5-Aza-7-deaza-2'-deoxyguanosine: Oligonucleotide duplexes with novel base pairs, parallel chain orientation and protonation sites in the core of a double helix

Article abstract of DOI:10.1002/(SICI)1099-0690(199902)1999:2<485::AID-EJOC485>3.0.CO;2-J

Oligonucleotides containing 5-aza-7-deaza-2'-deoxyguanosine (1) were synthesized. Solid-phase synthesis was performed with the phosphonate 15 or the phosphoramidite 5. The amino-unprotected phosphonate 4 was also employed. Hybridization studies of oligonucleotides containing 1 resulted in new base pairs leading to duplexes with parallel (ps) or antiparallel (aps) chain orientation. Among those with parallel chains a stable 'purine-purine' base pair was observed between 5-aza-7-deazaguanine and guanine or 7-deazaguanine. Antiparallel stranded duplexes are formed when 5-aza-7-deazaguanine pairs with cytosine. This base pair has only two hydrogen bonds under neutral conditions but is stabilized by a third one in acidic medium. A new base pair is also detected between the base of 1 and isoguanine (neutral medium).

Full text of DOI:10.1002/(SICI)1099-0690(199902)1999:2<485::AID-EJOC485>3.0.CO;2-J

FULL PAPER
5-Aza-7-deaza-2Ј-deoxyguanosine: Oligonucleotide Duplexes with
Novel Base Pairs, Parallel Chain Orientation and Protonation Sites in the
Core of a Double Helix
Frank Seela*[a] and Alexander Melenewski[a]
Keywords: 5-Aza-7-deaza-2Ј-deoxyguanosine / Oligonucleotides / Parallel DNA / Protonated base pairs / Tm values
Oligonucleotides containing 5-aza-7-deaza-2Ј-deoxyguano-
sine (1) were synthesized. Solid-phase synthesis was
performed with the phosphonate 15 or the phosphoramidite
was observed between 5-aza-7-deazaguanine and guanine
or 7-deazaguanine. Antiparallel stranded duplexes are
formed when 5-aza-7-deazaguanine pairs with cytosine. This
base pair has only two hydrogen bonds under neutral
conditions but is stabilized by a third one in acidic medium.
A new base pair is also detected between the base of 1 and
isoguanine (neutral medium).
5. The amino-unprotected phosphonate
4
was also
employed. Hybridization studies of oligonucleotides con-
taining 1 resulted in new base pairs leading to duplexes with
parallel (ps) or antiparallel (aps) chain orientation. Among
those with parallel chains a stable “purine-purine” base pair
Base-modified 2Ј-deoxyribonucleosides having nitrogen
patterns different from those of the naturally occurring
DNA constituents are of significant importance in DNA
research and biotechnology.[1] Due to their ability to restrict
the base pairing to certain motifs[2] or to form entirely new
base pairs[3] novel DNA structures are accessible. Modified
nucleosides are also valuable to probe the complex forma-
tion of DNA with ligands or proteins and to study other
biochemical interactions.
In the past a large number of base-modified purine 2Ј-
deoxyribonucleosides have been synthesized and incorpo-
rated into oligonucleotides.[4] Among them, the 7-deazapu-
rines play an important role.[5Ϫ9] The 5-aza-7-deazapurine
nucleosides are related to the latter. The 5-aza-7-deaza-2Ј-
deoxyisoguanosine is the only compound which has already
been incorporated into oligonucleotides.[10] The related 5-
aza-7-deaza-2Ј-deoxyguanosine (1, dZ)[11] which is the sub-
ject of this study, is a structural analogue of 2Ј-deoxyguano-
sine (3). Although the base, 5-aza-7-deazaguanine, has very
similar spatial requirements as guanine, N-7 as a proton
acceptor site is absent. On one hand, N-1 is now a proton
acceptor and no longer a proton donor as in the parent
guanine. On the other hand, the s-triazine ring of 1 rep- Scheme 1
resents the same WatsonϪCrick-type site as 2Ј-deoxyisocy-
tidine (2), and, therefore, it becomes similar to 3 when it is
and the protonated 5-aza-7-deazaguanine base with cyto-
sine, guanine and related bases.
protonated at N-1 (1-cation) (Scheme 1).
According to the changes of the donor-acceptor pattern,
various new base pairs are conceivable and novel nucleic
acid structures can be formed. This study describes building
blocks of 5-aza-7-deaza-2Ј-deoxyguanosine (1) for solid-
phase oligonucleotide synthesis (4, 5) (Scheme 2) and in-
vestigations concerning the pairing modes of the neutral
Building Blocks
5-Aza-7-deaza-2Ј-deoxyguanosine (1) was prepared ac-
cording to Rosemeyer and Seela[11] starting with compound
7, and was then converted into oligonucleotide building
blocks after precedent protection of the functional
groups.[12,13] Due to the different nitrogen pattern of 5-aza-
7-deazaguanine compared to guanine an altered reactivity
of the 2-amino function of 1 was anticipated. The protect-
[a]
Laboratorium für Organische und Bioorganische Chemie,
Institut für Chemie, Universität Osnabrück,
Barbarastraße 7, D-49069 Osnabrück, Germany
Fax: (internat.) ϩ 49(0)541/9692370
E-mail: FraSeela@rz.Uni-osnabrueck.de
Eur. J. Org. Chem. 1999, 485Ϫ496
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/0202Ϫ0485 $ 17.50ϩ.50/0
485

F. Seela, A. Melenewski
FULL PAPER
In order to circumvent this problem the following pro-
cedure was employed: (i) reaction of 1 with bis(4-methoxy-
phenyl)(phenyl)methyl chloride under formation of com-
pound 11. (ii) Subsequent reaction of 11 with N,N-dimeth-
ylacetamide dimethyl acetal[14] without isolation of the re-
action product 14. (iii) Further reaction of compound 14 to
either the phosphonate 15 or the phosphoramidite 5 using
standard conditions.[15,16]
Although the protection of the amino group was neces-
sary in the case of the phosphoramidite coupling, it was
unnecessary to introduce a protecting group when the pro-
tocol of phosphonate chemistry was employed.[17Ϫ19] For
this purpose compound 11 was directly converted into the
phosphonate 4 using a mixture of PCl3/N-methylmor-
pholine/1,2,4-triazole. Similarly, the phosphitylation of 11
with NC(CH2)2OP(Cl)[N(iPr)2] furnished the base-unpro-
tected phosphoramidite 16 (Scheme 5). During oligonucleo-
tide synthesis it turned out that both fully protected build-
ing blocks Ϫ the phosphonate 15 and the phosphoramidite
5 Ϫ as well as the amino-unprotected phosphonate 4 but
not the unprotected phosphoramidite 16 gave excellent
coupling yields in oligonucleotide synthesis.
Scheme 2
ing groups (see compounds shown in Table 1) (Schemes 3
and 4) were studied. Only the acetamidine 12 was found to
be suitable. However, even this compound is not stable and
forms the methoxy compound 13 during the workup pro-
cedure.
Table 1. Half-life values of deprotection of 2-deoxyribofuranosyl
derivatives of 8H-imidazo[1,2-a][1,3,5]triazin-4-one in 25% aq.
NH3 solution at 40°C
Compound λ [nm][a] τ [min] Compound λ [nm][a] τ [min]
Properties of the Monomers
6
7
8
295
295
300
< 1
> 500
3
10
12
13
298
300
300
< 1
43
4
The compounds described above were characterized by
1H-, 31P- and 13C-NMR spectra as well as by elemental
analyses (see Experimental Section). Table 2 summarizes
the 13C-NMR data of the 5-aza-7-deazapurine nucleosides
as well as of their protected derivatives. The signal assign-
ment of the starting material 1 has already been per-
formed.[11] However, in the case of the base only C-7, C-8
and the bridge-head C atoms could be assigned unambigu-
ously whereas the assignment of C-2 and C-6 remained ten-
tative. In the case of the amidine 8, the C-2 signal can be
identified by gated-decoupled 13C-NMR spectra. A small
coupling (6.8 Hz) between C-2 and the CH of the (dimeth-
ylamino)methylidene group is indicative for this position
(Table 3).
[a] Wavelength at which the kinetics was determined.
Scheme 3
Previous studies have shown that compound 1 is pro-
tonated on the s-triazine moiety exhibiting a pKBHϩ value
of 3.7.[11] Now, the site of protonation has been established
using 13C-NMR spectroscopy. Spectra were measured in
D2O within a pD range of 1Ϫ14. A significant downfield
shift of the C-2 and C-6 resonances (∆δ ϭ 8.4 ppm, both)
was observed when changing the pD from 5 to 1 while all
other chemical shifts remain almost constant (Table 2).
These experiments prove that N-1 is the protonation site.
This is different from 2Ј-deoxyguanosine (pKBHϩ ϭ 3.5)[20]
which is protonated at N-7. Only one transition in the UV
spectra of 1 at acidic pH was observed. The UV spectra of
1 in anhydrous dioxane has a maximum at 266 nm, whereas
in water the maximum is shifted to 258 nm (spectra not
shown). This phenomenon is not observed in the case of 7-
deaza-2Ј-deoxyguanosine. It implies that different tauto-
mers are formed in solvents of different polarity Ϫ a finding
which has already been proposed earlier based on 13C-
Scheme 4
486
Eur. J. Org. Chem. 1999, 485Ϫ496

Oligonucleotide Duplexes with Novel Base Pairs
FULL PAPER
Scheme 5
Table 2. 13C-NMR-chemical shifts of 2-deoxyribofuranosyl derivatives of 8H-imidazo[1,2-a][1,3,5]triazin-4-one[a]
[b]
C-2
C-2
C-4
C-6
C-6
C-7
C-7
C-8
C-8a
C-4
[c]
1
150.1
144.6
149.5
153.0
153.8
154.7
153.4
152.3
150.1
149.9
150.0
148.9
149.8
164.1
149.2
149.9
149.9
165.4
156.7
161.6
165.1
166.0
166.9
165.5
164.4
169.3
165.4
169.4
161.0
169.0
169.2
160.1
165.3
162.0
108.4
110.3
109.4
109.0
109.8
110.7
109.3
108.1
108.3
108.2
108.3
108.9
108.2
108.6
108.8
108.4
108.1
114.2
118.0
116.4
115.4
116.2
117.0
115.7
114.6
115.4
114.0
115.3
116.3
115.5
116.3
116.1
113.7
115.1
150.2
149.0
149.6
150.1
150.9
151.8
150.5
149.3
149.8
150.3
150.1
149.5
150.5
150.6
150.0
150.4
150.2
1[d]
1[e]
1[f]
1[g]
1[h]
1[i]
1[j]
6
8
9
10
12
13
11
4
15
C-1Ј
C-2Ј
C-3Ј
C-4Ј
C-5Ј
CϭN(O)
NϪCH3
CH3
OϪCH3
1
87.7
87.9
87.4
87.2
88.0
88.9
87.6
86.8
83.1
82.6
85.7
85.5
83.3
83.6
87.9
82.7
83.1
38.8
39.1
38.4
38.6
39.5
40.3
38.9
38.2
n.d.
38.6
40.6
n.d.
n.d.
n.d.
n.d.
37.7
37.2
70.6
71.1
71.0
71.0
71.9
72.7
71.3
70.2
70.4
70.1
70.1
70.0
70.5
70.3
70.4
72.3
72.1
82.8
85.1
84.4
84.0
84.9
85.7
84.4
83.1
87.8
85.6
82.9
83.7
87.7
88.0
83.4
85.6
85.6
61.5
61.8
61.6
61.6
62.5
63.3
62.0
61.0
61.4
63.9
63.9
63.9
61.5
61.3
61.4
63.7
63.6
1[d]
1[e]
1[f]
1[g]
1[h]
1[i]
1[j]
6
19.0
8
9
159.7
159.6
161.0
161.9
164.1
34.6
34.7
10
12
13
11
4
[k]
17.0
17.0
53.7
55.0
55.0
15
158.0
16.0
8.5
[a]
[b]
[c]
[d]
[e]
Measured in [D6]DMSO at 303 K. Ϫ
Systematic numbering. Ϫ Purine numbering. Ϫ pH ϭ 1.0. Ϫ pH ϭ 3.8. Ϫ [f] pH ϭ
5.0. Ϫ [g] pH ϭ 6.3. Ϫ [h] pH ϭ 9.0. Ϫ [i] pH ϭ 12.0. Ϫ [j] pH ϭ 14.0. Ϫ [k] Superimposed by DMSO.
Eur. J. Org. Chem. 1999, 485Ϫ496
487

F. Seela, A. Melenewski
FULL PAPER
NMR spectroscopy of compound
[D6]DMSO.[11]
The concept of preorganization Ϫ brought forth by Cram
in the analysis of small organic guest-host complexes[21]
1 in D2O and
Oligonucleotides with 5-Aza-7-deazaguanine
Opposite to Cytosine
Ϫ
was later discussed and applied to nucleic acids by Eschen-
moser et al.[22] as well as by Kool.[23] This concept can be
used to design a modification which rigidifies a ligand, e.g.
an oligonucleotide single strand prior to binding to its
complement so that it more resembles the bound confor-
mation. This would increase the binding strength. On the
other hand the importance of stereoelectronic effects of a
nucleobase on the sugar puckering of the nucleoside and
therewith on the preorganization of a corresponding oli-
gonucleotide secondary structure has been discussed.[24][25]
The results described above prompted us to examine the
sugar conformation of compound 1. This was performed
on the basis of seven vicinal 1 H,1H-coupling constants de-
An interesting feature of the 5-aza-7-deazaguanine is its
ability to act as hydrogen-bond donor at N-1 (analogous
to guanine) in the protonated form.[29,30] Appropriate base
pairing is possible with other bases providing a proton to
N-1 of the 5-aza-7-deazaguanine moiety. Stable, tridentate
dZϪdC WatsonϪCrick (WC) base pairs are therefore ex-
pected in acidic solution (Scheme 6, WC base pair II)
whereas under neutral conditions (Scheme 6, WC base pair
Ia or Ib) a lower stability is anticipated. Such a stabilization
of a base pair in acidic medium has been already studied
for triplexes formed between cytosine and a guanineϪcyto-
sine base pair.[31]
In order to realize such a duplex structures, oligodeoxy-
thymidylates containing one central dG or dZ residue (17
and 19) were synthesized and hybridized with oligodeoxya-
denylates. The latter possesses a central dC or 5-methyl-2Ј-
termined in D2O. In particular, the sugar puckering [3ЈT2Ј
2Ј
(N)
v
T
(S)] and the conformation about the
3Ј
C(4Ј)ϪC(5Ј) [γ(g) v γt v γϪ(g)] bond were determined ap-
plying the  (6.2) program of Altona and co-work-
ers[26] as well as the method of Westhof et al.[27] Using the
[H,H] coupling constants 1Ј,2Ј, 1Ј,2ЈЈ, 2Ј,3Ј, 2ЈЈ,3Ј, and 3Ј,4Ј
the N/S conformer populations were determined to be 37%
N and 63% S. Compared to dG (29% N, 71% S) the sugar
conformation is slightly shifted towards the N confor-
mation. The conformation at the C(4Ј)ϪC(5Ј) bond of 1
[γϩ(g) 48%, γt 33%, γϪ(g) 19%] is similar to that of dG [γϩ(g)
53%, γt 30%, γϪ(g) 17%]. This means that 5-aza-7-deazapu-
Table 3. J(C,H) values [Hz] of the nucleosides 1 and 8[a]
J(C,H)
1
8
CH, CH
Ϫ
Ϫ
179.4
6.8
C(2), CHϭN
C(6), H-C(6)
C(6), H-C(7)
C(7), H-C(7)
C(7,) H-C(1Ј)
C(1Ј), H-C(1Ј)
C(3Ј), H-C(3Ј)
C(4Ј), H-C(4Ј)
C(5Ј), H-C(5Ј)
205.2
205.4
10.9
10.7
200.7[b]
4.5[b]
167.7
150.6
147.6
140.5
200.8
4.2
165.9
148.3
148.0
140.3
Scheme 6
[a] Measured in [D6]DMSO at 303 K. Ϫ [b] See ref.[11]
deoxycytidine residue (m5Cd) opposite to dZ or dG when
hybridized with the oligothymidylates (Table 4). The Tm val-
rine exerts a slightly stronger electron-withdrawing influ- ues of the oligonucleotides were measured as a function of
ence on the 2Ј-deoxyribose moiety than guanine thereby the pH by using buffer solutions (10 m sodium cacodylate,
biasing the N/S equilibrium of the sugar towards that of a 10 m MgCl2, and 100 m NaCl) which had been adjusted
ribonucleoside. Although there are significant changes of to pH ϭ 5.0, 6.0, 7.0, 8.5, and 9.5 by adding either 0.1 
the sugar moiety of base-modified nucleosides which will HCl or 0.1  NaOH. In all cases cooperative melting pro-
affect the preorganization of the sugar-phosphate back- files were observed. The Tm values of the resulting duplexes
bone, there is no simple correlation between stereoelec- are summarized in Table 4. From the data it is apparent
tronic effects within a base-modified nucleoside and the sta- that protonation Ϫ either at dZ (pK ϭ 3.7), dC (pK ϭ 4.3)
bility of an oligonucleotide duplex.[28]
or m5Cd (pK ϭ 4.5), respectively Ϫ stabilizes the duplex
488
Eur. J. Org. Chem. 1999, 485Ϫ496

Oligonucleotide Duplexes with Novel Base Pairs
FULL PAPER
Table 4. Tm values and thermodynamic data of 5Ј- prising that the modified and protonated duplex of Table 5
d(TTTTTXTTTTTT) · 5Ј-d(AAAAAAYAAAAA) at pH ϭ 7.0, 5.0
do not reach the stability of the parent duplex (22 · 23). To
our best knowledge these protonated double helices are the
and 9.5[a]
first ones formed by purineϪpyrimidine base pairs of the
WatsonϪCrick type. Related structures of the Hoogsteen
type are long known from triplexes when a dGϪdC duplex
binds a third strand containing dG.
In conclusion, it was demonstrated that 5-aza-7-deazagu-
anine forms a stable base pair with cytidine in acidic solu-
tions under antiparallel chain orientation. In this case a
proton is inserted between N-1 of 5-aza-7-deazaguanine
and N-3 of cytosine forming an additional hydrogen bond.
X · Y
pH Tm[b] [°C] Postulated base pair involving Z
17 · 18 G · C
7.0 40
5.0 39
9.5 39
19 · 18 Z · C
7.0 27
5.0 35
9.5 22
base pair Ia or Ib, Scheme 6
base pair II, Scheme 6
base pair Ia or Ib, Scheme 6
19 · 20 Z · m5C 7.0 28
5.0 37
base pair Ia or Ib, Scheme 6
base pair II, Scheme 6
base pair Ia or Ib, Scheme 6
9.5 27
Table 5. Tm values and thermodynamic data of oligonucleotide du-
plexes
19 · 21 Z · G
7.0 30
5.0 28
9.5 24
wobble base pair III, Scheme 7
wobble base pair III, Scheme 7
wobble base pair III, Scheme 7
5Ј-d (T
•
3Ј-d (A
A
•
T
X
•
C
X
•
C
T
•
A
C
•
Y
A
•
T
A
•
T
T
•
A
A
•
T
C
•
Y
T)
•
A)
[a] Oligomer concentration 10 µ of single strands. Ϫ [b] Measured
in 0.1  NaCl containing 10 m MgCl2 and 10 m sodium cacody-
late.
XXY...Y
pH Tm[a] [°C] Postulated base pair involving dZ
GGG...G 22 · 23 5
46
45
7
ZZG...G 24 · 23 5
34
23
ZϪC: base pair II, Scheme 7
ZϪC: base pair Ia or Ib, Scheme 7
7
containing a dZϪdC (∆Tm ϭ ϩ8°C) or a dZϪm5Cd base
pair (∆Tm ϭ ϩ9°C). The situation is different, however, in
the case of 19 · 21. Here, a dGϪdZ base pair (Scheme 7,
Pur · Pur wobble pair III) with an antiparallel strand orien-
tation is conceivable even under neutral conditions which
contains two hydrogen bonds. This would explain the still
relatively high Tm value of 19 · 21 at pH ϭ 7 (30°C). Upon
protonation as well as upon deprotonation a destabilization
of the duplex is observed (Table 4, ∆Tm ϭ 2Ϫ6°C) which
is due to the fact that in both cases the number of hydrogen
bonds is reduced. The observed Tm decrease of the duplex
19 · 18 between pH ϭ 7.0 and 9.5 might be the result of
the relatively lower stability of the dZϪdC base pair com-
pared to those of dZϪm5Cd or dGϪdC. As a consequence,
the dT deprotonation (pH ഠ 9.5) might occur earlier when
it is close to a dZϪdC base pair.
GGZ...Z 22 · 25 5
37
23
CϪZ: base pair II, Scheme 7
7
CϪZ: base pair Ia or Ib, Scheme 7
ZZZ...Z 24 · 25 5
25
ZϪC and CϪZ: all base pair II,
Scheme 7
ZϪC and CϪZ: all base pair Ia or
Ib Scheme 7
7
<10
[a] Measured at 260 nm in 0.1  NaCl, 10 m MgCl2, and 10 m
Na-cacodylate at 7 µmol single-strand concentration.
Oligonucleotides with 5-Aza-7-deazaguanine
Opposite to Guanine or Isoguanine
According to the fact that 5-aza-7-deazaguanine has the
same WatsonϪCrick recognition site as isocytosine
Using the helix axis as a dyad axis, rotation of one strand (Scheme 1) and isocytosine forms parallel duplex structures
of an antiparallel duplex by 180° results in a parallel ar- with guanine it was anticipated that also 5-aza-7-deazagu-
rangement. Doing this, the conformation of the sugarϪ anine can form parallel duplexes with guanine (Scheme 7,
phosphate backbone must not be necessarily changed in or- Pur · Pur base pair IV). In order to realize such a structure ,
der to get a stable right-handed duplex. The oligonucleo- the oligonucleotide d(T4Z4T4) (30) was synthesized along
tides depicted in Table 4 might adopt such structures with with other oligomers, namely d(A4G4A4) (27), d(T4G4T4)
parallel as well as antiparallel strand orientation.[32] In or- (26) and d(A4C4A4) (31). These oligonucleotides exhibit
der to circumvent this problem another oligonucleotide se- only linear increases of the absorbance upon heating.
quence was chosen (see oligonucleotides of Table 5) which Therefore, self-pairing can be ruled out under these con-
can only form aps duplex structures. In this case the modi- ditions. Moreover, in contrast to d(T4G4T4) (26),[27] the oli-
fied duplexes contain either two or four dZϪdC base pairs. gomer d(T4Z4T4) (30) is not able to form a quartet struc-
In all modified duplexes a stabilization occurred under ture. When the oligomer 26 is hybridized with 27 a Tm value
acidic but not under neutral conditions.
of only 13°C is obtained (Table 6). Obviously, this duplex
From the structural point of view the protonated du- is mainly held together by eight dAϪdT base pairs. This
plexes 24 · 23, 22 · 25 and 24 · 25 are of particular interest. duplex becomes strongly stabilized (∆Tm ϭ ϩ10°C) when
They represent double helices with a spine of positive only one of the dG residues is replaced by a dZ moiety
charges in the core of the double helix. The additional hy- (duplexes 28 · 27 and 29 · 27), independently of the position
drogen bonds lead to duplex stabilization while the positive of substitution. The replacement of all four dG residues by
charges might reduce base stacking. Therefore, it is not sur- dZ (30 · 27) increases the Tm value to 55°C (∆Tm
ϭ
Eur. J. Org. Chem. 1999, 485Ϫ496
489

F. Seela, A. Melenewski
FULL PAPER
tention of the chain orientation (Table 6) (3 versus 2 hydro-
gen bonds, Scheme 6, WC base pair Ia or Ib).
The formation of dGϪdZ base pairs can also be demon-
strated in the case of the duplex 30 · 26 compared to
26 · 32. Both have dT tails at the 3Ј and the 5Ј termini
which normally do not form hydrogen bonds with each
other. From the Tm values of these duplexes (Table 6) it is
evident that the four dGϪdZ base pairs in the ps duplex 30
· 26 (Scheme 7, Pur · Pur base pair IV) are more stable than
the GϪC base pairs within the aps duplex 26 · 32. Obvi-
ously, dZ dictates the parallel strand orientation with three
hydrogen bonds within each of the four dGϪdZ base pairs.
The base pair formed between dZ and dG under neutral
conditions can be either a purineϪpurine analogue base
pair with three (Scheme 7, Pur · Pur base pair IV) or a base
pair with two hydrogen bonds (Scheme 7, Pur · Pur wobble
pair III) Ϫ the first with parallel, the second with antiparal-
lel orientation of the strands. Moreover, under acidic con-
ditions where dZ and/or dG are protonated on the base
moiety three different Hoogsteen type dGϪdZ base pairs
with either parallel or antiparallel strand orientation can be
formulated (Scheme 8).
Scheme 7
The strand orientation of oligonucleotide duplexes can
be deduced from hybridization studies performed on oli-
gonucleotides containing tracts of identical bases either at
their 3Ј- or at the 5Ј-termini.[33,34] For such studies the oli-
gomers 33 and 34 as well as the homo-dZ oligomer 35 were
synthesized, and their duplex formation was studied. In the
case of parallel chain orientation it is expected that neither
the oligomer 33 nor 34 form duplexes or at best duplexes
of low stability in the case of partial hybridization. A stable
duplex should be formed when 33 and 34 are hybridized. It
was observed that self-pairing of 33 as well as 34 occurs but
that the duplexes exhibit relatively low Tm values (Table 7).
ϩ42°C). As this duplex is even more stable than the duplex
26 · 31 (Tm ϭ 45°C) containing 4 dGϪdC and 8 dAϪdT
base pairs, it is concluded that also 30 · 27 represents a fully
matched hybrid with 12 base pairs (8 ϫ 2 ϩ 4 ϫ 3 H bonds)
and with a parallel strand orientation (Scheme 7, Pur · Pur
base pair IV). At this moment also for the oligonucleotides
28 · 27 and 29 · 27 a parallel strand orientation can be
tentatively postulated notwithstanding the fact that also
antiparallel dZϪdG base pairs are principally conceivable
even under neutral conditions (Scheme 7, Pur · Pur wobble
pair III). On the other hand, substitution of one dG base
by dZ within the antiparallel duplex d(T4G4T4) · d(A4C4A4)
The duplexes 33 · 33 and 34 · 34 can exhibit parallel or
(26 · 31) causes a destabilization by about 6°C under neu- antiparallel chain orientation by self-pairing (Scheme 9).
tral conditions d(T4ZG3T4) · d(A C4A4) (28 · 31) with re- For the self-paired oligomer 33 the antiparallel structure A
4
Table 6. Tm values and thermodynamic data of oligonucleotide duplexes[a]
Tm[b] [°C]
H [%]
5
Postulated base pair involving dZ
5Ј-d(T4GGGGT4)
3Ј-d(A4GGGGA4)
26 · 27
13
5Ј-d(T4ZGGGT4)
28 · 27
29 · 27
23
23
13
10
ZϪG: base pair IV, Scheme 8
ZϪG: base pair IV, Scheme 8
5Ј-d(T4GZGGT4)
5Ј-d(A4GGGGA4)
5Ј-d(T4Z Z Z ZT4)
30 · 27
26 · 31
55
45
14
14
ZϪG: base pair IV, Scheme 8
5Ј-d(T4GGGGT4)
3Ј-d(A4CCCCA4)
5Ј-d(T4GGGGT4)
28 · 31
30 · 26
39
25
13
8
ZϪC: base pair Ia or Ib, Scheme 7
ZϪG: base pair IV, Scheme 8
5Ј-d(T4 ZZZZ T4)
5Ј-d(T4GGGGT4)
5Ј-d(T4GGGGT4)
3Ј-d(T4CCCCT4)
26 · 32
15
15
[a] Oligomer concentration 10 µ of single strands. Ϫ [b] Measured in 1  NaCl containing 10 m MgCl2 and 10 m sodium cacodylate,
pH ϭ 7.0.
490
Eur. J. Org. Chem. 1999, 485Ϫ496

Oligonucleotide Duplexes with Novel Base Pairs
FULL PAPER
(base pair mode III, Scheme 9) is the most likely one (bold
face) as it contains the highest number of hydrogen bonds
(12 H bonds). The Tm value is 27°C (Table 7); upon pro-
tonation (pH ϭ 4) its stability is decreased to 22°C because
in this case the dZϪdG base pair is disturbed. The CD
spectrum of 33 · 33 is shown in Figure 1a and exhibits the
typical pattern of a B-type helix. Even upon protonation
(pH ϭ 4) the shape of the CD spectrum is maintained from
which it can be deduced that the B-DNA structure with aps
WatsonϪCrick-type base pairing and blunt ends is con-
served.
The Tm value of the self-paired duplex 34 · 34 amounts
to only 20°C which is 7°C lower than that of 33 · 33 (Table
7). This may be traced back either to a different secondary
structure, namely a parallel B-DNA with WatsonϪCrick-
type base pairing and sticky ends (Scheme 9: structure E,
mode IV) containing only nine hydrogen bonds or to the
inverted base stacking. Upon protonation (pH ϭ 4) the Tm
value of this self-paired duplex is significantly enhanced to
32°C (∆Tm ϭ 12°C). Moreover, the CD spectrum of 34 ·
34 is drastically changed (Figure 1b). In this case a reorien-
tation of the duplex from the ps structure E to an aps struc-
ture with Hoogsteen-type base pairing (Scheme 8, mode V)
might take place.
A mixture of the oligomers 33 and 34 gives rise to a much
more stable duplex compared to the self-paired ones with a
Tm value of 50°C (Table 7). The concentration dependence
of the Tm values indicates duplex melting (data not shown).
According to these findings the duplex 33 · 34 must adopt
a parallel strand orientation following the base pair mode
IV (Scheme 9, structure H, 18 H bonds, blunt ends). This
structure is conservative upon protonation which is demon-
Scheme 8
Table 7. Tm values and thermodynamic data of self-complementary oligonucleotides[a]
Tm[b] [°C]
pH
H [%]
Postulated base pair involving dZ
5Ј-d(Z-Z-Z-G-G-G)
33
27
7.0
18
ZϪG: wobble pair III, Scheme 9
5Ј-d(Z-Z-Z-G-G-G)
5Ј-d(Z-Z-Z-G-G-G)
33
33
27
22
5.0
4.0
19
28
ZϪG: wobble pair III, Scheme 9
ZϪG: wobble pair III, Scheme 9
5Ј-d(G-G-G-Z-Z-Z)
5Ј-d(G-G-G-Z-Z-Z)
5Ј-d(G-G-G-Z-Z-Z)
34
20
32
32
50
7.0
5.0
4.0
7.0
12
9
GϪZ: base pair IV, Scheme 8
GϪZ: base pair V, Scheme 8
GϪZ: base pair V, Scheme 8
ZϪG, GϪZ: base pair IV, Scheme 9
34
34
8
5Ј-d(Z-Z-Z-G-G-G)
5Ј-d(G-G-G-Z-Z-Z)
33 · 34
18
5Ј-d(Z-Z-Z-G-G-G)
5Ј-d(G-G-G-Z-Z-Z)
33 · 34
33 · 34
52
48
5.0
4.0
10
14
ZϪG, GϪZ: base pair IV, Scheme 9
ZϪG, GϪZ: base pair IV, Scheme 9
5Ј-d(Z-Z-Z-G-G-G)
5Ј-d(G-G-G-Z-Z-Z)
[c]
5Ј-d(Z)6
35
5Ј-d(Z)6
35 · 36
41
7.0
14
ZϪc7G: base pair IV, Scheme 9
5Ј-d(c7G)6
[c]
5Ј-d(c7G)6
36
5Ј-d(iGiGiG-C-C-C)[d]
37 · 38
47
7.0
7.0
Ϫ
Ϫ
5Ј-d(C-C-C-iGiGiG)
5Ј-d(G-G-G-Z-Z-Z)
34 · 37
41
20
ZϪiG: base pair IX, Scheme 10
[a] Oligomer concentration 10 µ of single strands. Ϫ [b] Measured in 1  NaCl containing 100 m MgCl2 and 60 m sodium cacodylate,
pH ϭ 7.0. Ϫ [c] Linear increase of absorbance at 260 nm. Ϫ [d] Ref.[28]
Eur. J. Org. Chem. 1999, 485Ϫ496
491

F. Seela, A. Melenewski
FULL PAPER
Scheme 9
strated by pH-dependent CD spectra (Figure 1c). The Tm
value is only slightly reduced (Table 7) when changing the
pH from 7 to 4 which is consistent with our structural as-
sumption.
The parallel orientation of oligonucleotide duplexes con-
taining dGϪdZ base pairs is supported by experiments per-
formed on the homooligonucleotides 5Ј-d(Z-Z-Z-Z-Z-Z)
(35) and 5Ј-d(c7G-c7G-c7G-c7G-c7G-c7G) (36). Both oli-
gonucleotides contain 7-deazapurine bases (7-deazaguanine
and 5-aza-7-deazaguanine), excluding Hoogsteen-type base
pairing. The Tm value of 35 · 36 (Table 7) was determined
UV-spectrophotometrically as well as by temperature-de-
pendent CD spectra. Both Tm values were found to be
41°C. According to this Tm value the duplex must follow
the WatsonϪCrick-type base pair IV with parallel chain
orientation (analogous structure H, Scheme 9); only here, a
Tm value which is in the range of that of the duplex 33 · 34
(50°C). The difference (∆Tm ϭ 9°C) can be attributed to
the altered sequence as well as to the presence of c7Gd in-
stead of dG.
As isocytosine and 5-aza-7-deazaguanine have the same
donor-acceptor pattern (Scheme 1), an antiparallel duplex
structure should also be formed in the case of an oligonu-
cleotide built up from 5-aza-7-deazaguanine-isoguanine
base pairs (mode IX, Scheme 10). Similarly, isoguanine
Scheme 10
pairs with isocytidine with aps chain orientation (mode X, value of 41°C was formed (Table 7). The corresponding ps
Scheme 10).[34,35] In order to prove this hypothesis the oli- duplex of 37 · 38 showed a Tm value of 47°C.
gonucleotides 5Ј-d(G3-Z3) (34) and 5Ј-d(isoG3-C3) (37) were
The experiments performed with oligonucleotides con-
hybridized. Also in this case a stable aps duplex with a Tm taining dZ opposite to Gd show that fully matched duplexes
492
Eur. J. Org. Chem. 1999, 485Ϫ496

Oligonucleotide Duplexes with Novel Base Pairs
FULL PAPER
with parallel chain orientation (Pur · Pur base pair IV) are
formed whereas those containing dZ opposite to isoGd
(base pair IX) form duplexes with antiparallel chains.
In general it was demonstrated that various new base
pairs can be constructed when 5-aza-7-deazaguanine re-
places guanine within oligonucleotide duplexes. Depending
on the complementary base parallel as well as antiparallel
hybrids are accessible. In particular, “purineϪpurine” base
pairs (dGϪdZ) (base pair IV)[36] are remarkably stable
when the chain orientation is parallel.
Experimental Section
General: Solvent systems for flash chromatography (FC) and thin-
layer chromatography (TLC): CH2Cl2/MeOH, 95:5 (A); CH2Cl2/
MeOH, 9:1 (B); CH2Cl2/MeOH, 8:2 (C); CH2Cl2/MeOH, 75:25
(D); CH2Cl2/MeOH/Et3N, 95:3:2 (E); CH2Cl2/MeOH/Et3N,
88:10:2 (F); CH2Cl2/MeOH/Et3N, 78:20:2 (G); CH2Cl2/EtOAc/
Et3N, 45:45:10 (H); CH3CN/H2O, 9:1 (I). Ϫ Elemental analyses
were performed by Mikroanalytisches Laboratorium Beller
(Göttingen, Germany). Oligonucleotide synthesis was carried out
with a DNA synthesizer, model 381 A (Applied Biosystems, Weiter-
stadt, Germany), on a 1-µmol scale. The enzymatic hydrolysis of
the oligomers was carried out as described.[37] The mixture was
analyzed on reversed-phase HPLC (RP-18, solvent system III).
Quantification of the resulting nucleosides was made on the basis
of the peak areas which were divided by the extinction coefficients
of the nucleoside constituents (λ260: dA 15400, dC 7300, dG 11700,
dT 8800, dZ 11500). Ϫ UV: Hitachi U 3200 UV-VIS spectrophoto-
meter. Ϫ NMR: Bruker AMX-500 (500.15 MHz and 125.4 MHz
for 1H and 13C, respectively). For 1H, [D6]DMSO as solvent δH
ϭ
2.50; for 13C NMR, [D6]DMSO δC ϭ 39.5. Bruker AC-250 (101.1
MHz for 31P, relative to 85% H3PO4 as external standard). All spec-
tra were measured at 30°C. Ϫ MALDI-TOF spectra were recorded
by Mrs. Julia Gross, Westfälische Wilhelms Universität Münster,
Institut für Medizinische Physik und Biophysik (Head: Prof. Dr. F.
Hillenkamp) with a home-built apparatus.
pKa Values: The pKa values were determined UV-spectrophoto-
metrically at 20°C in TeorellϪStenhagen buffer.[38]
HPLC Separation: HPLC was carried out according to Seela et
al.[39] The solvent gradients consisting of 0.1  (Et3NH)OAc, pH ϭ
7.0/MeCN 95:5 (A) and MeCN (B) were used in the following or-
der: gradient I, 3 min 15% B in A, 7 min 15Ϫ40% B in A, 10 min
40% B in A, 5 min 40Ϫ15% B in A; flow rate 1 mL/min; gradient
II, 20 min 0Ϫ20% B in A, flow rate 1 mL/min; gradient III, 20 min
100% A, flow rate 0.6 mL/min.
Tm Experiments: Melting curves were measured with a Cary-1/3
UV/VIS spectrophotometer (Varian, Australia). The temperature
of the solution was increased linearly with time at a rate of 60°C/
h using a Lauda PM-350 programmer and a Lauda RCS 6 bath
equipped with an R 22 unit (MWG Lauda, FRG). The actual tem-
perature was measured in the reference cell with a Pt-100 resistor.
The Tm values were calculated using the software package provided
by Dr. H. Apel, Varian, (Darmstadt, Germany).
Figure 1. pH-dependent CD spectra a) of self-complementary oli-
gomer d(ZZZGGG) (33), b) of self-complementary oligomer
d(GGGZZZ) (34) and c) of duplex d(ZZZGGG) Ϫ d(GGGZZZ)
(33 · 34), measured at 0°C in 60 m sodium cacodylate, 1  NaCl,
and 100 m MgCl2 at pH ϭ 4.0, 5.0 and 7.0; the oligomer concen-
tration was 10 µ of single-strands
8-(2-Deoxy-β--erythro-pentofuranosyl)-2-(isobutyrylamino)-8H-
imidazo[1,2-a][1,3,5]triazin-4-one (6): Compound 1[11] (50 mg, 0.19
mmol) was dried by co-evaporation with pyridine (3 ϫ 5 mL). The
residue was suspended in pyridine (5 mL), and Me3SiCl (0.48 mL,
3.75 mmol) was added at room temp. After 15 min of stirring, the
Eur. J. Org. Chem. 1999, 485Ϫ496
493

F. Seela, A. Melenewski
FULL PAPER
solution was treated with isobutyric anhydride (0.62 mL, 3.78
Reaction of Compound 1 with N,N-Dimethylacetamide Dimethyl
mmol) and maintained at room temp. for 3 h. The mixture was Acetal: Compound 1 (197 mg, 0.74 mmol) was suspended in DMF
cooled (ice bath) and water (1 mL) was added. Then, 25% aq. am-
monia solution (1 mL) was added and stirring was continued for
15 min. The solution was concentrated to give an oil. FC (silica
gel, column 20 ϫ 1.8 cm, 100 mL of CH2Cl2, F) furnished a main
zone which gave 6 (21 mg, 33%) as a colorless, amorphous solid.
TLC (silica gel, B): Rf ϭ 0.35. Ϫ UV (MeOH): λmax (lg ε) ϭ 260,
(3 mL). After 10 min, N,N-dimethylacetamide dimethyl acetal (0.7
mL 4.2 mmol) was added under stirring. The reaction was con-
tinued at room temp. for 3 d. After evaporation of the solvent, the
residue was submitted to FC (column: 15 ϫ 1.8 cm, silica gel).
Flash chromatography with solvent C furnished two zones.
8-(2-Deoxy-β--erythro-pentofuranosyl)-2-[(dimethylamino)ethyl-
idene]amino)]-8H-imidazo[1,2-a][1,3,5]triazin-4-one (12): From the
slow migrating zone compound 12 (116 mg, 47%) was obtained as
colorless amorphous solid; TLC (silica gel, C): Rf ϭ 0.3. Ϫ 1H
NMR ([D6]DMSO): δ ϭ 7.61 (d, J ϭ 2.8 Hz, 1 H, 7-H), 7.49 (d,
J ϭ 2.7 Hz, 1 H, 6-H), 6.25 (“t”, J ϭ 6.7 Hz, 1 H, 1Ј-H), 5.33 (m,
1 H, 3Ј-OH), 5.01 (“t”, J ϭ 5.3 Hz, 1 H, 5Ј-OH); 4.34 (s, br., 1 H,
3Ј-H), 3.83 (m, 1 H, 4Ј-H), 3.55 (m, 1 H, 5Ј-Hα), 3.54 (m, 1 H, 5Ј-
Hβ), 3.07Ϫ3.01 [m, 6 H, N(CH3)2], 2.46Ϫ2.40 (m, 1 H, 2Ј-Hα),
2.27Ϫ2.20 (m, 1 H, 2Ј-Hβ), 2.14 [s, 3 H, C(CH3)].
1
295 nm (4.09, 4.16). Ϫ H NMR ([D6]DMSO): δ ϭ 7.72 (m, 1 H,
7-H), 7.63 (m, 1 H, 6-H), (“t”, J ϭ 6.4 Hz, 1Ј-H), 5.32 (m, 1 H,
3Ј-OH), 5.00 (m, 1 H, 5Ј-OH), 4.41 (m, 1 H, 3Ј-H), 3.84 (m, 1 H,
4Ј-H), 3.63 (m, 2 H, 5Ј-H), 2.90 (m, 1 H, 2Ј-Hα), 2.27 (m, 1 H, 2Ј-
Hβ), 1.27 [s, 1 H, CH(CH3)2], 1.0 [d, J ϭ 6.7 Hz, 6 H, C(CH3)2].
8-(2-Deoxy-β--erythro-pentofuranosyl)-2-{[(dimethylamino)meth-
ylidene]amino}-8H-imidazo[1,2-a][1,3,5]triazin-4-one (8): To a sus-
pension of compound 1 (60 mg, 0.22 mmol) in MeOH (3 mL) N,N-
dimethylformamide diethyl acetal (0.16 mL, 1.07 mmol) was added
under stirring at room temp. The stirring was continued overnight.
After concentration, the residue was submitted to FC (silica gel,
column 15 ϫ 1.8 cm, C) yielding 8 as a colorless amorphous solid
which crystallized from MeOH in colorless needles (47 mg, 66%),
m.p. 178°C; TLC (silica gel, C): Rf ϭ 0.45. Ϫ UV(MeOH): λmax
8-(2-Deoxy-β--erythro-pentofuranosyl)-2-{[(methoxy)ethyl-
idene]amino)}-8H-imidazo[1,2-a][1,3,5]triazin-4-one (13): The faster
migrating zone furnished compound 13 (26 mg, 11%) as colorless
amorphous solid; TLC (silica gel, C): Rf ϭ 0.3. Ϫ 1H NMR
([D6]DMSO): δ ϭ 7.76 (m, 1 H, 7-H), 7.63 (s, br., 1 H, 6-H), 6.29
(“t”, J ϭ 6.3 Hz, 1 H, 1Ј-H), 5.36 (m, 1 H, 3Ј-OH), 5.06 (“t”, J ϭ
4.8 Hz, 1 H, 5Ј-OH), 4.35 (s, br., 1 H, 3Ј-H), 3.85 (m, 1 H, 4Ј-H),
3.71 (s, 3 H, OCH3), 3.60Ϫ3.53 (m, 2 H, 5-H2), 2.48Ϫ2.43 (m, 1
H, 2Ј-Hα), 2.28Ϫ2.25 (m, 1 H, 2Ј-Hβ), 1.99 (s, 3 H, CH3).
1
(lg ε) ϭ 259, 298 nm (4.16, 3.67). Ϫ H NMR ([D6]DMSO): δ ϭ
8.72 [s, 1 H, CHN(CH3)2], 7.60 (s, 1 H, 7-H), 7.48 (s, 1 H, 6-H),
6.32 (s, br., 1 H, 1Ј-H), 5.30 (s, 1 H, 3Ј-OH), 5.00 (s, br., 1 H, 5Ј-
OH), 4.35 (s, br., 1 H, 3Ј-H), 3.84 (s, 1 H, 4Ј-H), 3.56 (s, br., 2 H,
5Ј-H), 3.16 and 3.03 [2 s, 6 H, N(CH3)2], 2.50 (m, 1 H, 2Ј-Hα), 2.24
(m, 1 H, 2Ј-Hβ). Ϫ C13H18N6O4 (322.33): calcd. C 48.44, H 5.63,
N 26.07; found C 48.37, H 5.60, N 25.96.
2-Amino-8-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β--
erythro-pentofuranosyl}-8H-imidazo[1,2-a][1,3,5]triazin-4-one (11):
Compound 1 (197 mg, 0.74 mmol) was dried by repeated co-evap-
oration from absolute pyridine and then dissolved in absolute pyri-
dine (2 mL). Thereupon, bis(4-methoxyphenyl)(phenyl)methyl
chloride (325 mg, 0.96 mmol) was introduced under Ar. The solu-
tion was stirred for 3.5 h at ambient temp., 5% aq. NaHCO3 (40
mL) was added and the solution was extracted with CH2Cl2 (25
mL, twice). The combined organic extracts were dried (Na2SO4),
filtered, concentrated, and the residue was applied to FC (silica gel,
column 22 ϫ 1.8 cm, 100 mL of CH2Cl2, F). Concentration of the
main zone afforded 11 (240 mg, 57%) as colorless, amorphous
solid; TLC (silica gel, B): Rf ϭ 0.35. Ϫ 1H NMR ([D6]DMSO):
δ ϭ 7.37Ϫ7.34 (m, 3 H, 7-H, 6-H and aromatic H), 7.30Ϫ7.22 (m,
aromatic H), 6.95 (s, br., 2 H, NH2 ), 6.87Ϫ6.84 (m, aromatic H),
6.21 (“t”, J ϭ 6.5 Hz, 1 H, 1Ј-H), 5.35 (s, 1 H, 3Ј-OH), 4.36 (m, 1
H, 3Ј-H), 3.92 (m, 1 H, 4Ј-H), 3.74 (s, 6 H, OCH3); 3.15 (m, 2 H,
5Ј-H), 2.50 (m, 1 H, 2Ј-Hα), 2.28 (m, 1 H, 2Ј-Hβ). Ϫ C31H31N5O6
(569.62): calcd. C 65.37, H 5.49, N 12.29; found C 65.32, H 5.55,
N 12.37.
Reaction of 8 with Bis(4-methoxyphenyl)(phenyl)methyl Chloride:
Compound 8 (205 mg, 0.63 mmol) was dried by repeated co-evap-
oration with absolute pyridine and then dissolved in absolute pyri-
dine (3 mL). Then bis(4-methoxyphenyl)(phenyl)methyl chloride
(235 mg, 0.70 mmol) (Ar) was added and the solution was stirred
for 3.5 h at ambient temp. After addition of 5% aq. NaHCO3 (40
mL), the solution was extracted with CH2Cl2 (25 mL, twice). The
combined organic extracts were dried (Na2SO4), filtered, concen-
trated, and the residue was separated by FC into two zones (silica
gel, column 22 ϫ 1.8 cm, 100 mL of CH2Cl2, F).
8-{5-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β--erythro-
pentofuranosyl}-2-{[(dimethylamino)methylidene]amino}-8H-imida-
zo[1,2-a][1,3,5]triazin-4-one (9): From the slow migrating zone com-
pound 9 (155 mg, 39%) was obtained as colorless amorphous solid;
1
TLC (silica gel, C): Rf ϭ 0.3. Ϫ H NMR ([D6]DMSO): δ ϭ 8.73
[s, 1 H, CHN(CH3)2], 7.46 (d, J ϭ 2.6 Hz, 1 H, 7-H), 7.40 (d, J ϭ
2.7 Hz, 1 H, 6-H), 7.37Ϫ7.23 (m, aromatic H), 6.85 (m, aromatic
H), 6.34 (“t”, J ϭ 6.7 Hz, 1 H, 1Ј-H), 5.38 (m, 1 H, 3Ј-OH), 4.37
(s, br., 1 H, 3Ј-H), 3.95 (m, 4Ј-H), 3.74 (2 s, 6 H, 2 OCH3), 3.51
(m, 1 H, 5Ј-Hα), 3.17 [m, 4 H, 2Ј-Hα and N(CH3)], 3.05 [s, 3 H,
N(CH3)], 2.32 (m, 1 H, 2Ј-Hß).
2-Amino-8-{5-O-[bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β--
erythro-pentofuranosyl-3Ј-(phosphonato)]-8H-imidazo[1,2-a][1,3,5]-
triazin-4-one (4): To a solution of PCl3 (132 µl, 1.55 mmol) and N-
methylmorpholine (1.7 mL, 15.2 mmol) in absolute CH2Cl2 (14
mL) was added 1H-1,2,4-triazole (345 mg, 5.0 mmol). After stirring
for 20 min at room temp. and cooling to 0°C, a solution of com-
8-{5-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β--erythro- pound 11 (230 mg, 0.4 mmol) in CH2Cl2 (9 mL) was added slowly.
pentofuranosyl}-2-(formylamino)-8H-imidazo[1,2-a][1,3,5]triazin-4-
one (10): From the faster migrating zone compound 10 (23 mg, 6%)
Stirring was continued for 30 min at 0°C, then the mixture was
poured into 1  (Et3NH)HCO3 (TBK, pH ϭ 8.0, 17 mL), shaken
was obtained as a colorless foam; TLC (silica gel, E): Rf ϭ 0.4. Ϫ and separated. The aqueous phase was extracted with CH2Cl2 (12
1H NMR ([D6]DMSO): δ ϭ 10.8 (s, br., 1 H, NH), 9.34 (s, OCH),
7.61 (d, J ϭ 2.5 Hz, 1 H, 7-H), 7.53 (d, J ϭ 2.5 Hz, 1 H, 6-H),
7.36Ϫ7.19 (m, aromatic H), 7.86Ϫ7.82 (m, aromatic H), 6.30 (“t”,
mL, three times) and the combined organic extracts were dried
(Na2SO4) and concentrated. FC (silica gel, column 15 ϫ 1.8 cm, B
(100 mL); C (100 mL); then D) furnished a main zone which was
J ϭ 6.8 Hz, 1 H, 1Ј-H), 5.39 (m, 1 H, 3Ј-OH), 4.39 (m, 1 H, 3Ј- concentrated. The residue was dissolved in CH2Cl2 (20 mL), ex-
H), 3.96 (m, 1 H, 4Ј-H), 3.73 (2 s, 6 H, 2 OCH3), 3.18 (m, 2 H, 5Ј- tracted with aq. 0.1  TBK (10 ϫ 15 mL), dried with Na2SO4 and
H), 2.63Ϫ2.59 (m, 1 H, 2Ј-Hα), 2.35Ϫ2.31 (m, 1 H, 2Ј-Hβ).
concentrated. Compound 4 (119 mg, 40%) was obtained as yellow-
494
Eur. J. Org. Chem. 1999, 485Ϫ496

Oligonucleotide Duplexes with Novel Base Pairs
FULL PAPER
ish foam; TLC (silica gel, C): Rf ϭ 0.45. Ϫ 1H NMR ([D6]DMSO): J ϭ 6 Hz, 1 H, 1Ј-H), 6.03 (s, 1/2 PH); 4.72 (s, br., 1 H, 3Ј-H), 4.09
δ ϭ 7.36Ϫ7.17 (m, 11.5 H, 7-H, 6-H, PH, aromatic H), 6.98 (s, br., (s, 1 H, 4Ј-H), 3.73 (m, OCH3), 3.17 (s, 2 H, 5Ј-H), 3.03 (s, br., 2
2 H, NH2), 6.85 (m, 4 H, aromatic H), 6.20 (m, 1 H, 1Ј-H), 6.04 H, MeCH2), 2.95 [m, 6 H, N(CH3)2], 2.13 (s, CH3), 1.14 (m, 10 H,
(s, 1/2 H, PH), 4.71 (s, br., 1 H, 3Ј-H), 4.07 (s, 1 H, 4Ј-H), 3.73 (m,
3 H, OCH3); 3.15 (m, 3 H, 5Ј-H and CH2), 2.94 (m, 1 H, CH2),
MeCH2). Ϫ 31P NMR ([D6]DMSO): 2.27 [1J(P,H) ϭ 291.94 Hz;
.
3J(P,H) ϭ 9.01). Ϫ C41H54N7O8P3/5HCl (825.78): calcd. C 59.66,
2.40 (m, 1 H, 2Ј-Hα), 1.02 (m, 6 H, CH3).
Ϫ
31P NMR
H 6.59, N 11.87; found C 59.58, H 6.51, N 11.45.
([D6]DMSO): δ ϭ 2.01 [1J(P,H) ϭ 581.68 Hz; J(P,H) ϭ 8.27]. Ϫ
C37H47N6O8P (734.79): calcd. C 60.48, H 6.45, N 11.44; found C
60.60, H 6.63, N 11.18.
3
Phosphoramidite 5: Compound 11 (136 mg, 0.24 mmol) was sus-
pended in MeOH (1 mL). After 10 min, N,N-dimethylacetamide
diethyl acetal (0.35 mL, 2.43 mmol) was added under stirring. The
reaction was continued at 50°C overnight. After concentration, the
residue was co-evaporated with MeCN, dissolved in absolute
CH2Cl2 (9 mL) followed by addition of (iPr)2EtN (140 µL, 0.32
mmol). To the resulting mixture chloro(2-cyanoethoxy)(diisopropy-
lamino)phosphane (177 µL, 0.77 mmol) was added within 2 min at
room temp. After stirring for 30 min, the mixture was diluted with
CH2Cl2 (36 mL), cooled to 0°C, and quenched by adding a 5%
aqueous NaHCO3 solution (9 mL). Then the aqueous layer was
extracted with CH2Cl2 (3 ϫ 24 mL), the combined organic layers
were dried (Na2SO4), filtered, and concentrated. The residue was
applied to flash chromatography (silica gel column 8 ϫ 2 cm,
CH2Cl2/MeOH/ Et3N, 96:3:1); colorless foam (126 mg, 63%); TLC
(silica gel, CH2Cl2/MeOH/Et3N, 96:3:1): Rf ϭ 0.35. Ϫ 31P NMR
(CDCl3): δ ϭ 149.45, 149.38.
Phosphoramidite 16: To a solution of 11 (100 mg, 0.18 mmol) and
(iPr)2EtN (91 µL, 0.50 mmol) in anhydrous CH2Cl2 (2.3 mL),
chloro(2-cyanoethoxy)(diisopropylamino)phosphane (115 µL, 0.50
mmol) was added within 2 min at room temp. After stirring for 30
min, the mixture was diluted with CH2Cl2 (11 mL), cooled to 0°C
and quenched by adding a 5% aq. NaHCO3 solution (11 mL).
Then, the aqueous layer was extracted with CH2Cl2 (3 ϫ 6 mL),
the combined organic layers were dried (Na2SO4), filtered, and con-
centrated. The residue was applied to FC (silica gel, column 8 ϫ 2
cm, CH2Cl2/EtOAc/Et3N, 69:30:1). Colorless foam of 16 (81 mg,
58%); TLC (silica gel, CH2Cl2/EtOAc/Et3N, 69:30:1): Rf ϭ 0.27. Ϫ
31P NMR (CDCl3): δ ϭ 149.71, 149.64.
8-{5-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-β--erythro-
pentofuranosyl}-2-{[(dimethylamino)ethylidene]amino}-3Ј-(phospho-
nato)-8H-imidazo[1,2-a][1,3,5]triazin-4-one (15): 184 mg (0.32
mmol) of 11 was suspended in 6 mL of MeOH. After 10 min, N,N-
dimethylacetamide diethyl acetal (0.56 mL, 3.90 mmol) was added
under stirring. The reaction was continued at 50°C overnight. After
concentration, the residue was co-evaporated with MeCN and dis-
solved in absolute CH2Cl2 (11.5 mL). The resulting mixture was
added within 10 min to a previously prepared solution of PCl3
(105 µL, 1.24 mmol), 1,2,4-triazole (276 mg, 4.0 mmol) and N-
methylmorpholine (1.38 mL, 12.16 mmol) in absolute CH2Cl2 (11.5
Solid-Phase Synthesis of the Oligonucleotides 17؊38: The synthesis
of the oligonucleotides 17, 19, 30, 28 and 26 was performed using
3ЈЈ-phosphonates of [(MeO)2Tr]ib2Gd, [(MeO)2Tr]ib2c7Gd, [(Me-
O)2Tr]Bz4Cd, [(MeO)2Tr]Ad, and [(MeO)2Tr]Td, as well as com-
pounds 4 and 5. The synthesis followed the regular protocol of
the DNA synthesizer for 3Ј-phosphonates.[25] The synthesis of the
oligomers 18, 20, 21, 22؊25, 27, 26, 31, 33؊38 was carried out on
a 1-µmol scale in a automated DNA synthesizer (Applied Biosys-
mL, 0°C). After stirring for 20 min, the reaction mixture was hy- tems, ABI 392Ϫ08) using the regular phosphoramidite protocol.[19]
drolyzed by addition of TBK buffer (1 , pH ϭ 7.5, 14 mL). The The phosphoramidite 5 as well as those of the regular DNA con-
aqueous phase was extracted three times with CH2Cl2 (9 mL each) stituents were employed. The oligonucleotides were recovered from
and the combined organic extracts were dried (Na2SO4). After
evaporation of the solvent, the residue was submitted to FC (col-
the synthesizer as the 5Ј-[bis(4-methoxyphenyl)(phenyl)methyl]ated
derivatives. They were isolated, deprotected, and purified on oli-
umn: 15 ϫ 1.8 cm, solvents: B (100 mL); C (100 mL); then D). The gonucleotide cartridges.[40] Their purity was checked by reverse-
main zone was concentrated, the residue dissolved in CH2Cl2 (20
mL) and extracted three times with TBK buffer (0.1 ). After dry-
ing (Na2SO4) and concentration, compound 15 (160 mg, 61%) was
phase HPLC. The nucleoside composition of the oligomers was
determined from reverse-phase HPLC profiles, obtained after hy-
drolysis with snake-venom phosphodiesterase followed by alkaline
obtained as a yellowish foam; TLC (silica gel, C): Rf ϭ 0.45. Ϫ phosphatase. MALDI-TOF mass spectra have been measured for
UV (MeOH): λmax (lg ε) ϭ 237, 255, 275 nm (4.35, 4.22, 4.10). Ϫ several cases (Table 8 ). The oligomers were lyophilized in a Speed-
1H NMR ([D6 ]DMSO): δ ϭ 7.44 (s, 1 H, 6-H), 7.35Ϫ7.21 (m, 10.5 Vac evaporator to yield a colorless foam which was dissolved in
H, 7-H, 1/2 PH, aromatic H), 6.84 (m, 4 H, aromatic H), 6.23 (t, H2O (100 µL) and stored frozen at Ϫ18°C.
Table 8. Molecular weights determined by MALDI-TOF mass spectra of oligonucleotides
Yield
[A260 units]
Nucleoside
composition
Mϩ
(calcd.)
Mϩ
(found)
d(T5ZT6)
22
21
20
18
18
21
16
24
23
17
18
dT:dZ
11.3:1.0
1.1:1.0
3612
1913
1923
3685
3685
4017
1913
3644
3644
3644
3644
3612
1910
1915
3687
3686
4020
1913
3643
3651
3645
3641
d(G3Z3)
dG:dZ
d(Z3G3)
d(T4 Z4T4)
dG:dZ
dT:dZ
1.0:1.1
2.1:1.0
8.1:3.1:1.0
8.2:3.9:1.0
d(T4 GZGGT4)
d(T4 ZG4T4)
dT:dG:dZ
dT:dG:dZ
d(Z)6
Ϫ
[a]
d(TAGGTCAATACT)
d(AGTATTGACCTA)
d(TAZZTCAATACT)
d(AZTATTZACCTA)
[a]
[a]
[a]
[a] Not determined.
Eur. J. Org. Chem. 1999, 485Ϫ496
495

F. Seela, A. Melenewski
FULL PAPER
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Acknowledgments
We thank Drs. Natalya Ramzaeva and Helmut Rosemeyer for help-
ful discussion, Mrs. Elisabeth Feiling and Mrs. Cathrin Mittelbach
for the synthesis of oligonucleotides. Financial support by the Bun-
desministerium für Bildung, Wissenschaft, Forschung und Techno-
logie and the Fonds der Chemischen Industrie is gratefully
acknowledged.
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Received July 13, 1998
[O98316]
496
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