only 1.58 per modified residue (Table 3 and [5]). Contrarily, the bromo
compound 2b contributes a 4 ± 58 stabilization per modified residue, which represents
an outstandingly high value induced by a non-canonical base. The strength of this effect
is sequence-dependent, but the stability of the duplexes increases with an increasing
number of 2b incorporations, as shown in Fig. 2. When the iodo nucleoside 2c replaces
Fig. 2. Graph of the Tm values against the number of 2b residues

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Helvetica Chimica Acta ± Vol. 84 (2001)
Table 3. Tm Values and Thermodynamic Data of Duplex Formation of Oligonucleotides Containing the
Pyrazolo[3,4-d]pyrimidine Nucleosides 2a ± c Opposite to dTa)
Tm [8C]
DH8 [kcal/mol] DS8 [kcal/mol K]
DG8310 [kcal/mol]
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC CAG TTA TGA)-5' (12)
47
83.8
235.9
10.6
5'-d(TAG GTC 2a2aT ACT)-3' (13)
3'-d(ATC CAG TTA TGA)-5' (12)
50
50
51
54
56
59
63
67
93.3
263.09
279.63
278.58
280.74
251.66
251.25
274.04
285.02
268.47
283.41
254.58
260.43
263.29
268.72
284.63
284.73
11.7
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC C2aG TT2a TGA)-5' (14)
98.79
98.66
99.96
91.42
91.85
100.70
105.40
99.08
107.43
90.65
93.22
95.09
96.46
102.95
104.93
±
12.06
12.26
12.89
13.37
13.92
15.70
17.00
15.81
19.53
11.69
12.45
13.43
13.12
14.67
16.62
±
5'-d(TAG GTC 2a2aT ACT)-3' (13)
3'-d(ATC C2aG TT2a TGA)-5' (14)
5'-d(TAG GTC 2bAT ACT)-3' (15)
3'-d(ATC CAG TTA TGA)-5' (12)
5'-d(TAG GTC 2b2bT ACT)-3' (16)
3'-d(ATC CAG TTA TGA)-5' (12)
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC C2bG TT2b TGA)-5' (17)
5'-d(TAG GTC 2bAT ACT)-3' (15)
3'-d(ATC C2bG TT2b TGA)-5' (17)
5'-d(TAG GTC 2b2bT ACT)-3' (16)
3'-d(ATC C2bG TT2b TGA)-5' (17)
5'-d(T2bG GTC 2b2bT 2bCT)-3' (18) 64
3'-d(ATC CAG TTA TGA)-5 (12)
5'-d(T2bG GTC 2b2bT 2bCT)-3' (18) 75
3'-d(ATC C2bG TT2b TGA)-5' (17)
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC C2cG TTA TGA)-5' (19)
51
54
57
55
59
66
72
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC CAG TT2c TGA)-5' (20)
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC C2cG TT2c TGA)-5' (21)
5'-d(TAG GTC 2c2cT ACT)-3' (22)
3'-d(ATC CAG TTA TGA)-5' (11)
5'-d(TAG GTC 2c2cT ACT)-3' (22)
3'-d(ATC C2cGT T A TGA)-5' (19)
5'-d(TAG GTC 2c2cT ACT)-3' (22)
3'-d(ATC C2cGT T2c TGA)-5' (21)
5'-d(T2cG GTC2c2cT 2cCT)-3' (23)
3'-d(ATC C2cG TT2c TGA)-5' (21)
±
a) Thermodynamic parameters are derived from the fitting of melting curves measured at 260 nm in 0.1m NaCl,
10 mm MgCl2, and 10 mm Na-cacodylate buffer, pH 7.0, with 5 mm ‡ 5 mm single-strand concentration. The DG8
are taken directly from the program Meltwin 3.0 referring to 3108. Earlier reports from our laboratory obtained
with the fitting program refer to the same temperature and not to 2988 as indicated. The thermodynamic data
determined by the vanꢀt Hoff plots from the concentration dependence of the Tm values are consistent within
15% with those obtained from the curve fitting. The vanꢀt Hoff data of the formation of the duplex 11 ´ 12 are the
following: DH8 ˆ 86.8 kcal/mol; DS8 ˆ 243.7 cal/mol K; DG8310
ˆ
11.3 kcal/mol.

Helvetica Chimica Acta ± Vol. 84 (2001)
1055
the bromo compound 2b, a similar effect regarding duplex stabilization is observed
(Table 3). The effects of halogeno substituents introduced into the pyrazolo-
[3,4-d]pyrimidine derivatives of dG [3] or dA [2] amounts only to 28 per modified
residue.
A similar set of experiments as described for the duplexes containing the
halogenated 8-aza-7-deazapurine nucleosides 2b or 2c were performed with the
halogenated 2'-deoxyuridine derivatives 3b or 3c (Table 4). Neither the bromo
nucleoside 3b nor the iodo nucleoside 3c increases the stability of the duplexes
significantly compared to that of the nonhalogenated 3a or dT. Thus, only the base pairs
of type I (Fig. 3,a) with halogeno substituents located at the 7-position of the
Table 4. Tm Values and Thermodynamic Data of Duplex Formation of Oligonucleotides Containing the
Pyrimidine Nucleosides 3a ± c Opposite to dAa)
Tm [8C]
DH8 [kcal/mol]
DS8 [cal/mol K]
DG8310 [kcal/mol]
5'-d(TAG G3aC AA3a ACT)-3' (24)
3'-d(ATC CAG TTA TGA)-5' (12)
47
86.35
244.74
10.45
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC CAG 3a3aA TGA)-5' (25)
48
46
48
49
49
49
50
50
92.11
86.37
95.76
97.43
94.25
96.38
94.99
99.14
261.95
245.42
271.88
276.95
267.57
273.73
269.20
281.47
10.86
10.25
11.44
11.53
11.26
11.48
11.50
11.85
5'-d(TAG G3aC AA3a ACT)-3' (24)
3'-d(ATC CAG 3a3aA TGA)-5' (25)
5'-d(TAG G3bC AA3b ACT)-3' (26)
3'-d(ATC CAG TTA TGA)-5' (12)
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC CAG 3b3bA TGA)-5' (27)
5'-d(TAG G3bC AA3b ACT)-3' (26)
3'-d(ATC CAG 3b3bA TGA)-5' (27)
5'-d(TAG G3cC AA3c ACT)-3' (28)
3'-d(ATC CAG TTA TGA)-5' (12)
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC CAG 3c3cA TGA)-5' (29)
5'-d(TAG G3cC AA3c ACT)-3' (28)
3'-d(ATC CAG 3c3cA TGA)-5' (29)
a) See Table 3.
8-aza-7-deazapurine (pyrazolo[3,4-d]pyrimidine) moiety are stabilized by the halo-
geno substituents, while those of type II (Fig. 3,a) with the halogeno atoms attached at
the 5-positions of the pyrimidine base exert little influence compared to those with
dA ´ dT pairs.
As the stability of the base pairs of the halogenated pyrimidine nucleosides 3b or 3c
with dA was low compared to the halogenated pyrazolo[3,4-d]pyrimidine compounds
2b or 2c incorporated opposite to dT, tridentate base pairs are formed in which the
halogenated pyrimidine nucleosides 3a ± c are located opposite to the purine-2,6-
diamine nucleoside 1 instead of dA (Fig. 3,b; base-pair type III). In this case, a
strengthening of the base pair can be expected if the formation of a third H-bond is

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Helvetica Chimica Acta ± Vol. 84 (2001)
possible. However, the Tm values of those duplexes were also not influenced
significantly by replacement of the dA residues by the nucleoside 1 (Table 5). This
indicates that the 2-amino group does not participate in possible base pairs as shown in
IIIa ± c (Fig. 3,b), a finding similar to that observed for a base pair between 1 and dT
[5][6][24]. Similar behavior was found in the case of duplexes containing the base pair
IVa (Fig. 3,b) formed between nucleoside 2a and the pyrimidine nucleosides 3a ± c
(Table 5). Nevertheless, the duplexes incorporating the base pairs IVb,c are slightly
more stable than those containing the base pairs IIIb,c.
Significant duplex stabilization was observed when the halogenated 8-aza-7-
deazapurine nucleosides 2b or 2c were incorporated opposite to the pyrimidine
Fig. 3. Base-pair types I ± VI

Helvetica Chimica Acta ± Vol. 84 (2001)
1057
Table 5. Tm Values and Thermodynamic Data of Oligonucleotide Duplexes Containing the Halogenated
Pyrimidine Nucleosides 3a ± c Opposite to the Purine-2,6-diamine Nucleoside
d]pyrimidine-4,6-diamine Nucleoside 2aa)
1
or the Pyrazolo[3,4-
Tm [8C]
DH8 [kcal/mol]
DS8 [cal/mol K]
DG8310 [kcal/mol]
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC C1G TT1 TGA)-5' (30)
49
83.15
232.81
10.94
5'-d(TAG G3aC AA3a ACT)-3' (24)
3'-d(ATC C1G TT1 TGA)-5' (30)
48
49
49
48
48
48
48
49
50
51
49
51
50
73.03
73.84
77.60
54.74
60.03
60.09
62.35
87.32
89.11
81.32
90.30
93.05
90.58
202.44
203.94
215.48
144.65
161.49
160.37
167.33
245.54
250.34
225.60
253.96
261.91
254.11
10.25
10.59
10.76
9.88
5'-d(TAG G3bC AA3b ACT)-3' (26)
3'-d(ATC C1G TT1 TGA)-5' (30)
5'-d(TAG G3cC AA3c ACT)-3' (28)
3'-d(ATC C1G TT1 TGA)-5' (30)
5'-d(T1G GTC 11T 1CT)-3' (31)
3'-d(ATC CAG TTA TGA)-5' (12)
5'-d(T1G GTC 11T 1CT)-3' (31)
3'-d(ATC CAG 3a3aA TGA)-5' (25)
9.94
5'-d(T1G GTC 11T 1CT)-3' (31)
3'-d(ATC CAG 3b3bA TGA)-5' (27)
10.35
10.45
11.16
11.47
11.35
11.53
11.82
11.77
5'-d(T1G GTC 11T 1CT)-3' (31)
3'-d(ATC CAG 3c3cA TGA)-5' (29)
5'-d(TAG GTC 2a2aT ACT)-3' (13)
3'-d(ATC CAG 3a3aA TGA)-5' (25)
5'-d(TAG GTC 2a2aT ACT)-3' (13)
3'-d(ATC CAG 3b3bA TGA)-5' (27)
5'-d(TAG GTC 2a2aT ACT)-3' (13)
3'-d(ATC CAG 3c3cA TGA)-5' (29)
5'-d(TAG G3aC AA3a ACT)-3' (24)
3'-d(ATC C2aG TT2a TGA)-5' (14)
5'-d(TAG G3bC AA3b ACT)-3' (26)
3'-d(ATC C2aG TT2a TGA)-5' (14)
5'-d(TAG G3cC AA3c ACT)-3' (28)
3'-d(ATC C2aG TT2a TGA)-5' (14)
a) See Table 3.
nucleosides 3a ± c (Table 6 and Fig. 3,c; base-pair type V and VI). The Tm increase
amounts to ca. 4 ± 58 per incorporated residue of the halogenated nucleosides 2b or 2c.
All duplexes containing the halogenated nucleosides 2b or 2c (Fig. 3,c) give very
similar Tm values, no matter which pyrimidine monomer is located opposite to it. Thus,
only halogeno substituents attached at the modified purine residues lead to a duplex
stabilization, while halogeno atoms at the 5-position of the 2'-deoxyuridine moiety
contribute very little to the duplex stability [25][26].
2.3. Base Discrimination. ± To investigate the discrimination of the iodo nucleoside
2c towards the four canonical DNA constituents, hybridization experiments were
performed according to Table 7. As expected, the base pair 2c ´ dT is the strongest (11 ´
21, Tm 578), while those of the duplexes forming mismatches melt at a significantly

1058
Helvetica Chimica Acta ± Vol. 84 (2001)
Table 6. Tm Values and Thermodynamic Data of Oligonucleotides Containing the Pyrazolo[3,4-d]pyrimidine-
4,6-diamine Nucleosides 2b,c Opposite to dT and 3a ± ca)
Tm [8C]
DH8 [kcal/mol]
DS8 [cal/mol K]
DG8310 [kcal/mol]
5'-d(TAG GTC 2bAT ACT)-3' (15)
3'-d(ATC CAG 3a3aA TGA)-5' (25)
52
92.88
260.97
11.94
5'-d(TAG GTC 2b2bT ACT)-3' (16)
3'-d(ATC CAG 3a3aA TGA)-5' (25)
55
64
54
55
65
54
55
65
56
56
57
55
55
55
96.78
101.57
98.06
95.42
106.49
87.97
89.17
101.57
95.76
96.53
97.07
96.93
100.82
98.00
269.67
276.51
275.03
266.68
290.34
244.29
246.44
275.34
265.45
268.70
269.07
270.69
282.19
273.26
13.14
15.82
12.76
12.71
16.44
12.20
12.74
16.18
13.43
13.20
13.62
12.98
13.29
13.25
5'-d(T2bG GTC 2b2bT 2bCT)-3' (18)
3'-d(ATC CAG 3a3aA TGA)-5' (25)
5'-d(TAG GTC 2bAT ACT)-3' (15)
3'-d(ATC CAG 3b3bA TGA)-5' (27)
5'-d(TAG GTC 2b2bT ACT)-3' (16)
3'-d(ATC CAG 3b3bA TGA)-5' (27)
5'-d(T2bG GTC 2b2bT 2bCT)-3' (18)
3'-d(ATC CAG 3b3bA TGA)-5' (27)
5'-d(TAG GTC 2bAT ACT)-3' (15)
3'-d(ATC CAG 3c3cA TGA)-5' (29)
5'-d(TAG GTC 2b2bT ACT)-3' (16)
3'-d(ATC CAG 3c3cA TGA)-5' (29)
5'-d(T2bG GTC 2b2bT 2bCT)-3' (18)
3'-d(ATC CAG 3c3cA TGA)-5' (29)
5'-d(TAG G3aC AA3a ACT)-3' (24)
3'-d(ATC C2bG TT2b TGA)-5' (17)
5'-d(TAG G3bC AA3b ACT)-3' (26)
3'-d(ATC C2bG TT2b TGA)-5' (17)
5'-d(TAG G3cC AA3c ACT)-3' (28)
3'-d(ATC C2bG TT2b TGA)-5' (17)
5'-d(TAG G3aC AA3a ACT)-3' (24)
3'-d(ATC C2cG TT2c TGA)-5' (21)
5'-d(TAG G3bC AA3b ACT)-3' (26)
3'-d(ATC C2cG TT2c TGA)-5' (21)
5'-d(TAG G3cC AA3c ACT)-3' (28)
3'-d(ATC C2cG TT2c TGA)-5' (21)
a) See Table 3.
lower temperature (Table 7). The discrimination of the iodo nucleoside 2c is similar to
that of the canonical nucleosides, except that the duplex 33 ´ 21 (Tm 508) shows a 78
lower Tm value than the duplex 11 ´ 21 (Tm 578), while duplex 33 ´ 12 (Tm 468) has almost
the same stability as the parent duplex 11 ´ 12 (Tm 488). The high Tm value of 33 ´ 12 is
the result of the formation of the dG ´ dA Hoogsteen pair, which is obviously not formed
between dG and compound 2c.
The CD spectra of the duplexes containing the 8-aza-7-deazaadenin-2-amine
derivatives 2a,b were measured next. A B-like DNA structure can be deduced from the
curves displayed in Fig. 4,a. A positive Cotton effect around 270 to 290 nm and a
negative lobe at 250 nm are observed for the standard duplex 11 ´ 12. The CD spectrum

Helvetica Chimica Acta ± Vol. 84 (2001)
1059
Table 7. Tm Values of Oligonucleotides Containing the Pyrazolo[3,4-d]pyrimidine-4,6-diamine Nucleoside 2c
Opposite to the Four Canonical Nucleosidesa)
Tm [8C]
Tm [8C]
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC C2cG TT2c TGA)-5' (21)
57
5'-d(TAG GTC AAT ACT)-3' (11)
3'-d(ATC CAG TTA TGA)-5' (12)
48
5'-d(TAG GAC AAT ACT)-3' (32)
3'-d(ATC C2cG TT2c TGA)-5' (21)
45
50
45
5'-d(TAG GAC AAT ACT)-3' (32)
3'-d(ATC CAG TTA TGA)-5' (12)
38
46
36
5'-d(TAG GGC AAT ACT)-3' (33)
3'-d(ATC C2cG TT2c TGA)-5' (21)
5'-d(TAG GGC AAT ACT)-3' (33)
3'-d(ATC CAG TTA TGA)-5' (12)
5'-d(TAG GCC AATACT)-3' (34)
3'-d(ATC C2cG TT2c TGA)-5' (21)
5'-d(TAG GCC AATACT)-3' (34)
3'-d(ATC CAG TTA TGA)-5' (12)
a) See Table 3.
Fig. 4. a) CD Spectra of duplexes 11 ´ 12, 13 ´ 14, 16 ´ 17, and 31 ´ 30 (in 100 mm NaCl, 10 mm MgCl2, and 60 mm
Na-cacodylate (pH 7.0)). b) CD Spectra of duplexes 11 ´ 35, 13 ´ 35, 16 ´ 35, and 31 ´ 35 (in 100 mm NaCl, 10 mm
MgCl2, and 60 mm Na-cacodylate (pH 7.0)). Concentration of the oligonucleotides: 5 mm ‡ 5 mm (single-strand
concentration).
of the duplex 31 ´ 30 containing 1 shows significant differences similar to that of 16 ´ 17,
while the duplex 13 ´ 14 shows a stronger negative Cotton effect at 260 nm and a positive
one at 235 nm. Similar differences were reported for the oligonucleotides containing 7-
substituted 7-iodo-8-aza-7-deaza guanine [4].
2.4. DNA-RNA Hybrids. To study the influence of the 2,6-diamino-substituted
nucleosides 1 and 2a,b on the stability of DNA ´ RNA hybrids, the oligodeoxyribonu-
cleotides 16, 24, 26, 28, 31, and 11 were hybridized with the oligoribonucleotide 35.
Table 8 shows that the various modifications exert a significant influence on the duplex
stability of the DNA ´ RNA hybrids. While the incorporation of the nonhalogenated
nucleosides 1 or 2a stabilizes the DNA ´ RNA structure very little, the hybridization of
the oligonucleotide containing 2b with 35 (16 ´ 35) shows a significant increase of the
duplex stability as in DNA. Compounds 3b and 3c enhance the thermal stability of
DNA ´ RNA to a small degree, which is also observed for other modifications of 2'-
deoxyuridine [27]. The hypochromicities of the chimeric hybrids are slightly decreased

1060
Helvetica Chimica Acta ± Vol. 84 (2001)
Table 8. Tm Values and Thermodynamic Data of DNA ´ RNA Hybridsa)
Tm [8C]
DH8 [kcal/mol]
DS8 [cal/mol K]
DG8310 [kcal/mol]
5'-d(TAG GTC AAT ACT)-3' (11)
3'-(AUC CAG UUA UGA)-5' (35)
45
92.1
264.0
10.2
5'-d(T1G GTC 11T 1CT)-3' (31)
3'-(AUC CAG UUA UGA)-5' (35)
48
48
53
49
44
48
47
60.46
±
162.12
±
10.18
±
5'-d(TAG GTC 2a2aT ACT)-3' (13)
3'-(AUC CAG UUA UGA)-5' (35)
5'-d(TAG GTC 2b2bT ACT)-3' (16)
3'-(AUC CAG UUA UGA)-5' (35)
98.64
84.55
74.43
77.19
78.77
276.97
236.26
208.26
215.13
220.52
12.74
11.28
9.84
10.47
10.37
5'-d(TAG GTC 2c2cT ACT)-3' (22)
3'-d(AUC CAG UUA UGA)-5' (35)
5'-d(TAG G3aC AA3a ACT)-3' (24)
3'-(AUC CAG UUA UGA)-5' (35)
5'-d(TAG G3bC AA3b ACT)-3' (26)
3'-(AUC CAG UUA UGA)-5' (35)
5'-d(TAG G3cC AA3c ACT)-3' (28)
3'-(AUC CAG UUA UGA)-5' (35)
over those of the DNA ´ DNA duplexes (data not shown). From the CD spectra of
Fig. 3,b it can be seen that the DNA ´ RNA hybrids adopt the A-form [28].
2.5. Duplexes with Parallel Chain Orientation. The base pairing of the nucleosides 1,
2a ± c as well as 3b,c was also investigated in parallel-stranded DNA [29]. For this
purpose, it was necessary to replace the dC ´ dG pair by a m5iCd ´ iGd pair. The duplexes
11 ´ 36 and 37 ´ 12 served as standards [29]. Substitution of dA residues by the diamino
nucleoside 1 resulted in a slight stability decrease of the parallel-stranded (ps) duplexes
31 ´ 36 and 37 ´ 30 (Table 9). The incorporation of 2a ± c into the ps DNA resulted in a
significant increase of the Tm values. The incorporation of the substituted pyrimidine
nucleosides 3b,c in place of dT leads only to minor changes of the Tm values. From this,
it can be concluded that similar to DNAwith antiparallel chain orientation, the halogeno
substituents of the ꢀpurineꢁ nucleosides 2b,c stabilize these duplexes, while the halogeno
substituents of the pyrimidine nucleosides contribute very little to the duplex stability.
Conclusion. ± The halogeno substituents introduced at the 7-position of the 8-aza-7-
deazapurine 2'-deoxynucleoside (see 2b,c) or the 5-position of 2'-deoxyuridine residues
(see 3b,c) have very different influences on the stability of nucleic acid duplexes. In the
case of the 7-halogenated 8-aza-7-deazapurine 2'-deoxynucleosides 2b,c, each mono-
mer contributes ca. 4 ± 58 to the duplex stabilization, while the pyrimidine nucleosides
3b,c show very little influence or even no effect. This is surprising, as the halogeno
atoms in both series of nucleobases are directed into the major groove of B-DNA, both
being in a nonidentical but very similar environments. When both, the halogenated
pyrimidines and the halogenated 8-aza-7-deazapurine nucleosides are present in a DNA
duplex, only the latter make a contribution to the duplex stabilization. Consequently,
the increase of the hydrophobic character of the major groove induced by the lipophilic
halogeno substituents and the expelling of H2O molecules is not the major effect

Helvetica Chimica Acta ± Vol. 84 (2001)
1061
Table 9. Tm Values and Thermodynamic Data of Parallel-Stranded Oligonucleotide Duplexes Containing the
Nucleosides 1, 2a ± c, and 3a ± ca)b)
Tm [8C]
DH8 [kcal/mol]
DS8 [cal/mol K]
DG8310 [kcal/mol]
5'-d(TAG GTC AAT ACT)-3' (11)
5'-d(ATiC iCAiG TTA TiGA)-3' (36)
39
74.4
212.3
8.5
5'-d(T1G GTC 11T 1CT)-3' (31)
5'-d(ATiC iCAiG TTA TiGA)-3' (36)
36
41
45
37
36
44
39
45
48
43
42
48.12
61.77
66.59
50.64
55.09
85.0
129.68
170.95
183.94
137.87
152.14
242.0
7.90
8.75
9.54
7.88
7.90
10.0
8.38
10.3
10.56
9.19
9.15
5'-d(TAG GTC 2a2aT ACT)-3' (13)
5'-d(ATiC iCAiG TTA TiGA)-3' (36)
5'-d(TAG GTC 2b2bT ACT)-3' (16)
5'-d(ATiC iCAiG TTA TiGA)-3' (36)
5'-d(TAG G3bC AA3b ACT)-3' (26)
5'-d(ATiC iCAiG TTA TiGA)-3' (36)
5'-d(TAG G3cC AA3c ACT)-3' (28)
5'-d(ATiC iCAiG TTA TiGA)-3' (36)
5'-d(TiCA TAA iCTiG iGAT)-3' (37)
5'-d(AGT ATT GAC CTA)-3' (12)
5'-d(TiCA TAA iCTiG iGAT)-3' (37)
5'-d(AGT 1TT G1C CTA)-3' (30)
61.00
80
169.68
230
5'-d(TiCA TAA iCTiG iGAT)-3' (37)
5'-d(AGT 2aTT G2aC CTA)-3' (14)
5'-d(TiCA TAA iCTiG iGAT)-3' (37)
5'-d(AGT 2bTT G2bC CTA)-3' (17)
68.34
76.03
67.15
186.29
215.51
187.02
5'-d(TiCA TAA iCTiG iGAT)-3' (37)
5'-d(AGT A3b3b GAC CTA)-3' (27)
5'-d(TiCA TAA iCTiG iGAT)-3' (37)
5'-d(ACT A3c3c GAC CTA)-3' (29)
a) Measured at 260 nm in 1m NaCl, 100 mm MgCl2, 60 mm Na-cacodylate buffer, pH 7.0, with 5 mm ‡ 5 mm
b
oligomer concentration. ) d(iC) ˆ m5iCd ˆ 2'-deoxy-5-methylisocytidine.
induced by the halogenation of the major groove. Apparently, stacking inter-
actions between the nearest neighbors are strengthened in the case of 2b,c but not
with 3b,c.
Financial support by the Deutsche Forschungsgemeinschaft and the Roche Diagnostics GmbH is gratefully
acknowledged. We thank Dr. H. Rosemeyer and Y. He for the NMR measurements, Dr. T. Wenzel for the
MALDI-TOF spectra, H. Debelak for his support in the synthesis of 8a, H. Debelak and E. Feiling for the
drawings. Compound 10a was provided by Y. He.
Experimental Part
General. See [1][2]. Flash chromatography (FC): 0.4 bar, silica gel 60 H (Merck, Darmstadt, Germany).
Thin-layer chromatography (TLC): Aluminium sheets, silica gel 60 F254 (0.2 mm, Merck, Germany). Solvent systems
for FC and TLC: CH2Cl2/MeOH 9 :1 (A), CH2Cl2/MeOH 95 :5 (B), CH2Cl2/acetone 9 :1 (C), CH2Cl2/AcOEt
85 :15 (D), CH2Cl2/acetone 95 :5 (E). M.p.: Büchi SMP-20 apparatus (Büchi, Switzerland); uncorrected. NMR
Spectra: Avance DPX-250 and AMX-500 spectrometers (Bruker, Germany); d values in ppm downfield

1062
Helvetica Chimica Acta ± Vol. 84 (2001)
from internal SiMe4 (1H, 13C). Microanalyses were performed by Mikroanalytisches Labor Beller, Göttingen,
Germany.
Oligonucleotides. 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 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 by the
Meltwin 3.0 program [30]. CD Spectra: Jasco 600 (Jasco, Japan) spectropolarimeter with thermostatically
(Lauda RCS-6 bath) controlled 1-cm cuvettes. UV Spectra: 150-20 spectrometer (Hitachi, Japan); the following
7
extinction coefficients were used (e260): Gd 2700, dT 8800, dC 7300, dA 15400, dG 11700, 2a 8800, 2b 8700, 2c
8700, 3a 10000, 3b 4800, 3c 3700. Snake-venom phosphodiesterase (EC 3.1.15.1, Crotallus durissus) 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, Leipzig, Germany); Table 10.
‡
Table 10. Molecular Masses ([M ‡ H] ) of Oligonucleotides Measured by MALDI-TOF Mass Spectrometry
‡
‡
[M ‡ H] (calc.)
[M ‡ H] (found)
5'-d(TAG GTC AAT ACT)-3' (11)
5'-d(AGT ATT GAC CTA)-3' (12)
5'-d(TAG GTC 2a2aT ACT)-3' (13)
5'-d(AGT 2aTT G2aC CTA)-3' (14)
5'-d(TAG GTC 2b2bT ACT)-3' (16)
5'-d(AGT 2bTT G2bC CTA)-3' (17)
5'-d(T2bG GTC 2b2bT 2bCT)-3' (18)
5'-d(AGT ATT G2cC CTA)-3' (19)
5'-d(AGT 2cTT GAC CTA)-3' (20)
5'-d(AGT 2cTT G2cC CTA)-3' (21)
5'-d(TAG GTC 2c2cT ACT)-3' (22)
5'-d(T2cG GTC 2c2cT 2cCT)-3' (23)
5'-d(TAG G3aC AA3a ACT)-3' (24)
5'-d(AGT A3a3a GAC CTA)-3' (25)
5'-d(TAG G3bC AA3b ACT)-3' (26)
5'-d(AGT A3b3b GAC CTA)-3' (27)
5'-d(TAG G3cC AA3c ACT)-3' (28)
5'-d(AGT A3c3c GAC CTA)-3' (29)
3644.4
3644.4
3674.4
3674.4
3832.5
3832.5
4020
3786
3786
3926.5
3927
4206.3
3613.1
3613.1
3774.1
3774.1
3868.1
3868.1
3645
3645
3677
3675
3830
3832
4021
3787
3792
3927
3931
4210
3616
3615
3775
3772
3871
3871
Synthesis and Purification of Oligonucleotides. The synthesis was carried out on a 1-mmol scale with the 3'-
phosphoramidites of [(MeO)2Tr]ib2Gd, [(MeO)2Tr]bz6Ad, [(MeO)2Tr]bz4Cd, and [(MeO)2Tr]Td for the
synthesis of oligonucleotides containing 2a ± c, and [(MeO)2Tr]tac2Gd, [(MeO)2Tr]tac6Ad, [(MeO)2Tr]tac4Cd,
and [(MeO)2Tr]Td for the synthesis of oligonucleotides containing 3a ± c. After cleavage of the oligonucleotides
from the solid support, the former were deprotected in 25% aq. NH3 soln. for 12 ± 15 h at 608. The latter were
incubated in 25% aq. NH3 soln. for 1.5 ± 2 h at r.t. for deprotection. Purification of the 5'-(dimethoxytrityl)-
oligomers was performed by reversed-phase HPLC (RP-18). The following solvent gradient was used (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 with a flow rate of
1.0 ml/min. The concentrated oligonucleotide solns. 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 by reversed-phase
HPLC with the gradient: 20 min 0 ± 20% B in A with a flow rate of 1 ml/min. The oligomers were desalted on a
short column (RP-18, silica gel) and then lyophilized on a Speed-Vac evaporator to yield colorless solids, which
were frozen at 248.
Nucleoside-Composition Analysis. 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 then alkaline
phosphatase (3 ml) at 378 for another 30 min. The mixtures were analyzed on reversed-phase HPLC (RP-18, at
260 nm, gradient A, 0.7 ml/min). The retention time of 2a ± c and 3a ± c were used as standards (Fig. 1). The
extinction coefficients of the nucleosides and the peak areas were used for quantification of the composition of
the oligonucleotides (Fig. 1,a ± d).

Helvetica Chimica Acta ± Vol. 84 (2001)
1063
1-(2-Deoxy-b-d-erythro-pentofuranosyl)-3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (2c). A soln.
of 1-(2-deoxy-b-d-erythro-pentofuranosyl)-3-iodo-4-isopropoxy-1H-pyrazolo[3,4-d]pyrimidin-6-amine (4) [11]
(1 g, 2.3 mmol) in aq. 25% NH3 soln. (80 ml) was heated at 708 in an autoclave for 4 d. The solvent was
evaporated and the residue dissolved in hot H2O and crystallized. Colorless needles (640 mg, 71%). M.p. 1548.
TLC (A): Rf 0.2. UV (MeOH): 223 (31800), 260 (8700), 278 (9100). 1H-NMR ((D6)DMSO): 2.12
(m, Ha C(2')); 2.67 (m, Hb C(2')); 3.38, 3.44 (m, 2 H C(5')); 3.73 (m, H C(4')); 4.33 (m, H C(3')); 4.73
(t, J ˆ 5.7, OH C(5')); 5.17 (d, J ˆ 4.3, OH C(3')); 6.27 (ꢀtꢁ, J ˆ 6.5, H C(1')); 6.34 (br., NH2); 6.62 (br., NH2).
Anal. calc. for C10H13IN6O3 (392.2): C 30.63, H 3.34, N 21.43; found: C 30.91, H 3.61, N 21.27.
3-Bromo-1-(2-deoxy-b-d-erythro-pentofuranosyl)-N4,N6-bis(2-methylpropanoyl)-1H-pyrazolo[3,4-d]pyrim-
idine-4,6-diamine (5a). Compound 2b [5] (0.74 g, 2.14 mmol) was co-evaporated with anh. pyridine (3Â) and then
dissolved in anh. pyridine (5 ml), while stirring at r.t. Me3SiCl (1.37 ml, 10.8 mmol), and after 15 min, isobutyric
anhydride (3.56 ml, 21.5 mmol) were added. Stirring was continued for 3 h. The mixture was cooled in an ice-
bath and diluted with H2O (2.5 ml); 5 min later, aq. 25% NH3 soln. (4.3 ml) was added. After stirring for 30 min,
the mixture was evaporated and co-evaporated with toluene (3Â). The residue was purified by FC (CH2Cl2/
MeOH 9 :1), furnishing two zones. From the fast migrating zone, 5a was obtained as a colorless amorphous solid
(500 mg, 48%). Rf (A) 0.4. UV (MeOH): 284 (9500), 239 (33900). 1H-NMR ((D6)DMSO): 1.15 (m, 2 Me2CH);
2.25 (m, Ha C(2')); 2.75 (m, Hb C(2')); 2.86 (m, 2 Me2CH); 3.47 (m, 2 H C(5')); 3.81 (m, H C(4')); 4.44
(m, H C(3')); 4.73 (t, J ˆ 5.5, OH C(5')); 5.32 (d, J ˆ 4.3, OH C(3')); 6.54 (ꢀtꢁ, J ˆ 6.6, H C(1')); 10.59,
10.72 (2s, 2 NH). Anal. calc. for C18H25BrN6O5 (485.3): C 44.55, H 5.19, N 17.32; found: C 44.90, H 5.28,
N 16.81.
3-Bromo-1-(2-deoxy-b-d-erythro-pentofuranosyl)-N6-(2-methylpropanoyl)-1H-pyrazolo[3,4-d]pyrimidine-
4,6-diamine (6). The slower migrating zone from the above reaction afforded 6 as a colorless amorphous solid
(0.2 g, 22%). Rf (A) 0.36. UV (MeOH): 283 (10900), 237 (49500). 1H-NMR ((D6)DMSO): 1.04, 1.07
(m, Me2CH); 2.21 (m, Ha C(2')); 2.71 (m, Hb C(2')); 2.90 (m, Me2CH); 3.46 (m, 2 H C(5')); 3.78
(m, H C(4')); 4.38 (m, H C(3')); 4.72 (t, J ˆ 5.6, OH C(5')); 5.28 (d, J ˆ 4.3, OH C(3')); 6.42 (ꢀtꢁ, J ˆ 6.4,
H
C(1')); 6.96, 7.76 (br., NH2); 10.08 (br., NH). Anal. calc. for C14H19BrN6O4 (415.2): C 40.49, H 4.61, N 20.24;
found: C 40.58, H 4.72, N 19.93.
N4,N6-Dibenzoyl-1-(2-deoxy-b-d-erythro-pentofuranosyl)-3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-di-
amine (5b). Compound 2c (1.0 g, 2.55 mmol) was co-evaporated twice with toluene. The residue was dissolved
in anh. pyridine (40 ml), and Me3SiCl (3.25 ml, 25.5 mmol) was added while stirring. The mixture was stirred
under Ar, cooled to 08, and PhCOCl (3.0 ml, 25.8 mmol) was added dropwise within 30 min. After stirring
overnight at r.t., the mixture was diluted with AcOEt (200 ml), the org. phase washed with sat. aq. NaHCO3
soln. (200 ml) and ice-cold H2O (200 ml), the aq. phase extracted with AcOEt (2 Â 400 ml), the combined org.
phase evaporated, and the residue dissolved in THF/MeOH/H2O 5 :4 :1 (250 ml). The dark orange soln. was
cooled to 08, then 2n NaOH (25 ml) was added, and stirring was continued for another 40 min. The residue was
purified by FC (CH2Cl2/MeOH 98 :2 ! CH2Cl2/MeOH 95 :5): 5b (1.15 g, 75%). Amorphous solid. TLC (B): Rf
0.4. UV (MeOH): 244 (17400), 276 (14200). 1H-NMR ((D6)DMSO): 2.13 (m, Ha C(2')); 2.67 (m, Hb C(2'));
3.38, 3.52 (m, 2 H C(5')); 3.84 (m, H C(4')); 4.46 (m, H C(3')); 4.72 (t, J ˆ 5.7, OH C(5')); 5.29 (d, J ˆ 4.4,
OH C(3')); 6.66 (ꢀtꢁ, J ˆ 6.5, H C(1')); 7.51 ± 8.11 (m, arom. H); 10.54, 10.78 (s, 2 NH). Anal. calc. for
C24H21IN6O5 (586.4): C 48.01, H 3.53, N 14.00; found: C 47.93, H 3.53, N 14.05.
3-Bromo-1-[2-deoxy-5-O-(4,4'-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-N4,N6-bis(2-methylpropano-
yl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (7a). Compound 5a (0.5 g, 1.03 mmol) was co-evaporated with
anh. pyridine (3Â) and dissolved in pyridine (1.5 ml). (MeO)2TrCl (0.45 g, 1.33 mmol) was added, and the
mixture was stirred at r.t. for 3 h. The reaction was quenched by addition of MeOH, and the mixture evaporated
and co-evaporated with toluene (3Â). FC (CH2Cl2/MeOH 10 :1) gave 7a. Colorless foam (0.57 g, 70%). Rf (B)
0.3. UV (MeOH): 237 (52000), 283 (10500). 1H-NMR ((D6)DMSO): 1.04 ± 1.17 (m, 2 Me2CH)); 2.29
(m, Ha C(2')); 2.85 (m, Hb C(2')); 2.89 (m, 2 Me2CH)); 3.07 (m, 2 H C(5')); 3.71 (s, 2 MeO); 3.94
(m, H C(4')); 4.46 (m, H C(3')); 5.35 (m, OH C(3')); 6.57 (m, H C(1')); 10.57, 10.74 (s, 2 NH). Anal.
calc. for C39H43BrN6O7 (787.2): C 59.47, H 5.46, N 10.67; found: C 59.08, H 5.37, N 10.39.
3-Bromo-1-[2-deoxy-5-O-(4,4'-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-N4,N6-bis(2-methylpropano-
yl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine 3'-(2-Cyanoethyl Diisopropylphosphoramidite) (8a). To a soln.
of 7a (0.24 g, 0.3 mmol) in anh. CH2Cl2 (3 ml) under Ar, iPr2EtN (0.16 ml, 0.9 mmol) and 2-cyanoethyl
diisopropylphosphoramidochloridite (91 ml, 0.41 mmol) were added, and the mixture was stirred at r.t. for
30 min (TLC monitoring). Then, the mixture was diluted with CH2Cl2 and the soln. washed with 5% aq.
NaHCO3 soln. (2Â) and brine. The org. phase was dried (Na2SO4) and evaporated and the product separated by
FC: 8a (193 mg, 65%). Colorless foam. Rf (E) 0.63, 0.69. UV (MeOH): 282 (10000), 237 (49500). 1H-NMR

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Helvetica Chimica Acta ± Vol. 84 (2001)
((D6)DMSO): 1.11 ± 1.35 (m, 4 Me2CH); 2.51 (m, Ha C(2')); 2.66 (m, Hb C(2')); 2.95 ± 3.91 (m, 2 H C(5'),
2 Me2CH, CH2CH2); 3.80 (s, MeO); 4.26 (m, H C(4')); 4.82 (m, H C(3')); 6.71 (m, H C(1')); 6.75 ± 7.44
(m, arom. H); 8.39, 8.59 (br., 2 NH). 31P-NMR (CDCl3), 149.61, 149.65. Anal. calc. for C48H60BrN8O8P (921.2):
C 58.36, H 6.08, N 11.35; found: C 58.86, H 6.18, N 11.55.
N4,N6-Dibenzoyl-1-[2-deoxy-5-O-(4,4'-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3-iodo-1H-pyrazo-
lo[3,4-d]pyrimidine-4,6-diamine (7b). Compound 5b (450 mg, 0.75 mmol) was co-evaporated twice with anh.
pyridine. The residue was dissolved in pyridine (2 ml), and dimethoxytrityl chloride (305 mg, 0.9 mmol) was
added. After 4 h stirring, the soln. was diluted with 5 ml of MeOH and washed with 5% aq. NaHCO3 soln. (3 Â
20 ml). The org. phase was dried (Na2SO4) and evaporated. The residue was purified by FC (CH2Cl2/acetone
95 :5 ! CH2Cl2/acetone 9 :1): 375 mg (55%) of 7b. Colorless foam. Rf (C) 0.4. UV (MeOH): 244 (16900), 276
(16200). 1H-NMR ((D6)DMSO): 2.35 (m, Ha C(2')); 2.67 (m, Hb C(2')); 3.07, 3.09 (2m, 2 H C(5')); 3.70
(s, 2 MeO); 3.96 (m, H C(4')); 4.56 (m, H C(3')); (d, J ˆ 4.8, OH C(3')); 6.72 ± 8.11 (m, arom. H);
10.54, 10.78 (2s, 2 NH). Anal. calc. for C45H39IN6O7 (902.73): C 59.87, H 4.35, N 9.31; found: C 59.93, H 4.33,
N 9.39.
N4,N6-Dibenzoyl-1-[2-deoxy-5-O-(4,4'-dimethoxytrityl)-b-d-erythro-pentofuranosyl]-3-iodo-1H-pyrazo-
lo[3,4-d]pyrimidine-4,6-diamine 3'-(2-Cyanoethyl Diisopropylphosphoramidite) (8b). Compound 7b (330 mg,
i
0.37 mmol) was dissolved in THF (5 ml). Pr2EtN (186 ml, 1.07 mmol) and 2-cyanoethyl diisopropylphosphor-
amidochloridite (108 ml, 0.48 mmol) were added under Ar. After 30 min, the mixture was diluted with CH2Cl2
(20 ml) and 5% aq. NaHCO3 soln. (2 Â 20 ml). The mixture was extracted with CH2Cl2 (3 Â 15 ml), the
combined org. layer dried (Na2SO4) and evaporated, and the oily residue submitted to FC (CH2Cl2/AcOEt
85 :15): 280 mg (69%) of 8b. Colorless foam. Rf (D) 0.8. 31P-NMR (CDCl3): 149.37, 149.38.
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Received February 13, 2001