7-Deazaisoguanine quartets: Self-assembled oligonucleotides lacking the Hoogsteen motif

Article abstract of DOI:10.1039/a704866a

Oligonucleotides containing consecutive 7-deazaisoguanine residues form self-assembled quartets, indicating that the purine nitrogen-7 of isoguanine is not participating in the hydrogen bonding pattern.

Full text of DOI:10.1039/a704866a

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7-Deazaisoguanine quartets: self-assembled oligonucleotides lacking the
Hoogsteen motif
Frank Seela* and Changfu Wei
Laboratorium fu¨r Organische und Bioorganische Chemie, Institut fu¨r Chemie, Universita¨t Osnabru¨ck, Barbarastr. 7, D-49069
Osnabru¨ck, Germany
NH2
Cl
N
Oligonucleotides containing consecutive 7-deazaisoguanine
residues form self-assembled quartets, indicating that the
purine nitrogen-7 of isoguanine is not participating in the
hydrogen bonding pattern.
N
N
i (from 5)
iii
N
N
H2N
HO
N
1
H2N
RO
O
O
Oligodeoxyribonucleotides with consecutive isoguanine resi-
dues, e.g. d(T4isoG4T4),† form self-assembled structures.1,2
Such aggregates have also been observed in the case of the
homopolyribonucleotide poly(isoguanylic acid)3 and the mono-
meric ribonucleoside isoguanosine.4 Like the G-quartet the
aggregate of isoguanine has properties of an ionophore.5 The
stability of the metal complexes depend on the size of the
particular ion.6 Ion-exchange HPLC experiments showed that
the aggregate of d(T4isoG4T4) has the same number of negative
phosphate charges as that of d(T4G4T4). Due to this observation
a tetrameric structure was also proposed for d(T4isoG4T4).2 The
stoichiometry of this complex was established using iso-
guanine-containing oligonucleotides with different dT-tails at
the 3A- and 5A-termini.7 So far, the isoguanine-quartet shows
behaviour similar to that of guanine. Although the tetrameric
assembly of isoguanine has been verified, several structural
motifs have been proposed for the quartet structure.2–4
Base-modified nucleosides are useful tools to probe the
structure of such high-molecular assemblies, in particular those
which are held together by hydrogen bonds. Regarding this it
has already been shown that the base pairing pattern can be
restricted to the Watson–Crick motif when 7-deazaguanine is
replacing guanine.8 As a result, Hoogsteen quartets cannot be
formed and the oligonucleotides are unable to form aggregates.9
Based on this principle, 7-deaza-2A-deoxyisoguanosine 1
(c7iGd) has been employed to probe the oligonucleotide quartet
structure formed by 2A-deoxyisoguanosine 2 (isoGd). New
oligonucleotide quartets should be accessible when the purine
nitrogen-7 does not participate in the hydrogen bonding pattern.
For this purpose the oligonucleotide building blocks 3 and 4
were synthesised. Oligonucleotides with consecutive 7-deaza-
HO
RO
6
5 R = 4-MeC6H4C(O)
7 R = H
ii
Scheme 1 Reagents and conditions: i, 25% NH3–dioxane (1:1 v/v), 120 °C,
3 d, 88%; ii, 25% NH3–dioxane (1:1 v/v), 60 °C, 3 d, 80%; iii, NaNO2, 20%
aq. AcOH, 67%
isoguanine residues were prepared and their ability to form self-
assembled aggregates was studied.
7-Deaza-2A-deoxyisoguanosine 1 has already been prepared
(i) by convergent nucleoside synthesis10 or (ii) by photo-
substitution on 2-chloro-7-deaza-2A-deoxyadenosine.11 Now, a
more efficient route has been employed using glycosylation of
the 2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine anion12
with 2-deoxy-3,5-di-O-(4-methylbenzoyl)-a-d-erythro-pento-
furanosyl chloride. The resulting nucleoside 5 was treated with
conc. NH3–dioxane (autoclave, 120 °C, 3 d) to give 2-amino-
7-deaza-2A-deoxyadenosine 6.13 The deprotected compound 7
was also obtained (60 °C, conc. NH3–dioxane, 3 d). Selective
deamination of 6 (NaNO2 in aq. AcOH) furnished the
nucleoside 1 (Scheme 1). It is stable in acidic solution whereas
2A-deoxyisoguanosine 2 is an extremely labile nucleoside.1
For the preparation of 3 and 4, compound 1 was blocked on
the 2-oxo function with a Ph2NC(O) group (Ph2NCOCl,
Pri2NEt–pyridine, room temp.) to give derivative 8. The
isobutyryl residue was then introduced to protect the amino
group, yielding compound 9. Standard conditions were em-
ployed to block the 5A-hydroxy group with a DMT residue†
(9 ? 10).‡ The phosphonate 3 as well as the phosphoramidite 4
were prepared from 10 according to known procedures (Scheme
2).
As discussed above, DNA quartets have been constructed
from oligonucleotides containing short runs of isoguanine, e.g.
d(T4isoG4T4).1,2 These self-assembled structures were identi-
fied by HPLC ion exchange chromatography [Fig. 1(a)].2 Their
chromatographic behaviour was almost identical to that ob-
served for d(T4G4T4). The chromatogram shows two peaks, a
fast migrating peak for the monomeric oligonucleotide and a
slow migrating peak for the tetramer (Fig. 1). The same
experiment was then performed with oligonucleotides contain-
ing consecutive 7-deazaisoguanine residues.§ For this purpose,
the oligomers 5A-d(T4c7iG4T4) 11 and 5A-d(T2c7iG6T4) 12 were
prepared by solid-phase synthesis.
NH2
NH2
7
N
5
5
HN1
HN3
3
N
1
N
N 9
N7
O
O
O
O
HO
HO
HO
HO
purine numbering
systematic numbering
2
1
NHBui
N
NHBui
N
N
Ph2NCO2
(MeO)2TrO
N
The oligomer 5A-d(T4c7iG4T4) 11 gives only one peak when
chromatographed on an ion exchange HPLC column under the
conditions described for d(T4isoG4T4) [Fig. 1(b)].2 This fast
migrating peak contains only the single-stranded oligomer.
However, when the number of consecutive c7iGd-residues is
increased from four to six by replacing another two dT-residues
as in the oligomer d(T2c7iG6T4) 12, a slow migrating zone
N
Ph2NCO2
N
O
O
(MeO)2TrO
O
O
P
O
P
H
O– +NHEt3
NCCH2CH2O
NPri2
3
4
Chem. Commun., 1997
1869

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NHBui
NH2
NHBui
N
N
N
iv
i
ii
iii
N
N
N
Ph2NCO2
HO
N
O
Ph2NCO2
HO
N
Ph2NCO2
N
3 or 4
1
O
O
(MeO)2TrO
HO
HO
9
HO
8
10
Scheme 2 Reagents and conditions: i, Ph2NCOCl, 80%; ii, Me3SiCl, BuiCl, 80%; iii, DMTCl, 51%; iv (for 3) PCl3, N-methylmorpholine, 1H-1,2,4-triazole,
93%; (for 4) (NCCH2CH2O)(Pri2N)PCl, EtNPri2, 85%
(a)
(b)
(c)
7
d(T c iG T )
4
4 4
7
[d(T c iG T )]
6 4 4
[d(T iG T )]
4 4 4
2
4
7
d(T c iG T )
2
6 4
A
d(T iG T )
4
4 4
5
10 15 20 25 30 35
t / min
5
10 15 20 25 30 35
t / min
5
10 15 20 25 30 35
t / min
Fig. 1 Ion exchange HPLC profiles of (a) 5A-d(T4isoG4T4), (b) 5A-d(T4c7iG4T4) and (c) 5A-d(T2c7iG6T4) at 25 °C. Solvent systems: A = 25 mm Tris-HCl
(containing 1 mm EDTA buffer, pH 8.0)–MeCN, 90:10; B = A containing 1.0 m NaCl. Gradient: t = 0–30 min, 20 ? 80% B in A; t = 30–35 min, 80%
B in A.
(tR = 22 min) appeared along with the fast migrating peak
(tR = 14 min) [Fig. 1(c)]. The latter contains the single-stranded
oligomer, while the slow migrating peak represents the
aggregate. Consequently, the aggregate of d(T2c7iG6T4) 12
reflects a self-assembled structure. The almost identical mobil-
ities of compounds d(T4isoG4T4) and d(T2c7iG6T4) on ion-
exchange HPLC indicate that both aggregates are formed by
assembly of four strands. This is the result of an identical
number of negative phosphate charges, which are responsible
for the chromatographic mobility.
From model building it is obvious that the structure of the
isoguanine quartet has to be different from that of guanine. The
structure of the two bases differs with regard to the position of
the purine substituents, thereby changing their donor–acceptor
pattern. Various aggregate motifs have been suggested for the
polyribonucleotide poly(isoG),3 for the monomeric isoguano-
sine,4 and also for oligodeoxyribonucleotides.2,14 Some struc-
tures do not use the purine nitrogen-7 to form the supramo-
lecular assembly. Among them the most probable structure for
the quartet of 12 is A (Fig. 2). It follows a motif which has been
proposed for a monomeric isoguanosine derivative.4
good candidate to form ionophors with good chemical stabil-
ity.
Footnotes and References
* E-mail: fraseela@rz.uni-osnabrueck.de
† Abbreviations: isoGd or d(isoG), 2A-deoxyisoguanosine; c7iGd or d(c7iG),
7-deaza-2A-deoxyisoguanosine; DMT or (MeO)2Tr, 4,4A-dimethoxy-
triphenylmethyl.
‡ Selected data for 10 (systematic numbering): lmax(MeOH)/nm (e) 274
(9800), 294 (7600); dH ([2H6]DMSO) 1.13, 1.14 (2 s, 2 CH3), 2.30 [m,
H-C(2A)], 2.88 (m, CH), 3.16 [m, 2 H-C(5A)], 3.72 (s, 2 CH3O), 3.95 [m,
H-C(4A)], 4.38 [m, H-C(3A)], 5.37 [br s, HO-C(3A)], 6.58 [m, H-C(1A)], 6.83
[d, J 7.6, H-C(5)], 7.22–7.46 [m, H-C(6) and 23 arom. H]; calc. for
C49H47N5O8 (833.95): C, 70.57; H, 5.68; N, 8.40; found: C, 70.29; H, 5.65;
N, 8.33%.
§ General procedure. The samples for ion exchange HPLC were prepared
by preheating to 90 °C for 2 min, cooling to room temperature for 10 min,
and storing at 220 °C for 30 min. The sample was then brought to room
temperature and injected onto a NucleoPac PA-100 column. Elution was
performed with Tris-HCl buffer, pH 8.0, by increasing the NaCl
concentration from 0.2 to 0.8 m (ref. 2).
1 F. Seela and C. Wei, Helv. Chim. Acta, 1997, 80, 73.
2 F. Seela, C. Wei and A. Melenewski, Nucleic Acids Res., 1996, 24,
4940.
3 T. Golas, M. Fikus, Z. Kazimierczuk and D. Shugar, Eur. J. Biochem.,
1976, 65, 183.
4 J. T. Davis, S. Tirumala, J. R. Jenssen, E. Radler and D. Fabris, J. Org.
Chem., 1995, 60, 4167.
5 S. Tirumala and J. T. Davis, J. Am. Chem. Soc., 1997, 119, 2769.
6 F. Seela, C. Wei and A. Melenewski, unpublished work.
7 F. Seela, C. Wei and A. Melenewski, Origins Life Evol. Biosphere,
1997, in the press.
H
N
X
N
H
H
X
N
N
N
H
N
O
N
H
N
H
O
A X = CH
B X = N
M+
H
N
H
O
N
O
H
N
H
N
N
N
N
X
X
H
N
H
8 F. Seela, Q.-H. Tran-Thi and D. Franzen, Biochemistry, 1982, 21,
4338.
Fig. 2
9 F. Seela and K. Mersmann, Helv. Chim. Acta, 1993, 76, 1435.
10 F. Seela, S. Menkhoff and S. Behrendt, J. Chem. Soc. Perkin Trans. 2,
1986, 525.
11 Z. Kazimierczuk, R. Mertens, W. Kawczynski and F. Seela, Helv. Chim.
Acta, 1991, 74, 1742.
12 F. Seela, A. Kehne and H.-D. Winkeler, Liebigs Ann. Chem., 1983,
137.
13 F. Seela, H. Steker, H. Driller and U. Bindig, Liebigs Ann. Chem., 1987,
15.
The experiments described suggest that oligonucleotides
containing either 7-deazaisoguanine or isoguanine form similar
supramolecular structures with an identical hydrogen bonding
pattern (A or B). Nevertheless, due to the structural changes of
the heterocyclic base—a purine system is replaced by a
pyrrolo[2,3-d]pyrimidine heterocycle—the strength of the hy-
drogen bonding array results in somewhat lower stability for the
7-deazaisoguanine quartet A compared to that formed by
isoguanine (B). Unlike 2A-deoxyisoguanosine, which is very
sensitive to acidic conditions (glycosylic bond hydrolysis),
7-deaza-2A-deoxyisoguanosine is very stable. This makes it a
14 R. Krishnamurthy, S. Pitsch, M. Minton, C. Miculka, N. Windhab and
A. Eschenmoser, Angew. Chem., 1996, 108, 1619.
Received in Glasgow, UK, 8th July 1997; 7/04866A
1870
Chem. Commun., 1997