Pyrazolo[3,4-d][1,2,3]triazine DNA: Synthesis and base pairing of 7-deaza-2,8-diaza-2′-deoxyadenosine

Article abstract of DOI:10.1021/jo040150i

7-Deaza-2,8-diaza-2′-deoxyadenosine (4) was synthesized from 8-aza-7-deaza-2′-deoxyadenosine (1) via the 1,N⁶-etheno derivative 5. Ring opening with sodium hydroxide followed by ring closure in the presence of sodium nitrite formed the tricycli

Full text of DOI:10.1021/jo040150i

P yr a zolo[3,4-d ][1,2,3]tr ia zin e DNA: Syn th esis a n d Ba se P a ir in g of
7-Dea za -2,8-d ia za -2′-d eoxya d en osin e
Frank Seela,* Meike Lindner, Virginie Glac¸on, and Wenqing Lin
Laboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie, Universita¨t Osnabru¨ck,
Barbarastr. 7, 49069 Osnabru¨ck, Germany, and Center for Nanotechnology, Gievenbecker Weg 11,
48149 Mu¨nster, Germany
frank.seela@uni-osnabrueck.de
Received March 16, 2004
7-Deaza-2,8-diaza-2′-deoxyadenosine (4) was synthesized from 8-aza-7-deaza-2′-deoxyadenosine (1)
via the 1,N6-etheno derivative 5. Ring opening with sodium hydroxide followed by ring closure in
the presence of sodium nitrite formed the tricyclic intermediate 5 from which the transiently
introduced “etheno” moiety was removed with NBS. Compound 4 was converted to the phosphor-
amidite 11, which was employed in solid-phase oligonucleotide synthesis. Base pairing studies on
4, incorporated in a 12-mer duplex, showed that this adenine nucleoside analogue forms a strong
base pair with dG but not with dT. This novel base pair is as stable as that of the canonical dA-dT
pair. As a result of the absence of nitrogen-7 compound 4 is expected to form a face to face base
pair with dG.
In tr od u ction
The aza and deaza derivatives of purine nucleosides
attract attention because of their biological activities as
antifungal, antiviral, and anticancer agents.1-3 They are
also valuable tools in bioorganic chemistry, molecular
biology, and nanotechnology.4-9 Meanwhile, a variety of
such base-modified nucleosides are available either by
chemical synthesis or from natural origin. Among them
are the nucleosides modified in the imidazole moiety such
as the 7-deazapurine nucleosides,10,11 the 8-aza-7-de-
azapurine nucleosides and the 8-azapurine nucleo-
sides.12-18 Compounds such as 1 (Figure 1), which can
* To whom correspondence should be addressed. Tel. +49(0)541-
9692791. Fax +49(0)541-9692370.
(1) Simons, C. Nucleoside Mimetics, Their Chemistry and Biological
Properties; Gordon and Breach Science Publishers: Langhorne, PA,
2001; pp 83-102 and references therein.
(2) Isono, K. Pharmacol. Ther. 1991, 52, 269-286.
(3) De Clercq, E. Nat. Rev. Drug Discovery 2002, 1, 13-25.
(4) He, J .; Mikhailopulo, I.; Seela, F. J . Org. Chem. 2003, 68, 5519-
5524.
(5) Seela, F.; He, Y. J . Org. Chem. 2003, 68, 367-377.
(6) Seela, F.; Kro¨schel, R. Nucleic Acids Res. 2003, 31, 7150-7158.
(7) He, J .; Seela, F. Nucleic Acids Res. 2002, 30, 5485-5496.
(8) Kandimalla, E. R.; Bhagat, L.; Wang, D.; Yu, D.; Zhu, F.-G.;
Tang, J .; Wang, H.; Huang, P.; Zhang, R.; Agrawal, S. Nucleic Acids
Res. 2003, 31, 2393-2400.
F IGURE 1.
be considered to be isosteric to 2′-deoxyadenosine (2),
have been already incorporated in duplex DNA.19
The 2-azapurines, such as 2-azaadenine and 2-azahy-
poxanthine, have also long been known to inhibit the
growth of both microbial and mammalian cells.20-29
Although the physical properties of these nucleosides
have been studied,30 very few were incorporated in
oligonucleotides.31 Recently, 2-aza-2′-deoxyadenosine (3)
was synthesized by our laboratory, and its behavior
within oligonucleotide duplexes was studied.32,33 In con-
(9) Niemeyer, C. M. Angew. Chem., Int. Ed. Engl. 1997, 36, 585-
587.
(10) Robins, R. K.; Revankar, G. R. Med. Res. Rev. 1985, 5, 273-
296.
(11) Limbach, P. A.; Crain, P. F.; McCloskey, J . A. Nucleic Acids
Res. 1994, 22, 2183-2196.
(12) Montgomery, J . A.; Shortnacy, A. T.; Secrist, J . A., III. J . Med.
Chem. 1983, 26, 1483-1489.
(13) Mizuno, Y.; Itoh, T.; Nomura, A. Heterocycles 1982, 17, 615-
644.
(14) Seela, F.; Lampe, S. Helv. Chim. Acta 1993, 76, 2388-2397.
(15) Seela, F.; Mersmann, K. Helv. Chim. Acta 1993, 76, 2184-2193.
(16) Seela, F.; Mersmann, K. Helv. Chim. Acta 1992, 75, 1885-1896.
(17) Kazimierczuk, Z.; Bindig, U.; Seela, F. Helv. Chim. Acta 1989,
72, 1527-1536.
(18) Seela, F.; Mu¨nster, I.; Lo¨chner, U.; Rosemeyer, H. Helv. Chim.
Acta 1998, 81, 1139-1155.
(19) Seela, F.; Kaiser, K. Helv. Chim. Acta 1988, 71, 1813-1823.
10.1021/jo040150i CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/17/2004
J . Org. Chem. 2004, 69, 4695-4700
4695

Seela et al.
SCHEME 1a
tinuation of the work on 2-azapurine nucleosides and
oligonucleotides and as the result of the unusual base
pairing properties of 3, the related 7-deaza-2,8-diazapu-
rine (7H-pyrazolo[3,4-d][1,2,3]triazine) system is now
investigated (purine numbering is used throughout the
general part). This manuscript reports on the synthesis
of 7-deaza-2,8-diaza-2′-deoxyadenosine (4), its conversion
into a building block for oligonucleotide synthesis, and
its base pairing properties within 12-mer duplexes.
Resu lts a n d Discu ssion
1. Syn th esis of th e Mon om er s. Up to now, several
procedures have been developed for the synthesis of
2-azapurine nucleosides, such as ring annulation on
imidazole nucleosides21,28,34-43 and the glycosylation of the
2-azapurine base.32,44,45 The ribonucleoside of 7-deaza-2,8-
diazapurine has already been prepared by Montgomery
et al. in 1972.21,28 However, the synthesis of the 2′-
deoxyribonucleoside 4 has not been reported so far.
Herein the synthesis of 4 is performed using the nucleo-
side 134 as starting material, thereby forming its 1,N6-
etheno derivative 5 as central intermediate (Scheme 1).
This synthetic route has been elaborated earlier for other
compounds34-37 and was recently successfully used for the
synthesis of 3 by our group.33
a
Reagents and conditions: (a) ClCH2CHO, 1 M aq NaOAc
buffer, pH 4.5-5, 82%; (b) 1 N NaOH, 83%; (c) NaNO2, 80% aq
HOAc, 72%; (d) NBS, 1 M aq NaOAc buffer, pH 4-4.5, 66%.
TABLE 1. 13C NMR Da ta of Nu cleosid esa
C(2)b
C(4)b
C(5)b
C(6)b
C(7)b C(10)b C(11)b
134 155.7 153.6
100.4
103.3
157.3
139.4
132.8
5
140.3 145.0
146.6c
142.7
131.8 132.4c 112.9c
Reaction of 1 with chloroacetaldehyde at pH 4.5-5
gave 1,N6-etheno-8-aza-7-deaza-2′-deoxyadenosine (5) in
82% yield, which was treated with 1 M sodium hydroxide
at room temperature to give the derivative 6 (83% yield).
Subsequent treatment of 6 with sodium nitrite in 80%
6e
7
96.3c 142.5c 137.5 122.4c 122.4c
104.3
96.0
99.4
99.9
99.4
131.1
153.2
150.3
150.3
150.2
131.3 132.9c 115.3c
4
8
9
149.2
147.9
147.6
147.9
132.9
136.8
136.8
136.6
10
C(1′) C(2′) C(3′) C(4′) C(5′) O-CH3 CdO CH CH3
134 84.1 38.0 71.0 87.5 62.3
84.3 38.0 71.1 87.9 62.4
(20) Krawczyk, S.; Migawa, M. T.; Drach, J . C.; Townsend, L. B.
Nucleosides Nucleotides Nucleic Acids 2000, 19, 39-68.
(21) Montgomery, J . A.; Laseter, A. G.; Shortnacy, A. T.; Clayton,
S. J .; Thomas, H. J . J . Med. Chem. 1975, 18, 564-567.
(22) Wooley, D. W.; Shaw, E. J . Biol. Chem. 1951, 189, 401-410.
(23) Shaw, E.; Wooley, D. W. J . Biol. Chem. 1952, 194, 641-654.
(24) Biesele, J . J . Cancer 1952, 5, 787-791.
5
6e 86.0
d
d
d
d
d
d
72.2 88.0 63.2
70.8 88.2 62.1
71.0 87.9 62.3
70.7 87.9 62.0
70.7 87.9 62.0
70.4 85.7 64.0
7
4
8
9
85.7
84.9
84.8
84.9
(25) Fjelde, A. Z. Krebsforsch. 1956, 61, 364-367.
(26) Guthrie, R.; Lu, W. N. Arch. Biochem. Biophys. 1964, 108, 398-
402.
177.4 34.6 19.0
170.6 23.9
177.4 34.6 19.0
10 84.8
54.7
(27) Cappuccino, J . G.; George, M.; Merker, P. C.; Tarnowski, G. S.
Cancer Res. 1964, 24, 1243-1248.
a
b
Measured in DMSO-d6 at 303 K. Purine numbering. c Ten-
(28) Montgomery, J . A.; Thomas, H. J . J . Med. Chem. 1972, 15, 182-
tative. Superimposed by the signal of DMSO-d6. e DMSO-d6-0.4
aq NH4OAc.
d
187.
(29) Bennett, L. L., J r.; Allan, P. W.; Carpenter, J . W.; Hill, D. L.
Biochem. Pharmacol. 1976, 25, 517-521.
(30) Lu¨demann, H.-D.; Westhof, E. Z. Naturforsch. 1977, 32c, 528-
aqueous acetic acid afforded 1,N6-etheno-7-deaza-2,8-
diaza-2′-deoxyadenosine (7) in 72%. Afterward compound
7 was treated with N-bromosuccinimide at pH 4-4.5 to
result in 4 (66% yield). The overall yield amounts to 32%
for the four steps. The structures of all compounds were
538.
(31) Acedo, M.; De Clercq, E.; Eritja, R. J . Org. Chem. 1995, 60,
6262-6269.
(32) Kazimierczuk, Z.; Seela, F. Liebigs Ann. Chem. 1990, 647-651.
(33) Sugiyama, T.; Schweinberger, E.; Kazimierczuk, Z.; Ramzaeva,
N.; Rosemeyer, H.; Seela, F. Chem. Eur. J . 2000, 6, 369-378.
(34) Seela, F.; Steker, H. Helv. Chim. Acta 1985, 68, 563-570.
(35) Yamaji, N.; Kato, M. Chem. Lett. 1975, 311-314.
(36) Yip, K. F.; Tsou, K. C. Tetrahedron Lett. 1973, 33, 3087-3090.
(37) Tsou, K. C.; Yip, K. F.; Miller, E. E.; Lo, K. W. Nucleic Acids
Res. 1974, 1, 531-547.
1
confirmed by H and 13C NMR spectra and by elemental
analyses (see Table 1 and Experimental Section). The 13
C
NMR resonances were assigned with the help of gated-
decoupled spectra (Supporting Information).
(38) Yip, K. F.; Tsou, K. C. J . Org. Chem. 1975, 40, 1066-1070.
(39) Kawana, M.; Ivanovics, G. A.; Rousseau, R. J .; Robins, R. K. J .
Med. Chem. 1972, 15, 841-843.
(40) Panzica, R. P.; Townsend, L. B. J . Heterocycl. Chem. 1972, 9,
623-628.
(41) Ivanovics, G. A.; Rousseau, R. J .; Kawana, M.; Srivastava, P.
C.; Robins, R. K. J . Org. Chem. 1974, 39, 3651-3654.
(42) Andres, J . I.; Herranz, R.; Garcia Lopez, M. T. J . Heterocycl.
Chem. 1984, 21, 1221-1224.
(43) Rayner, B.; Tapiero, C.; Imbach, J . L. J . Heterocycl. Chem. 1982,
19, 593-596.
(44) Parkin, A.; Harnden, M. R. J . Heterocycl. Chem. 1982, 19, 33-
40.
(45) Bakulev, V. A.; Mokrushin, V. S.; Ofitserov, V. I.; Pushkareva,
Z. V.; Grishakov, A. N. Khim. Geterotsikl. Soedin. 1979, 6, 836-838.
Next, the stability of the N-glycosylic bond of 4 was
measured in aqueous hydrochloric acid (0.1 M, rt). The
reaction was monitored by UV spectroscopy at 291 nm.
The stability of 4 was found to be similar (τ ) 41 min) to
that of dA (2) (τ ) 45 min) but lower than that of z2Ad
(3) (τ ) 63 min). The UV maximum of 4 (λmax ) 310 nm)
shows a strong red shift compared to compound 1 (λmax
) 260 nm). In acidic medium, the maximum is blue
shifted from λmax ) 310 nm to λmax ) 290 nm. Next, the
pK value of protonation of compound 4 was measured
4696 J . Org. Chem., Vol. 69, No. 14, 2004

Pyrazolo[3,4-d][1,2,3]triazine DNA
SCHEME 2a
a
Reagents and conditions: (a) (i) (CH3)3SiCl, Py, (ii) (Bui)2O for 8 or Ac2O for 9, then (iii) 10% NH3, 70% for 8, 60% for 9; (b) (MeO)2TrCl,
i
i
Py, 80%; (c) Pr2NP(Cl)OCH2CH2CN, Pr2EtN, CH2Cl2, 72%.
and compared with that of 3.46 Both compounds show
very similar values (pKa ) 1.7 for 4 and 1.9 for 3), which
are quite different from those of 1 (pKa ) 4.0) and 2 (pKa
) 3.8).47 We assume that the site of protonation is the
same for compound 4 (N-1) as it is for dA.
For the synthesis of oligonucleotides the phosphor-
amidite 11 was prepared. It involves at first the protec-
tion of the amino group of 4. Previous studies have shown
that the benzoyl group is inappropriate in this case
because of its long half-life.33 To look for a more appropri-
ate group, the isobutyryl and acetyl groups were selected
for protection. Thus, compound 4 was treated with
trimethylsilyl chloride and isobutyric anhydride or acetic
anhydride as described by J ones et al.48 to give compound
8 and 9 in 70% and 60% yield, respectively (Scheme 2).
Then, the stability of these protecting groups was studied.
The τ values of hydrolysis were determined in concen-
trated aqueous ammonia (40 °C) and monitored by UV
spectroscopy at 235 nm showing that both the isobutyryl
and the acetyl group have appropriate half-lives of 51
min for 8 and of 31 min for 9. Because of the higher yield
of 8, it was used for further manipulations. The introduc-
tion of the 4,4′-dimethoxytrityl group followed the stan-
dard protocol, giving compound 10 in 80% yield.49 Sub-
sequent phosphitylation with chloro-(2-cyanoethoxy)-
N,N-(diisopropylamino)phosphine gave the phosphor-
amidite 11 (72% yield).50 All new compounds were
F IGURE 2. RP-18 HPLC-elution profile of enzymatic digests
of the oligonucleotides 5′-d(4 CGC GCG) (16) (a) and 5′-d(AGT
4TT G4C CTA) (15) (b).
shown in the following tables. For the investigation of
the base pairing selectivity and base pair stability of
compound 4, two oligonucleotides, 5′-d(TAG GTC AAT
ACT) (12) and 3′-d(ATC CAG TTA TGA) (13), were
synthesized to form the already known reference duplex
12‚13.18 For characterization, the oligonucleotides 14, 15,
and 16 were hydrolyzed with snake venom phosphodi-
esterase, followed by alkaline phosphatase19 to yield the
nucleosides, which were separated by RP-18 HPLC
(Figure 2 and Supporting Information). The oligonucle-
otides were also characterized by MALDI-TOF spectra
(Supporting Information). The masses were in good
agreement with the calculated values (Supporting Infor-
mation).
Initially, compound 4, as an analogue of dA, was
incorporated opposite to dT. As can be seen from Table
2, the incorporation of one 4-dT base pair reduces the
Tm of the duplex 17‚13 as well as of 12‚18 by 5 and 4 °C,
respectively. Incorporation of two modified base pairs
shows a decrease of the Tm value within the range of
8-13 °C (19‚13, 12‚15, 20‚13). In these cases the position
of incorporation results in slightly different stabilities due
to nearest neighbor influences. Consecutive incorporation
of 4-dT base pairs led to a decrease stronger than of that
of separated base pairs. The duplex 20‚13 possessing a
terminal modification has a minor effect on the Tm value
1
characterized by H and 13C NMR spectra and by ele-
mental analysis (see Table 1 and Experimental Section).
2. Syn th esis a n d Ba se P a ir in g P r op er ties of Oli-
gon u cleotid es. The oligonucleotide synthesis was per-
formed applying the phosphoramidite 11 in solid-phase
oligonucleotide chemistry in an automatized DNA syn-
thesizer on controlled pore glass (CPG) 500 serving as
solid phase.51 The oligomers were deprotected by incuba-
tion with a 25% aqueous NH3 solution (60 °C, 20 h) and
purified by RP-18 HPLC. The oligomers synthesized are
(46) Albert, A.; Serjeant, E. P. The Determination of Ionization
Constants; Chapman and Hall Ltd.: London, 1971; pp 44-64.
(47) Seela, F.; Ramzaeva, N.; Rosemeyer, H. Purines. In Science of
Synthesis; Shinkai, N., Yamamoto, Y., Eds.; Thieme: Stuttgart, 2003;
Vol. 16, pp 945-1108.
(48) Ti, G. S.; Gaffney, B. L.; J ones, R. A. J . Am. Chem. Soc. 1982,
104, 1316-1319.
(49) Schaller, H.; Weimann, G.; Lerch, B.; Khorana, H. G. J . Am.
Chem. Soc. 1963, 85, 3821-3827.
(50) Beaucage, S. L.; Caruthers, M. Tetrahedron Lett. 1981, 22,
1859-1862.
(51) McDowell, J . A.; Turner, D. H. Biochemistry 1996, 35, 14077-
14089.
J . Org. Chem, Vol. 69, No. 14, 2004 4697

Seela et al.
SCHEME 3a
TABLE 2. Tm Va lu es a n d Th er m od yn a m ic Da ta of
Oligon u cleotid e Du p lexes Con ta in in g Regu la r a n d
Ba se-Mod ified Nu cleosid e Resid u esa ,b
Tm
[°C]
∆G310
duplex
no.
∆Tm
[kcal/mol]d
c
5′-d(TAG GTC AAT ACT)
3′-d(ATC CAG TTA TGA)
(12)
(13)
47
0
-10.4
5′-d(TAG GTC A4T ACT)
3′-d(ATC CAG TTA TGA)
5′-d(TAG GTC AAT ACT)
3′-d(ATC C4G TTA TGA)
5′-d(TAG GTC 4 4 T ACT)
3′-d(ATC CAG TTA TGA)
5′-d(TAG GTC AAT ACT)
3′-d(ATC C4G T T4 TGA)
5′-d(T 4G GTC AAT 4CT)
3′-d(ATC CAG TTA TGA)
(17)
(13)
(12)
(18)
(19)
(13)
(12)
(15)
(20)
(13)
42
43
34
37
39
-5
-4
-8.8
-9.1
-7.0
-7.6
-8.0
-6.5
-5
-4
5′-d(TAG GTC AAT ACT)52
3′-d(ATC CAG TGA TGA)
(12)
(21)
44
48
0
0
-9.2
a
Face to face base pair motifs of (I) 4-dG and (II) 4-dT; (III)
alternative Hoogsteen motif for 3-dG base pair.
5′-d(TAG GGC AAT ACT)52 (22)
-10.5
3′-d(ATC CAG TTA TGA)
(13)
TABLE 3. Com p a r ison of Tm Va lu es a n d
5′-d(TAG GTC A4T ACT)
3′-d(ATC CAG TGA TGA)
5′-d(TAG GGC AAT ACT)
3′-d(ATC C4G TTA TGA)
5′-d(TAG GTC 4 4T ACT)
3′-d(ATC CAG GGA TGA)
5′-d(TAG GGC AAG ACT)
3′-d(ATC C 4G T T 4 TGA)
5′-d(T 4G GTC AAT 4 CT)
3′-d(AGC CAG TTA GGA)
(17)
(21)
(22)
(18)
(19)
(23)
(24)
(15)
(20)
(25)
46
48
46
46
48
+2
0
-9.6
-10.6
-9.2
Th er m od yn a m ic Da ta of Du p lex Con ta in in g z2c7z8Ad
a n d z2Ad 3a ,b
4
Tm
∆G310
c
duplex
no.
[°C]
∆Tm
+1
[kcal/mol]d
+1
+1
+1
5′-d(TAG GTC 4 4T ACT)
3′-d(ATC CAG GGA TGA)
5′-d(TAG GTC 3 3T ACT)
3′-d(ATC CAG GGA TGA)
5′-d(TAG GGC AAG ACT)
3′-d(ATC C 4G T T 4 TGA)
5′-d(TAG GGC AAG ACT)
3′-d(ATC C 3G T T 3 TGA)
5′-d(TAG GTC 4 4T ACT)
3′-d(ATC CAG TTA TGA)
5′-d(TAG GTC 3 3T ACT)
3′-d(ATC CAG TTA TGA)
5′-d(TAG GTC AAT ACT)
3′-d(ATC C 4G TT 4 TGA)
5′-d(TAG GTC AAT ACT)
3′-d(ATC C 3G TT 3 TGA)
(19)
(23)
(34)
(23)
(24)
(15)
(24)
(35)
(19)
(13)
(34)
(13)
(12)
(15)
(12)
(35)
46
46
46
45
34
37
37
37
-9.2
-10.1
-9.4
-11.5
-7.0
-7.7
-7.6
-7.6
-9.4
+1
+1
-10.0
5′-d(TAG GTC A4T ACT)
3′-d(ATC CAG TAA TGA)
5′-d(TAG GAC AAT ACT)
3′-d(ATC C4G TTA TGA)
5′-d(TAG GTC 4 4T ACT)
3′-d(ATC CAG AAA TGA)
5′-d(TAG GAC AAA ACT)
3′-d(ATC C 4G T T 4 TGA)
5′-d(TAG GTC A4T ACT)
3′-d(ATC CAG TCA TGA)
5′-d(TAG GCC AAT ACT)
3′-d(ATC C 4G TTA TGA)
5′-d(TAG GTC 4 4T ACT)
3′-d(ATC CAG CCA TGA)
5′-d(TAG GCC AAC ACT)
3′-d(ATC C 4G T T 4 TGA)
(17)
(26)
(27)
(18)
(19)
(30)
(31)
(15)
(17)
(28)
(29)
(18)
(19)
(32)
(33)
(15)
34
37
21
21
32
36
20
20
-13
-7.0
-7.8
-5.7
-5.3
-6.8
-7.4
-5.2
-5.3
+0.5
-6.5
-5
-10
-13
-13
-5
-15
-5
-11
a
4 ) z2c7z8Ad ) 7-deaza-2,8-diaza-2′-deoxyadenosine; 3 ) z2Ad
-13.5
-13.5
b
) 2-aza-2′-deoxyadenosine. Determined in 10 mM sodium ca-
codylate buffer, pH 7.0, containing 100 mM NaCl and 10 mM
MgCl2. c Per modified base pair. 1 cal ) 4.184 J .
d
a
b
4 ) z2c7z8Ad ) 7-deaza-2,8-diaza-2′-deoxyadenosine. Deter-
mined in 10 mM sodium cacodylate buffer, pH 7.0, containing 100
strong base pair with dG comparable to that of a dA-dG
pair (12‚21 and 22‚13) or that of dA-dT (12‚13).
mM NaCl and 10 mM MgCl2. c Per modified base pair. 1 cal )
d
4.184 J .
In the following experiments the nucleoside 4 was
introduced opposite to dA or dC residues. This results in
a dramatic decrease of the Tm values. One incorporation
opposite to dA (17‚26 and 27‚18) reduces the Tm by -13
and -10 °C, respectively, and opposite to dC (17‚28 and
29‚18) by -15 and -11 °C. In both cases, two incorpora-
tions (19‚30, 31‚15, 19‚32, and 33‚15) show a decrease
of the Tm values in the range of 26-27 °C. Regarding
these results, the base of compound 4 forms a strong base
pair with guanine but a much weaker one with thymine,
adenine, and cytosine with an order of relative stability
of dG > dT > dA = dC, which is similar to 3.33
It seems interesting to compare the duplex stability of
compounds 3 and 4. Because of the absence of nitrogen-7
in compound 4 this molecule cannot form a Hoogsteen
base pair, which makes the formation of a face to face
base pair as shown in motif (I) very likely (Scheme 3).
As seen from Table 3 the incorporation of the nucleosides
3 or 4 opposite to dG causes duplexes of almost identical
as a modification in a central position (12‚15). These
observations demonstrate that the 4-dT base pair is
significantly less stable than that of dA-dT.
Next, compound 4 was incorporated into a duplex
position opposite to dG. In these cases the duplexes of
12‚21 and 22‚13 are used as reference compounds.
Replacement of one dA-dG base pair by one 4-dG pair
(17‚21; 22‚18) resulted in no significant change of the
Tm value (∆Tm ) 1-2 °C). Incorporation of two 4-dG pairs
shows also no significant changes in the stability (∆Tm
) 1-2 °C). All consecutive central 4-dG pairs (19‚23),
separated central 4-dG pairs (24‚15), and peripheral 4-dG
base pairs (20‚25) exhibit similar stabilities with ∆Tm
)
1-2 °C. Moreover, no matter if one or two 4-dG were
introduced, the duplexes have the same stability as that
of the standard duplexes (12‚13, 12‚21, and 22‚13) with
a ∆Tm range of 1-2 °C. Thus, the nucleoside 4 forms a
4698 J . Org. Chem., Vol. 69, No. 14, 2004

Pyrazolo[3,4-d][1,2,3]triazine DNA
TABLE 4. Tm Va lu es a n d Th er m od yn a m ic Da ta of
to the 4-dG pair might result from a steric clash of the
2-oxo group of dT with nitrogen-2 of 4 as well as an
electronic repulsion of these residues. In contrary, at-
tracting forces (H-bonding) are expected to stabilize the
base pair of 3 or 4 with dG (see motif (I)). As the motif
(I) is formed by two “purine” bases one might argue that
this will destabilize the sugar phosphate backbone and
distort the double helix. However, this unfavorable
situation might be compensated by the stronger stacking
of the odd “purine-purine” base pairs as has been reported
for the face to face pair of dG-dA.53
Oligon u cleotid es w ith Da n glin g Nu cleotid esa ,b
Tm
[°C]
∆G310
duplex
no.
∆Tm
0
[kcal/mol]c
5′-d(CGC GCG)33
3′-d(GCG CGC)
(36)
(36)
(16)
(16)
(38)
(38)
(39)
(39)
(40)
(40)
(37)
(37)
46
57
53
55
53
57
-8.2
5′-d(4 CGC GCG)d
3′-d(GCG CGC 4)
5′-d(3 CGC GCG)33
3′-d(GCG CGC 3)
5′-d(A CGC GCG)33
3′-d(GCG CGC A)
5′-d(T CGC GCG)33
3′-d(GCG CGC T)
5′-d(c7A CGC GCG)33
3′-d(GCG CGC c7A)
+11
+7
-10.1
-9.7
+9
-10.1
-9.6
+7
Exp er im en ta l Section
+11
-10.0
Gen er a l. Solvents were technical grade and distilled before
use. Solvent systems for TLC and chromatography: (A) CH2-
Cl2/CH3OH 95:5 v/v; (B) CH2Cl2/CH3OH 9:1 v/v; (C) CH2Cl2/
CH3OH 85:15 v/v; (D) CH2Cl2/CH3OH 8:2 v/v; (E) H2O/iPrOH
9:1 v/v; (F) CH2Cl2/CH3COCH3 9:1 v/v; (G) CH2Cl2/CH3COCH3
85:15 v/v; (H) CH2Cl2/CH3COCH3 8:2 v/v. Flash chromatogra-
phy: 0.4 bar, silica gel 60H. TLC: aluminum sheet, silica gel
60 F254 (0.2 mm). NMR spectra: measured at 250.13 MHz for
1H and 125.13 MHz for 13C, δ values in ppm relative to internal
SiMe4 (1H, 13C) or external H3PO4.
7-(2-Deoxy-â-D-er yth r o-p en tofu r a n osyl)-im id a zo[1,2-c]-
7H-p yr a zolo[4,3-e]p yr im id in e (5). To a solution of com-
pound 134 (3.0 g, 12.00 mmol) in aqueous sodium acetate buffer
(1 M, pH 4.5-5.0, 70 mL) at 40-50 °C was added chloro-
acetaldehyde (50% aqueous solution, 15 mL). The solution was
stirred at room temperature for 70 h. The reaction mixture
was evaporated, chromatographed (silica gel, column 15 cm
× 3 cm, solvent system A f C), and the product was
crystallized from MeOH/EtOAc (1:1, v/v). Compound 5 (2.71
g, 82%) was isolated as colorless needles, mp 197-200 °C. TLC
(silica gel, solvent system B): Rf 0.4. UV (MeOH): λmax 278
nm (ꢀ 4500), 257 nm (ꢀ 8100), 232 nm (ꢀ 41000). 1H NMR
(DMSO-d6): δ 2.34, 2.88 (2m, 2H, H2-C(2′)), 3.54 (m, 2H, H2-
C(5′)), 3.86 (m, 1H, H-C(4′)), 4.49 (m, 1H, H-C(3′)), 4.78 (dd,
1H, OH-C(5′)), 5.35 (d, J ) 3.4 Hz, 1H, OH-C(3′)), 6.72 (“t”,
J ) 6.3 Hz, 1H, H-C(1′)), 7.52 (d, J ) 1.6 Hz, 1H, H-C(11)),
8.06 (d, J ) 1.6 Hz, 1H, H-C(10)), 8.40 (s, 1H, H-C(7)), 9.32
(s, 1H, H-C(2)). Anal. Calcd for C12H13N5O3 (275.26): C, 52.36;
H, 4.76; N, 25.44. Found: C, 52.61; H, 4.90; N, 25.15.
a
4 ) z2c7z8Ad ) 7-deaza-2,8-diaza-2′-deoxyadenosine; 3 ) z2Ad
) 2-aza-2′-deoxyadenosine; c7Ad ) 7-deaza-2′-deoxyadenosine.
Determined in 0.01 M Na2HPO4, 1 M NaCl, pH 7.0. c 1 cal )
4.184 J . Determined in 10 mM sodium cacodylate buffer, pH 7.0,
containing 100 mM NaCl and 10 mM MgCl2.
b
d
stability (see 19‚23, 15‚24 and 34‚23, 24‚35). Moreover,
these duplexes exhibit Tm values nearly identical to those
of the standard duplex 12‚13. Also the introduction of 3
or 4 opposite to thymine yields duplexes with a similar
reduced stability (19‚13, 12‚15 and 34‚13, 12‚35) and a
Tm decrease (∆Tm ) -5 °C) in comparison to the duplex
with canonical base pairs as in the duplex 12‚13. Thus,
the interchange of a nitrogen between the positions 7 and
8 has rather little influence on the duplex stability. It is
worth noting that all Tm values of duplex formation are
in good correspondence to the ∆G values. We have also
studied the influence of the nucleoside 4 forming a
dangling end at the terminus of d(G-C)3. This should
reflect the stacking ability in the absence of hydrogen
bonding. For this purpose the oligonucleotide duplexes
with overhanging ends were synthesized and compared
to related compounds from which the influence of the
dangling end was already reported (see duplexes of Table
4). According to this Table the stacking of compound 4
is at least as strong as those of other “purine” nucleosides.
5-Am in o-1-(2-d eoxy-â-D-er yth r o-p en tofu r a n osyl)-4-(im -
id a zol-2-yl)p yr a zole (6). Compound 5 (1.2 g, 4.36 mmol) was
stirred with aqueous NaOH (1 N, 20 mL) overnight at room
temperature. The reaction solution was acidified to pH 7.0 with
hydrochloric acid (2 N), concentrated, and purified by FC (silica
gel, column 10 cm × 3 cm, solvent system C) to yield the title
compound 6 as a yellowish foam (960 mg, 83%). TLC (silica
gel, solvent system C): Rf 0.3. UV (MeOH): λmax 254 nm (ꢀ
10500). 1H NMR (DMSO-d6): δ 2.11, 2.71 (2m, 2H, H2-C(2′)),
3.45 (m, 2H, H2-C(5′)), 3.80 (m, 1H, H-C(4′)), 4.37 (m, 1H,
H-C(3′)), 4.99 (dd, 1H, OH-C(5′)), 5.21 (d, J ) 3.8 Hz, 1H,
OH-C(3′)), 6.12 (“t”, J ) 6.4 Hz, 1H, H-C(1′)), 6.37 (s, 2H,
H-C(7), H-C(8)), 6.95 (s, 2H, NH2), 7.65 (s, 1H, H-C(3)),
11.94 (s, 1H, NH). Anal. Calcd for C11H15N5O3 (265.27): C,
49.81; H, 5.70; N, 26.40. Found: C, 49.83; H, 5.78; N, 26.36.
7-(2-Deoxy-â-D-er yth r o-p en tofu r a n osyl)-im id a zo[1,2-c]-
7H-p yr a zolo[4,3-e][1,2,3]tr ia zin e (7). A solution of com-
pound 6 (240 mg, 0.91 mmol) in 80% aqueous HOAc (20 mL)
was treated with sodium nitrite (70 mg, 1 mmol) during 1 h
in an ice bath. The reaction solution was evaporated, dissolved
in water, and evaporated repeatedly to remove HOAc. The
residue was then chromatographed on silica gel (column 15
cm × 3 cm, solvent system A f C) to give 7 as a yellow foam
(180 mg, 72%). TLC (silica gel, solvent system B): Rf 0.3. UV
Con clu sion
Compound 4 has the same capability to form strong
base pairs with dG and much weaker ones with dT as
was reported for other 2-azapurine nucleosides.33 The
strength of the 4-dG base pair is similar to that of the
dG-dA pair or that of dA-dT. The most likely motifs for
the base pairs are (I) for 4-dG and (II) for 4-dT (Scheme
3). The two 2-aza nucleosides show pKa values of 1.7 (4)
and 1.9 (3). In the absence of the nitrogen at the
2-position the pKa value is 3.8 for dA (2)47 and 4.0 for
compound 1. These differences reflect the weaker proton
acceptor capabilities of the 2-azapurines. Consequently,
they are also weaker proton acceptors in the 3-dT or 4-dT
pairs compared to those of compound 1 or 2 with dT. On
the other hand, the base pairs between 3 or 4 with dG
are very stable thus reaching the stability of a dA-dT
pair. According to the inability of compound 4 to form a
Hoogsteen pair as is possible in the case of compound 3
(motif (III)), the motif (I) is suggested for the 4-dG pair
and motif (II) for the pairing mode of 4 with dT.
Compound 3 is expected to form the same type of a base
pair motif. The lower stability of the 4-dT pair compared
(52) Seela, F.; Debelak, H. Nucleic Acids Res. 2000, 28, 3224-3232.
(53) Greene, K. L.; J ones, R. L.; Li, Y.; Robinson, H.; Wang, A. H.-
J .; Zon, G.; Wilson, W. D. Biochemistry 1994, 33, 1053-1062.
J . Org. Chem, Vol. 69, No. 14, 2004 4699

Seela et al.
(MeOH): λmax 295 nm (ꢀ 4300), 233 nm (ꢀ 34800). 1H NMR
(DMSO-d6): δ 2.47, 2.97 (2m, 2H, H2-C(2′)), 3.55 (m, 2H, H2-
C(5′)), 3.91 (m, 1H, H-C(4′)), 4.54 (m, 1H, H-C(3′)), 4.74 (“t”,
J ) 5.7 Hz, 1H, OH-C(5′)), 5.43 (d, J ) 4.7 Hz, 1H, OH-
C(3′)), 6.98 (“t”, J ) 6.1 Hz, 1H, H-C(1′)), 7.79 (d, J ) 1.6 Hz,
1H, H-C(10)), 8.70 (s, 1H, H-C(7)), 8.77 (d, J ) 1.6 Hz, 1H,
H-C(11)). Anal. Calcd for C11H12N6O3 (276.25): C, 47.83; H,
4.38; N, 30.42. Found: C, 48.00; H, 4.28; N, 30.05.
4-Am in o-7-(2-d eoxy-â-D-er yth r o-p en t ofu r a n osyl)-7H -
pyr a zolo[3,4-d][1,2,3]tr ia zin e (7-d ea za -2,8-d ia za -2′-d eoxy-
a d en osin e) (4). To a solution of compound 7 (276 mg, 0.97
mmol) in aqueous sodium acetate buffer (1 M, pH 4.0-4.5, 60
mL) at 40-50 °C was added N-bromosuccinimide (1.4 g, 7.87
mmol), the reaction mixture was stirred at room temperature
overnight. The solution was then evaporated. The residue was
dissolved in water and desalted with a Serdolit column (column
10 cm × 2 cm, solvent system H2O f E), purified by
chromatography (silica gel, column 6 cm × 3 cm, solvent
system B), and recrystallized from MeOH/H2O (1:1, v/v) to yield
4 as colorless crystals (161 mg, 66%), mp 178-181 °C. TLC
(silica gel, solvent system C): Rf 0.4. UV (MeOH): λmax 309
nm (ꢀ 6900), 260 nm (ꢀ 4100). 1H NMR (DMSO-d6): δ 2.36,
2.88 (2m, 2H, H2-C(2′)), 3.53 (m, 2H, H2-C(5′)), 3.85 (m, 1H,
H-C(4′)), 4.48 (m, 1H, H-C(3′)), 4.76 (“t”, J ) 5.7 Hz, 1H,
OH-C(5′)), 5.35 (d, J ) 4.4 Hz, 1H, OH-C(3′)), 6.78 (“t”, J )
6.3 Hz, 1H, H-C(1′)), 8.82 (s, 3H, NH2 and H-C(7)). Anal.
Calcd for C9H12N6O3 (252.23): C, 42.86; H, 4.80; N, 33.32.
Found: C, 42.78; H, 4.83; N, 33.18.
7-[2-Deoxy-5-O-(4,4′-d im et h oxyt r ip h en yl)m et h yl-â-D-
er yth r o-p en tofu r a n osyl]-4-isobu tyr yla m in o-7H-p yr a zolo-
[3,4-d ][1,2,3]tr ia zin e (10). Compound 8 (400 mg, 1.24 mmol)
was coevaporated twice with anhydrous pyridine and then
dissolved in anhydrous pyridine (15 mL). This solution was
treated with 4,4′-dimethoxytriphenylmethyl chloride (605 mg,
1.79 mmol) at room temperature for 4 h. Thereupon, MeOH
(3 mL) was added, and the stirring was continued for 10 min.
The solution was concentrated to half of the volume, and CH2-
Cl2 (70 mL) was added. This was washed twice with an
aqueous NaHCO3 (5%, 2 × 40 mL), twice with water (2 × 40
mL), and once with a saturated NaCl solution (40 mL). The
organic phase was dried over Na2SO4, filtered, and evaporated,
and the residue was chromatographed on silica gel (column
10 cm × 3 cm, solvent system F f H) to yield the nucleoside
10 as a yellow foam (620 mg, 80%). TLC (silica gel, solvent
system H): Rf 0.3. UV (MeOH): λmax 313 nm (ꢀ 8300), 267 nm
1
(ꢀ 12600), 236 nm (ꢀ 33600). H NMR (DMSO-d6): δ 1.18 (d,
6H, 2 CH3), 2.47 (m, 1H, HR-C(2′)), 2.98 (m, 1H, CH), 3.06 (m,
1H, Hâ-C(2′)), 3.68, 3.70 (2s, 6H, 2 OCH3), 3.73 (m, 1H,
H-C(5′)), 4.01 (m, 1H, H-C(4′)), 4.62 (m, 1H, H-C(3′)), 5.44
(d, J ) 5.0 Hz, 1H, OH-C(3′)), 6.70 (m, 4H, phenyl-H), 6.99
(m, 1H, H-C(1′)), 7.12, 7.24 (2m, 9H, phenyl-H), 8.61 (s, 1H,
H-C(7)), 11.94 (s, 1H, NH). Anal. Calcd for C34H36N6O6
(624.69): C, 65.37; H, 5.81; N, 13.43. Found: C, 65.67; H, 5.92;
N, 13.37.
7-[2-Deoxy-5-O-(4,4′-d im et h oxyt r ip h en yl)m et h yl-â-D-
er yth r o-p en tofu r a n osyl]-4-isobu tyr yla m in o-7H-p yr a zolo-
[3,4-d ][1,2,3]tr ia zin e-3′-[(2-cya n oeth yl)-N,N-d iisop r op y-
lp h osp h or a m id ite] (11). Compound 10 (400 mg, 0.64 mmol)
was coevaporated twice with anhydrous pyridine and then
dissolved in CH2Cl2 (20 mL). N,N-Diisopropylethylamine (210
µL, 1.21 mmol) and chloro-(2-cyanoethoxy)-N,N-diisopropy-
laminophosphine (210 µL, 0.94 mmol) were added under argon
atmosphere. After 20 min of stirring at room temperature,
CH2Cl2 (40 mL) was added. The organic phase was washed
twice with an aqueous solution of NaHCO3 (5%, 2 × 20 mL)
and once with a saturated solution of NaCl (20 mL). The
organic layer was dried over Na2SO4, filtered, and evaporated.
The residue was purified by FC (silica gel, column 10 cm × 3
cm, solvent system G) to furnish 11 as colorless foam (382 mg,
72%). TLC (silica gel, solvent system G): Rf 0.4. 31P NMR
(CDCl3): δ 149.9, 149.74. Anal. Calcd for C43H53N8O7P
(824.90): C, 62.61; H, 6.48; N, 13.58. Found: C, 62.93; H, 6.61;
N, 13.37.
7-(2-De oxy-â-D -er yt h r o-p e n t ofu r a n osyl)-4-isob u t y-
r yla m in o-7H-p yr a zolo[3,4-d ][1,2,3]tr ia zin e (8). After being
coevaporated twice with anhydrous pyridine, compound 4 (100
mg, 0.4 mmol) was suspended in anhydrous pyridine (1.6 mL)
and treated with trimethylsilyl chloride (260 µL, 2.05 mmol)
at room temperature during 15 min. To the reaction solution
was added isobutyryl anhydride (110 µL, 0.66 mmol), and the
mixture was stirred at room temperature for 3 h. The reaction
mixture was cooled in an ice-water bath and treated with H2O
(400 µL) for 15 min and then with aqueous NH3 (25% aq, 400
µL) for further 15 min. The mixture was then evaporated to
dryness and coevaporated with toluene. The residue was
purified over a silica gel column (10 cm × 1 cm, solvent system
B) to yield 8 as colorless foam (90 mg, 70%). TLC (silica gel,
solvent system B): Rf 0.3. UV (MeOH): λmax 306 nm (ꢀ 4700),
1
266 nm (ꢀ 7200). H NMR (DMSO-d6): δ 1.18 (d, 6H, 2 CH3),
2.43 (m, 1H, HR-C(2′)), 2.92 (m, 1H, CH), 2.97 (m, 1H, Hâ-
C(2′)), 3.53 (m, 2H, H2-C(5′)), 3.87 (m, 1H, H-C(4′)), 4.51 (m,
1H, H-C(3′)), 4.77 (“t”, J ) 5.4 Hz, 1H, OH-C(5′)), 5.39 (d, J
) 4.7 Hz, 1H, OH-C(3′)), 6.94 (“t”, J ) 6.1 Hz, 1H, H-C(1′)),
8.67 (s, 1H, H-C(7)), 11.92 (s, 1H, NH). Anal. Calcd for
Ack n ow led gm en t . We thank Dr. Helmut Rose-
meyer and Dr. Yang He for measuring the NMR spectra
and Mrs. E. Feiling for the oligonucleotide syntheses.
We also thank Mrs. E.-M. Becker for the pK measure-
ments and Mrs. M. Dubiel for her help during the
preparation of the manuscript. Financial support by the
European Community (Grant QLRT-2001-00506, “Fla-
vitherapeutics”) is gratefully acknowledged.
C
13H18N6O4 (322.32): C, 48.44; H, 5.63; N, 26.07. Found: C,
48.62; H, 5.55; N, 25.86.
4-Acetyla m in o-7-(2-d eoxy-â-D-er yth r o-p en tofu r a n osyl)-
7H-p yr a zolo[3,4-d ][1,2,3]tr ia zin e (9). The procedure de-
scribed for 8 was used but with acetic anhydride (110 µL, 1.17
mmol) instead of isobutyryl anhydride to yield a colorless foam
(70 mg, 60%). TLC (silica gel, solvent system B): Rf 0.3. UV
(MeOH): λmax 303 nm (ꢀ 3900), 265 nm (ꢀ 5600). 1H NMR
(DMSO-d6): δ 2.30 (s, 3H, CH3), 2.42, 2.92 (2m, 2H, H2-C(2′)),
3.45 (m, 2H, H2-C(5′)), 3.88 (m, 1H, H-C(4′)), 4.51 (m, 1H,
H-C(3′)), 4.77 (m, 1H, OH-C(5′)), 5.39 (m, 1H, OH-C(3′)),
6.93 (“t”, J ) 5.8 Hz, 1H, H-C(1′)), 8.62 (s, 1H, H-C(7)), 11.92
(s, 1H, NH). Anal. Calcd for C11H14N6O4 (294.27): C, 44.90;
H, 4.80; N, 28.56. Found: C, 45.05; H, 4.99; N, 28.42.
Su p p or tin g In for m a tion Ava ila ble: General experimen-
tal methods for oligonucleotides, J (H,C) coupling constant (Hz)
of nucleosides, coupling yields of oligo-2′-deoxyribonucleotides,
MALDI-TOF spectrum of the oligonucleotide 5′-d[TAG GTC
A4T ACT] (17), and composition of the oligonucleotides 14, 15,
and 16. This material is available free of charge via the
Internet at http://pubs.acs.org.
J O040150I
4700 J . Org. Chem., Vol. 69, No. 14, 2004