2'-O-Appended polyamines that increase triple-helix-forming oligonucleotide affinity are selected by dynamic combinatorial chemistry

Article abstract of DOI:10.1002/cbic.201000538

Stability leads to selection: Cationic appending groups on a triplex-forming oligonucleotide (TFO) designed to bind a DNA target can be rapidly selected from a small library of various amines and polyamines for their capacity to stabilize triple-helix formation.

Full text of DOI:10.1002/cbic.201000538

DOI: 10.1002/cbic.201000538
2’-O-Appended Polyamines that Increase Triple-Helix-Forming
Oligonucleotide Affinity are Selected by Dynamic Combinatorial Chemistry
Laurent Azꢀma,[a, b] Katell Bathany ,[b, c, d] and Bernard Rayner*[a, b]
The sequence-specific recognition of double-stranded DNA by
triplex-forming oligonucleotides (TFOs) has potential applica-
tion as therapeutics in the antigene strategy as well as tools in
molecular biology.[1] TFOs bind to the major groove of DNA
either in a parallel or antiparallel orientation according to their
base composition. TFOs composed of pyrimidine bases bind
parallel to the purine strand of oligopurine–oligopyrimidine
duplexes through T-AT and C+-GC triplet formation. However,
the use of TFOs under physiological pH and ionic strength is
hampered by their weak binding to DNA duplexes mainly due
to the requirement for cytosine protonation and charge repul-
sion between the three negatively charged strands. Spermine
and related polyamines are largely protonated at physiological
pH and are known to promote triplex stabilization both upon
external addition[2] as well as upon conjugation at the 5’-termi-
nus,[3] C5 of dU,[4] and C4 of 5-methyl-dC.[5] In contrast, attach-
ment of spermine on a 2’-position in a TFO was found to have
a deleterious effect on triplex stability.[6] In this case, replace-
ment of one internal dT unit by an ara U 2’-phosphorylpropyl-
spermine within an oligo dT was found to abolish its capacity
to form a triplex with an oligo-dA·oligo-dT duplex; this sug-
gests that tethering position, ara C2’, or/and the linker phos-
phorylpropyl was inappropriate. However, Cuenoud et al.
showed later that TFOs containing 2’-O-aminoethyl ribonucleo-
tides (2’-AE-TFOs) formed stabilized triplexes owing to dual
recognition of DNA targets by base–base contacts and con-
comitant salt-bridge formation between positively charged am-
monium groups on the TFO and DNA phosphates.[7a,b] This
dual recognition approach would be further improved if amino
groups present on 2’ positions of a 2’-AE-TFO were substituted
by polyamines, thus generating 2’-polycationic chains that are
able to strongly interact with several DNA phosphate groups
and bring additional stabilization to the triplex.
In this context, dynamic combinatorial chemistry (DCC) ap-
pears to be a method of choice for the identification of such
2’-O-appended polyamines.[8] DCC has attracted increasing in-
terest over recent years as an alternative approach to tradition-
al combinatorial chemistry that combines in a single step the
library build-up and screening processes. DCC involves the use
of reversible reactions between different building blocks to
generate an equilibrating mixture of compounds that is able
to respond through noncovalent interactions to the addition
of a target molecule. The preferential binding of one member
of the mixture to the target induces a shift in the equilibrium
towards that particular compound. Thus DCC offers in situ
screening of the combinatorial library simply by comparing its
composition in the absence and presence of the target. DCC
experiments have been performed by using various biological
targets including nucleic acids.[9] In previous studies, we have
established that DCC can be used to identify covalently ap-
pended small molecules that stabilize oligonucleotide com-
plexes.[9e,f] For that purpose, equilibrating imines formed from
2’-aminonucleotide incorporated into an oligonucleotide
ligand and a small set of aldehydes were submitted to the
template effect of a nucleic acid target. These studies were car-
ried out in the context of DNA and RNA duplexes as well as an
RNA–RNA kissing complex. In each case, after reduction of the
imines, a chemically stable conjugated ligand with an in-
creased affinity for its target was identified that corresponded
to the most amplified compound. Here, by using an “inverted”
imine reaction, we report an application of DCC for the screen-
ing of various amines and polyamines for their ability upon re-
action with a 2’-linked aldehyde group present in an internal ri-
bonucleoside unit of a TFO to stabilize the triplex formed with
a DNA target.
Eleven-base-long 2’-O-methyl TFO 1 bearing a central 2’-O-
(2-oxoethyl)uridine and able to form a parallel triplex with the
stem of DNA hairpin 2 was synthesized (Scheme 1A). TFO 1
was obtained by periodate-mediated oxidation of precursor 2’-
O-Me oligonucleotide containing a 2’-O-(2,3-dihydroxypropyl)-
uridine in position 6 and synthesized by the phosphoramidite
method.[10] The fully protected phosphoramidite of this latter
nucleoside was synthesized (Scheme 2) by using a slightly
modified procedure as described by Zatsepin et al.[11] Starting
from uridine, the less-expensive 3’,5’-di-tert-butyl disiloxane
protecting group was preferred to Markewicz’s reagent; pro-
tection of the 3-N position by pivaloyloxymethyl (Pom) group
was necessary for further selective 2’-O allylation. Palladium-
assisted allylation proceeded in good yield, and the vic-diol
was subsequently produced through oxidation by osmium
tetroxide/4-methylmorpholine N-oxide (NMO). The diol was
protected through acetylation, and the resulting uridine deriv-
ative was 3’-O phosphitylated.
[a] Dr. L. Azꢀma, Dr. B. Rayner
U869, INSERM
146 rue Lꢀo Saignat, 33076 Bordeaux (France)
Fax: (+33)5-57-57-45-65
E-mail: bernard.rayner@inserm.fr
[b] Dr. L. Azꢀma, Dr. K. Bathany , Dr. B. Rayner
Department of Technology for Health, University of Bordeaux
146 rue Lꢀo Saignat, 33076 Bordeaux (France)
[c] Dr. K. Bathany
European Institute of Chemistry and Biology
2 rue Robert Escarpit, 33607 Pessac (France)
[d] Dr. K. Bathany
Present address: Centre de Gꢀnomique Fonctionnelle
146 rue Lꢀo Saignat, 33076 Bordeaux (France)
Fax: (+33)5-57-57-16-84
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201000538.
ChemBioChem 2010, 11, 2513 – 2516
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template (the target) are rapid processes compared to their
reduction into stable secondary amines.[9e,12] Because formation
is a considerably slower process for triplex than for duplex[13]
and to avoid a possible kinetic bias, a triplex between TFO 1
and target 2 was allowed to form before amines A–G and
sodium cyanoborohydride were added.[14] Under these condi-
tions, interconvertion of imines is likely to occur on extensively
target-bound TFO in a similar way to that operating in the
tethering fragment-based drug-discovery method,[15] thereby
amplifying the template effect of DNA target 2. Attempts to
analyze the resulting mixture of conjugated TFOs 1A–G by RP-
or ion-paired RP-HPLC failed due to considerable peak overlap.
Therefore, we turned our attention to the use of MALDI-Tof
mass spectrometry under conditions similar to those reported
by Sarracino et al. for the quantitative detection of oligonucle-
otides.[16] Before analysis, samples were extensively desalted
and no internal standard was added because the relative quan-
tification of conjugates 1A–G was sufficient for our purpose.
Typical mass spectra obtained from conjugate mixture generat-
ed in the presence or absence of target 2 are presented in Fig-
ure 1A. Proportions of 1A–G were estimated from the corre-
sponding peak heights and their change induced by the pres-
ence of DNA target 2 are reported in Figure 1B. There was a
clear amplification of conjugates 1E (9%) and 1G (29%) de-
rived from (2-aminoethyl)guanidine and tris(2-aminoethyl)-
amine, respectively, which occurred at the expense of other
conjugates. Conjugates 1B–D were unchanged or moderately
repressed (1 to À8%) whereas conjugates 1A and 1F were
more strongly repressed (À33 and À29%, respectively).
Scheme 1. Structures of A) DNA hairpin target 2 and TFO 1 (italicized letters
indicate 2’-O-methyl-ribonucleotides); B) reacting amines (A–G) that were
used for generating the dynamic library.
Scheme 2. Synthesis of fully-protected 2’-O-(2,3-dihydroxypropyl)uridine
phosphoramidite. a) tBu2SiCl2, AgNO3, DMF; b) Pom-Cl, Bu4NHSO4, CH2Cl2/aq
Na2CO3; c) allylmethyl carbonate, tris(dibenzylidene acetone)Pd2, PPh3, THF;
d) NMO, OsO4, THF then Ac2O, N,N-dimethyl-4-aminopyridine, pyridine;
e) 3(HF)Et3N, THF; f) 4,4’-dimethoxytritylchloride (DMTr-Cl), pyridine;
g) (iPr)2NEt, chloro(2-cyanoethoxy)(diisopropylamino)phosphine, iPr2EtN,
CH2Cl2.
For the build up of the dynamic combinatorial library, we se-
lected seven amines A–G (Scheme 1B) differing in chain length
and number of positive charges present at physiological pH
and exhibiting various potential for hydrogen bonding and
electrostatic interactions. Previously reported DCC experiments
involving imine libraries were based on the assumption that
formation of interconverting imines and their interaction with
Figure 1. Expensions of the MALDI-Tof mass spectrum of A) untemplated
and templated dynamic combinatorial library after reduction by NaBH3CN
and B) relative amplifications (as estimated from relative MS peak heights)
of 2’-O-conjugated TFOs. Data shown represent the meanÆs.d. of three
DCC experiments
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ChemBioChem 2010, 11, 2513 – 2516

This discrimination suggests that electrostatic interactions
between positively charged 2’-O-appending chains at pH 6
and the polyanionic DNA target have mainly driven the selec-
tion. Moreover a terminal guanidinium group (in 1E) contrib-
utes more to this process than a terminal ammonium group
(in 1B–D). In addition, when comparing data corresponding to
conjugates 1B–D, which are derived from diamines, a chain-
length effect on selection is apparent that suggests a better fit-
ting of conjugates derived from 1,2-diaminoethane and 1,3-di-
aminopropane over the one derived from putrescine. Next, to
ascertain that the selection occurring during the DCC process
was related to the thermodynamic stabilities of the triplexes
formed with DNA target 2, conjugates 1A–G were separately
re-synthesized and UV-monitored melting experiments were
carried out.
upon conjugation with 1,2-diaminoethane was at variance
with the amplification ranking of 1B and might be tentatively
explained by the fact that although the selection operated on
unstable imines, binding properties were measured with the
corresponding reduced compounds 1A–G. This could also ac-
count for the discrepancy observed between a sharp difference
in amplification values for 1E and 1G (9 and 29%, respectively)
and their close Tm values.[8c] Finally, conjugation with methyl-
amine afforded the lowest stabilizing effect with a DTm value
consistent with previously reported data.[7a] In this latter case,
replacing the methyl group by a 4-methylbenzyl group as in
1F increased the stabilizing effect, suggesting that contribu-
tions to the stability of triplex other that electrostatic interac-
tions and H-bonding might occur with 2’-O-appending groups.
Binding properties of selected TFO 1G were further studied.
The stability at pH 5.5 of triplex 1G+2 containing a single tet-
ramine chain at micromolar concentration was higher than
that of triplex 1Me+2 in the presence of millimolar concen-
tration of externally added tetramine G (Tm 44.78C). In contrast,
the attachment of tetramine in 1G had no effect on the stabili-
ty of a duplex formed with complementary oligoribonucleotide
(data not shown); this illustrates the target specificity of the se-
lection that occurred during the DCC experiment. We also ex-
amined TFO 1G binding with a mismatched triplet by inverting
the AT base pair in the DNA target opposite to the tetramine-
linked U. This produced an unstable U-TA in place of U-AT. A
decrease in triplex Tm of 30.18C was observed that was higher
than that obtained with 1Me (24.98C; Table 1).
Of the two transitions observed during the melting of each
triplex, the one with the higher Tm value (around 728C), which
was the same for all complexes, corresponded to the melting
of the hairpin DNA target 2. The one with lower Tm value cor-
responded to the dissociation of the third strand (Figure 2). In
all cases, the presence of a 2’-O cationic chain in the third
strand of the triple helices had a stabilizing effect compared to
the starting triplex 1+2 which exhibited a Tm value (36.48C)
similar to that of the triplex obtained with the all-2’-O-methyl
version of 1, 1Me (U*=2’-O-methyl-U (Scheme 1) and 2 (Tm =
36.38C). The strongest effects were observed with TFOs 1E
(DTm =10.68C) and 1G (DTm =10.98C) in accordance with their
amplifications. The 2’-O chain present in TFO 1G resulted from
conjugation with tris(2-aminoethyl)amine (pKa values 8.56,
9.59, 10.29) and was the highest positively charged (+3 at
pHꢀ7) group submitted to the selection. The high DTm that
was observed reflected its capacity to interact with negatively
charged target phosphates. Although, only two positive charg-
es are expected on the 2’-O chain present in TFO 1E, the pres-
ence of a planar and highly basic guanidinium group (pKa =
12.5) capable of inducing directionality in H-bonding interac-
tions might account for efficient stabilization of triplexes.[17]
When considering the stabilizing effect of 2’-O linked diamines
in 1B–D, 1,3-diaminopropane was slightly more efficient than
putrescine as reflected by a higher amplification of 1C during
the selection. In contrast, the lower stabilizing effect observed
This is a clear demonstration that the increased thermal sta-
bility provided upon 2’-O conjugation of a TFO with tetramine
G does not compromise its selectivity. The binding of TFO 1G
was tested at pH 7. Upon these conditions, deprotonation of
cytosines is known to destabilize C-GC triplets. At neutral pH,
triplex 1G+2 was still observed with a Tm value of 20.78C,
whereas triplex 1+2 was no longer detected. The extra bind-
ing energy that resulted from interactions with a single 2’-O-
appended tetramine was sufficient to allow the formation of
the triple helix.
In conclusion, we have described the successful use of DCC
for the rapid identification of cationic 2’-O-appended groups
that increase TFO affinity. Remarkably, a good correlation of
the most amplified conjugates with the best binders was
found in spite of the fact that all conjugates subjected to se-
Table 1. Effects of pH and mismatch on triplex stability (Tm).[a]
TFO
pH 5.5
pH 7.0[b]
1Me[c]
1G
36.3 (11.4)[d]
47.2 (17.1)[d]
<5
20.7
[a] The experiments were performed as reported in Figure 2. [b] Buffer
pH 7.0: 10 mm cacodylate buffer (pH 7.0), 100 mm NaCl, 0.1 mm EDTA.
[c] 1Me (U*=2’-O-methyl-U, Scheme 1) corresponds to the full 2’-O-
methyl version of 1. [d] Values in brackets correspond to Tm of triplexes
formed with hairpin DNA target 2inv.
Figure 2. Triple-helix stabilizing effects obtained upon conjugation of TFO 1
with amines A–G. The Tm values (Æ0.58C) are averages of the values deter-
mined from three separate melting experiments by using a temperature in-
crease of 0.48C.minÀ1. Each strand concentration was 1 mm in 10 mm cacody-
late buffer (pH 5.5) with 100 mm NaCl and 0.1 mm EDTA).
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Experimental Section
DCC experiment: A mixture of amines A (250 mm), B (2 mm), C
(15 mm), D (52,5 mm), E (8 mm), F (70 mm), and G (4 mm) was ob-
tained from neutralized (pH 5–6, with diluted HCl) stock solutions
in H2O.[14] TFO 1 (0.23 mm) was incubated (1 min at 908C then 12 h
at 48C) in the absence or in presence of DNA target 2 (0.23 mm) in
cacodylate buffer (23 mm, pH 6.0, 85 mL), NaCl (23 mm), KCl
(165 mm), and EDTA (0.12 mm). A mixture of amines (10 mL) was
added and after 30 min at 258C a freshly prepared solution of
NaBH3CN (100 mm, 5 mL) in H2O was added. The resulting mixture
was stirred for 24 h at 258C. Aliquots (50 mL) from reaction mix-
tures were drop-dialyzed (twice) over V series membranes (Milli-
pore) against 0.1m aqueous ammonium citrate and then analyzed
on a MALDI-Tof mass spectrometer (Reflex III, Brucker) operated in
the reflectron mode with a 20 kV acceleration voltage and a 23 kV
reflector voltage. A mixture of oligonucleotides d(T12)-d(T18)
(Sigma) was used for external calibration. The matrix used was a
4:1 mixture of 2,4,6-trihydroxyacetophenone (10 mgmLÀ1) in EtOH
and 100 mm aq ammonium citrate. Samples (0.8 mL, aq. oligonucle-
otides (~50 mm)/matrix 1:1) were spotted on a stainless steel target
and air-dried before analysis. For each sample, a MALDI mass spec-
trum was acquired by accumulating the ion signals from 100 UV
laser shots with a constant laser power.
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Experimental details for synthesis of fully-protected 2’-O-(2,3-dihy-
droxypropyl)uridine 3’-O phosphoramidite, its incorporation into
oligonucleotides, synthesis and characterization of conjugated
TFOs, and UV-melting experiments are described in the Supporting
Information.
[14] Initial concentrations of amines were adjusted to compensate for their
differences in reactivity with TFO 1 and to roughly balance the distribu-
tion of interconverting imines thus providing significant proportion of
each conjugate. This allowed a more accurate measurement of the rela-
tive amplifications.
Acknowledgements
We thank Derrick R. Robinson for careful reading of the manu-
script. This work was supported by INSERM, France.
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Keywords: amination
recognition · oligonucleotides · self-assembly
·
combinatorial chemistry
·
DNA
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Received: September 10, 2010
Published online on November 19, 2010
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