Oligonucleotide duplexes containing N8-glycosylated 8-aza-7-deazaguanine and self-assembly of 8-aza-7-deazapurines on the nucleoside and the oligomeric level.

Article abstract of DOI:10.1039/b309485p

The 8-aza-7-deazaguanine N8-(2'-deoxy-beta-D-ribofuranoside) (1) was synthesized, converted into the phosphoramidite 4 and incorporated into oligonucleotides. Nucleoside 1 forms stable base pairs with 2'-deoxy-5-methylisocytidine in DNA with antiparallel chain orientation (aps) and with 2'-deoxycytidine in duplexes with parallel chains (ps). According to the CD spectra self-complementary oligonucleotides d(1-m5isoC)3 and d(1-C), form autonomous DNA-structures. Neither the nucleoside 1 nor the regularly linked 8-aza-7-deaza-2'-deoxyguanosine form G-like tetrads while the regularly linked 8-aza-7-deaza-2'-deoxyisoguanosine gives higher molecular assemblies which are destroyed by bulky 7-bromo substituents. This was verified on monomeric nucleosides by ESI-MS spectrometry and on oligonucleotides by HPLC analysis.

Full text of DOI:10.1039/b309485p

View Article Online / Journal Homepage / Table of Contents for this issue
Oligonucleotide duplexes containing N8-glycosylated 8-aza-7-
deazaguanine and self-assembly of 8-aza-7-deazapurines on the
nucleoside and the oligomeric level†
Frank Seela* and Rita Kröschel
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie,
Fachbereich Biologie/Chemie, Universität Osnabrück, Barbarastr. 7, D-49069 Osnabrück,
Germany in Cooperation with the Center for Nanotechnology (CeNTech),
Gievenbecker Weg 11, 48149 Münster, Germany. E-mail: Frank.Seela@uni-osnabrueck.de
Received 7th August 2003, Accepted 17th September 2003
First published as an Advance Article on the web 13th October 2003
The 8-aza-7-deazaguanine N 8-(2Ј-deoxy-β--ribofuranoside) (1) was synthesized, converted into the
phosphoramidite 4 and incorporated into oligonucleotides. Nucleoside 1 forms stable base pairs with 2Ј-deoxy-5-
methylisocytidine in DNA with antiparallel chain orientation (aps) and with 2Ј-deoxycytidine in duplexes with
parallel chains (ps). According to the CD spectra self-complementary oligonucleotides d(1-m5isoC)3 and d(1-C)3
form autonomous DNA-structures. Neither the nucleoside 1 nor the regularly linked 8-aza-7-deaza-2Ј-deoxy-
guanosine form G-like tetrads while the regularly linked 8-aza-7-deaza-2Ј-deoxyisoguanosine gives higher molecular
assemblies which are destroyed by bulky 7-bromo substituents. This was verified on monomeric nucleosides by
ESI-MS spectrometry and on oligonucleotides by HPLC analysis.
Introduction
Nucleosides with unusual glycosylation positions are naturally
occurring; pseudouridine is found in tRNA,1 the N7-glyco-
sylated α--ribofuranoside of adenine is a constituent of
vitamin B12. Various others have been isolated as side products
of glycosylation reactions.2 Oligonucleotides incorporating
nucleosides with unusual linkages between the nucleobases and
the sugar phosphate backbone show unexpected base pairing
properties.3 In order to extend our knowledge of the recog-
nition motifs formed by non-natural bases mimicking the shape
of natural ones we have already investigated the base pairing
properties of 8-aza-7-deazapurine nucleosides related to the
Scheme 1 Unusually linked 2Ј-deoxyribonucleosides.
canonical DNA constituents.4 This led us to duplex structures
with enhanced duplex stability and other favorable properties.
For most of the functions of DNA the nucleobases differ in
their structure and therefore in their recognition properties
while the sugar phosphate backbone remains unchanged. A
compares them with oligonucleotides containing the regularly
linked molecules. Also the possible formation of higher molecu-
lar assemblies incorporating the nucleosides 1, 5–7 (Scheme 2)
will be reported. For identification ESI-mass spectrometry is
wider view of possible biopolymers resembling the character-
used on the monomeric nucleosides and ion exchange HPLC on
istics of a DNA molecule is possible by changing the hetero-
the oligonucleotides.
cyclic moieties of the nucleobases, their substituent pattern
and/or their arrangement within the DNA-duplex. This can
lead to novel DNA-like molecules suitable for various appli-
cations in molecular biology and nanotechnology.
As the pyrazolo[3,4-d]pyrimidine (8-aza-7-deazapurine)
nucleobases contain a nitrogen at position-8, they became
targets for nucleosides with unusual glycosylation sites. Earlier,
we have shown that the 8-aza-7-deazaadenine N 8-(2Ј-deoxy-
ribofuranoside) (2, Scheme 1) or its 2-amino derivative develop
base pairing ambiguity against the four canonical DNA con-
stituents.5,6 The 7-deazaguanine C8-(2Ј-deoxyribofuranoside)
(3) forms stable base pairs in duplexes mimicking the properties
of 2Ј-deoxyisoguanosine.7
Scheme 2 Regularly linked 8-aza-7-deazapurine 2Ј-deoxyribonucleo-
This manuscript reports on the synthesis and the base pairing
of the 8-aza-7-deazaguanine N 8-(2Ј-deoxy-β--ribofuranoside)
1 which was converted into the phosphoramidite 4 and
incorporated in oligonucleotides. The investigation includes
duplexes with antiparallel and parallel chain orientation and
sides.
Results and discussion
1. Monomer synthesis and oligonucleotides containing N8-
glycosylated 8-aza-7-deazaguanine (1)
† Electronic supplementary information (ESI) available: expanded ver-
sions of Tables 2–5 and figure showing HPLC profiles of the enzymatic
hydrolysis of oligonucleosides 5Ј-d(1-C)3 and 5Ј-d(TAG 1 TC AAT
ACT). See http://www.rsc.org/suppdata/ob/b3/b309485p/
Earlier, the 8-aza-7-deazaguanine N 8-(2Ј-deoxy-β--ribo-
furanoside) (1) was synthesized from 2-amino-6-methoxy-8-
aza-7-deazapurine 2Ј-deoxyribofuranoside (8) by treatment
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 0 – 3 9 0 8
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
3900

View Article Online
Table 1 13C NMR Chemical shifts of 8-aza-7-deazapurine nucleosides (d6-DMSO, 25 ЊC)
C(3)a
C(7)b
C(3a)a
C(5)b
C(4)a
C(6)b
C(6)a
C(2)b
C(7a)a
C(4)b
Compound
C(1Ј)
C(2Ј)
C(3Ј)
C(4Ј)
C(5Ј)
HC᎐N
᎐
19
59
611
9
10
11
12
13
14
127.8
134.9
134.7
128.0
128.1
136.0
138.1
128.3
135.3
102.2
99.7
92.6
104.1
104.4
92.9
93.3
102.5
99.8
159.3d
157.4d
157.0d
160.2d
160.2d
157.4d
156.7d
159.3d
157.7d
153.5d
154.6d
155.2d
158.1d
158.0d
155.3d
154.2d
153.5d
154.9d
160.7
89.7
83.1
84.3
89.9
89.2
83.7
82.9
89.5
82.9
40.0
70.5
71.0
71.2
70.6
70.4
73.5
72.2
72.2
72.6
88.1
87.3
88.2
88.1
85.6
87.6
86.7
87.4
86.8
61.9
62.4
62.5
62.0
64.1
64.2
62.9
63.0
63.3
155.2
37.9
e
c
160.0d
39.8
158.0
157.9
160.1d
c
e
c
e
37.4
160.8d
155.7d
c
37.5
a Systematic numbering. b Purine numbering. c Superimposed by DMSO. d Tentative assignment. e Not detected.
with aqueous potassium hydroxide8,9 (purine numbering is used
throughout this manuscript). For solid-phase oligonucleotide
synthesis the phosphoramidite 4 was prepared. The amino
group of compound 1 was protected with a dimethylamino-
methylidene residue leading to compound 9 (Scheme 3). Regard-
ing oligonucleotide synthesis, the stability of the protecting
group was investigated and kinetic measurements were per-
formed in 25% aqueous ammonia solution at 40 ЊC. The reac-
tion was monitored UV-spectrophotometrically. The deter-
mined half life value for compound 9 was 5 minutes which is
suitable for further manipulations. Compound 9 was converted
into the 5Ј-O-(dimethoxytriphenylmethyl) protected (DMT)
derivative 10 employing standard conditions.10 Finally, the
phosphoramidite 4 was prepared from 10 using chloro(2-cyano-
ethoxy)(diisopropylamino)-phosphine in the presence of
N-ethyldiisopropylamine. All compounds were characterized
glycosylation position of the corresponding N8-derivatives 9, 10
and 13 was established.8
The oligonucleotides were prepared by solid-phase synthesis
on a 392 DNA-synthesizer (Applied Biosystems, Weiterstadt,
Germany) using the standard protocol of phosphoramidite
chemistry.12 The coupling efficiency of the modified phos-
phoramidite 4 was always higher than 98%. The nucleoside
composition of the oligonucleotides was proven by MALDI-
TOF mass spectrometry ( Table 7) as well as by enzymatic
hydrolysis using snake-venom phosphodiesterase followed by
alkaline phosphatase and RP-HPLC.
1.1. Non-self-complementary oligonucleotide duplexes with
antiparallel or parallel chain orientation. In order to investigate
the base pairing properties of the nucleoside 1 it was incorpor-
ated in the antiparallel duplex 5Ј-d(TAGGTCAATACT)ؒ3Ј-
d(ATCCAGTTATGA) (15ؒ16) replacing dG residues in one or
both strands. As we have recognized from experiments per-
formed on the related 7-deazaguanine C8-nucleoside 37 that a
change of the glycosylation site from nitrogen-9 to position-8
resulted in a change of the nucleobase recognition pattern
(guanine behaves like isoguanine), oligonucleotide duplexes
were constructed in which compound 1 was located opposite to
isoCd. Because of the low stability of isoCd under alkaline con-
ditions the 5-methyl derivative was used instead.13 From Table 2
it is apparent that the incorporation of compound 1 opposite to
m5isoCd results in duplexes which are only slightly less stable
than those containing dG–dC pairs (15ؒ16), while incorpor-
ation of compound 1 opposite to dC leads to a strong destabil-
ization. Thus compound 1 shows the base pairing properties as
iGd. Evidently, the change of the glycosylation site from N9 to
N8 (5 vs. 1) leads to a similar change of the base pair recog-
nition as the interchange of the substituents of the base moiety
1
by H and 13C NMR spectroscopy (see Table 1 and experi-
mental part) as well as by elemental analysis. As it has been
shown earlier that the change of the glycosylation position
from N9 to N8 results in a significant downfield shift of
C(5), C(6) and C(4) and in an upfield shift at C(7), the
(dG
iGd).
With regard to Tm-data of oligonucleotide duplexes contain-
ing 2Ј-deoxyisoguanosine as reference, the incorporation of one
nucleoside 1-residue shows a reduction of the Tm value of 3 ЊC
for 23ؒ18 and 6 ЊC for 19ؒ24, compared to the isoguanine con-
taining duplexes 17ؒ18 and 19ؒ20, demonstrating that the
duplex stability depends on the nearest neighbours. Incorpor-
ation of four modified nucleoside residues leads to a ∆Tm of
Ϫ5 ЊC per modification (25ؒ26). Oligonucleotide duplexes
of the same length containing an abasic site—which neither
develop stacking interactions nor hydrogen bonding—were
shown to induce a much stronger Tm-decrease (∆Tm = Ϫ15 ЊC
per modification)5 than the change of the glycosylation site
from base position-9 to position-8. This indicates that the
incorporation of compound 1 stabilizes duplex DNA by base
stacking and the formation of a tridentate base pair according
to the motif I (see duplexes 23ؒ18 and 19ؒ24). The motif I is
related to the already published motif II7 and shows also
relationships to motif III with regard to the formation of a base
pair with m5isoCd. The motifs I–III are all of that of a tri-
dentate base pair similar to the canonical motif IV (Scheme 4).
Scheme 3 Protecting group strategy for the synthesis of the phos-
phoramidite 4. Reagents and conditions: (i) N,N-dimethylformamide
dimethylacetal–methanol; rt; 48 h. (ii) DMT-Cl–pyridine; rt; 4.5 h.
(iii) chloro(2-cyanoethoxy)(diisopropylamino)phosphine–iPr2EtN; rt;
30 min.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 0 – 3 9 0 8
3901

View Article Online
Table 2 Tm Values and thermodynamic data of non-self-comple-
mentary antiparallel-stranded oligonucleotide duplexes containing
N8-linked 8-aza-7-deaza-2Ј-deoxyguanosine (1) hybridized against
d(iC) and dCa, b
Table 3 Tm Values and thermodynamic data of non-self-comple-
mentary antiparallel-stranded oligonucleotide duplexes containing
N8-linked 8-aza-7-deaza-2Ј-deoxyguanosine (1) hybridized against
dG, 1, dT and dAa
Duplex
Compound no.
Tm/ЊC
Duplex
Compound no.
Tm/ЊC
5Ј-d(TAG GTC AAT ACT)
3Ј-d(ATC CAG TTA TGA)
5Ј-d(TAG iGTC AAT ACT)14
3Ј-d(ATC iCAG TTA TGA)
5Ј-d(TAG GTiC AAT ACT)14
3Ј-d(ATC CAiG TTATGA)
5Ј-d(ATiC iCAiG TTA TiGA)
3Ј-d(TAiG iGTiC AAT AiCT)
15
16
17
18
19
20
21
22
51
54
54
60
5Ј-d(TAG 1 TC AAT ACT)
3Ј-d(ATC GAG TTA TGA)
5Ј-d(TAG 1 TC AAT ACT)
3Ј-d(ATC 1 AG TTA TGA)
5Ј-d(TAG 1 TC AAT ACT)
3Ј-d(ATC T AG TTA TGA)
5Ј-d(TAG 1 TC AAT ACT)
3Ј-d(ATC AAG TTA TGA)
23
27
23
28
23
29
23
30
48
46
44
33
5Ј-d(TAG 1 TC AAT ACT)
3Ј-d(ATC iCAG TTATGA)
5Ј-d(TAG GTiC AAT ACT)
3Ј-d(ATC CA 1 TTA TGA)
5Ј-d(ATiC iCA1 TTA T1A)
3Ј-d(TA 1 1TiC AAT AiCT)
5Ј-d(TAG 1 TC AAT ACT)
3Ј-d(ATC CAG TTA TGA)
5Ј-d(TAG GTCAAT ACT)
3Ј-d(ATC CA1 TTA TGA)
23
18
19
24
25
26
23
16
15
24
48
51
40
36
35
5Ј-d(TAG GTG AAT ACT)
3Ј-d(ATC CA 1 TTA TGA)
5Ј-d(TAG GT1 AAT ACT)
3Ј-d(ATC CA1 TTA TGA)
5Ј-d(TAG GTT AAT ACT)
3Ј-d(ATC CA1 TTA TGA)
5Ј-d(TAG GTA AAT ACT)
3Ј-d(ATC CA1 TTA TGA)
31
24
32
24
33
24
34
24
43
45
41
30
a Measured in 1 M NaCl, 0.1 M MgCl2, 60 mM sodium cacodylate
buffer, pH 7. 5 µM ϩ 5 µM Oligonucleotide concentration.
a Measured in 1 M NaCl, 0.1 M MgCl2, 60 mM sodium cacodylate
buffer, pH 7. 5 µM ϩ 5 µM oligonucleotide concentration. b d(iC) =
m5isoCd = 2Ј-deoxy-5-methylisocytidine.
dT (23ؒ30; 33ؒ24) are surprisingly stable. This observation was
also made on duplexes incorporating those compounds at a
different position of the oligomer. Also rather stable duplexes
are found when compound 1 was facing itself (23ؒ28; 32ؒ24).
Thus compound 1 shows ambiguous base pairing against dG,
dT and 1 but shows base pair discrimination against dA. This
behaviour shows similarities to that of the C8-linked 7-deaza-
guanine 2Ј-deoxyribonucleoside.7
According to the observation that compound 1 forms rather
base pairs with 2Ј-deoxy-5-methylisocytidine in duplexes with
antiparallel chain orientation, it was expected that stable
duplexes with parallel chain orientation are also formed when
compound 1 is incorporated in such duplexes and is located
opposite to dC. Hybridization experiments according to Table 4
were performed with the parallel-stranded isoguanine contain-
ing duplex 35ؒ16.14 Isoguanine was replaced by compound 1
and the four regular nucleosides as well as 2Ј-deoxy-5-methyl-
isocytidine were located on the opposite side in order to investi-
gate the duplex stabilities. Compared to 35ؒ16 the duplex 26ؒ16
shows a Tm decrease of 4.5 ЊC per modification with two con-
secutive 1–dC pairs. Although there is a lower duplex stability
when compound 1 was introduced in the case of non-self-
complementary duplexes—which might be due to a hetero-
geneous DNA structure—still a specific base pairing could be
observed. Nevertheless the duplex 26ؒ16 is significantly more
stable than those incorporating dA, dT and dG opposite to
compound 1, while that of 26ؒ36 still shows a rather high Tm-
value.
1.2 Self-complementary duplexes. Earlier it has been
demonstrated, that duplexes with alternating residues of the
8-aza-7-deazaadenine N 8-(2Ј-deoxyribofuranoside) (2) and dT
are extraordinarily stable with Tm-values higher than those of
oligonucleotides of the same length but incorporating alternat-
ing dA–dT.15 This prompted us to study self-complementary
hexanucleotides with alternating residues of the nucleoside 1
and dC or m5isoCd. From the results shown on self-comple-
mentary duplexes it is very likely that the chain orientation of
this duplex is parallel forming a base pair according to motif V
(Scheme 5). As a consequence base overhangs are expected
to be present at the 3Ј and 5Ј-termini. Interestingly, the duplex
5Ј-d(1-C)3 (37ؒ37) is rather stable and shows a similar Tm to
that of 5Ј-d(iG-C)3 (38ؒ38) ( Table 5). The related duplex
containing pyrazolo[3,4-d]pyrimidine isoguanine analogue
5Ј-d(6-C)3 is more stable while that of the 5Ј-d(40-C)3 contain-
Scheme 4 Base pair motifs of duplexes with antiparallel strand
orientation (aps).
Next, the base pair discrimination of compound 1 against
the canonical DNA-constituents dG, dT or dA was studied
(Table 3). While duplexes with compound 1 opposite to dA are
rather labile, the ones with 1 opposite to dG (23ؒ27; 31ؒ24) and
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 0 – 3 9 0 8
3902

View Article Online
Table 4 Tm Values and thermodynamic data of parallel-stranded
non-self-complementary oligonucleotide duplexes containing N8-linked
8-aza-7-deaza-2Ј-deoxyguanosine (1)a, b
Table 5 Tm Values and thermodynamic data of self-complementary
oligonucleotidesa with parallel and antiparallel chain orientation
Duplex
Compound no.
Tm/ЊC
Duplex
Compound no.
Tm/ЊC
5Ј-d(1-C-1-C-1-C)-3Ј
37
37
38
38
39
39
41
41
42
42
43
43
45
45
47
47
34
33
41
22
37
46
62
37
5Ј-d(TiCA TAA iCTiG iGAT) 13
5Ј-d(AGT ATT GAC CTA)
5Ј-d(TiCA TAA iCT1 1AT)
5Ј-d(AGT ATT GAC CTA)
5Ј-d(TiCA TAA iCT1 1AT)
5Ј-d(AGT ATT GA1 CTA)
5Ј-d(TiCA TAA iCT1 1AT)
5Ј-d(AGT ATT GA G CTA)
5Ј-d(TiCA TAA iCT1 1AT)
5Ј-d(AGT ATT GAT CTA)
5Ј-d(TiCA TAA iCT1 1AT)
5Ј-d(AGT ATT GAA CTA)
5Ј-d(TiCA TAA iCT1 1 AT)
5Ј-d(AGT ATT GAiC CTA)
35
16
26
16
26
36
26
27
26
29
26
30
26
18
45
36
32
23
25
23
28
5Ј-d(1-C-1-C-1-C)-3Ј
5Ј-d(iG-C-iG-C-iG-C)-3Ј 17
5Ј-d(iG-C-iG-C-iG-C)-3Ј
5Ј-d(6-C-6-C-6-C)-3Ј17 b
5Ј-d(6-C-6-C-6-C)-3Ј
5Ј-d(40-C-40-C-40-C)-3Ј17 c
5Ј-d(40-C-40-C-40-C)-3Ј
5Ј-d(1-iC-1-iC-1-iC)-3Ј
3Ј-d(iC-1-iC-1-iC-1)-5Ј
5Ј-d(G-C-G-C-G-C)-3Ј18
3Ј-d(C-G-C-G-C-G)-5Ј
5Ј-d(44-C-44-C-44-C)-3Ј18 d
3Ј-d(C-44-C-44-C-44)-5Ј
5Ј-d(46-C-46-C-46-C)-3Ј18 e
3Ј-d(C-46-C-46-C-46)-5Ј
a Measured in 1 M NaCl, 0.1 M MgCl2, 60 mM sodium cacodylate
buffer, pH 7. 5 µM ϩ 5 µM Oligonucleotide concentration. b d(iC) =
m5isoCd = 2Ј-Deoxy-5-methylisocytidine.
a Measured in 1 M NaCl, 0.1 M MgCl2, 60 mM sodium cacodylate
buffer, pH 7.0. 5 µM ϩ 5 µM Oligonucleotide concentration b 6: c7z8iGd:
8-Aza-7-deaza-2Ј-deoxyisoguanosine c 40: c7iGd: 7-Deaza-2Ј-deoxy-
isoguanosine d 44: c7z8Gd: 8-Aza-7-deaza-2Ј-deoxyguanosine e 46: c7iGd:
7-Deaza-2Ј-deoxyguanosine
Scheme 5 Base pair motifs of ps-duplexes containing compound 1 or
2Ј-deoxyisoguanine.
ing the corresponding pyrrolo[2,3-d]pyrimidine base shows a
lower stability. These findings correspond well to those
observed on 6-mer duplexes with regularly linked “guanine”
nucleosides such as 5Ј-d(G-C)3 vs. 5Ј-d(44-C)3 and 5Ј-d(46-C)3.
It is apparent that the duplexes with parallel chain orien-
tation contain only five base pairs, while those formed by anti-
parallel chains are hold together by six pairs. Thus the parallel
duplexes are expected to show a lower stability. Nevertheless the
terminal overhangs will stabilize the five base pair duplex struc-
ture by additional stacking interactions which will cause a posi-
tive effect on the duplex stability.16
Next the stability of the duplex 5Ј-d(1-m5isoC)3 42ؒ42 was
studied. In this case the chain orientation is expected to be
antiparallel. This duplex showed a slightly higher Tm-value as
that of 37ؒ37. Thus, the recognition pattern of guanine is
changed into that of isoguanine when the glycosylation site is
shifted from position-9 to position-8. Compound 1 is able to
form duplexes with parallel and antiparallel chain as it is
observed for isoguanine nucleosides. Such changes can be also
caused by changing the anomeric configuration which has
been recently investigated on anomeric 5-aza-7-deazaguanine
mimicking the base pair recognition site of isocytosine.19 In
order to obtain some information on the particular duplex
structure of the duplexes 37ؒ37 and 42ؒ42 with the unusually
linked backbones their CD-spectra were measured. According
to Fig. 1 this duplex forms an autonomous DNA-structure
being different to that from A- or B-DNA. A related CD-spec-
Fig. 1 CD-spectrum of the duplex (a) 5Ј-d(1-C)3 and (b) 5Ј-d(1-
m5iC)3.
trum of 37ؒ37 has been observed for a 12-mer duplex d(2-T)6.6
Moreover the CD-spectrum of 42ؒ42 shows similarities to a left
handed Z-DNA.3a
2. Supramolecular assemblies of 8-aza-7-deazapurine
2Ј-deoxyribonucleosides
Several investigations have shown that guanine and isoguanine
nucleosides as well as the corresponding base or sugar-modified
oligonucleotides are able to form supramolecular assemblies. In
the case of guanine nucleosides tetrameric structures (Scheme
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 0 – 3 9 0 8
3903

View Article Online
sodium cations.26 Now we have performed similar experiments
using 8-aza-7-deazapurine 2Ј-deoxynucleosides (1, 5–7). These
phenomena were studied by ESI-MS spectrometry (ESI-MS;
HP LC/MS 1100 Series with Chemstation Software) employ-
ing the lipophilic 3Ј, 5Ј-TBDMS-protected nucleosides 11–
14,48 (Scheme 7). The nucleoside derivatives were prepared
from the corresponding nucleosides by silylation with tert-
butyldimethylsilyl chloride (TBDMS-Cl). The sugar protected
nucleosides were dissolved in CHCl3 together with sodium
tetraphenylborate. The suspensions were stirred for 30 minutes
at room temperature, stored over night at Ϫ23 ЊC, and
employed for ESI-MS studies.
Scheme 7 3Ј, 5Ј-TBDMS protected 8-aza-7-deazapurine 2Ј-deoxy-
ribonucleosides.
Scheme 6 Structures of (a) dG-tetraplex, (b) iGd-tetraplex, and
(c) iGd-pentaplex.
Table 6 summarizes the outcome of these experiments. The
nucleoside 11 is able to build up a tetrameric aggregate with one
sodium cation complexed to four compound 11 residues (m/z =
2004.8). The sodium is expected to be located in the center
of the assembly. For steric reason the tetramer should form
a bowl shaped structure.27 A pentameric aggregate as found
for the isoguanine ribonucleoside20 was not detected for the
pyrazolo[3,4-d]pyrimidine nucleoside,22 which can be traced
back to the structural differences of compound 11 to the
isoguanine derivative. It has been reported that 8-aza-7-deaza-
purine nucleosides show a particular glycosylic bond conform-
ation (high-anti) compared to the anti-conformation of purine
nucleosides which can affect supramolecular aggregation.
Although compound 11 formed a tetrad, such a formation was
not observed when 8-aza-7-deaza-2Ј-deoxyisoguanosine was
carrying a 7-bromo substituent (12). Apparently, the higher
molecular assembly shown in Scheme 8 can not accommo-
date the bulky bromo residues. It might be possible that the
amino group of compound 12 forms a hydrogen bond with the
bromine substituent. Such a bond has been detected in the case
of a derivative lacking the 2-oxo group.28
6a) have been identified while isoguanine compounds tend to
form tetrameric (Scheme 6b) as well as pentameric structures
(Scheme 6c).20–22
In order to prevent such an aggregation, which obscures data
collection during the sequencing performed by gel electro-
phoresis or on arrays with immobilized DNA, base modified
guanine nucleosides have been used to impair the undesired
self-aggregation. Compounds showing this capability are
7-deaza-2Ј-deoxyguanosine and 8-aza-2Ј-deoxyguanosine.23,24
They can not form Hoogsteen base pairs due to the lack of the
nitrogen at position 7 thereby destroying the G-quartet. The
situation is different in the case of isoguanine assemblies.25 As
nitrogen-7 is not participating in the formation of the supra-
molecular aggregates 8-aza-7-deaza-2Ј-deoxyisoguanosine (6)
forms high molecular assemblies.22
It has been already shown on a sugar protected isoguanine
ribonucleoside by electrospray-ionization mass-spectroscopy
(ESI-MS) that both tetrameric as well as the pentameric
assemblies are formed simultaneously in the presence of
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 0 – 3 9 0 8
3904

View Article Online
Table 6 Relative peak areas (%) of 3Ј, 5Ј-TBDMS-protected nucleosides (N*) by ESI mass spectrometrya
Dimer
Monomer
[N* H]ϩ
Tetramer
Compound
[N*2 H]ϩ
[N*2 Na]ϩ
[N*4 Na]ϩ
11
12
48
13
14
100
69
14
25
100 (m/z: 2004.8)
100 (m/z: 2005.4)
100
100
100
9
a Nucleoside concentration: 10 mM in CHCl3, NaBPh4.
Scheme 8 8 Structure of the tetraplex formed by 8-aza-7-deaza-2Ј-
deoxyisoguanosine (6) (R = H), but does not occur in the case of 7
(R = Br).
The same experiments as performed with the N9 compounds
11 and 12 were undertaken with the N8-linked 8-aza-7-deaza-
2Ј-deoxyguanosine derivative (13) as well as with the regularly
linked nucleoside 14. In both cases aggregates are not formed,
which results from the absence of a nitrogen-7 as Hoogsteen
acceptor position in the case of compound 14 and/or the
unusual glycosylation site of compound 13 ( Table 6). In com-
parison, the purine nucleoside 2Ј-deoxyguanosine in form of
derivative 48 was shown to create a tetrameric species at 2005.4
m/z containing one sodium ion per four dG-residues.
In order to confirm these phenomena also on the oligo-
nucleotide level, the nucleosides of Scheme 2 were incorporated
in oligonucleotides. The following oligonucleotides were pre-
pared by solid-phase synthesis: 5Ј-d(T4G4T2) (49), 5Ј-d(T454T2)
(50), 5Ј-d(T464T2) (51), and 5Ј-d(T474T4) (52). Then the oligo-
nucleotides were investigated with regard to aggregate form-
ation by ion exchange chromatography (experiments performed
on compounds 49 and 51 were already reported earlier).22 For
this purpose the solutions were injected onto an ion-exchange
Gen-Pak Fax (49–51) or Dionex PA-100 (52) column thermo-
stated at 30 ЊC.
Earlier experiments performed on 5Ј-d(T4G4T2) (49) have
shown that the peak with the short retention time represents the
single-stranded oligomer while the slower migrating peak
results from the high molecular assembly (Fig. 2a). In contrast
to that, the 8-aza-7-deaza-2Ј-deoxyguanosine-containing oligo-
nucleotide 50 shows only one fast migrating peak indicating
that an aggregation does not take place. This is in accordance
with the mass spectrometric experiments performed on the
nucleoside derivatives dG and 14 (see above).
Fig. 2 Ion exchange HPLC elution profile (a) of 5Ј-d(T4G4T2) (47),
(b) 5Ј-d(T454T2) (48), (c) 5Ј-d(T464T2) (49), and (d) 5Ј-d(T474T4) (50) at
30 ЊC; conditions: see Experimental.
the N8-glycosylated compound 1, as is similarly found for
7-deaza-2Ј-deoxyguanosine.23 In the case of “isoguanine”
nucleosides, such as 7, aggregation is prevented by bulky
7-substituents.
Conclusions
Introduction of the 8-aza-7-deazaguanine N 8-(2Ј-deoxy-β--
ribofuranoside) 1 into oligonucleotide duplexes with anti-
parallel chain orientation leads to a change of the base
recognition, which is now comparable with that of 2Ј-deoxy-
isoguanosine. Antiparallel duplexes are rather stable when
While oligonucleotides incorporating short runs of 8-aza-7-
deaza-2Ј-deoxyguanosine do not form tetrameric assemblies,
8-aza-7-deaza-2Ј-deoxyisoguanine (6) builds up such structures
(Fig. 2c).21,22 However, the oligonucleotide incorporating the
7-bromo derivative 7 (52) does not aggregate (Fig. 2d, the reten-
tion time is slightly faster due to Dionex PA-100 column).
These observations correlate well with the findings on the
monomeric nucleoside level (see above). From this one can
deduce that the aggregation of guanine nucleosides is prevented
both by regularly linked 8-aza-7-deazaguanine (5) as well as by
compound 1 forms base pairs with 2Ј-deoxy-5-methyl-
isocytidine; towards dG and itself it shows ambiguous base
pairing. In parallel duplexes the most stable base pair is formed
when the nucleoside 1 base pairs with dC. These properties
observed on non-self-complementary duplexes with random
base pair composition can be also realized on self-comple-
mentary oligonucleotide duplexes such as the hexanucleotides
5Ј-d(1-C)3 and 5Ј-d(1-m5isoC)3.
Investigationofsupramolecularaggregatesofpyrazolo[3,4-d]-
pyrimidine nucleosides related to dG indicate that aggregation
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 0 – 3 9 0 8
3905

View Article Online
Table
7
Molecular weights determined by MALDI-TOF mass
spectroscopy of modified oligonucleotides
is prevented when nitrogen-7 is absent as electron donating
site both on the N9 and N8-glycosylated isomer 1 and 5, as
shown earlier for other 7-deazapurine nucleosides such as
7-deaza-2Ј-deoxyguanosine. Even, the weakly basic 7-nitrogen
of 8-aza-2Ј-deoxyguanosine nucleoside prevents the form-
ation of supramolecular assemblies.24 Isoguanine nucleo-
sides such as 8-aza-7-deaza-2Ј-deoxyisoguanosine still forms
self-assembled structures. However, the presence of a bulky
7-bromo substituent prevents aggregate formation also in this
case.
Oligonucleotide
MHϩ (calc)
MHϩ (found)
5Ј-d(TAG1TCAATACT)
5Ј-d(AGTATT1ACCTA)
5Ј-d(ATiCiCA1TTAT1A)
3Ј-d(ATCiCAGTTATGA)
5Ј-d(TAGGTiCAATACT)
21
22
23
18
19
3645.4
3645.4
3673.5
3657.7
3657.7
3645.5
3648.5
3673.4
3657.4
3659.8
Oligonucleotides
The oligonucleotide syntheses were carried out on an ABI
392–08 DNA synthesizer (Applied Biosystems, Weiterstadt,
Germany) in a 1 µmol scale using the phosphoramidite 4,
those of the regular 2Ј-deoxynucleosides (Applied Bio-
systems, Weiterstadt, Germany) together with the DPC- and
Experimental
General
Thin-layer chromatography (TLC) was performed on TLC
aluminium sheets silica gel 60 F254 (0.2 mm, VWR Inter-
national, Germany). Reversed-phase HPLC was carried out on
a 4 × 250 mm RP-18 (10 µm) LiChrosorb column (VWR Inter-
national) with a Merck-Hitachi HPLC pump (model 655 A-12)
connected with a variable wavelength monitor (model 655-A), a
controller (model L-5000), and an integrator (model D-2000).
UV-spectra were recorded on a U-3200 spectrophotometer
(Hitachi, Japan), λmax. in nm, ε in dm3 molϪ1 cmϪ1. Half-life
values (τ) were measured on U-3200 spectrophotometer
(Hitachi, Japan) connected with a temperature controller
(Lauda, Germany). NMR spectra were measured on an Avance
DPX 250 and an AMX 500 spectrometer (Bruker, Germany);
chemical shifts (δ) are in ppm downfield from internal TMS
(1H, 13C) or external 85% H3PO4 (31P). The J-values are given in
Hz. The solvents were purified and dried according to standard
procedures. MALDI-TOF was recorded on Biflex-III spectro-
meter (Bruker, Leipzig, Germany) in the reflector mode. The
average power of the nitrogen laser (337.1 nm) at 20 Hz was 3–4
mW (150–200 µJ/pulse) with a delay time of 600 ns. The
enzymatic hydrolysis of the oligomers was performed as
described below using the following extinction coefficients:
ε280: dA: 2400, dC: 6900, dT: 6300, m5isoCd: 1600, 1: 6600.
Snake-venom phosphodiesterase (EC 3.1.15.1, Crotallus adam-
anteus) and alkaline phosphatase (EC 3.1.3.1, E. coli) were gen-
erous gifts from Roche Diagnostics GmbH, Germany.
formamidine-protected phosphoramidite of 8-bromo-8-aza-
19
7-deaza-2Ј-deoxyisoguanosine
as well as the isobutyryl-
protected phosphoramidite of 8-aza-7-deaza-2Ј-deoxyguano-
sine following the synthesis protocol for 3Ј-β-cyanoethyl
phosphoramidites. Oligonucleotide 42 was synthesized with a
Universal support column carrying a 3Ј-terminal ribose moiety
(Universal support 500, Glen Research, Sterling, VA, USA).
After cleavage of the oligonucleotides from the solid support,
the former were deprotected in 25% aqueous ammonia solution
for 12–16 h at 60 ЊC. Purification of the 5Ј-dimethoxytrityl-
oligomers was performed by reversed-phase HPLC (RP-18)
with the following solvent gradient system (A, 0.1
M
(Et3NH)OAc (pH 7.0)–MeCN 95 : 5; B, MeCN): 3 min 20% B
in A with a flow rate of 1.0 mL minϪ1, 12 min 20–40% B in A
with a flow rate of 1 mL minϪ1. Treatment with 2.5% CHCl2-
COOH/CH2Cl2 for 5 min at room temperature to remove the
4,4Ј-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 minϪ1. Oligonucleotides
for investigation of aggregate formation were synthesized in the
trityl-off mode, deprotected in 25% aq. ammonia solution at
60 ЊC for 16 h followed by purification by ion exchange
chromatography (NucleoPac-column, 4 × 250 mm) with the
following solvent system (C: 1.5 M LiCl in aq. NaOH (pH
12.0)–D: aq. NaOH (pH 12.0): 5 min 5% D in E, 25 min 5–30%
D in E, 10 min 30–5% D in E with a flow rate of 1.0 ml minϪ1
.
Ion-exchange HPLC
The oligomers were desalted and lyophilized on a Speed-Vac
evaporator to yield colorless solids. MALDI-TOF mass spectra
of modified oligonucleotides are shown in Table 7.
The composition of oligonucleotides was proven by enzym-
atic hydrolysis: The oligonucleotides were dissolved in 0.1 M
Tris-HCl buffer (pH 8.3, 200 µl), and treated with snake-venom
phosphodiesterase (3 µl) at 37 ЊC for 45 min and alkaline phos-
phatase (3 µl) at 37 ЊC for another 30 min. The reaction mix-
tures were analyzed by HPLC (RP-18, at 260 and 280 nm, sol-
vent system A, 0.7 ml minϪ1).
The ion-exchange chromatography was performed on a 4.6 ×
100 mm Gen-Pak Fax Column (Waters, WAT015490, USA) or
a 4 × 250 mm NucleoPac-column (Dionex P/N 043010, USA)
using a Merck-Hitachi HPLC apparatus with a pump (model
L-7100), a UV-detector (model L-4250) connected with an
integrator (model D-2000). A column oven (model L-7350,
Merck, Germany) was used to control the temperature of the
ion-exchange column. The oligonucleotides were prepared as
follows. A sample of 0.1 A260-units was dissolved in 20 µL 1.0 M
NaCl-buffer. The solution was heated to 60 ЊC for 5 min.,
brought to room temperature and kept in a refrigerator
(Ϫ23 ЊC) for 16 h. Then, the sample was brought to room tem-
perature, diluted with 100 µL buffer A and injected into the
system, which had been preheated to 30 ЊC. The column was
eluted using the following systems: 0–40 min with 0–80% B in A
with a flow rate of 0.5 mL minϪ1 (A: 25 mM Tris-HCl, 1.0 mM
EDTA, pH 8.0; B: 1.0 M NaCl, 25 mM Tris-HCl, 1.0 mM
EDTA, pH 8.0).
2-(2-Deoxy-ꢀ-D-erythro-pentofuranosyl)-6-{[(dimethylamino)-
methylidene]amino}-2H-pyrazolo[3,4-d]pyrimidin-4-one 9
To a stirred suspension of 1 (200 mg, 0.75 mmol) in MeOH
(10 mL) N,N-dimethylformamide dimethylacetal (1 mL, 7.53
mmol) was added. The mixture was stirred at room temperature
for 48 h and evaporated to dryness. The residue was applied to
flash chromatography (FC; column 2 × 10 cm, CH2Cl2–MeOH
9 : 1 to 8 : 2) and eluted stepwise. Colorless foam of 9 (210 mg,
87 %) (Found: C, 48.36; H, 5.70; N, 26.01. C13H18N6O4 requires
C, 48.44; H, 5.63; N, 26.07%); TLC (CH2Cl2–MeOH 8 : 2): Rf
0.77; UV (MeOH): 233 (24300); 278 (13000); 338 (15500).
δH (250 MHz; d6-DMSO): 2.21 (1H, m, 2Ј-Hα), 2.50 (1H, m,
2Ј-Hβ), 3.03, 3.16 (6H, 2m, 2NCH3), 3.55 (2H, m, 5Ј-H2), 3.85
(1H, m, 4Ј-H), 4.37 (1H, s, 3Ј-H), 4.91 (1H, 5Ј-OH), 5.27 (1H,
m, 3Ј-OH), 6.51 (1H, ‘t’, J = 6.0, 1Ј-H), 8.42 (1H, s, 3-H) and
Electrospray-ionization mass spectrometry
The ESI-MS measurements were performed using HP LC/MS
1100 with Chemstation Software. To 1 mL of a 10 mM solu-
tion in CHCl3 0.05 mmol NaBPh4 was added. The suspension
was stirred for 30 minutes and incubated at Ϫ23 ЊC overnight.
The solid was removed, the solution filtered and used for the
ESI-MS measurements.
8.56 (1H, s, HC᎐N).
᎐
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 0 – 3 9 0 8
3906

View Article Online
2-[2-Deoxy-5-O-(4,4Ј-dimethoxytrityl)-ꢀ-D-erythro-pento-
furanosyl]-6-{[(dimethylamino)methylidene]amino}-2H-
pyrazolo[3,4-d]pyrimidin-4-one 10
mg, 72%) (Found: C, 46.02; H, 7.14; N, 12.10. C22H40N5O4Si2Br
requires C, 45.98; H, 7.02; N 12.19%); TLC (CH2Cl2–MeOH
9 : 1): Rf 0.70; UV (MeOH): 231 (18500), 296 (2600); δH (250
MHz; d6-DMSO): 0.04 (12H, m, CH3), 0.86 (18H, m, CH3),
2.17 (1H, m, 2Ј-Hα), 2.68 (1H, m, 2Ј-Hβ), 3.58 (2H, m, 5Ј-H2),
3.75 (1H, m, 4Ј-H), 4.55 (1H, m, 3Ј-H), 6.25 (1H, m, 1Ј-H), 7.62
(2H, s, NH2) and 10.83 (1H, s, NH).
Compound 9 (210 mg, 0.65 mmol) was dried by repeated
co-evaporation from anhydrous pyridine and dissolved in
anhydrous pyridine (1.5 mL). The solution was treated with
dimethoxytrityl chloride (286 mg, 0.84 mmol) at room temper-
ature under stirring (4.5 h). MeOH (1 mL) was added, and the
stirring was continued for 5 min. The mixture was poured into
5% aqueous NaHCO3 solution (5 mL) and extracted with
CH2Cl2 (3 × 5 mL). The combined organic layers were dried
(Na2SO4). After evaporation of the solvent, the residue was
applied to FC (silica gel, column 2 × 15 cm), which was pre-
washed with CH2Cl2 and eluted with CH2Cl2–acetone (stepwise
elution from 98 : 5 to 8 : 2) to give 10 (270 mg, 66%) as a
colorless foam (Found: C, 65.31; H, 5.76; N, 13.28. C34H36N6O6
requires C, 65.37; H, 5.81; N, 13.45%); TLC (CH2Cl2–acetone
9 : 1): Rf 0.60; UV (MeOH): 236 (31200), 319 (44800); δH (250
MHz; d6-DMSO): 2.28 (1H, m, 2Ј-Hα), 2.67 (1H, m, 2Ј-Hβ),
3.03, 3.16 (6H, m, NCH3), 3.34 (2H, m, 5Ј-H2), 3.69 (3H, m,
OCH3), 3.92 (1H, m, 4Ј-H), 4.43 (1H, m, 3Ј-H), 5.31 (1H, d,
J = 4.9, 3Ј-OH), 6.18 (1H, m, 1Ј-H), 6.74–7.34 (m, arom. H),
6-Amino-2-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-ꢀ-D-
erythro-pentofuranosyl]-2H-pyrazolo[3,4-d]pyrimidin-4-one 13
150 mg (0.56 mmol) Anh. 1 was dissolved in pyridine (2 ml).
530 mg (3.5 mmol) tert-Butyldimethylsilyl chloride and 400 mg
(5.9 mmol) imidazole were added and the reaction mixture was
stirred at room temperature for 48 h. Purification was per-
formed by flash chromatography (silica gel, column 3 × 15 cm,
CH2Cl2
CH2Cl2–MeOH 9 : 1) yielding 13 (180 mg, 65%) as a
colorless solid. (Found: C, 53.26; H, 8.28; N, 14.10. C22H41-
N5O4Si2 requires C, 53.30; H, 8.34; N 14.13%); TLC (CH2Cl2–
MeOH 9 : 1): Rf 0.50; UV (MeOH): 244 sh (6300), 319 (5600);
δH (250 MHz; d6-DMSO): 0.04 (12H, m, CH3), 0.82 (18H, m,
CH3), 2.27 (1H, m, 2Ј-Hα), 2.64 (1H, m, 2Ј-Hβ), 3.56 (br., 5Ј-H2,
4Ј-H), 4.54 (1H, m, 3Ј-H), 6.09 (1H, m, 1Ј-H), 6.23 (2H, s,
NH2), 8.39 (1H, s, 3-H) and 10.96 (1H, s, NH).
8.48 (1H, s, 3-H), 8.66 (1H, s, HC᎐N) and 10.96 (1H, s, NH).
᎐
2-[2-Deoxy-5-O-(4,4Ј-dimethoxytriphenylmethyl)-ꢀ-D-erythro-
pentofuranosyl]-6-{[(dimethylamino)methylidene]amino}-2H-
pyrazolo[3,4-d]pyrimidin-4-one 3Ј-(2-cyanoethyl-N, N-diiso-
propyl)phosphoramidite 4
6-Amino-1-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-ꢀ-D-
erythro-pentofuranosyl]-2H-pyrazolo[3,4-d]pyrimidin-4-one 14
250 mg (0.94 mmol) Anh. 5 was dissolved in pyridine (2.5 ml).
885 mg (5.87 mmol) tert-Butyldimethylsilyl chloride and
668 mg (9.8 mmol) imidazole were added and the reaction mix-
ture was stirred at room temperature for 48 h. Purification was
performed by flash chromatography (silica gel, column 3 ×
A solution of compound 10 (250 mg, 0.4 mmol) in CH2Cl2
(5 mL) was preflushed with argon and kept under argon atmos-
phere. Then, 2-cyanoethyldiisopropylphosphoramidochloridite
(130 µL, 0.58 mmol) and N,N-diisopropylethylamine (130 µL,
0.75 mmol) were added at room temperature. Stirring was
continued for 30 min. An aqueous solution of 5% NaHCO3
(10 mL) was added. The mixture was shaken, the layers separ-
ated, and the aqueous layer extracted with CH2Cl2 (3 × 15 mL).
The combined organic extracts were dried (Na2SO4), filtered,
evaporated, and applied to FC (silica gel, column 2 × 10 cm,
CH2Cl2–acetone 8 : 2) yielding a colorless foam (265 mg, 80%).
TLC (CH2Cl2–acetone 8 : 2): Rf 0.95. 31P NMR (CDCl3): 149.6,
149.9.
15 cm, CH2Cl2
CH2Cl2–MeOH 9 : 1) yielding a colorless
solid (230 mg, 50%) (Found: C, 53.26; H, 8.28; N, 14.10.
C22H41N5O4Si2 requires C, 53.30; H, 8.34; N 14.13%); TLC
(CH2Cl2–MeOH 9 : 1): Rf 0.50; UV (MeOH): 253 (13300);
δH (250 MHz; d6-DMSO): 0.04 (12H, m, CH3), 0.86 (18H, m,
CH3), 2.18 (1H, m, 2Ј-Hα), 2.80 (1H, m, 2Ј-Hβ), 3.56 (2H, m,
5Ј-H2), 3.97 (1H, m, 4Ј-H), 4.56 (1H, m, 3Ј-H), 6.30 (1H, m,
1Ј-H), 6.71 (2H, s, NH2), 7.82 (1H, s, 3-H) and 10.60 (1H, s,
NH).
Acknowledgements
4-Amino-1-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-ꢀ-D-
erythro-pentofuranosyl]-1H-pyrazolo[3,4-d]pyrimidin-6-one 11
We thank Mr Christoph Hess for the ESI-MS spectra, Dr Yang
He and Dr Helmut Rosemeyer for the NMR spectra, and Mrs
Elisabeth Feiling for the oligonucleotide synthesis. Financial
support by the Roche Diagnostics GmbH is gratefully
acknowledged.
200 mg (0.75 mmol) Anh. 6 was dissolved in pyridine (3 ml).
710 mg (4.7 mmol) tert-Butyldimethylsilyl chloride and 532 mg
(7.81 mmol) imidazole were added and the reaction mixture
was stirred at room temperature for 48 h. Purification was per-
formed by flash chromatography (silica gel, column 3 × 15 cm,
CH2Cl2
CH2Cl2–MeOH 9 : 1) yielding a colorless solid (210
References
mg, 57%) (Found: C, 53.41; H, 8.07; N, 14.52. C22H41N5O4Si2
requires C, 53.30; H, 8.34; N 14.13%); TLC (CH2Cl2–MeOH
9 : 1): Rf 0.50; UV (MeOH): 251 (5300), 290 (4500); δH (250
MHz; d6-DMSO): 0.02 (12H, m, CH3), 0.86 (18H, m, CH3),
2.13 (1H, m, 2Ј-Hα), 2.76 (1H, m, 2Ј-Hβ), 3.63 (2H, m, 5Ј-H2),
3.76 (1H, m, 4Ј-H), 4.56 (1H, m, 3Ј-H), 6.27 (1H, m, 1Ј-H), 7.92
(2H, s, NH2), 8.56 (1H, s, 3-H) and 10.83 (1H, s, NH).
1 R. Shapiro and R. W. Chambers, J. Am. Chem. Soc., 1961, 83, 3920–
3921.
2 H. Vorbrüggen and C. Ruh-Polenz, Organic Reactions, John Wiley
& Sons, Inc., New York, 2000, vol 55.
3 (a) F. Seela and P. Leonard, Helv. Chim. Acta, 1996, 79, 477–487;
(b) J. He and F. Seela, Org. Biomol. Chem., 2003, 1, 1873–1883.
4 F. Seela, Y. He, J. He, G. Becher, R. Kröschel, M. Zulauf and
P. Leonard, Base-modified Oligonucleotides with increased Duplex
Stability: Pyrazolo[3,4-d]pyrimidines replacing Purines, in Oligo-
nucleotide Synthesis: Methods and Applications, Ed. P. Herdewijn,
Humana Press Inc., Totowa, New Jersey, 2003, in press.
5 F. Seela and H. Debelak, Nucleic Acids Res., 2000, 28, 3224–3232.
6 J. He and F. Seela, Helv. Chim. Acta, 2002, 85, 1340–1354.
7 F. Seela and H. Debelak, J. Org. Chem., 2001, 66, 3303–3312.
8 H. Steker, PhD Thesis, Paderborn, 1988.
9 F. Seela and H. Steker, Helv. Chim. Acta, 1986, 69, 1602–1613.
10 M. Smith, D. H. Rammler, I. H. Goldberg and H. G. Khorana,
J. Am. Chem. Soc., 1962, 84, 430–440.
11 Z. Kazimierczuk, R. Mertens, W. Kawczynski and F. Seela,
Helv. Chim. Acta, 1991, 74, 1742–1748.
4-Amino-3-bromo-1-[2-deoxy-3,5-di-O-(tert-butyldimethylsilyl)-
ꢀ-D-erythro-pentofuranosyl]-1H-pyrazolo[3,4-d]pyrimidin-6-one
12
100 mg (0.29 mmol) Anh. 4-amino-3-bromo-1H-pyrazolo-
[3,4-d]pyrimidin-6-one was dissolved in pyridine (3ml). 355 mg
(2.4 mmol) tert-Butyldimethylsilyl chloride and 266 mg
(3.91 mmol) imidazole were added and the reaction mixture
was stirred at room temperature for 48 h. Purification was per-
formed by flash chromatography (silica gel, column 3 × 15 cm,
CH2Cl2
CH2Cl2–MeOH 9 : 1) yielding a colorless solid (120
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 0 – 3 9 0 8
3907

View Article Online
12 Users’ Manual of the DNA Synthesizer; Applied Biosystems,
Weiterstadt, Germany; p 392.
13 F. Seela and Y. He, Helv. Chim. Acta, 2000, 83, 2527–2540.
14 F. Seela and C. Wei, Helv. Chim. Acta, 1999, 82, 726–745.
15 F. Seela and K. Kaiser, Helv. Chim. Acta, 1988, 71, 1813–
1823.
16 H. Rosemeyer and F. Seela, J. Chem. Soc., Perkin Trans. 2, 2002,
746–750.
17 F. Seela, C. Wei, A. Melenewski, Y. He, R. Kröschel and E. Feiling,
Nucleosides Nucleotides, 1999, 18, 1543–1548.
20 M. Cai, A. L. Marlow, J. C. Fettinger, D. Fabris, T. J. Haverlock,
B. A. Moyer and J. T. Davis, Angew. Chem., 2000, 112, 1339–1341.
21 J. C. Chaput and C. Switzer, Proc. Natl. Acad. Sci. USA, 1999, 96,
10614–10619.
22 F. Seela and R. Kröschel, Bioconjugate Chem., 2001, 12, 1043–1050.
23 S. Mizusawa, S. Nishimura and F. Seela, Nucleic Acids Res., 1986,
14, 1319–1324.
24 F. Seela and S. Lampe, Helv. Chim. Acta, 1994, 77, 1003–1017.
25 F. Seela and C. Wei, Chem. Commun., 1997, 1869–1870.
26 J. T. Davis, personal communication.
18 F. Seela and H. Driller, Nucleic Acids. Res., 1989, 17, 901–910.
19 F. Seela, S. Amberg, A. Melenewski and H. Rosemeyer, Helv. Chim
Acta, 2001, 84, 1996–2014.
27 M. Meyer and J. Sühnel, J. Phys. Chem. A, 2003, 107, 1025–1031.
28 F. Seela, M. Zulauf, H. Reuter and G. Kastner, Acta Crystallogr.,
Sect. C., 1999, 55, 1947–1950.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 3 9 0 0 – 3 9 0 8
3908