Stabilisation of duplex DNA by 7-halogenated 8-aza-7-deazaguanines

Article abstract of DOI:10.1039/a804414g

Oligonucleotides containing 7-halogenated 8-aza-7-deaza-2'-deoxyguanosine (c⁷z⁸G_d) derivatives such as d(Br⁷c⁷z⁸G-C)4 8 (T_m = 88 deg C) and d(I⁷c⁷z⁸

Full text of DOI:10.1039/a804414g

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Stabilisation of duplex DNA by 7-halogenated 8-aza-7-deazaguanines
Frank Seela*† and Georg Becher
Laboratorium für Organische und Bioorganische Chemie, Institut für Chemie, Universität Osnabrück, Barbarastr.7, D-49069
Osnabrück, Germany
Oligonucleotides containing 7-halogenated 8-aza-7-deaza-
Their base composition was confirmed by enzymatic hydroly-
sis. Furthermore, their thermodynamic stability was determined
by temperature dependent UV–melting profiles. Table 1
7
8
7 7 8
2
A-deoxyguanosine (c z G
G-C) 8 (T = 88 °C) and d(I c z G-C)
significantly more stable than d(G-C) 5 (T
d
) derivatives such as d(Br c z
7
7 8
4
m
4
9 (T
m
= 84 °C) are
= 59 °C).
4
m
m
summarises T values as well as thermodynamic data (Melt-
12
Win ) of the duplex formation of the self-complementary
The introduction of 7-halogenated 7-deazapurines (pyrrolo-
2,3-d]pyrimidines A) into oligonucleotides reveals that these
oligonucleotides 5, 7–9 and 12–14 as well as of the correspond-
7
[
ing oligomers containing 7-deazaguanine (c G
11).
d
) (6, 10 and
2
residues are well accommodated in the major groove of the
duplex DNA. Furthermore, this modified DNA is stabilised and
the particular DNA structure is retained.1 Consequently, the 7
position of 7-deazapurines is an ideal site for the introduction of
functional residues into the DNA which can serve later as
reporter groups, cleaving agents or residues useful in sequenc-
According to Table 1 it is apparent that the 8-aza-
7-deazaguanine moiety which has no 7-substituent stabilises the
,2
7
8
oligonucleotide duplex [d(c z G-C)
pared to the parent 2A-deoxyguanosine [d(G-C)
59 °C, DT = 13 °C), while the 7-deaza-2A-deoxyguanosine in
m
] 6 shows a destabilisation (T =
4
] 7 (T
m
= 72 °C) com-
4
m
] 5 (T =
m
3
7
ing by mass spectrometry or by atomic force microscopy. In
[d(c G-C)
4
m
= 53 °C, DT
2
the series of modified nucleobases related to purines, the 8-aza-
26 °C). These findings are in agreement with earlier observa-
7-deazapurines (pyrazolo[3,4-d]pyrimidines B) depict another
tions made on other oligonucleotides as well as on polynucleo-
tides.1
3–15
The octanucleotides with halogenated 8-aza-7-deaza-
heterocyclic system which can be derivatised at the same
position while retaining the Watson–Crick recognition site of a
purine base (C), but not showing the unfavourable properties of
7
7 8
guanine residues [d(Br c z G-C)
4
] 8 (T
m
= 88 °C) and
7
7 8
[d(I c z G-C) ] 9 (T = 84 °C) show considerable duplex
4 m
8
-substituted purine residues.4
stabilisation. The stability of these duplexes was even higher
than those of the related oligomers with the 7-halogenated
7-deazaguanine residues (10 and 11). Due to the high T values
m
of 8 and 9 it was not possible to obtain a complete melting
profile, which is necessary to determine the thermodynamic
data. Therefore, a set of hexanucleotides was measured showing
O
O
O
N
R
N
R
N
N
N
HN
HN
HN
N
R
H2N
N
H2N
N
H2N
ca. 15 °C lower T
m
values. Again, the iodo compound 14
A
B
C
O
O
R
R
Regular pyrazolo[3,4-d]pyrimidine nucleosides have already
4
3
6
7
5
been incorporated into oligonucleotides.5–7 Also, the mono-
5 HN
6
1 HN
N2
N 8
meric 7-bromo and 7-iodo derivatives of 8-aza-7-deaza-
i
N 1
i
2
4
N 9
H2N
N
7
Bu HN
N
8
2
A-deoxyguanosines (1a,b) have been synthesised. As we
3
wanted to prove whether compounds 1a,b show the same
favourable properties as the corresponding 7-deazapurines,
building blocks for oligonucleotide solid-phase synthesis were
prepared. For this purpose the isobutyryl residue was introduced
as an amino protecting group yielding the protected nucleosides
O
O
HO
HO
HO
systematic numbering
HO
purine numbering
2
a (64%) and 2b (66%) (Scheme 1). The protecting group
stability was determined UV spectrophotometrically at 300 nm
in conc. NH at 40 °C. The half-lives of 7-bromo-8-aza-
1
a R = Br
2a R = Br
b R = I
b R = I
3
ii
7
7 8
7
8
d
-deaza-2A-deoxyguanosine 2a (Br c z G ; 38 min) and 7-iodo-
7
7 8
O
R
O
R
d
-aza-7-deaza-2A-deoxyguanosine 2b (I c z G ;41 min) were
similar to that of the parent 8-aza-7-deaza-2A-deoxyguanosine
HN
HN
7
8
7
(
d
c z G ; 37 min). Subsequently, 4,4A-dimethoxytrityl (DMT)
N
N
groups were introduced under standard conditions furnishing
the 5A-protected compounds 3a and 3b (74 and 71% yield),
respectively. They were then converted into the phosphor-
amidites 4a and 4b (80 and 76%). All monomeric compounds
iii
i
N
N
i
N
N
Bu HN
Bu HN
O
O
DMTO
DMTO
1
13
31
were characterised by H, C and P NMR spectra as well as
by elemental analyses.9
HO
O
P
7
7 8
Next, the self-complementary hexanucleotides d(Br c z G-
3a R = Br
b R = I
NCCH2CH2O
NPri2
7
7 8
C)
3 3
13 and d(I c z G-C) 14 as well as the octamers
7
7 8
7 7 8
d(Br c z G-C)
4
8 and d(I c z G-C)
4
9 were prepared using the
4a R = Br
b R = I
building blocks 4a,b. The oligonucleotides were removed from
3
the solid support (conc. aq. NH ), deprotected and purified on
Scheme 1 Reagents and conditions: i, HMDS, Bui
2
O, DMF, room temp.,
OPC cartridges.10 Their purity was proven by ion-exchange
1
3 h, 64% (2a), 66% (2b); ii, DMTCl, py, room temp., 4 h, 74% (3a), 71%
(3b); iii, (Pr 2N)(NCCH CH O)PCl, THF, room temp., 30 min, 80% (4a),
76% (4b)
chromatography on a 4 3 50 mm NucleoPac PA-100 column
i
2
2
(
DIONEX), and MALDI-TOF mass spectra11 were obtained.
Chem. Commun., 1998
2017

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Table 1 T
m
values and thermodynamic data of duplex formation of oligonucleotidesa,b
2
1
DS/cal K
DG298/kcal
1
21
21
Compound
d(G-C)
m
T /°C
DH/kcal mol2
mol
mol
4
·d(G-C)
4
5·5
4
59
53
72
88
84
67
67
47
73
71
267
265
2179
2178
211.9
210.2
7
7
d(c G-C)
d(c z G-C)
4
·d(c G-C)
6·6
·d(c z G-C)
7
8
7 8
4
4
8
7·7
274
2193
214.5
7
7
8
7
7
c
c
c
4 4
d(Br c z GC) ·d(Br c z GC) 8·8
7
7
8
7
7
8
c
c
c
d(I c z G-C)
4
·d(I c z G-C)
4
9·9
7
7
7 7
d(Br c G-C)
4
·d(Br c G-C)
·d(I c G-C)
4
10·10
271
266
255
270
263
2188
2171
2150
2181
2162
213.0
212.6
28.4
214.3
212.9
7
7
7 7
d(I c G-C)
d(G-C) ·d(G-C)
4
4
11·11
3
7
3
12·12
7
8
7 7 8
d(Br c z G-C)
3
·d(Br c z G-C)
3
13·13
7
7
8
7 7 8
d(I c z G-C)
3 3
·d(I c z G-C) 14·14
a
. b Measured in 10 m NaCl. c Not measurable.
M Na cacodylate, 10 mM MgCl , 0.1 M
2
Oligonucleotide conc. is 10 m
M
exhibits a somewhat lower T
m
value than the bromo derivative
logenated compounds is the change of the pK values of
deprotonation (7-deaza-8-aza-2A-deoxyguanosine = 9.3; com-
pounds 1a,b = 9.0). This effect also increases the N-glycosylic
13 (Table 1). When comparing the thermodynamic data of the
duplexes 13·13 and 14·14 with the unmodified duplex 12·12 it
is apparent that a more favourable enthalpy term leads to duplex
stabilisation. This effect was much more pronounced in the
series of oligonucleotides containing 7-halogenated 8-aza-
8
bond stability and is most likely explained by the electron-
withdrawing effect caused by the 7-substituents. As a result, the
hydrogen bonds within the G-C base-pair are strengthened and
the duplex becomes stabilised. It was also shown that the
triphosphates of 8-aza-7-deazaguanine nucleosides are effi-
7
-deazaguanine than in those containing halogenated 7-deaza-
guanine.
From the chromatographic behaviour of the 7-halogenated
1
6
ciently incorporated into DNA using DNA polymerases; they
are useful for introducing reporter groups into a sterically
unproblematic position of the DNA molecule.
nucleosides 1a,b on RP-18 HPLC (Fig. 1) as well as of the
corresponding oligonucleotides 8, 9, 13 and 14 it is apparent
that the halogen substituents make the compounds more
hydrophobic. Thus, the major grooves of such DNA duplexes
become hydrophobic and water molecules, normally being
present in these grooves, are expelled. This can influence both
the enthalpy and the entropy of duplex formation. However,
enthalpic changes play the major role.
We thank Dr N. Ramzaeva for helpful discussions. Financial
support by the Bundesministerium für Bildung, Forschung und
Technologie (BMBF) is gratefully acknowledged.
Notes and References
Another difference which is observed for the 7-halogenated
-aza-7-deazaguanine nucleosides compared to the non-ha-
†
E-mail: Fraseela@rz.uni-Osnabrueck.de
8
1
2
3
F. Seela and H. Thomas, Helv. Chim., Acta, 1995, 78, 94.
N. Ramzaeva and F. Seela, Helv. Chim. Acta, 1996, 79, 1549.
C. W. Siegert, A. Jacob and H. Köster, Anal. Biochem., 1996, 243,
5
5.
4
5
S. N. Rao and P. A. Koliman, J. Am. Chem. Soc., 1986, 108, 3048.
F. Seela, N. Ramzaeva and M. Zulauf, Nucleosides Nucleotides, 1997,
1
6, 963.
6
C. R. Petrie, A. D. Adams, M. Stamm, J. Van Ness, S. M. Watanabe and
R. B. Meyer, Bioconjugate Chem., 1991, 2, 441.
7
8
9
F. Seela and H. Driller, Helv. Chim. Acta, 1988, 71, 1191.
F. Seela and G. Becher, Synthesis, 1998, 207.
2
Selected data for 3a: d
-C(2A)], 2.75 [m, H
.95 [m, H-C(4A)], 4.44 [m, H-C(3A)], 5.30 [d, J 4.5, HO-C(3A)], 6.37 [t,
J 6.5, H-C(1A)], 6.77–7.16 (2m, 13 arom. H), 11.86 (s, NH), 11.97 (s,
H
([ H
6
]DMSO) 1.03 [d, J 6.7, (CH
3
)
2
], 2.25 [m,
H
3
b
a
-C(2A)], 3.05 [m, H
2
-C(5A)], 3.70 (s, 2 CH
3
O),
2
NH). For 4a: d
J 6.7, (CH ], 2.25 [m, H
-C(5A)], 3.70 (s, 2 CH O), 3.92 [m, H-C(4A)], 4.44 [m, H- C(3A)], 5.29
d, J 4.2, HO-C(3A)], 6.37 [t, J 5.5, H-C(1A)], 6.76–7.33 (2m, 13 arom.
H), 11.83 (s, NH), 11.92 (s, NH). For 4b: d (CDCl ) 148.4, 148.5.
P
(CDCl
3
) 148.4, 148.5. For 3b: d
H
([ H
6
]DMSO) 1.12 [d,
3
)
2
b a
-C(2A)], 2.77 [m, H -C(2A)], 3.03 [m,
H
2
3
[
P
3
1
1
0 Oligonucleotide Purification Cartridges, Applied Biosystems, Weiter-
stadt, Germany.
7
8
+
4
1 Selected data for d(c z GC) 7: Calc.: 2412. Found (M ): 2412. For
7
7
8
+
7 7 8
d(Br c z GC)
Calc. 2918. Found (M ) 2915.
2 J. A. McDowell and D. H. Turner, Biochemistry, 1996, 35, 14077.
4 4
8: Calc.: 2727. Found (M ): 2728. For d(I c z GC) 9:
+
1
Fig.
1
HPLC profile of (a) 6-amino-1H-pyrazolo[3,4-d]pyrimidin-
13 F. Seela and H. Driller, Nucleic Acids Res., 1989, 17, 901.
14 F. Seela and S. Lampe, Helv. Chim. Acta, 1994, 77, 1003.
15 L. J. P. Latimer and J. S. Lee, J. Biol. Chem., 1991, 266, 13 849.
16 K. Mersmann and F. Seela, unpublished results.
4
1
(5H)-one, (b) 8-aza-7-deaza-2A-deoxyguanosine, (c) 1a and (d) 1b on a RP-
8 (200 3 10 mm) column. The following solvent systems were used: 0.1
M
3
(Et NH)OAc (pH 7.0)–MeCN (95:5) (A) and MeCN (B). They were used
according to the following profile: 20 min 5–20% B in A, 35 min 20–50%
B in A, 35 min 5% B in A.
Received in Glasgow, UK, 10th June 1998; 8/04414G
2018
Chem. Commun., 1998