Synthesis and characterization of oligonucleotide conjugates bearing electroactive labels

Article abstract of DOI:10.1016/j.bmcl.2012.12.057

Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3′ or 5′ end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3′ end and an electrochemical label at 5′ end.

Full text of DOI:10.1016/j.bmcl.2012.12.057

Bioorganic & Medicinal Chemistry Letters 23 (2013) 955–958
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and characterization of oligonucleotide conjugates bearing
electroactive labels
a,
b
Julie Moreau a, , Nabil Dendane b, , Bernd Schöllhorn c, Nicolas Spinelli b, , Claire Fave , Eric Defrancq
⇑
⇑
a Institut de Topologie et de Dynamique des Systèmes (ITODYS), UMR 7086 CNRS/Université Paris Diderot, Sorbonne Paris Cité, 15 rue Jean-Antoine de Baïf, 75205 Paris Cedex, France
b Département de Chimie Moléculaire, UMR CNRS 5250/Université Joseph Fourier, BP 53-38041, Grenoble Cedex 9, France
c Laboratoire d’Electrochimie Moléculaire, UMR 7591 CNRS/Université Paris Diderot, Sorbonne Paris Cité, 15 rue Jean-Antoine de Baïf, 75205 Paris Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochem-
ical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were pre-
pared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides
either at 30 or 50 end. Both labels were found to not disturb the recognition properties of the oligonucleo-
tide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates
with a disulfide at 30 end and an electrochemical label at 50 end.
Received 27 November 2012
Revised 12 December 2012
Accepted 15 December 2012
Available online 22 December 2012
Keywords:
Conjugation
Oxime
Ó 2012 Elsevier Ltd. All rights reserved.
Oligonucleotides
Ferrocene
Viologen
Oligonucleotide based biosensors1 play an important role in
diagnosis since they are used for detecting a large panel of analytes
including nucleic acids,2 proteins,3 small organic molecules,4 met-
als5 as well as whole cells.6 For these reasons, a large number of re-
search projects are devoted to the development of DNA-based
biosensors for which the transduction of the recognition event is
performed through optical,7 gravimetric8 or electrochemical meth-
ods.9 The latter provide high sensitivity and have the advantage of
needing only simple and compact apparatus.
One strategy for electrochemical monitoring of biomolecular
recognition is the comparison of the signal due to an electroactive
label chemically linked to the oligodeoxyribonucleotide (ODN) in
absence and in presence of the target. The most studied redox label
is certainly ferrocene (Fc) since this molecule allows synthetic
modifications thanks to the high chemical stability of the iron II
g5 complex. Moreover, the value of the oxidation potential is not
in the range of the O2 reduction and cyclovoltammetry (CV) can
be performed without further precautions. Preparations of ferro-
cene–ODN conjugates have been described10 including direct
incorporation of a Fc phosphoramidite during ODN synthesis,11
amidative oxidation of H-phosphonate linkage12 or enzymatic
incorporation of a Fc nucleotide triphosphate.13 Fragment coupling
strategy,14 that is, chemoselective coupling of reporter and ODN
bearing mutually-reactive functional moieties, is of great interest
because each fragment can be separately prepared with the most
efficient chemistry available. Up to date, only few applications of
this strategy for ferrocene labeling of ODN have been reported such
as the coupling of a ferrocenyl activated ester with an amino mod-
ified ODN,15 or a CuAAC (copper catalyzed alkyne cycloaddition)
reaction between oligonucleotide bearing an alkyne group and an
azido-modified Fc.16
Viologens (1,10-disubstituted 4,40-bipyridinium dications) rep-
resent another class of redox active molecules, showing low redox
potentials17 far from those of nucleobases and thus are excellent
candidates for the development of electrochemical nucleic acids
biosensors. However, viologen cannot be directly incorporated
during the automated DNA synthesis because of incompatible
reactivities. Indeed degradation of viologen can occur during
ammonia treatment required for deprotecting ODNs.18 So far there
is only one report describing conjugation of viologen with oligonu-
cleotides in solution, the method involving the reaction between
an activated ester of a methyl viologen derivative and an amino
containing ODN.19 This strategy showed certain drawbacks be-
cause the coupling reaction involves carbodiimide activated inter-
mediate, which is usually unstable in aqueous medium. This could
be overcome by using a large excess of activated ester of viologen.
With the aim of developing an efficient and reliable strategy for
synthesizing oligonucleotide conjugates with either ferrocene (Fc)
or methyl viologen (MV) labels, we focused on the use of oxime
bond formation. This approach has been proved to be efficient
⇑
Corresponding authors. Tel.: +33 45 652 0833 (N.S.); tel.: +33 15 727 7208
(C.F.).
E-mail addresses: Nicolas.Spinelli@ujf-grenoble.fr (N. Spinelli), claire.fave@univ-
paris-diderot.fr (C. Fave).
These authors contributed equally to this research.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.057

956
J. Moreau et al. / Bioorg. Med. Chem. Lett. 23 (2013) 955–958
for ODN conjugation with various reporters including peptides,
carbohydrates or fluorescent labels.20 In the context of electro-
chemical labeling of ODNs the strategy requires the preparation
of ODN carrying an aldehyde group for subsequent coupling reac-
tion with aminooxy containing redox labels.21 Herein we report on
the preparation of aminooxy ferrocene 122 and aminooxy viologen
2 (Fig. 1) and the subsequent coupling reaction with aldehyde con-
taining oligonucleotides either at 30 and 50 end. The hybridization
and the electrochemical properties of the conjugates are also
described.
Protected precursor 5 of aminooxy ferrocene 1 was synthesized
in three steps starting from N-(5-Hydroxy-3-oxapentyl)ferro-
cenoylamide 3 (Scheme 1).23 The hydroxy group was converted
into tosylate 4, which was substituted with ethyl acetohydroxa-
mate under phase transfer catalysis (PTC) conditions leading to
aminooxy-protected ferrocene 5 with an overall yield of 72% from
3. PTC was used for preventing b-elimination of the tosylate group
under the basic conditions required for substitution.24
Scheme 1. Synthesis of N-[5-aminooxy-3-oxapentyl] ferrocenoylamide 1.
Precursor 9 of aminooxy-methyl-viologen 2 was synthesized
according to the following procedure (Scheme 2). Trityl protected
aminooxy alcohol 625 was first converted to mesylate 7, which
was used for preparing the monocation 8 through nucleophilic
substitution of mesylate group by 4,40-bipyridine; the methane
sulfonate counter ion was then exchanged with iodide. Addition
of iodomethane led to trityl protected aminooxy viologen 9.
The aminooxy protecting groups were kept for storage and were
removed just before the coupling reaction with ODNs. Free amino-
oxy compounds 1 and 2 were obtained in quantitative yield after
treatment of 5 and 9, respectively, with a 45/45/5 (v/v/v) CH3CN/
H2O/TFA solution and freeze drying. Compounds 1 and 2 were used
for ODN conjugation without further purification.
The efficiency and versatility of the conjugation were first eval-
uated on model undecamers bearing an aldehyde group either at
the 30 end (50CGC ACA CAC GC X30 10, with X = CHO) or at the 50
end (50Y CGC ACA CAC GC30 11, with Y = CHO). These ODNs were
synthesized according to previously reported methods (see the
Supplementary data).26 The conjugation reactions were carried
out in ammonium acetate buffer (0.4 M, pH 4.6) by using a slight
excess of aminooxy derivatives 1 or 2 (Scheme 3). The course of
the reaction was followed by reverse phase HPLC and the reaction
proceeded essentially to completion within 6–8 h to yield the con-
jugates 10a–b and 11a–b as depicted in Scheme 3. The conjugates
were almost obtained with satisfactory yields after RP-HPLC
purification.27
Crude reaction mixtures were found cleaner when the conjuga-
tion was performed at the 50 end (11a and 11b) rather than at the
30 end (10a and 10b). This could be due to the transposition of the
30 diol during ODN synthesis leading to loss of the diol group via
dephosphorylation.28 The conjugates 10a,b and 11a,b were charac-
terized by ESI-MS. In all cases, the experimentally determined
molecular weights were in excellent agreement with the calcu-
lated values (Table 1).
Scheme 2. Synthesis of 1-[6-aminooxyhexyl]-10-methyl-4,40-bipyridinium 2.
For demonstrating that the method might be useful for the elab-
oration of electrochemical biosensors, the compatibility with a 30-
disulfide moiety on oligonucleotides was studied. Indeed, disulfide
is the precursor of thiol function commonly used for anchoring oli-
gonucleotides on gold surfaces.29 A model 30-disulfide 50-benzalde-
hyde30 ODN was synthesized (50X GCA GTA TCT TCT ATT TCT CCA
CAC TGC Y30, X = 50-benzaldehyde, Y = 30-disulfide, 12). Benzalde-
hyde moiety was chosen instead of 1,2-diol because of the instabil-
ity of the disulfide linkage upon periodate treatment required for
generating aldehyde from diol.31 This ODN was conjugated with
the two aminooxy electrochemical labels 1 and 2 to obtain conju-
gates 12a and 12b, respectively (Scheme 3, C).
RP-HPLC profiles showed almost quantitative conjugation reac-
tion (Scheme 3, C) and the conjugates 12a,b were purified by RP-
HPLC27 and unambiguously characterized by ESI-MS (Table 1).
In order to demonstrate that the electrochemical properties of
the labels were not affected, CV of conjugates 12a and 12b were re-
corded in a HEPES buffer containing 1M of sodium perchlorate on
screen-printed carbon electrodes (Fig. 2). Conjugate 12a showed a
reversible redox signal (E1/2 = 0.4 V vs Ag/AgCl) consistent with the
ferrocene bearing an amido group. The CV of conjugate 12b was re-
corded under inert atmosphere in order to avoid oxygen reduction
and showed two reversible one-electron reduction peaks, one at
ꢀ0.47 V (vs Ag/AgCl) and one at ꢀ0.74V (vs Ag/AgCl) consistent
with the presence of the viologen.
The hybridization properties of conjugates 10a,b and 11a,b
were investigated by thermal denaturation and circular dichroism
experiments. This study suggests that the redox labels have neither
destabilizing nor perturbation effects on the conformation proper-
ties of the ODN recognition process (see the Supplementary data).
Figure 1. Structures of N-[5-aminooxy-3-oxapentyl] ferrocenoylamide 1 and 1-[6-
aminooxyhexyl]-10-methyl-4,40-bipyridinium 2.

J. Moreau et al. / Bioorg. Med. Chem. Lett. 23 (2013) 955–958
957
Scheme 3. Syntheses of conjugates (A: 10a,b, B: 11a,b, C: 12a,b) and RP-HPLC (260 nm) profiles of unlabeled ODN and of crude conjugates.
Table 1
tides with both an electrochemical label and an anchoring func-
tion. We believe that this strategy might be useful for
elaborating electrochemical nucleic acids biosensors using ferro-
cene and viologen labels. Indeed both labels possess distinct redox
potentials which could be exploited for simultaneous detection of
different analytes.
ESI-MS data for conjugatesa
Conjugate
m/z Calcd
m/z Found
10a
10b
11a
11b
12a
12b
3707.3
3660.8
3747.7
3702.8
9010.9
8963.6
3707.1
3659.2
3749.0
3701.5
9011.1
8963.8
Acknowledgments
a
Analyses were carried out in the negative mode. CH3CN/H2O/Et3N (50:50:2, v/v/
This work was supported by Agence Nationale pour la Recher-
che (ANR ECSTASE Project). The authors thank the scientific struc-
ture ‘Nanobio’ at Grenoble for providing facilities.
v) was used as the eluent at a flow rate of 8
l
L minꢀ1
.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
12.057.
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