Synthesis and properties of oligo-2′-deoxyribonucleotides containing internucleotidic phosphoramidate linkages modified with pendant groups ending with either two amino or two hydroxyl functions

Article abstract of DOI:10.1016/S0968-0896(03)00273-6

Single and multiple incorporations of stereochemically pure modified dinucleoside-phosphoramidates involving substituent groups ending with bis-hydroxyethyl and bis-aminoethyl groups have been performed into pyrimidic triple helix-forming oligo-2′-deoxyribonucleotides designed to bind parallel to the purine strand of the DNA target. The ability of these modified oligo-2′-deoxyribonucleotides to form triple helices has been studied by UV-melting curve analyses, and circular dichroism. Only the oligonucleotides involving modified phosphate groups with the Rp configuration formed more stable triple helices than did the parent phosphodiester sequences. Incorporating the modifications into the third oligonucleotide strands has little effect on the structure of the triplexes. At pH 7, the incorporation of two, three or four modified phosphate groups into the third strands stabilizes the triplexes, as compared to the unmodified oligonucleotide. Stronger stabilization was observed with compounds containing linkers ending with amino functions. Stability increases with the number of modifications without being fully additive. This might be due to the different environments of the phosphate groups inside the sequence.

Full text of DOI:10.1016/S0968-0896(03)00273-6

Bioorganic & Medicinal Chemistry 11 (2003) 3499–3511
Synthesis and Properties of Oligo-20-deoxyribonucleotides
Containing Internucleotidic Phosphoramidate Linkages Modified
with Pendant Groups Ending with either Two Amino
or Two Hydroxyl Functions
Ulysse Asseline,* Marcel Chassignol, Jolanta Draus,
Maurice Durand and Jean-Claude Maurizot
´ ´
Centre de Biophysique Moleculaire, CNRS UPR 4301, affiliated with the University of Orleans and with INSERM,
´
Rue Charles Sadron, 45071 Orleans Cedex 02, France
Received 18 October 2002; accepted 16 April 2003
Abstract—Single and multiple incorporations of stereochemically pure modified dinucleoside-phosphoramidates involving sub-
stituent groups ending with bis-hydroxyethyl and bis-aminoethyl groups have been performed into pyrimidic triple helix-forming
oligo-20-deoxyribonucleotides designed to bind parallel to the purine strand of the DNA target. The ability of these modified oligo-
20-deoxyribonucleotides to form triple helices has been studied by UV-melting curve analyses, and circular dichroism. Only the
oligonucleotides involving modified phosphate groups with the Rp configuration formed more stable triple helices than did the
parent phosphodiester sequences. Incorporating the modifications into the third oligonucleotide strands has little effect on the
structure of the triplexes. At pH 7, the incorporation of two, three or four modified phosphate groups into the third strands stabi-
lizes the triplexes, as compared to the unmodified oligonucleotide. Stronger stabilization was observed with compounds containing
linkers ending with amino functions. Stability increases with the number of modifications without being fully additive. This might
be due to the different environments of the phosphate groups inside the sequence.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
C.GxG and T.AxA base triplets. A pyrimidine third
strand binds parallel to the purine strand of the double-
+
.
.
Numerous applications have been found for oligo-
nucleotides, because of their hybridization properties, in
various areas such as biotechnology and diagnostics.
Their potential use as therapeutics in regulating gene
expression following the antisense, antigene, ribozyme
and aptamers strategies has stimulated a great deal of
research work.1ꢀ6 The antigene strategy relies on the
formation of triple-helical nucleic acid structures invol-
ving synthetic oligonucleotides that recognize short
stranded target to form T AxT and C GxC iso-
morphous triplets via Hoosgsteen hydrogen bonding
(where C+ indicates protonated cytosine and the sym-
.
bols and x stand for Watson–Crick and Hoogsteen
hydrogen bonds, respectively). The binding stability of
the C/T-containing pyrimidine third strand is pH sensi-
tive because its cytosine bases can only form two
Hoogsteen bonds when protonated, rendering the tar-
.
geting of C G stretches difficult due to the charge-charge
repulsion between contiguous protonated cytosines. A
third purine strand binds in an anti-parallel orientation
.
oligopyrimidine oligopurine runs in double-stranded
DNA via Hoogsteen or reverse Hoogsteen hydrogen
bond formation with purine bases in the major groove.
The triple helix formation with short third strand
oligonucleotides was first reported for pyrimidine oligo-
nucleotides,7,8 then for oligonucleotides containing G
and T,9 G and A,10,11 and T, C and G.12 The recog-
nition of oligopurine runs by the above reported oligo-
nucleotides involves the formation of T.AxT, C.GxC+,
.
.
forming C GxG and T AxA base triplets via reverse
Hoogsteen hydrogen bonding. G- and T-containing
oligonucleotides can also form triplexes whose third
strand orientation is sequence dependent.
The stability of triple-stranded RNA and DNA could
be increased when the electrostatic repulsion among the
polyanionic single strands was reduced by partial or
complete replacement of the negatively charged phos-
phodiester by either neutral or positively charged
groups. Along these lines, several families of modified
*Corresponding author. Tel.: +33-2-3825-5597; fax: +33-2-3863-
1517; e-mail: asseline@cnrs-orleans.fr
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00273-6

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U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
oligonucleotides have been developed that can be sepa-
rated into the following two broad structural classes:
those that retain the nucleoside and phosphorus backbone
of DNA and incorporate a pendant group at the phos-
phorus atom either neutral, as in the case of methylphos-
phonate, phosphotriester, or involving amino function(s)
that can be protonated at physiological pH via phos-
phonates or phosphoramidates linkages,13ꢀ27 and those
that replace the phosphate internucleotide linkage with
other chemical units such as formacetal, MMI, guani-
dinium or methylthiourea.28ꢀ36 More important modifi-
cations of the oligonucleotide backbone involving the
replacement of sugar and phosphate units have been
reported such as in the PNA37 and the pyrrolidine-amide
oligonucleotide mimic.38 Among these different families
of modified oligonucleotides, we have focused our atten-
tion on those that incorporate a pendant group at the
phosphorus atom. The replacement of one non-bridging
oxygen atom by any substituent induces the formation of
a chiral center at the phosphorus atom which leads to a
mixture of diastereoisomers. It has been shown that the
stability of complexes formed between modified oligonu-
cleotides and their complementary target sequences
depends on the stereochemistry of the P-chiral inter-
nucleoside linkages.15,16,22,24 In addition, steric hindrance
of the substituent at a phosphorus atom also has a nega-
tive effect on the stability of the complexes.
of positively charged groups, their location along the
sequence, the structure and length of the linkers used to
connect the positively charged pendant groups to the
ODN as well as the stereochemistry when the substitu-
tion is performed at the phosphorus atom level. We
chose to introduce at internucleotidic positions via
phosphoramidate linkages, pendant groups involving a
tertiary amino group and two other groups, either two
amino or two hydroxyl functions (Fig. 1). In addition,
we chose to change the length of the linker between the
phosphoramidate linkage and the tertiary amino func-
tion by using either ethyl or propyl groups. The syn-
thesis of the linkers and the modified oligonucleotides
was performed as depicted in Schemes 1 and 2, respec-
tively. As this modification introduces chirality at the
phosphorus atom, stereochimically pure Rp and Sp
dinucleoside-phosphoramidates with either of the above
described linkers were prepared and incorporated into
the oligonucleotides for evaluation of their effect on
binding stability. Identification of the stereochemistry of
the modified internucleotidic linkage confirmed that
only the oligonucleotides with the Rp configuration at
the modified phosphorus form slightly more stable tri-
plexes than do the unmodified oligonucleotides, used as
reference. Results also indicated that slightly more
stable triplexes were obtained with a bis-hydroxylated
linker involving a propyl chain between the phosphorus
atom and the tertiary amino function, as well as with a
bis-aminated linker involving the ethyl group. Multiple
incorporations (2, 3 and 4) of dinucleoside-phosphor-
amidates involving the selected linkers with the Rp
configuration, have been performed into 17-mer oligo-
nucleotides. As a target for triple helix formation, we
We report here single and multiple incorporations of a
series of modified dinucleoside-phosphoramidates
involving substituent groups, ending with either two
hydroxyl or two amino functions, into 22-mer and
17-mer pyrimidic TFOs, respectively. These modified
TFOs are designed to bind in a parallel orientation rela-
tive to the purine strand of the DNA target. For triple
helix formation we have chosen, as a model, a 24-bp long
.
chose a 22-bp oligopyrimidine oligopurine sequence
which is depicted in Figure 2 as part of a synthetic
24-bp long duplex bridged by two hexaethylene glycol
linkers.39,40 Triplex-forming oligonucleotides (TFOs)
specific to this oligopurine run are aligned below it.
.
target involving a 22-bp oligopyrimidine oligopurine
sequence, present on the exon 11 of BRCA1 gene. This
sequence involves a run of five thymines but is devoid of
consecutive cytosines in order to avoid the problem of
charge repulsion between positively charged cytosines.
Both strands of the target sequence were joined at their
extremities with hexaethylene glycol linkers in order to
enhance the thermal stability of the double-stranded DNA
target. The ability of these modified oligonucleotides to
form triple helices has been studied using UV-melting
curve analysis and circular dicroism spectroscopy.
Results and Discussion
Experimental design
A survey of the literature shows that among the various
methods used to increase the affinity of oligonucleotides
for their double-stranded target is the possibility of
introducing an amino function, that can be protonated,
at physiological pH, so as to reduce the electrostatic
repulsion between the third strand oligonucleotide and the
DNA target. Another approach consists in introducing
groups that are able to form additional hydrogen bond-
ing with the target. Binding properties of the reported
modified ODNs depend on many parameters: the number
Figure 1. Structures of the modified internucleotidic linkages.

U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
3501
Scheme 1. Synthesis of substituent groups 19, 20, 30 and 31 used for the internucleotidic modifications.
as described in Scheme 2. First, dinucleoside-30-H-
phosphonates 32a and 32b were separately reacted with
an excess of hydroxylated linkers 19 (n=2) and 20
(n=3) in the presence of CCl4 and pyridine to give two
pairs of dinucleosides, 33a, 33b and 34a, 34b, involving
a pendant group ending with hydroxyl functions. These
hydroxyl functions were then protected by benzoylation
to give two pairs of fully protected dinucleosides 35a,
35b and 36a, 36b, respectively. The latter were desily-
lated by treatment with tetrabutylammonium fluoride to
afford two pairs of dinucleosides 37a, 37b and 38a, 38b.
Preparation of the linkers
The bis-hydroxylated linkers 19 and 20 were obtained as
described in Scheme 1 by reaction of N-(2-bromo-
ethyl)phthalimide 14 or N-(3-bromopropyl)phthalimide
15 with diethanolamine (9 equiv) 16 followed by treat-
ment with hydrazine hydrate. The bis-aminated linkers
30 and 31 were obtained following a four-step pro-
cedure as described in Scheme 1. First, both amino
functions of diethylenetriamine 21 were trifluoro-
acetylated by reaction with ethyltrifluoro-acetate 22 to
give compound 23.41 The latter was then reacted in
acetonitrile with one equivalent of either bromoethanol
24 or bromopropanol 25 in the presence of excess tri-
ethylamine (1.5 equiv) at 50 ꢁC for 24 h. The obtained
hydroxylated linkers 26 and 27 were transformed sepa-
rately into azido derivatives 28 and 29 by reaction with
triphenylphosphine (1.5 equiv) and lithium azide (3
equiv), in the presence of carbon tetrabromide (1.5 equiv).
Aminated linkers 30 and 31 were obtained by hydrogena-
tion in the presence of 10% Pd on activated charcoal.
Preparation of modified dinucleosides involving protected
bis-aminated linkers 43a, 43b, 44a and 44b. Starting
from each pure diastereoisomer 32a and 32b of the fully
protected dinucleoside H-phosphonate 32 (see above),
these compounds were obtained following a two-step
procedure as described in Scheme 2. First, dinucleoside-
30-H-phosphonates 32a and 32b were separately reacted
with an excess of aminated linkers 30 (n=2) and 31
(n=3) in the presence of CCl4 and pyridine to give two
pairs 41a, 41b and 42a, 42b of the dinucleosides invol-
ving a pendant group ending with two protected amino
functions. The latter were desilylated by treatment with
tetrabutylammonium fluoride to give two pairs 43a, 43b
and 44a, 44b.
Synthesis of modified dinucleosides 37a, 37b, 38a, 38b,
43a, 43b, 44a and 44b and preparation of their phosphor-
amidite derivatives 39a, 39b, 40a, 40b, 45a, 45b, 46a and
46b
Preparation of modified dinucleosides involving protected
bis-hydroxylated linkers 33a–38b. Starting from each
pure diastereoisomer 32a and 32b of the fully protected
dinucleoside-30-H-phosphonate 32, obtained following a
procedure adapted from the literature,42 these com-
pounds were synthesized following a three-step procedure
Preparation of phosphoramidite derivatives 39a–40b and
45a–46b of modified dinucleosides 37a–38b and 43a–44b.
Starting from dinucleosides 37a–38b and 43a–44b, the
phosphoramidite derivatives were obtained following a
procedure adapted from the literature.43 In the case of
compounds 45a–46b, the purification step on silica gel

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U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
column was omitted in order to prevent partial loss of the
trifluoroacetyl groups. These modified phosphoramidite
derivatives were purified by precipitation in cold hexane.
1-mmol scale. For the coupling of modified dinucleoside-
30-phosphoramidites 39a, 39b, 40a, 40b, 45a, 45b, 46a or
46b, described above, the concentration of amidites was
1.5 times that of the unmodified nucleoside-30- phos-
phoramidites and the coupling and detritylation (after
incorporation of the modified dimer) times were
increased by 20%. Purification was performed by ion
exchange chromatography. After the desalting step, the
purity of the modified oligonucleotides was verified by
both ion-exchange and reversed-phase chromatography
analyses. The results of ion exchange analyses per-
formed at pH 7 clearly show that the modified oligo-
nucleotides with pendant groups ending with hydroxyl
Synthesis of oligonucleotides bearing one 2a, 2b, 3a, 3b,
4a, 4b, 5a, 5b, or several 7–12 appended linker(s) ending
with either two hydroxyl or two amino functions at the
internucleotidic position
Oligo-20-deoxyribonucleotides containing either a single
2a, 2b, 3a, 3b, 4a, 4b, 5a, 5b or multiple incorporations
7–12 of the modified dinucleosides were assembled via
phosphoramidite chemistry on a CPG support at a
Scheme 2. Synthesis of modified dinucleoside-30-phosphoramidites 39a–40b and 45a–46b. R=tert-butyldimethylsilyl, Bz=benzoyl.

U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
3503
Figure 2. Chemical structures of triple-helical complexes. The sequences of the circularized 24-bp long double-stranded target containing the 22-bp
.
long oligopyrimidine oligopurine sequence and the third strands are depicted.
functions are more retained that those with pendant
groups ending with amino groups (see Experimental for
retention times). These results are consistent with the
possibility of protonation of the amino functions at pH
7. Oligonucleotides 2a, 2b, 3a, 3b, 4a, 4b, 5a and 5b were
characterized by nuclease degradation analysis which
confirmed the presence and the number of the modified
dinucleosides. Electrospray Mass Spectroscopy Analysis
of oligonucleotides 2a to 5b and 7–12 confirmed their
mass (Table 1). The purity of the modified oligonucleo-
tides was also confirmed by polyacrylamide gel electro-
phoresis analyses in denaturing conditions (Figs 3 and 4).
Dinucleoside isomers 32a and 32b were purified and
separated by silica gel chromatography. A part of each
pure dinucleoside-H-phosphonate isomer 32a and 32b
was separately transformed (with retention of configur-
ation at the phosphorus center) into pure dinucleoside-
phosphorothioate by treatment with S8 in a pyridine/
CS2 mixture.42 After deprotection and purification by
reversed-phase chromatography, aliquots of each pure
dinucleoside-phosphorothioate isomer was treated
separately with P1 endonuclease and snake venom
phosphodiesterase (SVP) and then with alkaline phos-
phatase (AP). Treatment with P1+AP led to the full
degradation of the Sp isomer into dT while the Rp iso-
mer remained unchanged. On the contrary, treatment
with SVP+AP led to full degradation of the Rp isomer
into dT while the Sp isomer remained unchanged.45 Our
results showed that only the dinucleoside-phosphoro-
thioate from dinucleoside-H-phosphonate 32a was
cleaved by P1+AP treatment, while only the dinucleo-
Determination of the configuration of isomers 2a, 2b, 3a,
3b, 4a, 4b, 5a and 5b of modified 22-mer oligonucleotides
This was achieved as previously reported.44 The fully
protected dinucleoside-H-phosphonate 32 was obtained
following a procedure adapted from the literature.42

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U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
side-phosphorothioate
from
dinucleoside-H-phos-
oligonucleotide 6. In the presence of 5 mM MgCl2 and
phonate 32b was cleaved by SVP+AP treatment. Since
sulfurization of H-phosphonate is a stereoselective pro-
cess, according to the Cahn-Ingold-Prelog rules, the
dinucleoside-H-phosphonate 32a possesses the Rp con-
figuration and the dinucleoside-H-phosphonate 32b the
Sp configuration. Since the conversion of dinucleoside-
H-phosphonates 32a and 32b to dinucleosides 33a–34b
and 41a–42b occurs with inversion of configuration,46
we were able to conclude that oligonucleotides 2a, 3a,
4a and 5a exhibit the Sp configuration and oligo-
nucleotides 2b, 3b, 4b, and 5b the Rp configuration.
140 mM KCl in 10 mM sodium cacodylate buffer, pH 7,
two transitions were observed for all the oligonucleo-
tides studied reflecting the dissociation of the TFO at
low temperature, then the melting of the double-stran-
ded target at a higher temperature. At pH 7, while
oligonucleotides 2a, 4a and 5a formed triple helices with
slightly lower stability than did the unmodified 22-mer 6
used as reference, the oligonucleotide 2b, 3b, 4b formed
triple helices with slightly higher stability (Table 2).
Oligonucleotide 3a formed triple helix with stability
equivalent to that obtained with the parent phos-
phodiester sequence 6, while the modifications incorpo-
rated into the oligonucleotides 5a and 5b have a slight
destabilizing effect on the triplex stability. These results
allowed us to select the modified compounds which
induce the strongest stabilization in each series in order
to proceed to multiple incorporations. In both series,
the modified oligonucleotides with the Rp configuration
at the phosphorus atom were the best candidates. In the
case of linkers ending with hydroxyl groups, compound
3b (with n=3) was retained while in the case of linkers
ending with amino functions modified oligonucleotide
4b with the shortest linker (n=2) was more efficient in
stabilizing the triplex structure. We then studied oligo-
nucleotides 7, 8, 9, 10, 11 and 12, involving multiple
internucleotidic linkages, at pH 7 and 5.5 in the pres-
ence of 5 mM MgCl2 and 140 mM KCl in 10 mM
sodium cacodylate buffer. Two types of behavior were
observed depending on the pH (Table 2). At pH 7, in any
case the modified oligonucleotides formed triple helices
with higher stability than did the unmodified oligo-
nucleotide 13, used as reference (Fig. 5 shows the melting
profile for the triplex containing oligonucleotide 12 at pH
Binding analysis: Tm measurements by UV absorption
In a first step oligonucleotides bearing a single incor-
poration of modified internucleotide linkages, 2a, 2b,
3a, 3b, 4a, 4b, 5a, 5b, were compared with unmodified
Figure 3. Denaturing polyacrylamide (20%) gel electrophoresis of
modified and unmodified 22-mer oligo-20-deoxyribonucleotides
stained with methylene blue: lane 1: TFO 2a, lane 2: TFO 2b, lane 3:
TFO 3a, lane 4: TFO 3b, lane 5: TFO 6, lane 6: TFO 4a, lane 7: TFO
4b, lane 8: TFO 5a, lane 9: TFO 5b.
Table 1. Mass analyses of modified oligonucleotides 2a–5b and 7–12
Oligonucleotides
22-mers
Mass Mass
calculated observed
Oligonucleotides
17-mers
Mass Mass
calculated observed
2a
2b
3a
3b
4a
4b
5a
5b
6670.4
—
6684.4
—
6668.5
—
6682.5
—
6669.2
—
6683.8
—
6668.2
—
6682.6
—
7
8
9
10
11
12
5337.75
5481.96
5626.18
5305.75
5433.97
5562.21
5337.29
5481.37
5625.36
5306.95
5434.92
5562.72
Table 2. Melting temperatures for triplexes
Third
strand
T am (ꢁC)
pH=7
Third
strand
T am (ꢁC)
pH=7
T bm f(ꢁC)
pH=5.5
2a
2b
3a
3b
4a
4b
5a
5b
33.7
38.1
36.9
38.1
35.4
38.6
35.4
36.1
37
7
8
9
10
11
12
27.5
28.3
29.8
2
2
30
51.5
54.2
54
6
9
53
56
58
52.7
13 (ref)
25
6 (ref)
Triplex stability: Tm values of the triplexes formed by the various TFOs (2a–6)
and (7–13) and the 24-bp long double-stranded target 1 (see Fig. 2 for sequen-
ces). The Tm measurements were conducted in 10 mM sodium cacodylate, 140
mM KCl and 5 mM MgCl2 as described in Materials and methods. The pH of
the buffer was either 7 (Tam) or 5.5 (Tmb ). The oligonucleotide concentrations were
either 1 mmol (Tam) or 1.2 mmol (Tmb ). Tms are given in ꢁCꢂ0.5 ꢁC.
Figure 4. Denaturing polyacrylamide (20%) gel electrophoresis of
modified and unmodified 17-mers oligo-20-deoxyribonucleotides stained
with methylene blue: lane 1: TFO 7, lane 2 : TFO8, lane 3: TFO 9, lane 4:
TFO 13, lane 5: TFO 10, lane 6: TFO 11, lane 7: TFO 12.

U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
3505
Figure 5. UV-melting profile for the triplex containing oligonucleotide 12 at pH 5.5. The ordinate shows the first derivative of the melting curve dA/
dT, while the insert shows the melting curve.
5.5). We observe that in both series, either with oligo-
nucleotides 7, 8, 9 (with pendant groups ending with
hydroxyl functions) or with oligonucleotides 10, 11, 12
(with pendant groups ending with amino groups) the
stability of the triplexes increased with the number of
modifications whereas the Tm increase observed was not
additive (probably due to the position of the modifi-
cations inside the sequence). The results are in accor-
dance with the fact that the net global negative charge
number of modified oligonucleotides decreased with the
number of modified phosphates thus reducing the
electrostatic repulsion between the third oligonucleotide
strand and the double-stranded target. At pH 5.5, the
results obtained were different. (We have verified the
stability of the P–N linkage at pH 5.5 by incubating the
oligonucleotide 12 in the buffer used for the hybridiza-
tion studies. After 66 h incubation, the reversed-phase
analysis indicated no degradation of the oligonucleo-
tide.) In any case, the Tm values were largely higher than
those observed at pH 7. However, the stabilization
observed when using reference 13 confirmed that this is
in part due to the protonation of the four isolated
cytosines present in the sequence which remains the
major contribution to the triplex stabilization. As
observed at pH 7, the presence of the modified inter-
nucleotide linkages increased the stability of the tri-
plexes except in the case of the oligonucleotide 2a. In
addition more stable triplexes were obtained with
oligonucleotides involving amino functions on the pen-
dant groups. These results are consistent with the pos-
sibility of a more extended protonation of these
functions. The possibility of protonation at pH 7 of the
terminal primary functions of the pendant groups in the
ODNs 10, 11 and 12 is corroborated by the ion-
exchange analysis data (see Experimental). However,
when comparing the Tm values in both series it is easy to
see that the increase in stability due to the presence of
the aminated pendant groups is weak. Examples of
incorporation of large pendant groups at phosphorus
have been previously reported.21ꢀ27 Among them
N,N-(dimethylaminopropyl)-phosphoramidate groups
provided good triplex stabilization.24 Different hypo-
theses can be raised to explain our results: (a) a problem
of steric hindrance; (b) hydration changes in the major
groove; (c) a loss of flexibility of the third strand and
difficulty for the latter to accommodate in the major
groove of the duplex; and (d) although circular dichro-
ısm studies have confirmed that the presence of the
pendant groups in modified ODNs induced very little
structural changes (see below), it is possible that the
amino functions are interacting with the phosphates
groups inducing slight deformations that are not
favourable to the triplex stabilization.
Circular dichroism studies
Circular dichroism spectroscopy was used to compare
the conformations of modified and unmodified trip-
lexes. Chemical modifications do not introduce any new
chromophores in the oligonucleotide. Therefore any cd
change is expected to reflect a conformational change.
Figure 6 shows cd spectra of the triplex containing the
unmodified third strand 13 together with those of the
triplexes containing oligonucleotides 9 and 12 at pH 5.5

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U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
Figure 6. Cd spectra of the triplexes containing the modified oligonucleotide 9 (~), 12 (&), and unmodified oligonucleotide 13 (&) at pH 5.5 and 20ꢁC.
and 20 ꢁC. It clearly shows that the conformations of
the three triplexes are quite similar and that the presence
of the substituent groups on several phosphates does
Experimental
General methods
not modify the helix geometry. Similar results were
obtained with oligonucleotides bearing two or three
modifications.
All solvents used were dried, distilled and stored as
described in ref 49. All chemicals were used as obtained
unless otherwise stated. 50-O-(4,40-Dimethoxytrityl)-b-
thymidine-30-H-phosphonate was purchased from Dis-
tribio. Benzoyl chloride, diethanolamine, N-(2-bromo-
ethyl)phthalimide, N-(3-bromopropyl)-phthalimide, tert-
butyldimethylsilyl chloride, hydrazine hydrate, diethylene-
Conclusion
We have described a single incorporation into a 22-mer
pyrimidine TFO of a modified dinucleoside-phosphor-
amidate substituted with (N,N-bis-aminoethyl)-1,3-
aminopropane-diamine, (N,N-bis-aminoethyl)-1,2-amino-
ethane-diamine, (bis-hydroxyethyl)-1,3-aminopropane-
diamine, and (bis-hydroxyethyl)-1,2-aminoethane-diamine
groups. Stereochimically pure Rp and Sp dinucleoside
isomers were obtained and incorporated into 22-mer
pyrimidine TFO. Tm measurements indicated that
modified oligonucleotides involving (N,N-bis-aminoethyl)-
1,2-aminoethanediamine and (bis-hydroxyethyl)-1,3-
aminopropanediamine with the Rp configuration at the
phosphorus atom are the best suited compounds to
increase the triplex stability. Multiple (2, 3 and 4)
incorporations of modified dinucleoside-phosphor-
amidate involving (N,N-bis-aminoethyl)-1,2-amino-
ethane-diamine or (bis-hydroxyethyl)-1,3-aminopropane-
diamine substituent groups with the Rp configuration
were performed into a 17-mer pyrimidine TFO. The
binding results obtained with these 17-mer pyrimidine
oligonucleotides involving a few stereochimically pure
phosphate groups substituted with an (N,N-bis-amino-
ethyl)-1,2-aminoethane-diamine group and a double-
stranded DNA target in the presence of 140 mM KCl,
at pH 7 indicated that a slight increase in stability
occurred and that the stabilization increased with the
number of modified phosphate groups. However, this
stabilizing effect is weak. Nevertheless, these results
confirm literature reports20,24,47,48 in that triplex forma-
tion is a P-stereodependant process when the third
strand involves P-chiral internucleotidic modifications.
triamine,
ethyltrifluoroacetate,
2-bromoethanol,
3-bromopropanol, acetic acid, acetic anhydride,
triphenylphosphine, CBr4, pivaloyl chloride and LiN3
were purchased from Aldrich. Hydrogen was from Air
liquide. Triethylamine and sodium sulfate were pur-
chased from Merck, 2-chloro-5,6-benzo-1,3,2-dioxa-
phosphorin-4-one
from
Fluka,
pyridine
and
dichloromethane from SDS, and acetonitrile from
Labo-Standa. Analytical thin-layer chromatography
(TLC) was performed on precoated alumina plates
(Merck silica gel 60F 254 (ref 5554). For flash chroma-
tography, Merck silica gel 60 (70–230 mesh) (ref 7734)
or Lichroprep Si 60, 15–25 mM (ref 109336) were used.
All 4,40-dimethoxytrityl-containing substances were
identified as orange-colored spots on TLC plates by
spraying with a 10% perchloric acid solution. Amino-
containing substances were identified as purple-colored
spots on TLC plates by spraying with a 0.1% ninhydrin
solution in n-butanol. Oligonucleotide syntheses were
performed on an Expedite Nucleic Acid Synthesis sys-
tem 8909 from Perseptive Biosystems. Analyses and
purifications by ion-exchange chromatography were
carried out on a Pharmacia FPLC with a DEAE col-
umn (8 mM, 100 mmꢃ10 mm, Waters) with a linear
gradient of NaCl in 25 mM Tris/HCl buffer, pH 7 (or
pH 5.5), containing 10% CH3CN. Reversed-phase
chromatography analysis was performed on a 600 E
(System Controller) equipped with a photodiode array
detector (Waters 990) using a Lichrospher 100 RP (5
mM) column (125 mmꢃ4 mm) from Merck with a linear
gradient of CH3CN in 0.1 M aqueous triethylammo-

U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
3507
nium acetate, pH 7, with a flow rate of 1 mL/min. Mass
analysis ion-molecular weights of the oligonucleotides
was performed by Electrospray Mass Spectroscopy
added. The heating was continued for 24 h, then the sol-
vent was removed by evaporation and the residue was
dissolved with CHCl3. A small amount of insoluble mate-
rial was removed by filtration and the filtrate concentrated
to dryness. The residue was purified by silica gel chroma-
tography using a dichloromethane/methanol mixture as
eluent (97:3, v/v to 94:6, v/v). Compounds 26 and 27
1
using a Quattro II (Micromas) instrument. H NMR
spectra were recorded on a Bruker AM 300WB or on a
Varian Unity 500 Spectrometer and 31P NMR spectra on
a Varian Unity 500 Spectrometer. Absorption spectra
were recorded with a Uvikon 860 spectrophotometer.
The concentrations were determined using the following
molar absorption coefficients e260=173,400 Mꢀ1 cmꢀ1
were obtained with 50% yield. Rf =0.38, Rf =0.35
using a dichloromethane/methanol mixture as27 eluent
26
(90:10, v/v).
for modified and unmodified 22-mer,50 e260
=
134,700 Mꢀ1 cmꢀ1 for modified and unmodified
17-mer,50 260=507,600 Mꢀ1 cmꢀ1 for circular double-
Selected data: compound 26. 1H NMR (CDCl3), d ppm:
2.7 (m, 6H, –(CH2)3 N), 3.0 (br s, 1H, OH), 3.4 (q,
J=5.5 Hz, 4H, CH2NHCO), 3.65 (t, J=5.2Hz, H2 ,
CH2OH), 7.82(br s, 2H, CONH). Mass analysis. I.S.
polarity positive. Compound 26 calculated for C10 H15
F6 N3O3. M+H=339. Found 339.1. Compound 27
Calculated for C11 H17 F6 N3O3. M+H=354. Found
354.7.
e
stranded DNA target. Nucleases were purchased from
Roche Biochemicals.
Preparation of the circular double-stranded target 1.
Synthesis was performed according to published proce-
dures using hexaethylene glycol linkers to tether both
strands.39,40,51
Compounds 26 and 27 (5.9 mmol each) were dried in a
dessicator overnight as well as triphenylphosphine and
lithium azide, separately. The next day, compound 26 or
27 (5.9 mmol), triphenylphosphine (8.8 mmol) and
lithium azide (17.7 mmol, 3 equiv) were placed with
anhydrous DMF in a round-bottomed flask. Carbon
tetrabromide (8 mmol, 1.5 equiv) solubilized in DMF (5
mL) were added dropwise to the flask. The reaction was
exothermic. After 2h reaction at room temperature,
MeOH (2mL) was added and the reaction mixture was
evaporated to dryness. The residue was purified on a
silica gel column using a dichloromethane/ethylacetate
mixture as eluent (90:10, v/v to 80:20, v/v). Compounds
28 and 29 were obtained as oil. Yield 70%. Rf =0.60,
Preparation of the bis-hydroxylated linkers 19 and 20.
N-(2-Bromoethyl)phthalimide 14 or N-(3-bromopropyl)-
phthalimide 15 (39, 4 mmol) and diethanolamine 16
(0.35 mol, 9 equiv) were heated at 50–60 ꢁC for 2.5 h.
Silica gel TLC analysis using a dichloromethane/
methanol mixture as eluent (85:15, v/v) showed com-
plete disappearence of the starting material (Rf =0.9,
Rf =0.9) with formation of new spots correspon1d4ing to
co1m5 pounds 17 and 18 (Rf =0.44,and Rf =0.46). The
reaction mixture was purified on a silica gel column
using a dichloromethane/methanol mixture as eluent
(98:2, v/v to 92:8, v/v). Yield 50%. Compounds 17 and
18 were reacted with hydrazine hydrate (2.2 equiv) in
refluxing methanol for 2.5 h. During the process a white
precipitate appeared. The reaction mixture was con-
centrated to dryness and the residue was purified on a
silica gel column using methanol/concentrated ammonia
mixture as eluent (97:3, v/v to 85:15, v/v). Compounds
19 and 20 were obtained as a pale yellow-colored oil
with 80% yield. Selected data. Compound 19. 1H NMR
(CDCl3), d ppm: 3.71 (s, 3H, OH+NH2), 3.54 (t, J=9.78
Hz, 4H), 2.75 (t, J=9.78 Hz), 2.54 (m, 6H). Mass analy-
sis. I.S. polarity positive. Compound 19. Calculated for
C6 H16 N2O2: M+H=149, 21. Found 148.9.
17
18
and Rf =0.57 using a dichloromethane/acetone2m8 ixture
29
1
as eluent (80:20, v/v). Selected data. Compound 28. H
NMR (CDCl3), d ppm: 2.7 (m, 6H, (CH2)3 N), 3.4 (m,
6H, 2CH 2NHCO, CH2N3), 7.2(br s, H2 , CONH).
Mass analysis. I.S. polarity positive. Compound 28:
Calculated for C10H14F6N6O2. M+H=365.25. Found
365.1. Compound 29: Calculated for C11H16F6N6O2.
M+H=379.25. Found 379.
Compounds 28 and 29 (4.12mmol) were separately
dried by co-evaporation with anhydrous acetonitrile
(4ꢃ15 mL) and then solubilized in MeOH (30 mL).
Then under argon, 10% palladium on activated
charcoal (0. 15 g) was added and hydrogenation was
performed for 18–24 h under magnetic stirring. The
catalyst was removed by filtration and the filtrate was
concentrated to dryness to give compounds 30 and 31
as pale yellow-colored oils. Yields 70%. Positive test
with ninhydrin. Products were used in the next steps
without purification. Mass analysis. I.S. polarity posi-
tive. Compound 30. Calculated for C10H16F6N4O2.
M+H= 339.25. Found 339.1. Compound 31.
Calculated for C11H18F6N4O2. M+H=353. Found
353.1.
Preparation of bis-protected aminated linkers 30 and 31.
Diethylene triamine 21 (50 mmol) and ethyltri-
fluoroacetate 22 (170 mmol, 3.5 equiv) were refluxed in
acetonitrile (100 mL) containing water (1.1 mL, 1.2
equiv) for 18 h. The reaction mixture was concentrated
to dryness and the residue was then taken in CH2Cl2
(100 mL). Compound 23 appeared as a white precipitate
and was recovered by filtration and dried over P2O5.
Yield 95%. Rf =0.70 using a dichloromethane/metha-
1
23
nol mixture as eluent (80:20, v/v). H NMR (CDCl3), d
ppm: 2, 58 [m, 4H, (CH2)2N], 3, 38 (m, 4H, 2ꢃ
CH2NHCO).
Compound 23 (9.8 mmol) was placed in CH3CN (15
mL) and excess triethylamine was added (14.7 mmol,
1.5 equiv). The mixture was heated to 50 ꢁC and bromo-
ethanol 24 (9.8 mmol, 1 equiv) or bromopropanol 25 was
Preparation of the modified dinucleosides 32a and 32b.
The synthesis was achieved as reported in ref 42. The
purification and separation of the dinucleosides 32a and
32b were performed in a single step on a silica gel col-

3508
U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
umn (60H Merck) using ethylacetate/acetic acid mixture
as eluent (99.9:0.1, v/v). The isomer 32a was eluted first.
Thin-layer chromatography analysis confirmed the full
mixture as eluent (90:10, v/v) showing the formation of
new products with lower Rf values (Rf =0.40,
Rf =0.44, Rf =0.39, and Rf38b=0.42). When the
37a
37b
38a
separation of the isomers. Rf =0.5, Rf =0,34 using a
reaction was completed, the reaction mixture was
hydrolyzed with water (0.4 mL), extracted with di-
chloromethane. The organic phase was washed with
water, dried over Na2SO4, evaporated to dryness and
the residue was purified on a silica gel column using a
dichloromethane/methanol mixture (97:3, v/v to 95:5,
v/v) containing 0.1% triethylamine as eluent. Yield
80%. Mass analysis. I.S. polarity positive. Compounds
37a. and 37b. Calculated for C61H67N6O17P.
M+H=1187. Found: 37a 1187.4 and 37b 1187.4.
32b
dichloromethane/methanol m3i2xature as eluent (90:10, v/v).
The purity of each isomer was confirmed by 31P NMR
analysis in CDCl3 (external reference H3PO4). One sin-
gle peak was obtained for each isomer d32a=7.25 ppm
and d32b=8.54 ppm.
Preparation of modified dinucleosides involving protected
bis-hydroxylated linkers 33a–38b. Each pure isomer 32a
and 32b of the dinucleoside-H-phosphonate 50-O-(4,40-
dimethoxytrityl)-thymidylyl-30-O-tert-butyl-dimethylsilyl-
thymidine-30-H-phosphonate (1g each, 1.05 mmol),
previously dried by co-evaporation with anhydrous
pyridine (5 mL, three times), was dissolved in a
CH3CN/pyridine mixture (50:50 v/v) (8 mL) and treated
separately with bis-hydroxylated linkers 19 (n=2) or 20
(n=3) (7.0 mmol) solubilized by an anhydrous pyridine/
CCl4 mixture (50:50, v/v) (5 mL) for 10 min over argon
atmosphere. Silica gel TLC analysis using a dichloro-
methane/methanol mixture as eluent (90:10, v/v)
showed complete reaction with formation of a new spot.
The reaction mixture was concentrated to dryness and
solubilized with CH2Cl2 (30 mL). The organic phase
was washed with NaHCO3 (5 mL) and dried over
Na2SO4. Silica gel TLC analysis using a dichloro-
methane/acetone-/triethylamine mixture as eluent
(50:50:10, v/v/v) (two elutions) showed complete reac-
tion with the formation of a new spot (Rf =0.46,
Rf =0.55, Rf =0.36, and Rf =0.43). The33aorganic
Compounds
38a.
and
38b.
Calculated
for
C62H69N6O17P. M+H=1201. Found: 38a 1201.6 and
38b 1201.4.
Preparation of phosphoramidite derivatives 39a–40b of
the modified dinucleosides 37a–38. Compounds 37a–38b
(0.32mmol) were dried by co-evaporation with anhy-
drous pyridine (5 mL) then with anhydrous CH3CN (5
mL, three times) and left in a dessicator under vacuum
overnight. The next day, the dessicator was filled with
argon before its opening. The residue was solubilized
with 1,2-dichloroethane (8 mL) and diisopropylethyl-
amine (0.2mL, 0.09 g, 3 equiv, 0.7 mmol) was added
and then 2-cyanoethyl diisopropylchlorophosphor-
amidite (0. 094 mL, 0.1 g, 1.3 equiv, 0.42mmol) was
added dropwise under stirring at room temperature. After
11.5 h, silica gel TLC analysis using a ethylacetate/acetone/
triethylamine mixture as eluent (75:25:5, v/v/v) showed
nearly complete reaction with the formation of two new
spots corresponding to both isomers of the phosphor-
amidite derivative. The reaction mixture was diluted with
cold ethyl acetate (30 mL) previously washed with a cold
aqueous 10% sodium carbonate solution. The organic
phase was washed with a cold 10% aqueous sodium car-
bonate solution (10 mL) and with a cold saturated aqu-
eous sodium chloride solution (10 mL), dried over Na2SO4
and concentrated to dryness. The residue was purified on a
silica gel column using ethylacetate/acetone/triethylamine
mixture as eluent (75:25:5, v/v/v). After precipitation from
cold (ꢀ78 ꢁC) hexane, compounds 39a–40b were obtained
as a white powder. Yields 70–75%.
33b
34a
34b
phase was concentrated to dryness and the residue was
purified on a silica gel column using a dichloromethane/
methanol mixture (97:3, v/v to 85:15, v/v) containing
0.1% triethylamine as eluent. Yields 55%.
Dinucleosides 33a, 33b, 34a, and 34b (0.55 mmol) were
co-evaporated with anhydrous pyridine (20 mL, three
times) and during the last evaporation, 10 mL of pyri-
dine were kept in the flask. The solution was cooled to
0 ꢁC and benzoyl chloride (1.25 mmol) was added
dropwise under stirring and argon atmosphere. After 30
min, the reaction was allowed to react at room tem-
perature for 3 h. The reaction was monitored by TLC
analysis using a CH2Cl2/MeOH mixture as eluent
(90:10, v/v) showing the formation of new products with
higher Rf values (Rf =0.44, Rf =0.46, Rf =0.53,
Preparation of the modified dinucleosides involving the
protected bis-aminated linkers 41a–44b. The fully pro-
tected dinucleotides 41a–42b were obtained as reported
for the preparation of dinucleotides 33a–34b by repla-
cing the bis-hydroxylated linkers 19 (n=2) or 20 (n=3)
with the bis-aminated linkers 30 (n=2) or 31 (n=3).
Silica gel TLC analysis using a dichloromethane/
methanol mixture as eluent (90:10, v/v) showed com-
plete reaction with a new spot (Rf =0.40, Rf =0.44,
35a
35b
and Rf =0.56).[(Rf =0.17, Rf =0.20, Rf36a=0.13,
36b
33a
33b
34a
and Rf =0.15]. When the reaction was completed, the
reaction34bmixture was hydrolyzed with NaHCO3 (4 mL),
extracted with dichloromethane. The organic phase was
dried over Na2SO4, evaporated to dryness and the
residue was purified on a silica gel column using a
dichloromethane/methanol mixture (98:2, v/v to 93:7,
v/v) containing 0.1% triethylamine as eluent. Yield
75%.
41a
41b
Rf =0.41, Rf =0.45). Yield=70% after purification
42a
on a silica gel c4o2blumn using a dichloromethane/methanol
mixture as eluent (98:2, v/v to 95:5, v/v). 1H NMR
(CDCl3), d ppm: 7.76–6.78 (m, 21H Ar), 5.11 (m, 1H,
H-10), 4.93 (br s, 1H, NH), 4.36 (d, 2H, J=6.77 Hz,
O-CH2), 4.0–3.98 (m, 1H, OH), 3.77–3.66 (m, 6H, H-30,
H-40, H-50, H-500+CH2O), 3.75 (s, 6H, OCH3), 3.12–
3.07 (m, 2H, CH2N), 2.17–2.02 (m, 2H, H-20, H-200),
1.61–1.56 (m, 2H, CH2).
The fully protected dinucleosides 35a, 35b, 36a, and 36b
(0.40 mmol) were treated under stirring with a 1.1 M
tetrabutylammonium fluoride solution in tetra-
hydrofuranne (5 equiv) for 1.5 h. The reaction was
monitored by TLC analysis using a CH2Cl2/MeOH

U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
3509
Starting from dinucleotides 41a–42b the desilylated
dinucleotides 43a–44b were obtained as reported for the
preparation of dinucleotides 37a–38b. Purification was
performed on a silica gel column using a dichloro-
methane/methanol (98:2, v/v to 97:3, v/v) mixture con-
taining 0.3% triethylamine as eluent. Yield 70–75%.
[Rf =0.30, Rf =0.32, Rf =0.27, Rf =0.30 using a
at room temperature. Acetic acid and tritanol were then
removed following standart conditions. The purity of all
the oligo-20-deoxyribonucleotides described was verified
by reversed-phase analysis using a Lichrocart system
(125ꢃ4 mm) packed with Lichrospher RP 18 (5 mm)
from Merck eluted with a linear gradient of acetonitrile
from 0 to 27.5% over 30 min, in a 0.1 M aqueous trie-
thylammonium acetate buffer, pH 7, with a flow rate of
1 mL/min. Ion exchange analysis was also performed on
a Mono Q column (HR 5/5, Pharmacia) using a linear
gradient of NaCl (0.0–0.6 M over 60 min) in a 25 mM
Tris/HCl buffer containing 10% CH3CN, pH 7. As
expected unmodified ODN 13 was eluted with the high-
est retention time 38 min 18 s. and ODNs containing the
pendant groups with amino functions were less retained
43b
44a
dic4h3aloromethane/methanol mixture as el4u4bent (90:10, v/v)].
Mass analysis. I.S. polarity positive. Compounds 43a
and 43b. Calculated for C51H59F6N8O15P. M+H=
1169. Found: 43a: 1169.5 and 43b: 1169.3. Compounds
44a. and 44b. Calculated for C52H61F6N8O15P.
M+H=1183. Found: 44a: 1183.3 and 44b: 1183.5.
Preparation of phosphoramidite derivatives 45a–46b of
the modified dinucleosides 43a–44b. Starting from di-
nucleosides 43a–44b and proceeding as reported for the
preparation of phosphoramidite derivatives 39a–40b,
except for the purification on silica gel column which
was omitted in order to prevent partial loss of the tri-
fluoroacetyl groups, the phosphoramidite derivatives
45a–46b were obtained with 75% yield. Foam. CCM:
Rf=0.65 using a mixture of ethyl acetate, acetone and
triethylamine as eluent (75:25:5, v/v/v).
than those containing the hydroxyls functions (Rt =29
min 36 s; Rt =26 min 18 s; Rt =25 min 24 s; Rt =19
7
10
11
min 6 s; Rt =18 min 18 s; R8t =9 min 30 s). These
9
12
results are in accordance with the possible protonation
of the primary amino functions at the ends of pendant
groups in oligonucleotides 10, 11 and 12. Analysis by
20% polyacrylamide gel electrophoresis using denaturing
conditions (7 M urea) indicated (Figs 3 and 4) the pre-
sence of only one band for each modified oligonucleo-
tide. In the both series, either with oligonucleotides 7, 8,
9 (with pendant groups ending with hydroxyl functions)
or with the oligonucleotides 10, 11, 12 (with pendant
groups ending with amino groups) mobilities were dif-
ferent according to the number of modifications (Fig.
4). Modified oligonucleotides with the greatest number
of modifications were more retained in the gel and tak-
ing into consideration the same number of modifi-
cations, oligonucleotides involving the pendant groups
with terminal amino functions were more retained than
the corresponding hydroxylated oligonucleotides. The
overall yield after the synthesis and purification steps is
close to 40% for oligonucleotides with a single phos-
phate modification and 30–35% for oligonucleotides
involving multiple phosphate modifications.
Preparation of oligonucleotides 2a–5b and 7–12. Oligo-
nucleotides were synthesized on a 1-mmol scale on an
Expedite Nucleic Acid Synthesizer using standard
b-cyanoethyl phosphoramidite chemistry.52 Reagents
were prepared as described in the user manual and a
standard concentration of a phosphoramidite solution
was used for all the umodified nucleotides. Concentra-
tions of the modified dinucleoside-phosphoramidites
involving varied substituent groups on the inter-
nucleotidic phosphate were increased up to 1.5 times
that of the umodified monomers. The coupling time for
the modified monomers and the detritylation time after
their incorporation were increased by 50 and 20%,
respectively. Coupling efficiency was estimated from
trityl assays and were >95% per step for the modified
dinucleosides. After the chain assembly (trityl-on
mode), oligomers 4a–5b and 10–12 attached to the sup-
port were treated with tert-butylamine in acetonitrile, to
selectively remove the b-cyanoethyl groups from the
phosphates so as to prevent the formation of acrylo-
nitrile adducts on the primary amino functions of the
pendant groups. They were then removed from the solid
support and deprotected at base level by concentrated
aqueous ammonia treatment (28% aq NH3) (4 mL) at
room temperature overnight. Oligonucleotides 2a–3b
and 7–9 were only treated by 28% aq NH3 (4 mL) at
room temperature overnight. The ammonia solution
was then removed by evaporation. Purification of the
tritylated oligonucleotides was performed by ion
exchange chromatography on a DEAE column (8 mM,
100 mmꢃ10 mm, Waters) using a linear gradient of
NaCl (0.075–0.525 M over 30 min) in a 25 mM Tris/HCl
buffer containing 10% CH3CN, pH 7. The desalting
step was performed on a HR 10/10 column (Pharmacia)
packed with Lichroprep PR 18 (Art 13900 from Merck).
After the desalting step, oligonucleotides were detrity-
lated by aqueous acid acetic treatment (80%) for 20 min
Full deprotection and nucleoside composition of the
modified oligonucleotides 2a–5b were ascertained by
nuclease degradation. An aliquot of the oligonucleotide
was digested with snake venom phosphodiesterase
(Pharmacia Biotech) and alkaline phosphatase (Boeh-
ringer) in a 0.1 M Tris–HCl buffer, pH 8.2, at 37 ꢁC.
After inactivation of the enzyme at 90 ꢁC for 2min, the
digestion products were analyzed by reversed-phase
chromatography using a Lichrocart system (125ꢃ4 mm)
packed with Nucleosil 100-5 C18 from Macheray-Nagel
equilibrated with a 0.1 M aqueous triethylammonium
acetate buffer, pH 7. The column was eluted at a flow
rate of 1 mL/min with 0.1 M aqueous triethylammo-
nium acetate buffer, pH 7, for 10 min and then with a lin-
ear gradient of 0–24% of acetonitrile in a 0.1 M aqueous
triethylammonium acetate buffer, pH 7, for 30 min and
with a linear gradient of 24–56% of acetonitrile in a
0.1 M aqueous triethylammonium acetate buffer, pH 7,
for 20 min. Detection was performed at 260 nm. Oligo-
nucleotides were totally degraded. Three peaks were
obtained. Comparison with natural nucleosides and
modified dinucleosides (obtained after full deprotection
of sample dinucleosides 37a–38b and 43a–44b) allowed

3510
U. Asseline et al. / Bioorg. Med. Chem. 11 (2003) 3499–3511
us to identify the different peaks. Mass analysis results
confirmed the calculated mass values for each oligonu-
cleotide (Table 1).
N. T.; Helene, C. Proc. Natl. Acad. Sci. U.S.A. 1992, 89,
8631.
13. Miller, P. S.; Yano, J.; Yano, E.; Carrol, C.; Jayaraman,
K.; Ts’o, P. O. P. Biochemistry 1979, 18, 5134.
14. Asseline, U.; Thuong, N. T.; Helene, C. J. Biol. Chem.
1985, 260, 8936.
Tm measurements by UV absorption experiments
15. Lancelot, G.; Guesnet, J. L.; Asseline, U.; Thuong, N. T.
Biochemistry 1988, 27, 1265.
16. Durand, M.; Maurizot, J. C.; Asseline, U.; Barbier, C.;
Thuong, N. T.; Helene, C. Nucleic Acids Res. 1989, 17,
1823.
17. Hau, J. F.; Asseline, U.; Thuong, N. T. Tetrahedron Lett.
1991, 32, 2497.
18. Froehler, B. C.; Ng, P.; Matteucci, M. D. Nucleic Acids
Res. 1988, 16, 4831.
19. Ozaki, H.; Kitamura, M.; Yamana, K.; Shimidzu, T. Bull.
Chem. Soc. J. 1990, 63, 1929.
20. Fathi, R.; Huang, Q.; Coppola, G.; Delaney, W.; Teasdale,
R.; Krieg, A. M.; Cook, A. F. Nucl. Acids Res. 1994, 22, 5416.
21. Letsinger, R. L.; Singman, C. N.; Histand, G.; Salunkhe,
M. J. Am. Chem. Soc. 1988, 110, 4470.
22. Jung, P. M.; Histand, G.; Letsinger, R. L. Nucleosides and
Nucleotides 1994, 13, 1597.
23. Horn, T.; Chatuverdi, S.; Balasubramanian, T. N.; Let-
singer, R. L. Tetrahedron Lett. 1996, 37, 743.
24. Chatuverdi, S.; Horn, T.; Letsinger, R. L. Nucleic Acids
Res. 1996, 24, 2318.
Changes in absorbance with temperature of 1 mM tri-
plexes in a 10 mM sodium cacodylate buffer (pH 5.5, or
pH 7) containing 5 mM MgCl2 and 140 mM KCl were
measured at l=260 nm in a Uvikon 941 cell changer
spectrophotometer equipped with a Huber PD 415
temperature programer connected to a cryothermostat
ministat circulating water bath (Huber). A cacodylate
buffer was chosen due to its limited dependence of pH
on temperature. The temperature was regulated at a rate
of 0.2 ꢁC/min from 0 to 80 ꢁC. To monitor triplex-to-
duplex transition, the samples were first heated and then
cooled. Oligonucleotide concentrations were 1 mM of
the circular duplex, and either 1 or 1.2 mM of the third
strand (see sequences in Fig. 2). The absorbance at 260
and 340 nm was recorded every 5 min. Corrections for
spectrophotometric instability were made by substracting
the absorbance at 340 nm from that at 260 nm. The Tm
values were determined using the first derivative. Esti-
mated accuracy of the melting temperature is ꢂ0.5 ꢁC.
25. Laurent, A.; Naval, M.; Debart, F.; Vasseur, J. J.; Ray-
ner, B. Nucleic Acids Res. 1999, 27, 4151.
26. Ehrenmann, F.; Vasseur, J.-J.; Debart, F. Nucleosides
Nucleotides Nucleic Acids 2001, 20, 797.
Circular dichroism experiments
Cd measurements were carried out on a Jobin Yvon
Mark IV dichrograph in optical cells with a pathlength
of 0.5 or 1 cm. Each cd spectrum was an average of two
scans with the buffer blank subtraction. The concen-
tration used to calculate the cd intensity was that of the
nucleotide unit.
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Acknowledgements
We thank H. Labbe (Centre de Biophysique Molecu-
laire, Orleans) for recording NMR spectra and C. Bure
(Centre de Biophysique Moleculaire, Orleans) for run-
ning the electrospray mass spectroscopy.
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