M. Naval et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1435–1438
1437
Table 2. Influence of a single mismatch on the Tm of duplexes with phosphodiester b-oligonucleotide 6 and PNHME a-oligonucleotides 7 and 8
(ÁTm=Tm mismatchÀTm match)
ODNs
Tm ꢀC (ÁTm) DNA target, X=
Tm ꢀC (ÁTm) RNA target, X=
C
A
G
C
A
G
6
7
8
30.5 (À14.9)
38.0 (À17.0)
44.0 (À10.0)
32.3 (À13.1)
39.3 (À15.7)
43.0 (À11.0)
37.7 (À7.7)
41.5 (À13.5)
43.0 (À11.0)
33.3 (À11.3)
35.5 (À10.5)
43.0 (À5.3)
33.8 (À10.8)
35.0 (À11.0)
36.8 (À12.0)
36.8 (À7.8)
34.3 (À11.7)
35.3 (À13.0)
term of affinitywith the perfectlymatched duplexes
(X=T for DNA and U for RNA targets, respectively).
As reported, these modified oligonucleotides form stable
parallel-stranded duplexes. Introduction of one 2-
aminoadenine modification (a-ODN 8) had a destabi-
lizing effect (ÁTm À1 ꢀC) on the DNA hybrid (X=T),
whereas the replacement of three a-dA in a-ODN 7 by
three 2-amino a-dA in a-ODN 9 slightlystabilized the
The data obtained with a-ODN 8 targeted against DNA
showed that the introduction of a 2-amino group in
adenine reduced discrimination between mismatches
(average ÁTm/mismatch À10.8 ꢀC) when compared to
adenine in PNHME a-ODN 7 (average ÁTm/mismatch
À15.4 ꢀC) as well as in phosphodiester b-ODN 6 (aver-
age ÁTm/mismatch À11.9 ꢀC). With a-ODN 8/RNA
hybrids, the most stable mismatch was C NH2A and the
least stable was G NH2A. Thus the stabilityof base pairs
decreased as U NH2A> >C NH2A>A NH2A> >G
NH2A. The discrimination between U NH2A and A (or
G) NH2A base pairs was enhanced compared to the dis-
crimination of U A and A (or G) A in PNHME a-ODN
7 or PO b-ODN 6/RNA duplexes. On the contrary, we
observed a significant reduction of discrimination
against C pairing with NH2A when compared with A in
ODNs 6 and 7/RNA duplexes. Formation of an addi-
tional hydrogen bond between cytosine and 2-amino-
adenine could be responsible of this lower specificityof
NH2A compared to A. Further investigations including
NMR and molecular modeling studies will be needed to
evaluate the structural features of the 2-aminoadenine
containing PNHME a-ODNs duplexes which result in
the observed stabilization effects particularlywith RNA
target.
corresponding duplex (ÁTm/NH2
A
+0.8 ꢀC). With
RNA duplexes (X=U), a strong positive effect on
binding was found when one (ÁTm+2.3 ꢀC) and three
2-amino-a-dA (ÁTm +6 ꢀC) were incorporated into
PNHME a-oligonucleotides.
This different behavior of 2-aminoadenine towards
DNA and RNA duplex formation was also reported for
phosphodiester b-oligonucleotides2 and for N30!P50
phosphoramidate ODNs.4 Thus, PNHME a-ODN 9
containing 2-aminoadenine formed more stable
duplexes with DNA target (ÁTm +12.1 ꢀC) and RNA
target (ÁTm +7.4 ꢀC) than did the natural PO b-ODN 6.
The sequence specificityof binding of PO b-ODN 6 and
PNHME a-ODNs 7 (containing A) and 8 (containing
one 2-amino A) was determined bycomparison of sin-
glymismatched duplexes (DNA and RNA, X=A, G,
C) to the perfectlymatched duplexes (DNA, X=T;
RNA, X=U) (Table 2). The stabilities of base pairs
formed with the PO b-ODN 6 decreased as T (or U)
A> >G A>A A> >C A. According to the literature,14
the most stable mismatches were those containing gua-
nine and the least stable were those containing cytosine.
In conclusion, we have described a new efficient syn-
thesis of 2-amino a-dA using a phase transfer glycosy-
lation method. The exocyclic amino functions of this
nucleoside were suitablyprotected bydiisobutylforma-
midine groups to be converted in its 30-H-phosphonate
synthon which was successfully incorporated into
PNHME a-ODNs. The replacement of adenine by2-
aminoadenine in PNHME a-ODNs reallyimproved
their affinityto RNA while keeping a good specifityfor
nucleic acids. To explain these data, a full structural
elucidation is currentlyunder investigation. Such mod-
ified oligonucleotides are verypromising for antisense
and diagnostic purposes in terms of binding to nucleic
acid targets. Therefore, we are presentlyevaluating their
cellular uptake for further biological applications.
Concerning PNHME a-ODN 7/DNA duplexes, the
stabilityof base pairs followed same grading than
observed for the PO b-ODN 6. However, a higher dis-
crimination between T A and X A base pairs was
observed for the modified a-ODN 7 (average ÁTm/mis-
match À15.4 ꢀC) compared to the natural one 6 (aver-
age ÁTm/mismatch À11.9 ꢀC). The most noticeable
effect was the difference between discrimination of T
and G byA in a-ODN 7 (ÁTm À13.5 ꢀC) compared to
the same discrimination in b-ODN 6 (ÁTm À7.7 ꢀC).
Surprisingly, with the RNA target, there was only a
verylittle influence on the thermal stabilityregardless
which mismatch was opposite to a-dA in PNHME
a-ODN 7. Here again, a difference between a-ODNs 6
and 7 was noticeable with the G A mismatch, more
stable for the regular PO ODN and less stable for the
modified one.
Acknowledgements
This work was supported bygrants from the Associ-
ation pour la Recherche contre le Cancer (ARC,
France). N.M. thanks the ARC for the award of a
research studentship.