8-Aza-7-deazapurine-pyrimidine base pairs: The contribution of 2- and 7-substituents to the stability of duplex DNA

Article abstract of DOI:10.1016/S0040-4020(02)00406-4

The 8-aza-7-deazapurin-2,6-diamine 2′-deoxyribonucleoside (2a)-dT base pair is more stable than the 2-amino-2′-deoxyadenosine (1)-dT pair. Halogeno or propynyl substituents introduced in the 7-position of the 8-aza-7-deazapurine system (2b-d) lead to an a

Full text of DOI:10.1016/S0040-4020(02)00406-4

TETRAHEDRON
Pergamon
Tetrahedron 58*2002) 4535±4542
8-Aza-7-deazapurine±pyrimidine base pairs: the contribution
of 2- and 7-substituents to the stability of duplex DNA
Junlin He and Frank Seelap
È È È È
Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie, Universitat Osnabruck, Barbarastrasse 7,
È
D-49069 Osnabruck, Germany
Received 21 January 2002; revised 15 March 2002; accepted 4 April 2002
AbstractÐThe 8-aza-7-deazapurin-2,6-diamine 20-deoxyribonucleoside *2a)±dT base pair is more stable than the 2-amino-20-deoxy-
adenosine *1)±dT pair. Halogeno or propynyl substituents introduced in the 7-position of the 8-aza-7-deazapurine system *2b±d) lead to
an additional duplex stabilization. Pyrazolo[3,4-d]pyrimidine nucleosides related to 20-deoxyadenosine or 20-deoxyguanosine show a similar
behavior. The base pair stabilization is comparably small when identical substituents are present in the 5-position of 20-deoxyuridine. The
extraordinary stability of the 2b±d±dT base pairs is caused by better proton donor properties of the two amino groups of the 8-aza-7-
deazapurin-2,6-diamine moiety and the better base stacking induced by the 7-substituents compared to that of 2-aminoadenine. q 2002
Elsevier Science Ltd. All rights reserved.
1. Introduction
introduced in position-7. Then, the base pairs between the
8-aza-7-deazapurin-2,6-diamine nucleosides 2b±d with dT
approach the stability of the tridentate dG±dC base pair.21,22
They show a similar base selectivity as dA±dT and do not
form stable odd base pairs as it was reported for the
pyrrolo[2,3-d]pyrimidine analogue of 1.23 This manuscript
reports on the duplex stability of oligonucleotides incorpo-
rating various 8-aza-7-deazapurine 20-deoxyribonucleo-
sides. The unusual contribution of the 2-amino group of
the 8-aza-7-deazapurin-2,6-diamine nucleosides will be
discussed by comparing their 7-substituted derivatives and
the nucleosides 1 and 3 *Scheme 1).24 Also, the stabilizing
effects of the 7-substituents in the series of nucleosides
containing 8-aza-7-deazapurin-2,6-diamine *2b±d), 8-aza-
7-deazaadenine *4a±c),25,26 and 8-aza-7-deazaguanine
*5a±c)26 will be reported. Finally, the behavior of the
major groove directed 7-substituents of the 8-aza-7-deaza-
purine nucleosides will be related to the 5-substituents of the
pyrimidine compounds 6a±d.
2-Amino-20-deoxyadenosine *1, n2Ad) *Scheme 1) has
attracted much attention because of its capability to form
a tridentate base pair with dT. The base pair is expected to
show a similar stability as that of dG±dC but retaining the
recognition characteristics of the dA±dT system.1±6 Thus, it
was suggested that the n2Ad±dT base pair makes a similar
contribution to the DNA duplex stability as the tridentate
dG±dC pair. Surprisingly, the Tm-values of DNAs incorpo-
rating compound 1 are not increasing linearly by an
increasing number of n2A residues.7±13 Also, they are
generally lower than expected for a tridendate base pair.
This was veri®ed on synthetic polynucleotides as well as
on naturally occurring S-2L DNA. This DNAÐcontaining
70% dG±dC and 30% n2Ad±dTÐshows a similar Tm-value
as an oligomer incorporating dA in place of n2Ad.3,14±16
According to these discrepancies, the in¯uence of the
1ÐdT pair has been studied extensively by CD and
NMR spectroscopy as well as by thermodynamic data
analysis.3,11,12,15,17±20
2. Results and discussion
Our laboratory has reported that the duplexes are signi®-
cantly more stable when the purin-2, 6-diamine nucleoside
1 is replaced by the 8-aza-7-deazapurin-2, 6-diamine
nucleoside 2a *Scheme 1) *purine numbering is used
throughout the discussion).21 The base pair of 2a with dT
becomes even more stable when halogeno substituents are
2.1. Synthesis and properties of the monomers
The phosphoramidites of the nucleosides 2a±c have been
described earlier.21,22 The synthesis of the phosphoramidite
of the propynyl compound 2d is now reported *Scheme 2).
The iodo nucleoside 2c was used as starting material for the
synthesis of the propynyl compound which was obtained
by the Pd*0)-catalyzed cross-coupling reaction.27,28 After-
wards, 2d was protected to give the N,N0-bis-isobutyryl
derivative 7.29 Further conversion afforded the DMT
Keywords: pyrazolo[3,4]pyrimidine; purin-2,6-diamine; nucleosides;
oligonucleotides; major groove substituents; base pair stability.
p
Corresponding author. Tel.: 149-541-9692791; fax: 149-541-9692370;
e-mail: frank.seela@uni-osnabrueck.de
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020*02)00406-4

4536
J. He, F. Seela / Tetrahedron 58 12002) 4535±4542
Scheme 1. Nucleosides.
compound 8.21 Phosphitylation gave the phosphoramidite 9,
which was then employed in solid-phase oligonucleotide
synthesis. The 13C NMR data of the monomers are
summarized in Table 1. For other details, see Section 4.
2.2. The stability of duplexes incorporating the base
pairs of 2-amino-20-deoxyadenosine *1) or of the 8-aza-7-
deazapurin-2,6-diamine 20-deoxyribonucleoside *2a)
with dT
It has been reported that 2-amino-20-deoxyadenosine *1)
contributes very little to the DNA±DNA or DNA±RNA
duplex stability.30 Six incorporations of the nucleoside 121
into the duplex 10´11 increase the Tm value by only 38C
*!duplex 12´13) which corresponds to a 10.58C Tm
increase per 1±dT base pair *Table 2). From this obser-
vation, it is obvious that the presumed tridentate base pair
I *Scheme 3) is rather weak or even not formed. However,
when the nitrogen-7 of compound 1 is shifted to position-8
*2a), six incorporations of 2a result in a Tm increase of
1108C, which averages to 11.78C per 2a±dT base pair
*!duplex 14´15, bp IIa, Scheme 3). This indicates that the
altered nitrogen pattern of the pyrazolo[3,4-d]pyrimidine
system causes a duplex stabilization.
In order to elucidate the role of the 2-amino group of
the 7-substituted pyrazolo[3,4-d]pyrimidin-2,6-diamine
20-deoxynucleoside 2a±d,22 the related `20-deoxyadeno-
sine' derivatives 4a±c25 lacking the 2-amino group were
incorporated in oligonucleotide duplexes at exactly the
same positions *Table 2). The replacement of one or several
dA-residues by compound 4a *!duplex 24´25, bp IVa,
Scheme 4) has no in¯uence on the Tm values even in the
case of six incorporations.26 The Tm value of 24´25 is only
38C lower than that of 12´13 with six incorporations of
nucleoside 1, but 108C lower than that of the duplex 14´15
Scheme 2. *a) Me3SiCl, isobutyric anhydride, pyridine, room temperature,
*b) DMT-Cl, pyridine, room temperature, *c) *NCCH2CH2O)*iPr)2NPCl,
*iPr)2EtN, CH2Cl2.

J. He, F. Seela / Tetrahedron 58 12002) 4535±4542
Table 1. C NMR chemical shifts of pyrazolo[3,4-d]-pyrimidin-4,6-diamine 20-deoxyribonucleosides and derivativesa
4537
CH3
13
C*2)a,b C*6)b,c
C*4)a,b C*7a)b,c
C*5)a C*3a)c
C*6)a,b C*4)b,c
C*7)a C*3)c
CuC
2a21
2c22
2d
7
156.9
157.0
157.8156.7
155.5
153.6
158.3
157.6
95.6
153.6
155.5
95.5
91.2
162.7
104.6
104.5
162.7
162.2
127.3
155.5
157.9
133.3
98.3
72.0, 91.5
129.2
126.4
4.2
72.5, 91.0
72.7, 90.84.2
4.2
8
C*10)
C*20)
C*30)
C*40)
C*50)
CvO
2a21
2c22
2d
7
83.3
83.1
83.0
83.5
83.5
38.0
37.6
37.6
37.5
37.9
71.3
70.9
71.0
70.8
70.5
87.4
87.3
87.3
87.6
85.5
62.7
62.4
62.5
62.5
64.1
175.2,176.0
175.2,175.8
8
Measured in *D6) DMSO at 298K.
Purine numbering.
a
b
Tentative.
Systematic numbering.
c
containing 2a. This means that the interchange of N-7 and
0
nucleosides 2a±c exhibit 10±188C higher Tm values than
those incorporating compounds 4a±c. Only a tridentate
base pair of 2a±d with dT can explain this ®nding, while
such a base pair is rather weak in the case of 1±dT.
C-8of 2 -deoxyadenosine exerts no in¯uence on the base
pair stability of dA±dT pair, a ®nding which is different
from the effect of the 2-amino group-containing nucleosides
1 vs 2a.
In order to evaluate the contribution of the 2-amino group
separately, the effect of the nucleoside 3 containing the
2-amino group as the only base substituent was studied
*bp III, Scheme 4). Here, a Tm decrease of 258C *22´23,
428C) *Table 2) was observed compared to compound 4a
carrying a 6-amino group *24´25).26 This phenomenon
indicates that the presence of a 2-amino group is sterically
unfavorable in compound 3. The incorporation of a
2-aminopurine instead of an adenine moiety results in
only a slight increase in Tm. However, when a 2a±dT base
pair is formed both amino groups of the 8-aza-7-deazapurin-
2,6-diamine 2a are able to form hydrogen bonds not
observed in the case of the purin-2,6-diamine nucleoside 1.
When H-7 of compound 4a is substituted by a bromo or iodo
substituent, the Tm increase of the duplex is in the same
range as with 2a *1188C for 2b, and 1158C for 2c
compared to 2a); the 7-propynyl residue of 2d increases
the Tm value by 188C *Table 2). The incorporation of 4b
and 4c enhances the Tm values by 10 and 118C compared to
4a. This indicates that the 7-substituents have a positive
effect in both series of pyrazolo[3,4-d]pyrimidine nucleo-
sides, no matter whether they carry a 2-amino function
or not. A similar phenomenon is observed in the series
of pyrrolo[2,3-d]pyrimidine nucleosides.31±33 However, a
comparison of the two series of oligonucleotide duplexes
shows that those containing the 2-amino functionalized
Table 2. Tm values of duplexes containing six modi®ed nucleoside residues
*1±4)
Duplex
Tm *8C)
47
50-d*TAG GTC AAT ACT) *10)
30-d*ATC CAG TTA TGA) *11)
50-d*T1G GTC 11T 1CT) *12)21
30-d*ATC C1G TT1 TGA) *13)
50-d*T2aG GTC 2a2aT 2aCT) *14)
30-d*ATC C2aG TT2a TGA) *15)
50-d*T2bG GTC 2b2bT 2bCT) *16)22
30-d*ATC C2bG TT2b TGA) *17)
50-d*T2cG GTC 2c2cT 2cCT) *18)
30-d*ATC C2cGT T2c TGA) *19)
50-d*T2dG GTC 2d2dT 2dCT) *20)
30-d*ATCC2d G TT2d T GA) *21)
50
57
75
72
75
50-d*T3G GTC 33T 3CT) *22)25
30-d*ATC C3G TT3 TGA) *23)
50-d*T4aG GTC 4a4aT 4aCT) *24)
30-d*ATC C4aG TT4a TGA) *25)
50-d*T4bG GTC 4b4bT 4bCT) *26)
30-d*ATC C4bG TT4b TGA) *27)
50-d*T4cG GTC 4c4cT 4cCT) *28)
30-d*ATC C4cGT T4c TGA) *29)
42
47
57
58
Measured at 260 nm in 100 mM NaCl, 10 mM MgCl2, and 10 mM
Na-cacodylate *pH 7.0) with 515 mM oligonucleotide concentration.
Scheme 3. The base pairs I and II.

4538
J. He, F. Seela / Tetrahedron 58 12002) 4535±4542
Figure 1. Graph of the DTm vs. the number of nucleoside 2d incorporations.
*a) 50-d*TAG GTC 2dAT ACT)´30-*ATC CAG TTA TGA), *b) 50-d*TAG
GTC AAT ACT)´30-*ATC C2dG TT2d TGA), *c) 50-d*TAG GTC 2dAT
ACT)´30-*ATC C2dG TT2d TGA), *d) 35´21, *e) 20´21.
Scheme 4. The base pairs III and IV.
two amino groups. As a result, they are now both in plane
with the heterocycle and in a more favorable situation for
base pairing and/or stacking. The amide character of the
amino groups is also in line with the ease of the removal
of amino protecting groups *acyl groups). They are much
more dif®cult to be split off from the purin-2,6-diamine
nucleoside 121,22 than from 2a±d.21,22,34,35
The reason for the different behavior of 2-amino groups
present in the purine nucleoside 1 and the pyrazolo[3,4-d]-
pyrimidine nucleosides 2a±d results from the differences
of steric and electronic properties of the nucleobases. The
2-amino group of compound 1 is out of plane of the hetero-
cyclic system.16 Moreover, this group which is located in the
minor groove of B-DNA can cause steric strain onto the
helix in the case of multiple incorporations. As a result,
the formation of the tridentate base pair is problematic.
These unfavorable properties are overcome by the use of
the pyrazolo[3,4-d]pyrimidine nucleosides 2a±d. The
presence of nitrogen-8allows the formation of mesomeric
structures *Scheme 5) increasing the amide character of the
The contribution of the 7-propynyl substituent of 2d to the
duplex stability goes along with the formation of tridentate
base pair with dT as found for 2b, c *Table 2).22 Each
incorporation of a 2b±dT, 2c±dT or 2d±dT base pair
contributes 13.58C *bp IIb, Scheme 3), 13.88C *bp IIc),
or 148C *bp IId) to the Tm increase *six incorporations!
duplexes 16´17, 18´19, and 20´21) *Table 2). The
Scheme 5. Mesomeric structures.
Table 3. Tm values of duplexes containing four 2a±d/dT base pairs in place of dG±dC
0
DG310 *kcal/mol)
0
DDG310 *kcal/mol)
Duplex
Tm *8C)
51
DH0 *kcal/mol)
DS0 *cal/mol K)
2279
50-d*TAG GTC 2a2aT ACT) *30)22
30-d*ATC C2aG TT2a TGA) *15)
50-d*TAG GCC GGC ACT) *31)
30-d*ATC CGG CCG TGA) *32)
299
212.3
66
n.d.
n.d.
n.d.
50-d*TAG GTC 2b2bT ACT) *33)
30-d*ATC C2bG TT2b TGA) *17)
50-d*TAG GTC 2c2cT ACT) *34)
30-d*ATC C2cGT T2c TGA) *19)
50-d*TAG GTC 2d2dT ACT) *35)
30-d*ATC C2dG TT2d TGA) *21)
67
66
68
2105
2104
2111
2285
2285
2300
217.0
216.6
218.0
24.7
24.3
25.7
Conditions see Table 2. Thermodynamic parameters are calculated from the program Meltwin 3.0; the standard error for DH0 and DS0 is 15%; n.d.ˆnot
detected.

J. He, F. Seela / Tetrahedron 58 12002) 4535±4542
4539
Table 4. Tm values of duplex formation of oligonucleotides containing the
5a±c±dC base pairs
2.3. The stability of the base pairs between 8-aza-7-
deaza-20-deoxyguanosine *5a) and its 7-substituted
derivatives 5b±c with dC
Duplex
Tm *8C)
47
50-d*TAG GTC AAT ACT) *10)
30-d*ATC CAG TTA TGA) *11)
50-d*TA5a 5aTC AAT ACT) *36)25
30-d*ATC CA5a TTA T5aA) *37)
50-d*TA5b 5bTC AAT ACT) *38)
30-d*ATC CA5b TTA T5bA) *39)
50-d*TA5c 5cTC AAT ACT) *40)
30-d*ATC CA5c TTA T5cA) *41)
The stabilizing effects of the 8-aza-7-deazapurine nucleo-
sides 2a±d and 4a±c prompted us to undertake similar
studies on duplexes containing the 7-substituted pyra-
zolo[3,4-d]pyrimidine analogues of 20-deoxyguanosine
*5a±c).25 The Tm values of duplex melting are listed in
Table 4. Contrary to the 4a±dT base pair, the introduction
of the 5a±dC base pair *bp Va±c in Scheme 6) has a
positive in¯uence on the duplex stability *!36´37) when
compared to that of dG±dC. An averaged Tm increase of
10.758C per base pair modi®cation is observed *Table 4).
This means that the shift of nitrogen-7 to position-8
strengthens the tridentate base pairs as it is observed for
the 2a±dT pair. Apparently, in both cases, the 2-amino
group becomes a better proton donor thereby increasing
the base pair stability.
50
55
54
Conditions see Table 2.
2.4. The duplex stability increase induced by the
pyrimidine nucleosides 6a±d vs the `purine' nucleoside
4a±c
The substituents at the 5-position of pyrimidine nucleosides
6a±d protrude into the major groove of B-DNA as it is
found for the 7-substituents of the 8-aza-7-deazapurine
nucleosides 4a±c *bp VIa±d vs IVa±c, Schemes 4 and
6). Thus, it was expected that the hydrophobic character
of the major groove would be increased in both series of
compounds. However, the Tm data of Table 5 clearly show
that the Tm increase caused by one incorporation is signi®-
cantly smaller for 6a±c *0±0.758C) than for 4a±c *0.75±
1.758C).36,37 Therefore, other factors have to be taken into
account. From the shape of the nucleobases it is obvious that
the overlap of a `purine' moiety is larger than for a pyrimi-
dine base. Moreover, it was shown that the polarizibility38
is related to the Tm-increase. The polarizabilities of
the 5-substituted pyrimidine bases are 9.91£10224 for 6a,
12.99£10224 cm3 for 6b, 15.32£10224 cm3 for 6c, 14.92£
10224 cm3 for 6d,39 while those values for the 7-substituted
dA-derivatives are 14.68£10224 cm3 for 4a, 17.73£
10224 cm3 for 4b, and 19.79£10224 cm3 for 4c. The corre-
sponding values for the diamino compounds 2a±d are
16.36£10224 cm3 for 2a, 19.41£10224 cm3 for 2b, 21.47£
10224 cm3 for 2c and 20.12£10224 cm3 for 2d). This indi-
cates that a high polarizibility goes along with a high
Tm-value. Apart from that, the substitution at the 7-position
of the nucleobases induces a stereoelectronic effect on the
sugar moiety which can alter its conformation, making the
Scheme 6. The base pairs V and VI.
replacement of four dA±dT pairs of the duplex 10´11 by
either four 2b±dT, four 2c±dT or four 2d±dT pairs
*duplexes 33´17, 34´19, 35´21, bp IIb±d, Scheme 3)
enhances the Tm values to that of the duplex 31´32 contain-
ing eight tridentate dG±dC pair *Table 3). In other words,
these pyrazolo-[3,4-d]pyrimidine±dT base pairs reach the
stability of the dG±dC pair.19 Also, a linear relationship
between the number of 2d-incorporations vs the DTm is
observed as it is found for dG±dC *Fig. 1).
Table 5. Tm values of duplex formulation of oligonucleotides containing nucleosides 6a±d
0
DDG310 *kcal/mol)
Duplex
Tm *8C)
46
DH0 *kcal/mol)
286
DS0 *cal/mol K)
2245
DG3100 *kcal/mol)
210.3
50-d*TAG G6aC AA6a ACT) *42)22
30-d*ATC CAG 6a6aA TGA) *43)
50-d*TAG G6bC AA6b ACT) *44)
30-d*ATC CAG 6b6bA TGA) *45)
50-d*TAG G6cC AA6c ACT) *46)
30-d*ATC CAG 6c6cA TGA) *47)
50-d*TAG G6dC AA6d ACT) *48)
30-d*ATC CAG 6d6dA TGA) *49)
49
294
2268
211.3
21.0
21.6
21.7
50
299
2282
211.9
52
289
2247
212.0
Conditions see Table 2.

4540
J. He, F. Seela / Tetrahedron 58 12002) 4535±4542
nucleosides more or less favorable for base stacking inter-
actions. The high-anti conformation of the pyrazolo[3,4-d]-
pyrimidine nucleosides demonstrated by X-ray data
analyses40 might also play a role in the stability of
those base pairs. However, it is yet not known how these
phenomena effect base stacking in detail.41
with a ¯ow rate of 1.0 ml/min. *A) 0.1 M *Et3NH)OAc *pH
7.0)/MeCN 95:5; *B) MeCN. Then the mixture was
evaporated to dryness and the residue was treated with
2.5% CHCl2COOH/CH2Cl2 for 5 min at room temperature
to remove the 4,40-dimethoxytrityl residues. The detrityl-
ated oligomers were puri®ed by reversed-phase HPLC
with the gradient: 20 min 0±20% B in A with a ¯ow rate
of 1 ml/min. The oligomers were desalted *RP-18, silica
gel) and lyophilized on a Speed-Vac evaporator to yield
colorless solids which were frozen at 2248.
Next, the thermodynamic data *DH0, DS0, DG0) of the
duplex formation were calculated using the program
Meltwin 3.0.42 The free energies correspond to the
Tm-values. The entropy change is always unfavorable during
duplex formation. By comparing the thermodynamic
parameters of duplexes formed by the halogenated nucleo-
sides 2b, c with that of the propynyl compound 2d, the latter
shows a more favorable enthalpy change. This might result
from the more lipophilic character of the propynyl group
and/or the conjugation of the triple bond with the hetero-
cyclic base. The latter will enlarge the total surface area of
the heterocycle. This mesomeric effect is not possible with
the bromo or iodo substituents.
The oligonucleotides were characterized by: *i) enzymatic
hydrolysis as described21 with snake-venom phospho-
diesterase *EC 3.1.15.1, Crotallus adamenteus) and alkaline
phosphatase *EC 3.1.3.1, E. coli from Roche Diagnostics
GmbH, Germany) and *ii) by MALDI-TOF-spectra
measured on a Bi¯ex III spectrometer *Bruker Saxonia,
Leipzig, Germany). The melting temperatures were
measured with a Cary-1/3 UV/VIS spectrophotometer
*Varian, Australia) equipped with a Cary thermo electrical
controller.
4.1.1.
1-*2-Deoxy-b-d-erythro-pentofuranosyl)-3-pro-
*2d).
3. Conclusion
pynyl-1H-pyrazolo[3,4-d]pyrimidin-4,6-diamine
To a solution of compound 2c *0.6 g, 1.54 mmol)22 in
anhydrous DMF *3.2 ml) which was kept under Ar atmos-
phere. CuI *66 mg, 0.36 mmol), tetrakis*triphenyl-phos-
phine)palladium*0) *207 mg, 0.29 mmol), and anhydrous
Et3N *0.77 ml, 5.5 mmol) were added. The reaction mixture
was cooled *ice-water bath) and saturated with propyne
*30 min). After stirring for 24 h at room temperature, a
second portion of the reagents *same amounts) was added
to the mixture. The propyne treatment was repeated for
another 30 min under cooling and the reaction was kept
for another 24 h at room temperature. Then, the mixture
was adsorbed on silica gel *25 g) and applied to FC
*CH2Cl2/MeOH 20:1). A white solid *0.43 g, 92%) was
obtained after evaporation. Rf *CH2Cl2/MeOH 9:1) 0.38.
The replacement of the purine nucleoside 2-amino-20-
deoxyadenosine *1) by the pyrazolo[3,4-d]pyrimidine
analogues 2a±d increases the dA±dT base pair stability
extraordinarily because of the formation of tridentate base
pairs and the in¯uence of the 7-substituents. The effect of
stabilization is not observed for the base pair of 2-amino-20-
deoxyadenosine *1) with dT. The stabilizing effects of the
7-substituents do not depend on the particular Watson±
Crick recognition site of the base. The tridentate base
pairs 2b±d±dT reach the stability of the tridentate dG±dC
pair. The contribution of identical 5-substituents of
20-deoxyuridine residues on the base pair stability is
comparably small or can even be neglected.
1
UV *MeOH): lmax 269 *12,000), 289 *8400). H NMR
**D6)DMSO), 2.12 *s, CH3); 2.14, 2.67 *2m, H2±C*20));
3.43 *m, H2±C*50)); 3.76 *m, H±C*40)); 4.36 *m,
H±C*30)); 4.77 *t, Jˆ5.7 Hz, OH±C*50)); 5.20 *d, Jˆ
4.3 Hz, OH±C*30)); 6.28*s, NH 2); 6.32 *t, Jˆ6.5 Hz,
H±C*10)). Anal. calcd for C13H16N6O3 *304.21): C 51.32,
H 5.30, N 27.63; found: C 51.28, H 5.16, N 27.55.
4. Experimental
4.1. General
Monomers. Thin-layer chromatography *TLC) was
performed on aluminum sheets, silica gel 60 F254 *0.2 mm,
Merck, Germany). Flash chromatography *FC) was carried
out *0.4 bar) on silica gel 60 H *Merck, Darmstadt,
Germany). NMR Spectra were measured on an Avance
-DPX-250 spectrometer *Bruker, Germany), at 250.13
4.1.2. 1-*2-Deoxy-b-d-erythro-pentofuranosyl)-4,6-diiso-
butyrylamino-3-propynyl-1H-pyrazolo[3,4-d]pyrimidine
*7). Compound 2d *0.64 g, 2.1 mmol) was co-evaporated
three times with anhydrous pyridine and then dissolved in
anhydrous pyridine *10 ml), followed by addition of
Me3SiCl *1.34 ml, 10.6 mmol). After 15 min, isobutyric
anhydride *1.75 ml, 7.5 mmol) was added to the solution.
After 3 h the reaction mixture was cooled in an ice-bath and
diluted with H2O *5.7 ml); 5 min later aq. ammonia *25%,
5.7 ml) was added and the stirring was continued for another
30 min. The reaction mixture was evaporated to dryness, the
residue was puri®ed by FC *CH2Cl2/MeOH 20:1) furnishing
a colorless solid *0.46 g, 49%); Rf *CH2Cl2/MeOH 9:1) 0.40.
1
MHz for H and 125.13 MHz for 13C, d values are in ppm
relative to internal SiMe4 *1H, 13C) or external H3PO4.
Microanalyses were performed by Mikroanalytisches
È
Labor Beller *Gottingen, Germany). Chemicals were
purchased from ACROS, Fluka, or Sigma-Aldrich. Solvents
of technical grade were distilled before use.
Oligonucleotides. The oligonucleotides were synthesized on
an ABI 392-08synthesizer in the trityl-on mode. The syn-
theses followed the standard protocol. Complete deprotec-
tion was conducted in 25% aq. NH3 for 12±18h at 60 8C.
After deprotection the DMT-containing oligonucleotides
were puri®ed by reversed-phase HPLC *RP-18); the
gradients are: 3 min 20% B in A, 12 min 20±40% B in A
1
UV *MeOH): lmax 241 *32,200), 280 *13,500). H NMR
**D6)DMSO), 1.14 *m, CH*CH3)2); 2.07 *s, CH3); 2.28,
2.76 *2m, H2±C*20)); 2.85 *m, CH*CH3)2); 3.48*m,
H2±C*50)); 3.82 *m, H±C*40)); 4.42 *m, H±C*30)); 4.72

J. He, F. Seela / Tetrahedron 58 12002) 4535±4542
4541
*m, OH±C*50)); 5.31 *d, Jˆ4.3 Hz, OH±C*30)); 6.56 *t,
Jˆ6.4 Hz, H±C*10)); 10.49, 10.60 *2s, NHCO). Anal.
calcd for C21H28N6O5 *444.39): C 56.76, H 6.31, N 18.92;
found: C 56.66, H 6.46, N 18.82.
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4.1.3. 1-[2-Deoxy-5-O-*4,40-dimethoxytriphenylmethyl)-
b-d-erythro-pentofuranosyl]-4,6-diisobutyrylamino-3-
propynyl-1H-pyrazolo[3,4-d]pyrimidine *8). Compound
7 *0.42 g, 0.94 mmol) was co-evaporated with anhydrous
pyridine *three times) and then dissolved in pyridine
*1.5 ml). To this solution DMT-Cl *0.48g, 1.41 mmol)
was added, and the mixture was stirred at room temperature
for 3 h. The reaction was quenched by addition of MeOH,
and the mixture was evaporated to dryness. It was dissolved
in CH2Cl2 *5 ml) and subjected to FC *CH2Cl2/MeOH 20:1)
to give a colorless foam *0.6 g, 85%). Rf *CH2Cl2/MeOH
9:1) 0.7. UV *MeOH): lmax 237 *48,800), 282 *13,100). 1H
NMR **D6)DMSO), 1.15 *m, CH*CH3)2); 2.07 *s, CH3);
2.30, 2.77 *2m, H2±C*20)); 2.85±3.06 *m, CH*CH3)2,
H2±C*50)); 3.71 *s, CH3O); 3.93 *m, H±C*40)); 4.49 *m,
H±C*30)); 5.34 *d, Jˆ4.7 Hz, OH±C*30)); 6.57 *m,
H±C*10)); 6.75±7.33 *m, arom. H); 10.45, 10.63 *s,
NHCO). Anal. calcd for C42H46N6O7 *746.23): C 67.56, H
6.17, N 11.26; found: C 67.64, H 6.30, N 10.96.
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1
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We thank Ms E. Feiling for the synthesis of oligonucleo-
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meinschaft and the Roche Diagnostics GmbH is gratefully
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