7-Deaza-2,8-diazaadenine containing oligonucleotides: Synthesis, ring opening and base pairing of 7-halogenated nucleosides

Article abstract of DOI:10.1016/j.tet.2005.05.046

Oligonucleotides containing 7-bromo-7-deaza-2,8-diaza-2′- deoxyadenosine (3) and 5-amino-3-bromo-4-carbamoyl-1-(2′-deoxy-β-d- erythro-pentofuranosyl)pyrazole (4) were synthesized. Compound 3 was prepared from 7-bromo-8-aza-7-deaza-2′-deoxyadenosine (5) vi

Full text of DOI:10.1016/j.tet.2005.05.046

Tetrahedron 61 (2005) 7520–7527
7-Deaza-2,8-diazaadenine containing oligonucleotides: synthesis,
ring opening and base pairing of 7-halogenated nucleosides
*
Wenqing Lin, Kuiying Xu and Frank Seela
a
¨ ¨ ¨ ¨
Laboratorium fur Organische und Bioorganische Chemie, Institut fur Chemie, Universitat Osnabruck, Barbarastr. 7,
¨
49069 Osnabruck, Germany
b
¨
Center for Nanotechnology, Gievenbecker Weg 11, 48149 Munster, Germany
Received 25 January 2005; revised 18 May 2005; accepted 19 May 2005
Available online 17 June 2005
Dedicated to the late Professor John A. Montgomery
Abstract—Oligonucleotides containing 7-bromo-7-deaza-2,8-diaza-20-deoxyadenosine (3) and 5-amino-3-bromo-4-carbamoyl-1-(20-
deoxy-b-D-erythro-pentofuranosyl)pyrazole (4) were synthesized. Compound 3 was prepared from 7-bromo-8-aza-7-deaza-20-deoxyade-
nosine (5) via the 1,N6-etheno derivative 6 and was converted into the phosphoramidite 11. The 7-bromo substituent of 3 increases
oligonucleotide duplex stability compared to the non-halogenated nucleoside. Oligonucleotides incorporating 3 are transformed to those
containing 4 during long time deprotection at elevated temperature (25% aq ammonia, 60 8C, 30 h). Compound 3 forms a strong base pair
with dG. The base pair stability decreases in the order dGOdTOdAOdC. Similar recognition selectivity is observed for the pyrazole
nucleoside 4, however, due to decreased stacking and higher flexibility of the pyrazole moiety, duplexes are less stable than those
containing 3.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
in Watson–Crick base pairing. On the other hand, it has been
reported that 7-halogeno substituents introduced in
7-deazapurine or the 8-aza-7-deazapurine moieties induce
oligonucleotide duplex stabilization.13–21 The six-mem-
bered ring of 2-azapurines is prone to ring opening under
alkaline conditions thereby forming substituted imidazole
derivatives; the same is observed on the corresponding
7-deaza-2,8-diazapurine nucleosides making them appli-
cable as precursors for substituted pyrazole moieties within
an oligonucleotide chain. Recent experiments indicate that
the ring opening reaction is accelerated when 7-halogeno
substituents are present in the 7-deaza-2,8-diazapurine
system. This manuscript reports on the synthesis of
7-bromo-7-deaza-2,8-diaza-20-deoxyadenosine (3), its con-
version into a building block for solid phase synthesis and
the preparation of corresponding oligonucleotides. Oligo-
nucleotides containing 3 are transformed to those containing
5-amino-3-bromo-4-carboxamidopyrazole (4) residues
under alkaline conditions (Scheme 1). As it was observed
that imidazole 4-carboxamide and pyrazole 4-carboxamide
form either a dA- or a dI-like hydrogen bonding motif
within oligonucleotide duplexes22–24 the base pairing
properties of duplexes containing compounds 3 and 4 are
studied.
2-Azapurines and their nucleosides attracted attention not
only because of their broad biological activity,1–9 but also of
their unusual base pairing properties when they are
constituents of oligonucleotides.10,11 Up to now, only a
few 2-azapurine nucleosides were incorporated into
oligonucleotides because of their paucity and instability in
alkaline solution.10–12 Previous studies of our laboratory on
oligonucleotides containing 2-aza-20-deoxyadenosine (1)10
and 7-deaza-2,8-diaza-20-deoxyadenosine (2)11 have shown
that 2-azaadenines form strong base pairs with guanine and
weaker ones with thymine with a relative stability order of
dGOdT[dAwdC (purine numbering is used throughout
the general part). A likely explanation for this is the
presence of an additional proton acceptor site at the
2-position of the purine moiety. The additional nitrogen
generates also an increased p-electron deficiency within the
aromatic system, in particular at nitrogen-1 being involved
Keywords: Oligonucleotides; Base pairing; Duplex stability; Nucleosides;
Azapurine; Pyrazole.
Corresponding author. Tel.: C49 541 9692791; fax: C49 541 9692370;
e-mail: frank.seela@uni-osnabrueck.de
*
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.05.046

W. Lin et al. / Tetrahedron 61 (2005) 7520–7527
7521
nucleoside via 1,N6-etheno adducts.27–29 Herein, the
synthesis of compound 3 is performed using 7-bromo-7-
deaza-8-aza-20-deoxyadenosine (5)21 as starting material
(Scheme 2). Reaction of 5 with chloroacetaldehyde at pH
4.5–5 yields 1,N6-etheno-7-bromo-8-aza-7-deaza-20-deoxy-
adenosine (6) (83%), which was treated with 1 M sodium
hydroxide at room temperature to give the derivative 7
(47%). Subsequent treatment of 7 with sodium nitrite in
80% aqueous acetic acid afforded 1,N6-etheno-7-bromo-7-
deaza-2,8-diaza-20-deoxyadenosine (8) in 88% yield. After-
wards compound 8 was treated with N-bromosuccinimide at
pH 4–4.5 to result in 3 (60%, overall yield: 20%). All new
compounds were characterized by 1H and 13C NMR spectra
and by elemental analyses (see Table 1 and Section 4). The
13C NMR resonances were assigned based on gated-
decoupled spectra.
Next, the phosphoramidite 11 was prepared. Previous
studies have shown that isobutyryl and acetyl groups are
more suitable than the benzoyl residue for the protection of
the amino group of 2-azapurine nucleosides.11 However,
when compound 3 was treated with trimethylsilyl chloride
and isobutyric anhydride under the conditions of transient
protection,38 the resulting material was impure after
extensive purification by column chromatography, as
shown by the 1H NMR spectra. This problem was overcome
by using the dimethylaminomethylidene residue as protect-
ing group. Treatment of 3 with dimethylformamide
dimethylacetal provided 5-bromo-7-(2-deoxy-b-D-erythro-
pentofuranosyl)-4-(dimethylaminomethylidene)-7H-pyra-
zolo[3,4-d][1,2,3]triazine (9) in 79% yield (Scheme 3). The
4,40-dimethoxytritylation followed the standard protocol
Scheme 1.
2. Results and discussion
2.1. Synthesis of the monomers
Among the several procedures developed for the synthesis
of 2-azapurine nucleosides2,8,25–37 the most attracting
protocol is the one starting with the corresponding adenine
Scheme 2. Reagents and conditions: (i) ClCH2CHO, 1 M aq NaOAc, pH 4.5–5, rt; (ii) 1 M NaOH, rt; (iii) NaNO2, 80% aq HOAc, 0 8C–rt; (iv) NBS, 1 M aq
NaOAc buffer, pH 4–4.5, rt.
13
Table 1. C NMR chemical shifts of 20-deoxyribonucleosides measured in (D6) DMSO at 298 K
C(2)a
C(4)a
C(5)a
C(6)a
C(7)a
C(5)b
C(3)c
C(9)d
C(10)a
C(11)a
C(10)
C(20)
C(30)
C(40)
C(50)
C(7a)b
C(5)c
C(4a)b
C(4)c
C(4)b
C(100)c
C(9b)d
C(300)c
C(2)d
C(400)c
C(3)d
C(5)d
C(6a)d
C(9a)d
e
2(11)
3
4
6
7
149.2
149.6
151.8
145.6
147.8f
143.0
150.7
151.3
96.0
95.9
95.2
103.5
94.0f
104.8
101.9
103.1
153.2
152.3
164.4
138.1
139.7f
129.9
155.2f
155.6
132.9
118.8
123.8
117.9
115.5f
117.4
120.5
121.3
84.9
85.0
84.3
84.4
84.6
86.0
84.1
86.3
71.0
70.6
70.8
70.7
70.8
70.5
70.6
72.9
87.9
87.9
87.5
87.8
87.5
88.2
87.9
85.1
62.3
62.0
62.2
62.1
62.2
61.8
62.0
64.2
38.0
37.6
38.0
37.8
38.3
38.0
38.6
141.4
132.6f
126.9f
133.3f
113.2f
121.9f
115.8f
8
9
10g
a Purine numbering.
b Systematic numbering for compounds 3, 9 and 10.
c Systematic numbering for compounds 4 and 7.
d Systematic numbering for compounds 6 and 8.
e Superimposed by the signal of DMSO-d6.
f
Tentative.
g Measured in CDCl3.

7522
W. Lin et al. / Tetrahedron 61 (2005) 7520–7527
Scheme 3. Reagents and conditions: (i) Me2NCH(OMe)2, MeOH, 40 8C; (ii) (MeO)2TrCl, pyridine, rt; (iii) iPr2NP(Cl)OCH2CH2CN, iPr2EtN, CH2Cl2, rt.
Table 2. Molecular masses of oligonucleotides determinated by MALDI-TOF mass spectroscopy
Oligonucleotides
[MCH]C (calcd) [Da]
[MCH]C (found) [Da]
30-d(ATC C3G TTA TGA) (12)
30-d(ATC C4G TTA TGA) (13)
50-d(TAG GTC 4AT ACT) (14)
30-d(ATC 4AG TTA TGA) (15)
30-d(AT4 CAG TTA TGA) (16)
30-d(ATC CAG TT 4 TGA) (17)
3725
3715
3715
3739
3739
3715
3725
3714
3715
3739
3740
3715
3Z7-Bromo-7-deaza-2,8-diaza-20-deoxyadenosine, 4Z5-amino-3-bromo-pyrazole-4-carboxamide 20-deoxy-b-D-ribofuranoside.
yielding compound 10 in 83% yield.39 Subsequent
phosphitylation with chloro-(2-cyanoethoxy)-N,N-(diiso-
propylamino)phosphine resulted in the phosphoramidite
was worked-up in the usual way two strong peaks are
detected by HPLC. Enzymatic digestion of the peak
contents with snake venom phosphodiesterase followed by
alkaline phosphatase showed that only the faster migrating
main peak contained the ring-opened nucleoside 4 while the
modification of the second peak was not identified. To
confirm the conversion on the oligonucleotide level the ring
opening was performed on 4-amino-5-bromo-7-(2-deoxy-b-
D-erythro-pentofuranosyl)-7H-pyrazolo[3,4-d][1,2,3]tria-
zine (7-bromo-7-deaza-2,8-diaza-20-deoxyadenosine) 3
which was converted to 5-amino-3-bromo-1-(2-deoxy-b-D-
erythro-pentofuranosyl)-4-carbarmoyl-1H-pyrazole (4).
Compound 3 was heated in a sealed vessel in 25% aq
NH3 at 60 8C for 16 h. After work-up the ring-opened
nucleoside was isolated in 63% yield. The structure was
1
11 (79%).40 All compounds were characterized by H and
13C NMR spectra and by elemental analysis (see Table 2
and Section 4).
2.2. Synthesis and characterization of the
oligonucleotides
The oligonucleotide synthesis was performed on an ABI
392-08 DNA synthesizer (Applied Biosystems, Weiterstadt,
Germany) in a 1-mmol scale applying the phosphoramidite
11 in solid phase oligonucleotide chemistry.41 Initially, the
regularly protected canonical phosphoramidites were
employed. After cleavage from the solid support, the
oligonucleotides were deprotected according to the standard
procedure (25% aq NH3 solution, 60 8C, 16 h). However, it
was difficult to obtain a pure oligonucleotides because of the
instability of the 2-azapurines in alkaline medium at
elevated temperature. This problem was overcome by
using the tBPA-protected phosphoramidites and employing
ultra mild deprotection conditions (25% aq NH3, room
temperature, 12 h). By this means the oligonucleotide 12
containing the nucleoside 3 residue was obtained.
1
proven to be 4 by H NMR, 13C NMR spectroscopy and
elemental analysis (Scheme 4, Table 1 and Section 4).
In all cases the purity of the oligonucleotides was confirmed
by the detection of a single peak (O99% area) in the HPLC
profile. Furthermore, MALDI-TOF spectra (Table 2) and
enzymatic analysis with snake venom phosphodiesterase
followed by alkaline phosphate confirmed the composition
of oligonucleotides (for the digest protocol see Section 4).
The oligomers synthesized are shown in Table 3.
To get the oligonucleotides containing pyrazole nucleoside
4 the oligonucleotides were deprotected by incubation with
a 25% aq NH3 solution at 60 8C for a period of 30 h. The
deprotected oligonucleotides were purified by RP-18 HPLC.
After removal of the 4,40-dimethoxytrityl residue, the
oligonucleotides were applied to RP-18 HPLC. The content
of the main peak was separated from side products and was
desalted to give the oligonucleotides 13–17 containing
compound 4.
When the pure oligonucleotide 12 containing nucleoside 3
was treated with aqueous ammonia at 60 8C for 30 h and
Scheme 4. Reagents and conditions: (i) NH4OH, 60 8C, 16 h.

W. Lin et al. / Tetrahedron 61 (2005) 7520–7527
7523
Table 3. Tm-values and thermodynamic data of oligonucleotide duplexes containing the base modified nucleosides 2, 3 and 4a,b
DG310
Duplex
Tm (8C)
DTm
DG310
c
c
Duplex
Tm
(8C)
DTm
(kcal/mol)d
(kcal/mol)d
50-d(TAGGTCAATACT)
30-d(ATCCAGTTATGA)
50-d(TAGGTCAATACT)
30-d(ATCC2GTTATGA)
50-d(TAGGTCAATACT)
30-d(ATCC3GTTATGA)
50-d(TAGGTCAATACT)
30-d(ATCC4GTTATGA)
50-d(TAGGCCAATACT)
30-d(ATCCAGTTATGA)
50-d(TAGGCCAATACT)
30-d(ATCC2GTTATGA)
50-d(TAGGCCAATACT)
30-d(ATCC3GTTATGA)
50-d(TAGGCCAATACT)
30-d(ATCC4GTTATGA)
18
19
18
21
18
12
18
13
47
43
45
42
0
K10.4
K9.1
24
23
24
21
24
12
24
13
36
36
35
35
0
0
K4
K2
K5
K7.4
K7.4
K7.7
K10.0
K9.2
K1
K1
50-d(TAGGGCAATACT)
30-d(ATCCAGTTATGA)
50-d(TAGGGCAATACT)
30-d(ATCC2GTTATGA)
50-d(TAGGGCAATACT)
30-d(ATCC3GTTATGA)
50-d(TAGGGCAATACT)
30-d(ATCC4GTTATGA)
50-d(TAGGTC4ATACT)
30-d(ATCCAGTTATGA)
50-d(TAGGGCAATACT)
30-d(ATCCAGTT4TGA)
50-d(TAGGTC4ATACT)
30-d(ATCC4GTTATGA)
50-d(TAGGGCAATACT)
30-d(ATC4AGTTATGA)
20
19
20
21
20
12
20
13
46
48
50
45
0
K10.5
K10.6
K11.6
K10.1
14
19
20
17
14
13
20
15
36
40
35
39
K11
K6
K7.7
K8.7
K7.7
K8.5
C2
C4
K1
K12
K7
50-d(TAGGACAATACT)
30-d(ATCCAGTTATGA)
50-d(TAGGACAATACT)
30-d(ATCC2GTTATGA)
50-d(TAGGACAATACT)
30-d(ATCC3GTTATGA)
50-d(TAGGACAATACT)
30-d(ATCC4GTTATGA)
50-d(TAGGGCAATACT)
30-d(AT4CAGTTATGA)
22
23
22
21
22
12
22
13
38
37
41
34
0
20
16
36
K10
K7.7
K1
C3
K4
K7.8
K8.7
K7.3
a Determined in 10 mM sodium cacodylate-buffer, pH 7.0, containing 100 mM NaCl and 10 mM MgCl2.
b 3Z7-bromo-7-deaza-2,8-diaza-20-deoxyadenosine, 4Z5-amino-3-bromo-pyrazole-4-carboxamide-20-deoxyfuranoside.
c Per modified base pair.
d 1 calZ4.184 J.
2.3. Duplex stability and base pairing selectivity
duplexes containing the bromo compound 3 over that of the
non functionalized nucleoside 2. For the assembly of base
pairs of compound 4 with dT or dG, the most plausible
motifs are iii and iv. As the nucleobase of compound 4 has a
smaller surface area than that of compound 3, stacking
interactions are expected to decrease while conformational
freedom increases. This results in lower Tm values in the
case of 4 compared to 3 (see Table 3). It is worth to mention
that the duplex containing two adenine-like moieties facing
each other shows an exceptionally high Tm value (duplex
22$12). Such a phenomenon has already been described for
duplexes in which two identical purine or purine-like bases
are located opposite to each other. This is correlated to the
intra-strand stacking observed for the purine–purine
nucleobases.43
For the investigation of the duplex stability and the base
pairing selectivity of oligonucleotides containing com-
pounds 3 or 4, the duplex 50-d(TAG GTC AAT ACT)
(18)$30-d(ATC CAG TTA TGA)(19)42 was modified in a
central position (see Table 3). Within these duplexes the
modified nucleosides were positioned opposite to the four
canonical DNA-constituents. As it can be seen from the data
of Table 3, compound 3 shows a different base recognition
than the canonical 20-deoxyadenosine. Compared to dA,
compound 3 forms a weaker base pare with dT (duplex
18$12), but stronger base pairs with dG (duplexes 20$12).
Compound 2 shows the same trend and compound 4 forms
weaker base pairs in all cases. In cases when dA or dC was
located opposite to 2, 3 or 4, the duplexes were significantly
less stable (Table 2) than those with dG or dT opposite to 2,
3, or 4. Thus, the base pairing preference of the compounds
3 and 4 is in the relative order dGOdT[dAwdC.
Compound 4 was also incorporated in various positions of
oligonucleotide duplexes showing that the stability was
depending on the position of incorporation (only shown for
the 4-dT pair; Table 3).
3. Conclusion
Oligonucleotides containing 7-bromo-7-deaza-2,8-diaza-20-
deoxyadenosine (3) are obtained when nucleobase protect-
ing groups are chosen which avoid harsh alkaline treatment
at elevated temperature during deprotection. As base-labile
protecting groups the dimethylaminomethylidene residue
was selected for the nucleoside 3 and p-tert-butylphenoxy-
acetyl residues for the canonical phosphoramidites dA, dG
and dC.44 Oligonucleotides containing 7-bromo-7-deaza-
2,8-diaza-20-deoxyadenosine (3) are transformed into those
incorporating 5-amino-3-bromo-4-carbarmoyl-1-(2-deoxy-
b-D-erythro-pentofuranosyl)-1H-pyrazole (4) when they
were treated with 25% aq ammonia at 60 8C for 30 h.
Similar reaction conditions (25% aq ammonia at 60 8C for
16 h) did not convert the non-halogenated nucleoside 2 into
Regarding base pairing we have already suggested motifs11
including those for the base pairs of 2 with dT as well as that
of 2 with dG. Similar motifs (i and ii) are now considered for
compound 3 (Scheme 5). In both motifs, the bulky bromo
substituents are well accommodated in the major groove of
B-DNA. At the same time the bromo residues will increase
base stacking as it has been observed in the case of
pyrazolo[3,4-d]pyrimidine nucleosides. Consequently, the
Tm values are increased in the case of oligonucleotide

7524
W. Lin et al. / Tetrahedron 61 (2005) 7520–7527
Scheme 5.
its ring opened derivative.11 Oligonucleotide duplexes
containing compounds 3 or 4 show base pairing selectivity
when located opposite to the canonical DNA constituents.
Nucleoside 3 forms a strong base pair with dG and a slightly
weaker one with dT as it was reported for other 2-azapurine
nucleosides such as 7-deaza-2,8-diaza-20-deoxyadeno-
sine.11 Oligonucleotide duplexes incorporating the haloge-
nated nucleoside 3 are thermally more stable than those
containing the non-functionalized nucleoside 2. Duplexes
containing the pyrazole nucleoside 4 show a similar base
pairing selectivity as those incorporating 3. The pyrazole
containing oligonucleotides are less stable than those
containing the ‘purine’ base 3 which is related to a
decreased surface area resulting in weaker stacking
interactions and the higher conformational flexibility of
the pyrazole moiety.
a delay time of 600 ns. -Solvents: technical grade, distilled
before use. Chemicals were purchased from ACROS, Fluka,
or Sigma-Aldrich (Sigma-Aldrich Chemie GmbH, Deisen-
hofen, Germany).
Oligonucleotides. The oligonucleotide synthesis was per-
formed applying phosphoramidite chemistry on a solid
support in an automated DNA-synthesizer with Controlled
Pore Glass (CPG) 500 serving as solid phase, to which the
starting nucleoside was bound via its O-30-position. The
4-tert-butylphenoxyacetyl-protected canonical phosphora-
midites (Millipore) were used for the oligonucleotides
synthesized. The syntheses were performed in a 1-mmol
scale according to standard conditions41 except that 145 s
coupling time was used for the modified phosphoramidite
(instead of 25 s) and the capping reagent CAP A contained
4-tert-butylphenoxyacetic anhydride instead of acetic
anhydride. After cleavage from the support, the oligomers
were incubated with a 25% aq NH3 solution. The following
conditions were employed: (i) room temperature, 12 h for
oligonucleotides containing 3; (ii) 60 8C, 30 h for oligo-
nucleotides containing 4. The deprotected oligomers were
purified on a 4!250 mm RP-18 HPLC column (10 mm)
with the following solvent gradient system: gradients of
MeCN (A) and 0.1 M Et3NHOAc (pH 7.0)/MeCN 95:5 (B);
gradient: 3 min 15% A in B, 7 min 15–40% A in B, 10 min
40% A in B and 5 min 15% A in B, and with flow rate of
1.0 ml minK1. The content of the main peak was collected
and evaporated to dryness, and the residue was treated with
2.5% CHCl2COOH/CH2Cl2 for 3 min at room temperature
to remove the 4,40-dimethoxytrityl residues. The detrityl-
ated oligomers were purified by reversed-phase HPLC
(RP-18, 4!250 mm, 10 mm) with the following solvent
gradient systems: gradients of MeCN (A) and 0.1 M
Et3NHOAc (pH 7.0)/MeCN 95:5 (B); gradient i: 25 min
0–20% A in B, 5 min 20% A in B, 5 min 20–0% A in B and
5 min B, with flow rate of 1 ml minK1 for oligonucleotide 5;
gradient ii: 30 min 0–15% A in B, 5 min 15–20% A in B,
5 min 20–0% A in B and 5 min B, with flow rate of
1 ml minK1 for oligonucleotides 6–10. All the oligomers
were desalted (RP-18, 4!100 mm, silica gel) and lyophi-
lized on a Speed-Vac evaporator to yield colourless solids
which were frozen at K24 8C.
4. Experimental
4.1. General
Monomers. Flash chromatography (FC) was performed at
0.4 bar on silica gel 60 H (VWR, Darmstadt, Germany).
Thin-layer chromatography (TLC) was carried out on TLC
aluminium sheet covered with silica gel 60 F254 (0.2 mm,
VWR, Germany). Reversed-phase HPLC was performed on
a 4!250 mm RP-18 LiChrosorb column (10 mm, VWR,
Germany) with a Merck-Hitachi HPLC pump (model 655
A-12) connected with a variable wavelength monitor
(model D-2000). UV spectra were recorded on a U3200
spectrophotometer (Hitachi, Japan). lmax in nm, 3 in
dm3 molK1. NMR spectra were measured on an Avance
DPX-250 spectrometer or an AMX-500 spectrometer
(Bruker, Rheinstetten, Germany) at 250.13 and
1
500.14 MHz for H nuclei and 62.90 and 125.13 MHz for
13C nuclei. Chemical shifts (d) are in ppm relative to internal
SiMe4 (1H, 13C) or external 85% H3PO4 (31P). Microanalyses
¨
were performed by Mikroanalytisches Labor Beller (Got-
tingen, Germany). MALDI-TOF mass spectra were recorded
ona Biflex-III spectrometer(Bruker, Leipzig, Germany) inthe
reflector mode. The average power of the nitrogen laser
(337.1 nm) at 20 Hz was 3–4 mW (150–200 mJ/pulse) with

W. Lin et al. / Tetrahedron 61 (2005) 7520–7527
7525
The nucleoside composition of oligonucleotides was
determined by enzymatic hydrolysis. The oligonucleotides
were dissolved in 0.1 M Tris–HCl buffer (pH 8.3, 200 ml),
and treated with snake-venom phosphodiesterase (EC
3.1.15.1, Crotallus adamanteus) (3 ml) at 37 8C for 45 min
and alkaline phosphatase (EC 3.1.3.1, E. coli from Roche
Diagnostics GmbH, Germany) (3 ml) at 37 8C for another
30 min. The reaction mixture was analyzed by HPLC
(RP-18, at 260 nm) solvent system 0.1 M (Et3NH)OAc,
pH 7.0/MeCN 95:5) showing the peaks of the modified
and the unmodified nucleosides. The oligonucleotides
were also characterized by MALDI-TOF-spectra. The
masses were in agreement with the calculated values
(Table 2).
repeatedly to remove HOAc. The residue was then applied
to FC (silica gel, column 2.5!6 cm, CH2Cl2–MeOH 20:1)
to give 8 as yellowish crystals (0.20 g, 88%); mp 184 8C
(dec.); Rf (CH2Cl2–MeOH 15:1) 0.18. UV (MeOH): lmax
1
235 (27,400). H NMR (D6 (DMSO)), 2.48, 2.88 (2m, 2H,
H2–C(20)); 3.39, 3.52 (2m, 2H, H2–C(50)); 3.87 (m, 1H,
H–C(40)); 4.51 (m, 1H, H–C(30)); 4.70 (t, JZ5.6 Hz, 1H,
OH–C(50)); 5.41 (d,0JZ4.7 Hz, 1H, OH–C(30)); 6.72 (t, JZ
5.9 Hz, 1H, H–C(1 )); 7.83 (d, JZ1.6 Hz, 1H, H–C(11));
8.81 (d, JZ1.6 Hz, 1H, H–C(10)). Anal. Calcd for
C11H11BrN6O3 (355.15): C 37.20, H 3.12, N 23.66; found:
C 36.98, H 3.30, N 23.51.
4.1.4. 4-Amino-5-bromo-7-(2-deoxy-b-D-erythro-pento-
furanosyl)-7H-pyrazolo[3,4-d][1,2,3]triazine (7-bromo-
7-deaza-2,8-diaza-20-deoxyadenosine) (3). To a solution
of compound 8 (135 mg, 0.38 mmol) in aq sodium acetate
(1 M, pH 4.0–4.5, 30 ml) N-bromosuccinimide (0.65 g,
3.65 mmol) was added at 40–50 8C, and the reaction
mixture was stirred at room temperature overnight.
After evaporation the residue was applied to FC (silica
gel, 2.5!8 cm, CH2Cl2–MeOH 15:1 to 9:1), and the
content of the main zone was collected and crystallized
from H2O to yield yellow crystals (76 mg, 60%); mp
189 8C (dec.); Rf (CH2Cl2–MeOH 6:1) 0.44. UV
(MeOH): lmax 318 (6900), 255 (5700). 1H NMR (D6
(DMSO)), 2.36, 2.82 (2m, 2H, H2–C(20)), 3.38, 3.52 (m,
2H, H2–C(50)); 3.85 (m, 1H, H–C(40)); 4.46 (m, 1H, H–
C(30)); 4.74 (t, JZ5.7 Hz, 1H, OH–C(50)); 5.35 (d, JZ
4.7 Hz, 1H, OH–C(30)); 6.74 (t, JZ6.2 Hz, 1H, H–
C(10)); 7.55, 8.60 (2br, 2H, NH2). Anal. Calcd for
C9H11BrN6O3 (331.13): C 32.65, H 3.35, N 25.38; found:
C 32.67, H 3.32, N 25.21.
The melting curves were measured with a Cary-1/3 UV/VIS
spectrophotometer equipped with a Cary thermoelectrical
controller. The thermodynamic data were calculated with
the program Meltwin 3.0.45
4.1.1. 9-Bromo-7-(2-deoxy-b-D-erythro-pentofuranosyl)-
imidazo[1,2-c]-7H-pyrazolo[4,3-e]pyrimidine (6). To a
stirred solution of compound 5 (0.63 g, 1.91 mmol) in
aqueous sodium acetate (1 M, pH 4.5–5.0, 70 ml) chloro-
acetaldehyde (50% aqueous solution, 12 ml) was added at
40–50 8C. Stirring was continued at room temperature for
3 days. The reaction mixture was evaporated, and the
residue was applied to FC (silica gel, 4!8 cm, CH2Cl2–
MeOH 100:2 to 15:1) to give compound 6 (0.56 g, 83%) as
colourless crystals (MeOH); mp 227 8C (dec.); Rf (CH2Cl2–
MeOH 15:1) 0.25. UV (MeOH): lmax 283 (3300), 260
1
(4800), 234 (29,600). H NMR (D6 (DMSO)), 2.35, 2.84
(2m, 2H, H2–C(20)); 3.39, 3.53 (2m, 2H, H2–C(50)); 3.86
(m, 1H, H–C(40)); 4.47 (m, 1H, H-C(30)); 4.73 (t, JZ5.6 Hz,
1H, OH–C(50)); 5.34 (d, JZ4.5 Hz, 1H, OH–C(30)); 6.68
(t, JZ6.2 Hz, 1H, H–C(10)); 7.57 (s, 1H, H–C(11)); 8.09
(s, 1H, H–C(10)); 9.35 (s, 1H, H–C(2)). Anal. Calcd for
C12H12BrN5O3 (354.16): C 40.70, H 3.42, N 19.77; found:
C 40.74, H 3.52, N 19.60.
4.1.5. 5-Bromo-7-(2-deoxy-b-D-erythro-pentofuranosyl)-
4-(dimethylaminomethylidene)-7H-pyrazolo[3,4-d]
[1,2,3]triazine (9). To a solution of compound 3 (0.12 g,
0.36 mmol) in MeOH (30 ml) N,N-dimethylformamide
dimethylacetal (0.18 ml, 1.34 mmol) was added while
stirring at 40 8C for 2.5 h. The solution was evaporated,
and the residue was purified on a silica gel column (2.5!
8 cm, CH2Cl2–MeOH 20:1 to 15:1) to yield a colourless
foam (0.11 g, 79%). Rf (CH2Cl2–MeOH 15:1) 0.2. UV
4.1.2. 5-Amino-3-bromo-1-(2-deoxy-b-D-erythro-pento-
furanosyl)-4-(imidazol-2-yl)-1H-pyrazole (7). A solution
of compound 6 (0.53 g, 1.50 mmol) in aqueous NaOH
(0.8 M, 38 ml) was stirred overnight at room temperature.
The solution was acidified to pH 7.0 with hydrochloric acid
(2 M), concentrated and purified by FC (silica gel, 4!6 cm,
CH2Cl2–MeOH 20:1 to 15:1) to yield a colourless foam
(0.24 g, 47%). Rf 1(CH2Cl2–MeOH 6:1) 0.3. UV (MeOH):
lmax 260 (9300). H NMR (D6 (DMSO)), 2.11, 2.66 (2m,
2H, H2–C(20)); 3.40, 3.47 (2m, 2H, H2–C(50)); 3.78 (m, 1H,
H–C(40)); 4.35 (m, 1H, H–C(30)); 4.85 (m, 1H, OH–C(50));
5.22 (d, JZ3.1 Hz, 1H, OH–C(30)); 6.10 (t, JZ6.3 Hz, 1H,
H–C(10)); 6.72 (s, 2H, NH2); 7.00 (d, JZ19.2 Hz, 2H,
H–C(7), H–C(8)); 11.34 (s, 1H, NH). Anal. Calcd for
C11H14BrN5O3 (344.16): C 38.39, H 4.10, N 20.35; found:
C 38.56, H 4.23, N 20.46.
1
(MeOH): lmax 339 (20,900), 226 (10,500). H NMR (D6
(DMSO)), 2.37 (m, 1H, Ha–C(20)); 2.85 (m, 1H, Hb–C(20));
3.26, 3.31 (2s, 6H, 2CH3); 3.54 (m, 1H, H–C(50)); 3.85 (m,
1H, H–C(40)); 4.45 (m, 1H, H–C(30)); 4.74 (t, JZ5.6 Hz,
1H, OH–C(50)); 5.36 (d, JZ4.6 Hz, 1H, OH–C(30)); 6.76 (t,
JZ6.3 Hz, 1H, H–C(10)); 9.16 (s, 1H, CH). Anal. Calcd for
C12H16BrN7O3 (386.20): C 37.32, H 4.18, N 25.39; found:
C 37.38, H 4.26, N 25.24.
4.1.6. 5-Bromo-7-[2-deoxy-5-O-(4,40-dimethoxytriphen-
ylmethyl)-b-D-erythro-pentofuranosyl]-4-(dimethylami-
nomethylidene)-7H-pyrazolo[3,4-d][1,2,3]triazine (10).
Compound 9 (77 mg, 0.2 mmol) was coevaporated twice
with anhydrous pyridine, dissolved in anhydrous pyridine
(2 ml) and treated with 4,40-dimethoxytriphenylmethyl
chloride (84 mg, 0.25 mmol) at room temperature for 2 h
while stirring. Thereupon, MeOH (1 ml) was added and the
stirring was continued for 10 min. The solution was
concentrated to half of the volume and CH2Cl2 (70 ml)
was added. The organic phase was washed with aqueous
4.1.3. 9-Bromo-7-(2-deoxy-b-D-erythro-pentofuranosyl)-
imidazo[1,2-c]-7H-pyrazolo[4,3-e][1,2,3]triazine (8).
A solution of compound 7 (0.22 g, 0.64 mmol) in 80% aq
HOAc (20 ml) was treated with sodium nitrite (47 mg,
0.68 mmol) during one hour in an ice bath. The reaction
solution was evaporated, dissolved in water and evaporated

7526
W. Lin et al. / Tetrahedron 61 (2005) 7520–7527
NaHCO3 (twice, 5%, 2!15 ml), with water (twice, 2!
15 ml) and once with a saturated NaCl (20 ml). The organic
layer was separated and dried over Na2SO4, filtered,
evaporated, and the residue was chromatographed on silica
gel (column 2.5!6 cm, CH2Cl2–MeOH 200:1) to yield a
yellowish foam (114 mg, 83%). Rf (CH2Cl2–MeOH 20:1)
References and notes
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0.35. UV (MeOH): lmax 339 (17,000), 2032 (25,700). H
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(157 mg, 0.47 mmol) was stirred with 25% aq ammonia
(20 ml) for 16 h at 60 8C in a sealed vessel. The reaction
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MeOH 20:1 to 15:1) to yield a colourless foam (96 mg,
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We thank Dr Xiaomei Zhang for measuring the
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MALDI-TOF mass spectra and Mrs. Elisabeth Michalek
for the oligonucleotide syntheses. We also appreciate the
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