5-Propynylamino α-deoxyuridine promotes DNA duplex stabilization of anionic and neutral but not cationic α-oligonucleotides

Article abstract of DOI:10.1016/j.bmcl.2006.11.052

Incorporation of 5-propynylamino and 5-propynyl α-2′-deoxyuridine into α-oligonucleotides (α-ON) allows high-affinity targeting of complementary DNA for α-ON with anionic and neutral backbone but not for cationic α-ON, revealing clues on the role of the a

Full text of DOI:10.1016/j.bmcl.2006.11.052

Bioorganic & Medicinal Chemistry Letters 17 (2007) 951–954
5-Propynylamino a-deoxyuridine promotes DNA duplex
stabilization of anionic and neutral but not cationic
a-oligonucleotides
Gaelle Deglane, Franc¸ois Morvan, Franc¸oise Debart and Jean-Jacques Vasseur*
¨
LCOBS, UMR 5625 CNRS-UM II, Universite´ Montpellier II, 34095 Montpellier, France
Received 30 October 2006; revised 14 November 2006; accepted 14 November 2006
Available online 18 November 2006
This paper is dedicated to Professor Jean-Louis Imbach on the occasion of his 70th birthday.
Abstract—Incorporation of 5-propynylamino and 5-propynyl a-20-deoxyuridine into a-oligonucleotides (a-ON) allows high-affinity
targeting of complementary DNA for a-ON with anionic and neutral backbone but not for cationic a-ON, revealing clues on the
role of the amino group of the propynylamino on the formation of DNA duplexes.
Ó 2006 Elsevier Ltd. All rights reserved.
a-Oligonucleotides (a-ON) have a nonnatural nucleo-
side a-anomery and are a class of ON exhibiting unique
hybridization properties. Unlike b-ON which hybridize
to ss DNA or RNA with an antiparallel orientation,
a-ON do it in a parallel manner without loss of affinity
and specificity for their target.1 Moreover, although the
replacement of the phosphodiester backbone with any
chiral phosphorus modification in b-ON induces a
destabilization of their hybrids formed with nucleic
acids,2 the introduction of neutral3 and cationic4,5 inter-
nucleoside phosphoramidates into a-ON stabilizes them.
The main dramatic stabilizing effect was observed with
cationic linkages due to the electrostatic attraction be-
tween phosphates of natural targets and the cationic
linkages of the modified a-ON. This strong affinity
was particularly useful to employ cationic a-ON as steric
blocking antisense agents.4
of the 5-propynylamino uracil with the positively
charged 20-aminoethoxy sugar modification10 into the
same b-ON has a synergetic effect on the hybrid
stability.11,12
The aim of the present work was to determine the effect
of 5-propynylamino a-dU X and 5-propynyl a-dU Y on
the stability of duplexes formed between a-ON and a
complementary parallel DNA sequence. This effect was
studied with anionic phosphodiester (I, PO), neutral
methyl phosphoramidate (II, PNHMe) and methoxy-
propyl phosphoramidate (III, PNHPrOMe), and with
cationic dimethylaminopropyl phosphoramidate (IV,
PNHPrNMe2) a-ON (Fig. 1).
NH2
HO
HO
O
O
N
N
OH
OH
To further increase the affinity of a-ON, we considered
introducing the modified nucleobase 5-propynylamino
uracil. Indeed, the propynyl amino group attached to
C5 of the uracil is one of the most promising modifica-
tions introduced in phosphodiester b-ON to form stable
duplexes6,7 and triplexes.8,9 Moreover, the combination
O
N
O
O
O
N
H
H
O
O
O
O
P
P
P
P
NH
O
NH
NH
I, PO
IV, PNHPrNMe2
II, PNHMe
III, PNHPrOMe
H
+
O
N
Figure 1. 5-Propynylamino a-dU X, 5-propynyl a-dU Y, and phos-
phodiester I, methyl phosphoramidate II; methoxypropyl phospho-
Keywords: Oligonucleotides; a-Anomery; Phosphoramidates; Bas
modification; Hybridization.
*
ramidate
internucleoside linkages.
III;
dimethylaminopropyl
phosphoramidate
IV
Corresponding author. Tel.: +33 4 67 14 33 20; fax: +33 4 67 04 20
29; e-mail: vasseur@univ-montp2.fr
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.052

952
G. Deglane et al. / Bioorg. Med. Chem. Lett. 17 (2007) 951–954
Table 1. Target and oligonucleotides synthesized
O
N
I
a
Sequence 50-30
Anomery Internucleoside
backboneb
NH
TolO
ON
O
TolO
O
i
O
ii
I
iii
5-I dU
N
OTol
Target
I
AAG AGG AAG AAA
TTC TCC TTC TTT
TTC TCC XTC TTT
TTC XCC TTC XTT
XTC XCC XTC XTT
b
a
a
a
a
PO
PO
PO
PO
PO
OTol
O
N
O
1
2
H
O
I–X1
I–X2
I–X4
F3C
O
NH
HO
DMTrO
DMTrO
O
O
O
I
I
v
iv
N
N
N
OH
OH
OH
I–Y1
I–Y2
I–Y4
TTC TCC YTC TTT
TTC YCC TTC YTT
YTC YCC YTC YTT
a
a
a
PO
PO
PO
3
O
N
H
O
O
N
O
N
4
O
H
5
H
O
v', iv, vi
O
vi
II
TTC TCC TTC TTT
TTC TCC XTC TTT
TTC XCC TTC XTT
XTC XCC XTC XTT
a
a
a
a
PNHMe
PNHMe
PNHMe
PNHMe
F3C
NH
DMTrO
DMTrO
II–X1
II–X2
II–X4
O
N
N
O
O
O
P
H
O
P
H
O
Et3NH
O
Et3NH
O
N
O
N
H
H
III
TTC TCC TTC TTT
TTC TCC XTC TTT
TTC XCC TTC XTT
XTC XCC XTC XTT
a
a
a
a
PNHPrOMe
PNHPrOMe
PNHPrOMe
PNHPrOMe
O
O
7
6
III–X1
III–X2
III–X4
Scheme 1. Preparation of 6 and 7. Reagents and conditions: (i) p-
TolCl, py, rt, 85%; (ii) a—HMDS, toluene, reflux; b—TMSOTf,
CH3CN, rt, 45%; (iii) MeONa, MeOH, rt, 92%; (iv) DMTrCl, py, rt,
90%; (v) HC„CCH2NHCOCF3, CuI, Et3N, Pd(PPh3)4, DMF, rt,
82%; (v0) propyne, CuI, Et3N, Pd(PPh3)4, DMF, rt, (Ref. 12); (vi) a—
H3PO3, PivCl, py, rt; b—aq TEAB, 81%.
IV
IV–X1
IV–X2
TTC TCC TTC TTT
TTC TCC XTC TTT
TTC XCC TTC XTT
a
a
a
a
a
PNHPrNMe2
PNHPrNMe2
PNHPrNMe2
PNHPrNMe2
PNHPrNMe2
IV–X20 TTC TCC XXC TTT
IV–X4
XTC XCC XTC XTT
IV–Y1
IV–Y2
IV–Y4
TTC TCC YTC TTT
TTC YCC TTC YTT
YTC YCC YTC YTT
a
a
a
PNHPrNMe2
PNHPrNMe2
PNHPrNMe2
Synthesis
of
5-propynylamino-20-a-deoxyuridine
(Scheme 1) was started with p-toluoylation of the
hydroxyls of commercially available 5-iodo-20-deoxyur-
idine yielding 1. Epimerization of 1 into the a-anomer
2 was performed with hexamethyldisilane in toluene at
reflux followed by a treatment with TMSOTf13 yielding
a ꢀ4:1 a/b separable mixture. The a-anomer 2 was then
deprotected with sodium methanolate to afford 5-iodo-
20-a-deoxyuridine 3 which was then tritylated to give
4. The aminopropynyl moiety protected as trifluoroace-
tamide was next introduced into compound 5 under pal-
ladium coupling reaction.11 Sonogashira reaction of 3 to
obtain 5-propynyl a-dU Y was performed as previously
reported.14 Finally, 20-deoxy-a-ribonucleoside 30-O-H-
phosphonates 6 and 7 were prepared according to pub-
lished procedure.3
a Sequences of ON where X or Y are the position at which 5-propy-
nylamino a-dU X or 5-propynyl a-dU Y were incorporated.
b PO, phosphodiester; PNHMe, methyl phosphoramidate; PNH-
PrOMe, methoxypropyl phosphoramidate; PNHPrNMe2, dimeth-
ylaminopropyl phosphoramidate internucleoside linkages.
The effect of the 5-propynylamino a-dU on DNA du-
plex stability was evaluated by UV thermal denaturation
experiments (Table 2) with a complementary b-se-
quence. At 0.1 M NaCl, the introduction of the 5-propy-
nylamino uracil in anionic a-ON I–X induces a
significant stabilization of the hybrids compared to the
slight effect that 5-propynyl a-dU has (I–Y). The higher
the number of base modifications, the larger was the sta-
bilization (DTm from 2.5 to 6.7 °C for I–X1 to I–X4).
However, the average stabilization per base modifica-
tion slightly decreases as the number of modifications
increases (DTm/modif. from 2.5 to 1.7 °C for 1–4 mod-
if.). This behavior reported for b-ON duplex containing
5-propynylamino b-dU6,7 was explained by a destabili-
zation resulting from the repulsion between cationic
propynylamino groups. However, it is not probable here
that propargylamino groups separated by two or more
base pairs could interact together and the relatively
low pKa of the amine (pKa propargylamine6 ꢀ8.2),
which becomes harder to protonate as the number of
propynylamino groups is increased, could simply ex-
plain this drop in DTm/modif. The electrostatic contri-
bution to the stabilization resulting from the
protonation of the propynylamino is confirmed with
melting experiments performed at 1 M NaCl. Indeed,
the stabilizing effect observed at high salt concentration
decreases with increasing number of propynylamino
groups (DTm(1M NaCl–0.1M NaCl) = from 14.5 °C for I to
10.8 °C for I–X4). When the NaCl concentration
The elongation of a-ON dodecamers (Table 1) was per-
formed on a CPG solid-support using H-phosphonate
chemistry.3 The stepwise coupling yield for H-phospho-
nates 6 and 7, using pivaloyl chloride as the coupling
agent, was comparable to the other protected a-nucleo-
side 30-H-phosphonates (98%). At the end of the elonga-
tion, the oxidation of the oligonucleotide H-
phosphonates was performed with standard iodine–pyr-
idine–water to generate a-ON phosphodiesters, and with
anhydrous CCl4-pyridine-amine (methylamine, 3-meth-
oxypropylamine or 3-dimethylaminopropylamine) to
create phosphoramidate internucleoside linkages
(PNHMe, PNHPrOMe, and PNHPrNMe2, respectively)
using a reported protocol.3 After cleavage of the ON
from the solid-support and their deprotection with con-
centrated ammonia, their purification was performed by
RP-HPLC for the anionic a-PO and the neutral
a-PNHMe and a-PNHPrOMe, whereas the cationic
a-PNHPrNMe2 was purified by cationic exchange chro-
matography as already reported.4,5

G. Deglane et al. / Bioorg. Med. Chem. Lett. 17 (2007) 951–954
953
Table 2. Melting temperatures (Tm values) obtained during thermal denaturation studies at different concentrations of NaCla
a-ON
Backbone
0.1 M NaCl
1 M NaCl
Tm (°C)
DTm (°C)
DTm/modif.
Tm (°C)
DTm (°C)
DTm/modif.
I
PO
40.5
43.0
44.3
47.2
—
—
55.0
56.5
56.5
58.0
—
—
I–X1
I–X2
I–X4
2.5
3.8
6.7
2.5
1.9
1.7
1.5
1.5
3.0
1.5
0.8
0.8
I–Y1
I–Y2
I–Y4
PO
41.0
41.0
42.0
0.5
0.5
1.5
0.5
0.3
0.4
56.0
56.0
56.0
1.0
1.0
1.0
1.0
0.5
0.3
II
PNHMe
48.5
52.5
54.0
56.5
—
—
47.0
49.5
48.5
49.5
—
—
II–X1
II–X2
II–X4
4.0
5.5
8.0
4.0
2.8
2.0
2.5
1.5
2.5
2.5
0.8
0.6
III
PNHPrOMe
PNHPrNMe2
PNHPrNMe2
48.0
51.5
52.5
56.0
—
—
46.0
47.5
47.5
47.0
—
—
III–X1
III–X2
III–X4
3.5
4.5
8.0
3.5
2.3
2.0
1.5
1.5
1.0
1.5
0.8
0.3
IV
74.5
71.5
69.5
nd
—
—
53.5
50.0
49.0
nd
—
—
IV–X1
IV–X2
IV–X4
ꢁ3.0
ꢁ5.0
nd
ꢁ3.0
ꢁ2.5
nd
ꢁ3.5
ꢁ4.5
nd
ꢁ3.5
ꢁ2.3
nd
IV–Y1
IV–Y2
IV–Y4
72.0
71.0
69.0
ꢁ2.5
ꢁ3.5
ꢁ5.5
ꢁ2.5
ꢁ1.8
ꢁ1.4
53.0
52.0
50.5
ꢁ0.5
ꢁ1.5
ꢁ3.0
ꢁ0.5
ꢁ0.8
ꢁ0.8
a The melting temperatures (Tm) for dissociation of the duplexes were measured as the maximum of the first derivative of the melting curve (A260nm vs
temperature) recorded in 10 mM sodium cacodylate buffer, pH 7.0, using 2 lM concentrations of the two complementary strands, and a micro-
molar extinction coefficient of 3000 for monomer X. DTm, change in Tm value calculated relative to the nonbase-modified ON-duplex; DTm/modif.,
average change in Tm value per modified base.
augments, the sodium cation competes with the ammo-
nium of the propynylamino lowering the favorable elec-
trostatic effects resulting from the interaction with PO
linkages. In comparison, as expected the stability of
the duplexes formed with the propynyl a-ON I–Y1, 2,
4 is as prone as the unmodified a-ON I to the change
lower salt concentration (Tm 56 °C), while the Tm of
the a-ON III do not diverge drastically (Tm 46 and
48 °C in 1 M and 0.1 M NaCl, respectively), are clearly
in favor of the behavior of protonated species. In this
case, the higher the number of base modifications,
the higher the sensitivity to the ionic strength
(DTm(1M NaCl–0.1M NaCl) = ꢁ4 °C for III–X1 and ꢁ9 °C
for III–X4). A comparable behavior is observed for
a-ON II. The stabilizing effect of the propynylamino
in a-ON II and III probably results from electrostatic
interactions between the amino groups and the phos-
phates of the complementary strand.
in the ionic strength (DTm(1M
15 °C).
ꢀ14–
NaCl–0.1M NaCl)
When incorporated into neutral a-ON II and III, the 5-
propynylamino a-dU X has a stronger effect on the sta-
bility of the duplexes (Tm ꢀ 3.5–4 °C for 1 modif.) than
when integrated into anionic a-ON I (Tm ꢀ 2.5 °C). Like
for ON I, introduction of more than one X modification
has a lower but still significant impact on the Tm (DTm/
modif. 2 °C for 4 modif.). The stabilizing effect of the
propynylamino cannot be explained here by a favorable
electrostatic interaction with the neighboring phos-
phates of the same strand as established for natural b-
ON PO.6,7,12 Furthermore, the similar Tm observed for
a-ON II and III with phosphoramidate linkages bearing
short methyl or longer methoxypropyl chains suggest no
steric interaction between the propynylamino group and
the neutral backbones. It has been also reported for tri-
plex formation, that the main reason for the stabiliza-
tion induced by the propynylamino comes up
primarily from enhanced base stacking interactions
due to the amino electronegative group attached to the
propyne and not from electrostatic interactions.15 How-
ever, the lower Tm obtained at a high salt concentration
(Tm 47 °C for III–X4) compared to those obtained at a
As regards to a possible enhanced stabilizing effect on
DNA duplex hybridization, we investigated the oppor-
tunity of combining the potential stabilizing effects of
cationic PNHPrNMe2 linkages and the 5-propynylami-
no uracil base modification in an ON with improved
hybridization properties. Indeed, we recently showed
by molecular modeling simulations that amino groups
borne by the phosphoramidate internucleoside linkages
of a-ON interact efficiently with PO groups of the com-
plementary DNA strand bridging the minor groove of
duplex.16 Considering that the 5-propynylamino is
directed through the major groove2, the two modifica-
tions are not inclined to interact together. Despite these
considerations, the introduction of the base modifica-
tions into a cationic a-ON IV induces destabilization
of the resulting hybrids (DTm ꢁ3 °C for IV–X1). This
destabilization did not depend on the position of the
base modifications within the ON as two distributed

954
G. Deglane et al. / Bioorg. Med. Chem. Lett. 17 (2007) 951–954
modifications in IV–X2 had the same effect on Tm as two
contiguous modifications in IV–X20 (not showed). It was
not possible to determine the Tm value with the ON IV–
X4 bearing four propynylamino residues due to a low
cooperativity and low hyperchromicity. It is not sure
that this effect arises from the proximity of positively
charged groups.
destabilization with cationic a-ON arises from a conflict
in the double helix between rigidity induced by the base
modification and bending due to the phosphate
modification.
Supplementary data
As written before, the two amines are located in different
positions of the duplex. Moreover, due to the differences
of pKa between the two amino functions (pKa N-dimeth-
ylamino propane17 and propargylamine6 ꢀ10 and ꢀ8.2,
respectively), it is not probable that the amino of the
propynylamino is protonated in a-ON IV–X. The fact
that the neutral 5-propynyl induces a similar destabiliza-
tion in ON IV–Y reinforces this assumption. Moreover,
the neutrality of the propynylamino group in these cat-
ionic a-ON is demonstrated by the similar sensitivity of
the stability to the ionic strength of all duplexes what-
ever the number of propynylamino or propynyl modifi-
cations (DTm(1M NaCl–0.1M NaCl) ꢀ ꢁ21 °C for ON IV–X
and ꢀ ꢁ19 °C for ON IV–Y). A simple reason for the
destabilization induced by the two modified bases would
be that protonated and nonprotonated propynylamino
behave differently. Another reason could be that the
interactions between cationic phosphoramidates and
phosphates of the target narrow the minor groove con-
tracting and bending the double helix.16 In contrast, due
to enhanced base stacking, it has been reported for b-
ON that the propynyl and propynylamino groups rigid-
ify the double helix18 and lengthen it.19 If a-ON contain-
ing propynyl and propynylamino groups behave
similarly, this rigidity may be somehow unfavorable to
the flexibility required to accommodate the interactions
in the duplex between cationic PNHPrNMe2 linkages on
one strand and PO on the other strand. However, if in-
creased ionic strength from 0.1 M to 1 M NaCl, that
reduces the interaction between the two backbones,
was able to lessen the propynyl negative effect (DTm
ꢁ3.5 to ꢁ1.5 °C IV–Y2), this was not the case with the
destabilization caused by the propynylamino which re-
mained almost identical at the two salt concentrations.
This could be explained by the stronger rigidity due to
enhanced pp stacking induced by 5-propynylamino ura-
cil X compared to its propynyl analogue Y.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.11.052.
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We concluded that the 5-propynylamino a-dU modifica-
tion induces various effects depending on the nature of
the charge owned by the backbone of a-ON. In anionic
and neutral a-ON, it resulted in stabilization, whereas a
destabilization was observed with cationic a-ON. We
have shown the importance of the electrostatic interac-
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