A novel heterobifunctional linker for facile access to bioconjugates

Article abstract of DOI:10.1039/b518151h

A convenient synthesis of a novel heterobifunctional linker molecule is described. The linker contains a thiol-reactive nitropyridyl disulfide group (Npys) and an aldehyde-reactive aminooxy group with a propensity to form disulfide and oxime linkages. The

Full text of DOI:10.1039/b518151h

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
A novel heterobifunctional linker for facile access to bioconjugates†
Yashveer Singh, Nicolas Spinelli, Eric Defrancq* and Pascal Dumy
Received 5th January 2006, Accepted 8th February 2006
First published as an Advance Article on the web 2nd March 2006
DOI: 10.1039/b518151h
A convenient synthesis of a novel heterobifunctional linker molecule is described. The linker contains a
thiol-reactive nitropyridyl disulfide group (Npys) and an aldehyde-reactive aminooxy group with a
propensity to form disulfide and oxime linkages. The utility of the linker molecule to cross-link different
biomolecules has been demonstrated by employing it in the efficient preparation of a peptide–
oligonucleotide conjugate. The linker reported herein could be a useful tool for cross-coupling of
different but appropriately functionalised biomolecules.
the synthesis of heterobifunctional linkers that can be utilized
Introduction
to cross-link biomolecules through chemoselective linkages has
assumed greater significance. Several such cross-coupling agents
have been developed and studied earlier for the preparation
of bioconjugates.8 For instance, a linker with reactive groups
like succinimidyl ester and maleimide has been used to cross-
couple proteins and enzymes.9 More recently, the conjugation of
carbohydrates to proteins was achieved by using an aminooxy-
thiol containing linker.10 Herein, the thiol moiety was reacted to a
bromoacetylated protein to form a thioether linkage.
Bioconjugation has generated significant research interest as it
provides an easy way to couple two or more molecules with distinct
properties thereby producing a novel complex structure (biocon-
jugate) that possesses the combined properties of its individual
components.1,2 This usually involves the joining (by chemical
or biological methods) of molecules such as antibodies (their
fragments), nucleic acids and their analogues, liposomal compo-
nents, polysaccharides, hormones, proteins and peptides to drugs,
radionuclides, toxins, fluorophores or photoprobes, inhibitors,
enzymes and haptens.3 Bioconjugation may also involve the simple
joining of two or more different biomolecules to combine their
useful properties. Examples in this category include peptide–
oligonucleotide,4 peptide–carbohydrate5 or oligonucleotide–
carbohydrate conjugates6 and so on. Over the years, several
bioconjugates have been prepared for use in research, therapeutics
and diagnostics. The results from these studies show that biocon-
jugation indeed adds significant benefits to the biomolecules being
conjugated. For instance, it may result in an increase in stability
to chemical or proteolytic degradation, reduced immunogenicity,
or improved pharmacokinetics, biodistribution and targeting.1,2,7
The conjugation of two or more biomolecules is mostly achieved
by incorporating mutually reactive groups into the individual
components, followed by their coupling in solution, leading to
the formation of stable chemical linkages like amides, thioethers,
disulfides or oximes.1,2 Such strategies have found wide acceptance
because of the high coupling efficiency and ease of purification. In
this context, the design and synthesis of appropriately derivatised
homo/heterofunctional linkers that can be utilized to cross-couple
two or more individual components (multiple conjugation) is
important. The requirements for bioconjugation are stringent,
for instance the use of a largely aqueous medium, physiological
pH, ambient temperature, short reaction time, equimolar pro-
portions of the components and formation of stable linkages.
This has spurred an interest in the synthesis of bioconjugates
based on chemoselective ligation techniques1–3 and therefore
The linker containing a thiol-reactive nitropyridyl disulfide
group (Npys) and an aldehyde-reactive aminooxy group is of
particular importance because such a linker molecule would
provide the possibility of generating two very versatile chemical
linkages, disulfide and oxime, in a single molecule. It would
be relevant to mention that the oxime bond formation has
been extensively used earlier to prepare a wide variety of
bioconjugates such as peptide–oligonucleotide,11 carbohydrate–
peptide,12 and carbohydrate–oligonucleotide conjugates.13 It has
also been used to anchor oligonucleotides and carbohydrates
onto glass surfaces with potential applications in the area of
microarray technology.14 The oxime ligation is chemoselective,
gives high coupling efficiency without additives and the oxime
bond is stable over a pH range and yet can be hydrolysed under
harsh conditions. The routine method employed for oxime bond
formation involves an addition–elimination reaction between an
aminooxy group and an aldehyde/ketone.15 Similarly, the disulfide
bond represents another linkage of choice for the conjugation
of biomolecules because it is stable and yet reversible in a
reducing environment where it is converted to a free sulfhydryl.16
This last property allows the selective release of biomolecules
in the intracellular medium. A thiol-reactive group is useful as
a potential precursor for the formation of the disulfide bond
because under physiological conditions a thiol group is more
nucleophilic than an amine. Several thiol-reactive reagents have
been used earlier to modify peptides and proteins at specific sites17
but the advantage associated with the use of a pyridyl disulfide
group is that it is thiol-specific under acidic as well as basic
conditions. Moreover, the pyridyl disulfide undergoes exchange
by oxidative coupling to a free sulfhydryl group to provide a
disulfide linkage and pyridine thione is released as a side product,
which is non-reactive and hence prevents contamination of the
disulfide.
LEDSS, UMR CNRS 5616, ICMG FR 2607, Universite´ Joseph Fourier,
301 rue de la Chimie, BP 53, F 38041, Grenoble Cedex 9, France. E-mail:
Eric.Defrancq@ujf-grenoble.fr
† Electronic supplementary information (ESI) available: Melting and CD
studies of 4, spectroscopic characterisation of compounds 1, 4–10 and
DTT treatment of 10. See DOI: 10.1039/b518151h
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We herein report on the synthesis and characterisation of a new
heterobifunctional linker molecule 1 that contains a thiol-reactive
nitropyridyl disulfide group and an aldehyde-reactive aminooxy
group (Fig. 1). The efficacy of this linker to cross-couple two
different biomolecules has been illustrated by employing it for the
preparation of a peptide–oligonucleotide conjugate.
important insofar as improvement in the therapeutic properties
of synthetic oligonucleotides is concerned. The cyclopentapeptide
used in the present work contains an arginine–glycine–aspartic
acid (RGD) tripeptide motif and is known for its selective and
powerful binding to avb3 integrin receptors.19 The oligonucleotide
conjugate of this peptide has been investigated for tumour
targeting20 and DNA delivery.21 The key question was to ascertain
whether the disulfide linkage is stable in the reaction conditions
employed for oxime bond formation and vice versa. It was therefore
decided to employ two synthetic routes for the preparation of
the conjugate 4. In the first approach, the linker 1 was coupled
to a peptide thiol followed by reaction to an oligonucleotide
aldehyde (Route A) whereas in the second approach, linker 1
was first reacted to an oligonucleotide aldehyde followed by
reaction to a peptide thiol (Route B). The reaction conditions and
their influence on the stability of other linkages were thoroughly
investigated.
Fig. 1 Heterobifunctional linker 1.
Cross-coupling through disulfide linkage formation followed by
oxime linkage formation (Route A). The preparation of RGD
peptide 5 containing the thiol functionality has been described
earlier.11a Similarly, the preparation of oligonucleotide 8 contain-
ing the 3ꢀ-aldehyde moiety (5ꢀ-CGCACACACGCX-3ꢀ where X =
aldehydic linker) has also been reported.11c The RGD peptide 5
was reacted with a slight excess of linker 1 in DMF–phosphate
buffer (pH = 4.8) at room temperature for one hour to obtain
linker–peptide 6. As expected, the reaction was found to be
fast and efficient. The HPLC profile of the crude conjugation
reaction mixture showed almost complete consumption of the
peptide thiol (Fig. 2A). The peptide derivative 6 was used in
the next step without further purification. It was treated with
50% trifluoroacetic acid in dichloromethane in the presence of
scavengers (triisopropyl silane–water) to remove the Boc protec-
tion. The reaction was carried out at room temperature for about
an hour and complete removal of the Boc group was achieved. The
linker–peptide derivative 7 containing the free aminooxy group
was obtained in a satisfactory yield after HPLC purification.
The second conjugation to the oligonucleotide was carried out
next. The linker derivative 7 was reacted with oligonucleotide-3ꢀ-
aldehyde 8 in a solution of ammonium acetate buffer (pH = 4.8)
at room temperature for 6–8 h. The slightly acidic conditions were
used for the reaction because these are conducive to the formation
of oxime bonds.1 The HPLC profile of the conjugation reaction
mixture is shown in the accompanying figure and two facts are
clearly evident from it (Fig. 2B). First, the conjugation reaction
is highly efficient as is known for oxime ligations and second,
no unaccounted side products are present (HPLC/mass analysis)
thereby suggesting that the reaction conditions used for the oxime
bond formation do not influence the disulfide linkage holding the
peptide. The conjugate 4 was obtained in a satisfactory yield after
HPLC purification. The derivatives 4 and 5–8 described above
were characterised by ESIMS analysis and an excellent agreement
between the calculated and the observed molecular weight was
observed.
Results and discussion
Syntheses
The synthesis of linker 1 was carried out in two straight steps
starting from the commercially available N-a-Boc-S-(3-nitro-2-
pyridylthio)-L-cysteine 2 (Scheme 1). The Boc protection on the
a-amino group in 2 was removed by treatment with 50% aqueous
trifluoroacetic acid at room temperature for 4–5 h to obtain 3 after
ether precipitation. This derivative was found to be sufficiently
pure (on TLC) and hence used in the next step without further
purification. The protected aminooxy moiety was incorporated
into 3 by an overnight reaction with the N-hydroxysuccinimide
ester of N-Boc-O-(carboxymethyl)-hydroxylamine in the presence
of N-methyl morpholine in anhydrous DMF at room temperature.
It would be relevant to note that the quantity of the base added
must be carefully controlled because the Npys group is known to
be unstable in basic conditions. The linker 1 was obtained in good
yield after purification on a silica gel column and characterized by
satisfactory 1H, 13C NMR, mass and elemental data.
Scheme 1 Preparation of the linker 1. (a) Aqueous TFA–CH2Cl2,
r.t., 73%; (b) N-hydroxysuccinimide ester of N-Boc-O-(carboxymethyl)-
hydroxylamine, N-methyl morpholine (1 equiv.) in DMF, overnight, r.t.,
89%.
The efficiency of the linker 1 to cross-couple different ap-
propriately functionalised biomolecules was investigated next. A
peptide–oligonucleotide conjugate 4 was prepared by using the
linker 1 (Scheme 2). Peptide–oligonucleotide conjugate was chosen
because such conjugates have been reported to enhance cellular
uptake efficiency, cell-specific targeting, stability to degradation
and binding affinity towards the target sequence of synthetic
oligonucleotides.18 Synthesis of such conjugates is considered
Cross-coupling reaction through oxime linkage formation fol-
lowed by disulfide linkage formation (Route B). Once the syn-
thesis of the peptide–oligonucleotide conjugate was successfully
achieved, the opposite route for the synthesis of the conjugate 4
was also attempted. Consequently, the linker 1 was treated with
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Scheme 2 Preparation of peptide–oligonucleotide conjugate 4 by using the linker 1. (a) DMF–phosphate buffer, 1 h, r.t.; (b) TFA–CH2Cl2–triisopropyl
silane–H2O, 1 h, r.t.; (c) ODN-3ꢀ-aldehyde 8, ammonium acetate buffer, 6–8 h, r.t.; (d) aqueous TFA, 3 h, r.t.; (e) peptide 5, DMF–phosphate buffer, 1 h,
r.t.
an aqueous solution of trifluoroacetic acid (50%, v/v) at room
temperature for 3 h to remove the Boc protecting group masking
the aminooxy functionality. The linker 9 with a free aminooxy
group was used in the next step without purification. It was
reacted with a slight excess of 11-mer-oligonucleotide-3ꢀ-aldehyde
8 (5ꢀ-CGCACACACGCX-3ꢀ) in a solution of ammonium acetate
buffer (pH = 4.6) at room temperature for 5–6 h. An efficient
conjugation led to the formation of conjugate 10, which was
purified by HPLC (Fig. 3A and 3B). Dual wavelengths of detection
were used to monitor this reaction: 260 nm for the oligonucleotide
and 400 nm for Npys. The ESIMS analysis however suggested the
loss of the Npys group. To ascertain whether the Npys group is
lost during the course of the conjugation reaction/purification or
during the mass analysis, conjugate 10 was treated with DTT. It
was found that peaks corresponding to Npys-on or Npys-off
products are well resolved (see ESI†) and hence possibly could
not be collected together during the purification of the conjugate
10. In our case, this loss of the Npys group could probably
be due to the basic conditions employed during the ESIMS
analysis of the conjugate (CH3CN–H2O–Et3N, 49 : 49 : 2). The
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Fig. 2 RP-HPLC profiles of crude conjugation reaction mixtures. (A) Linker 1 with RGD peptide 5 (disulfide bond formation); (B) linker–peptide 7
with oligonucleotide-3ꢀ-aldehyde 8 (oxime bond formation).
Fig. 3 RP-HPLC profiles of crude reaction mixtures. (A–B) Linker 9 with oligonucleotide-3ꢀ-aldehyde 8 (oxime bond); (C–D) peptide 5 with
linker–oligonucleotide 10 (disulfide).
linker–oligonucleotide conjugate 10 was next treated with a slight
excess of peptide 5 at room temperature for about an hour.
An efficient conjugation led to the formation of peptide–linker–
oligonucleotide conjugate 4 (Fig. 3C and 3D). Two different
wavelengths of detection as described above were also employed
to monitor this conjugation reaction. It was not possible to detect
the formation of the product at 260 nm but well-resolved peaks
for the disappearance of Npys on the oligonucleotide (RT =
12.6 min) and the appearance of a pyridine thione peak (RT =
9.2 min) were obtained when the reaction was monitored at
400 nm. The conjugate 4 was purified by HPLC. The derivatives
4, 9 and 10 were characterised by satisfactory ESIMS data. This
preparation of conjugate 4 conclusively established that an oxime
bond can be formed in the presence of a disulfide bond and
vice versa.
This is crucial because oligonucleotide conjugates showing duplex
instability and/or conformational perturbations may not be
desirable for various therapeutic applications where a high degree
of sequence specificity and affinity is demanded. Consequently, the
oligonucleotide duplexes with the complementary sequence were
prepared and investigated by thermal denaturation and circular
dichroism studies. The Tm value for the duplex formed by the
bioconjugate 4 with the complementary sequence was estimated
as 62.1 ◦C whereas the Tm value for the natural duplex was
found to be 61 ◦C (see ESI†). This suggests that conjugation
of an oligonucleotide to a peptide through the linker molecule
stabilizes the duplex slightly (DTm = 1.1 ◦C). A small to moderate
increase in the stability of the duplex has been observed earlier with
various other peptide–oligonucleotide conjugates.18 It is therefore
difficult to ascertain whether the slight increase in stability is
due to the presence of the peptide or the linker. However, it
is important to note that the linker does not destabilize the
duplex. Similarly, the CD spectrum for the duplex formed by the
conjugate 4 was found to be identical to the CD spectrum of
the natural duplex. The individual spectrum showed negative and
positive excitations respectively at 255 nm and 275 nm thereby
suggesting an unperturbed B-type duplex conformation (see
ESI†). An unperturbed duplex conformation is consistent with
several earlier studies with peptide–oligonucleotide conjugates13a,15
Biophysical studies
A linker employed to cross-couple biomolecules could be consid-
ered efficient if it does not diminish the useful and characteristic
biological features of the individual components. For instance, in
the present case, it is of the utmost importance to ascertain whether
the presence of the linker 1 in the conjugate induces an instability
and/or conformational perturbations in oligonucleotide duplexes.
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and the presence of linker 1 does not appear to have any significant
influence.
material on TLC) at ambient temperature. The reaction mixture
was concentrated under vacuum and further evaporated with
toluene to completely remove the TFA from the reaction mixture.
The residue so obtained was re-dissolved in acetonitrile and
precipitated with ether. The precipitate was collected and dried
in vacuum. This crude product (0.85 g, 73%) was found to be
sufficiently pure and was hence used in the next step without
further purification. m/z (DCI) 276 (20%, (M + H)+), 157 (100),
139 (100).
Conclusion
Thus, an efficient preparation of a novel heterobifunctional linker
with a propensity to generate disulfide and oxime linkages is
reported. The linker reported herein could be of general utility
to cross-couple potentially any two appropriately functionalised
biomolecules. This has been demonstrated in the present work
by utilizing the linker molecule for the preparation of a peptide–
oligonucleotide conjugate. The disulfide linkage could be formed
first followed by the formation of the oxime linkage and vice versa.
The introduction of a linker does not influence the stability and/or
the conformation of the duplex. The linker 1 also contains a free
carboxylic group, which can be used for on-support modification
of peptides and this makes it compatible with solid phase synthetic
procedures. The heterobifunctional linker reported herein could be
further explored to cross-couple various other biomolecules such
as proteins, carbohydrates, enzymes, drugs or fluorescent labels
and hence could be of immense use in the field of bioconjugation.
Heterobifunctional linker (1). 3 (250 mg, 0.64 mmol) was
dissolved in anhydrous DMF (12.0 mL) and N-methyl mor-
pholine (65 mg, 70 ll, 0.64 mmol) was added followed by
the N-hydroxysuccinimide ester of N-Boc-O-(carboxymethyl)-
hydroxylamine (222.5 mg, 0.77 mmol). The reaction mixture was
stirred overnight at ambient temperature under anhydrous con-
ditions. Afterwards, the reaction mixture was concentrated under
vacuum to remove the DMF and the residue so obtained was re-
dissolved in CH2Cl2. The organic layer was washed with 0.1 N HCl
and dried over anhydrous sodium sulfate. The linker 1 (258 mg,
89%) was obtained after purification by column chromatography
on a silica gel column using 10–20% MeOH in CH2Cl2 as the eluent
(found: C, 38.25; H, 4.25; N, 11.65. C15H19N4O8S2Na requires C,
38.30; H, 4.1; N, 11.1%); dH(300 MHz, CD3CN–D2O 6 : 1) 8.87
Experimental
=
(1H, dd, J 1.5, 4.6 Hz, CHNpys N), 8.71 (1H, dd, J 1.5, 8.2 Hz,
General
=
=
CHNpys–C(NO2) ), 7.50 (1H, dd, J 4.6, 8.2 Hz, CH CHNpys–CH),
4.77 (1H, dd, J 5.3, 7.9 Hz, CH(CO2H)–NH), 4.28 (2H, 2 x d, J
16.1 Hz, CH2–O–NH), 3.40 (1H, dd, J 5.3, 14.1 Hz, S–CHA(HB)–
CH), 3.22 (1H, dd, J 7.9, 14.1 Hz, S–CHB(HA)–CH), 1.45 (1H, s,
OC(CH3)3); dC (300 MHz, CD3CN) 170.4 (Cq), 168.8 (Cq), 157.4
(Cq), 155.4 (Cq), 153.8 (CH), 142.8 (Cq), 134.0 (CH), 121.5 (CH),
81.8 (Cq), 74.9 (CH2), 51.1 (CH), 39.1 (CH2), 27.1 (CH3); m/z
(DCI) 448.8 (60%, M−), 392.8 (100, (M − tBu)−), 348.8 (86, (M −
Boc)−).
All solvents and reagents used were of the highest purity available.
N-a-Boc-S-(3-nitro-2-pyridylthio)-L-cysteine was obtained from
Bachem. H and 13C NMR spectra were recorded on a Bruker
1
AVANCE 300 spectrometer. Mass spectra (DCI mode) for 1
and 3 were recorded on a Thermoquest spectrometer. ESI mass
spectra were measured on an Esquire 3000 spectrometer from
Bruker. The analysis was performed in positive mode for 5–7
and in negative mode for 4, 8–10. Aqueous acetonitrile (50%)
was used as the eluent for 5–7 and 2% Et3N was added for 4,
8–10. RP-HPLC analysis and purification were performed on
a Waters RP-HPLC system equipped with a dual wavelength
detector. For RP-HPLC analysis of 1, 5–7 and 9 a Nucleosil C18
column (Macherey Nagel, 30 × 4 mm, 3 lm) was used with the
following solvent system. Solvent A, 0.1% TFA in water; solvent
B, acetonitrile containing 10% water and 0.1% TFA; flow rate
of 1.3 mL min−1; a linear gradient of 5–100% B was applied in
15 minutes. The RGD peptide 5 and peptide–linker conjugate
7 were purified using a Delta Pak C18 column (Waters, 25 ×
200 mm, 15 lm) at a flow rate of 22 mL min−1. RP-HPLC
analysis of 4, 8 and 10 was performed on a Nucleosil C18 column
(Macherey Nagel, 250 × 4.6 mm, 5 lm) using the following solvent
system. Solvent A, 20 mM ammonium acetate buffer containing
5% acetonitrile; solvent B, acetonitrile containing 5% water; flow
rate of 1 mL min−1; a linear gradient of 0–30% B was applied in
20 minutes. These compounds were purified on a Nucleosil C18
column (Macherey Nagel, 10 × 250 mm, 7 lm) at a flow rate of
4 mL min−1.
Peptide RGD–cysteine (5). The procedure to prepare this pep-
tide has been described earlier.11a Linear peptide H-Asp(OtBu)-
Phe-Lys(Aloc)-Arg(Pmc)-Gly-OH was prepared by using stan-
dard solid-phase peptide synthesis protocols. Head to tail cycliza-
tion was effected in the presence of PyBOP in DMF. The Aloc
moiety was then cleaved by using the standard Pd0 procedure and
the peptide was purified by HPLC. Finally, the peptide was reacted
with Fmoc-Cys(Trt)–OH/PyBOP to introduce the cysteine group.
The Fmoc group was removed with piperidine in DMF. The tBu-
and Pmc-protecting groups were removed by using 90% TFA with
ethanedithiol as a scavenger to obtain 5.
Boc-aminooxy linker–peptide (6). 5 (10.0 mg, 0.014 mmol) was
dissolved in DMF–phosphate buffer (75 : 25 v/v; 1.0 mL; pH
= 4.8). A solution of linker 1 in DMF (0.015 mmol, 6.9 mg)
was added. The reaction mixture was stirred at room temperature
under argon for 3 h (monitored by HPLC). After, the reaction
mixture was concentrated and used in the next step without further
purification. m/z (ESI) 999.3 (M + H)+.
Aminooxy linker–peptide (7). The crude 6 obtained from the
above step was dissolved in a solution of 50% TFA in CH2Cl2
containing 5% triisopropylsilane and 5% water as scavengers.
The reaction mixture was stirred at room temperature for 1 h
(monitored by HPLC). The reaction mixture was concentrated
in a speed vac and the residue was re-dissolved in 50% aqueous
Syntheses
Amino-S-(3-nitro-2-pyridylthio)-L-cysteine (3). 2 (1.0 g, 2.66
mmol) was dissolved in CH2Cl2 (20 mL) and 50% aqueous TFA
(40.0 mL) was added. The reaction mixture was stirred for 4–5 h
(until the reaction shows complete consumption of the starting
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acetonitrile. The crude product was purified by HPLC. The pure
product (5.2 mg, 41% with respect to 5) was obtained as a white
powder after freeze-drying the appropriate fractions. m/z (ESI)
899.2 (M + H)+.
Thermal denaturation (Tm) studies
The melting curves (absorbance versus temperature) were recorded
at 260 nm on a UV–visible spectrophotometer equipped with a
temperature controller. The melting experiments were carried out
by mixing the equimolar amount of two oligonucleotide strands in
a solution of sodium phosphate buffer (10 mM, pH 7.0) containing
EDTA (1 mM) and NaCl (100 mM). The ODN concentration was
kept at 12 lM. ◦The absorbance was recorded in the temperature
range of 10–80 C at a sweep rate of 1 ◦C min−1. All experiments
were done in triplicate.
Oligonucleotide-3ꢀ-aldehyde (8). The preparation of the 11-
mer oligonucleotide (5ꢀ-CGCACACACGC-3ꢀ) containing 3ꢀ-
aldehyde functionality has been described by us.11d Briefly, auto-
mated synthesis of the oligodeoxynucleotide (ODN) was carried
out on an Expedite DNA synthesiser (Perkin-Elmer) by using
standard b-cyanoethyl nucleoside phosphoramidite protocols at
a 1 lM scale. The automated synthesis was carried out on
a modified solid support, (3-[(4,4ꢀ-dimethoxytrityl)-glyceryl-1-
succinyl]) long chain alkylamino controlled pore glass obtained
from Eurogentec. This support introduces a protected diol group
(an aldehyde precursor) into ODNs. Afterwards, the ODNs were
cleaved from the solid support and released into the solution by
treatment with 28% ammonia (1.5 mL) for 2 h and were finally
deprotected by keeping in the ammonia solution for 16 h at
Circular dichroism (CD) studies
The CD experiments were carried out by using similar solutions
as described above for the thermal denaturation studies.
Acknowledgements
◦
The Centre National de la Recherche Scientifique (CNRS) and
the Re´gion Rhoˆne-Alpes supported this work. The “Institut
Universitaire de France” is also acknowledged for financial
assistance.
55 C. The oligonucleotides containing free 3ꢀ-diol groups were
obtained after HPLC purification. The 5ꢀ-dimethoxytrityl group
was removed by treatment with 80% aqueous acetic acid for 1 h at
room temperature (standard procedure). These ODN-3ꢀ-diols were
subjected to oxidative cleavage with aqueous sodium-m-periodate
to obtain ODNs 8 with 3ꢀ-aldehyde functionality after HPLC
purification.
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Aminooxylinker (9). Linker 1 (20mg, 44.6 lmol)was dissolved
in a solution of 50% TFA in water (1 mL). The reaction mixture
was stirred at room temperature for 3 h (monitored on HPLC).
After, the reaction mixture was concentrated and the residue was
re-dissolved in water. The aminooxy linker 9 was obtained after
freeze-drying and the crude product was used in the next step
without further purification. m/z (ESI) 346.8 (M + H)+.
Linker–oligonucleotide conjugate (10). Oligonucleotide 3ꢀ-
aldehyde 8 (0.36 mg, 0.105 lmol) was dissolved in 0.1 M
ammonium acetate buffer (95 ll; pH = 4.6) and an aqueous
solution of aminooxy linker 9 was added (10 ll, 0.195 lmol).
The reaction mixture was stirred at room temperature for 8 h
(monitored by HPLC). The linker–oligonucleotide conjugate 10
(0.26 mg, 67% with respect to 8) was obtained after RP-HPLC
purification. m/z (ESI) 3721.2 (M − H)−, 3565.0 (M − Npys)−
(see ESI†).
Peptide–linker–oligonucleotide conjugate (4).
A) From linker–peptide conjugate (7). Oligonucleotide alde-
hyde 8 (0.7 mg, 0.206 lmol) was dissolved in a solution of 0.1 M
ammonium acetate buffer (0.7 mL, pH = 4.6) and an aqueous
solution of linker–peptide conjugate 7 (0.74 mg, 0.824 lmol)
was added. The reaction mixture was stirred at ambient temper-
ature for 7–8 h (monitored by RP-HPLC). The peptide–linker–
oligonucleotide conjugate 4 (0.48 mg, 55%) was obtained after
RP-HPLC purification. m/z (ESI) 4273.5 (M − H)−.
B) From linker–oligonucleotide conjugate (10). A solution of
peptide 5 (100 ll, 0.118 lmol) in phosphate buffer (0.1 M,
pH = 4.6) was added to the linker–oligonucleotide conjugate
10 (0.36 mg, 0.097 lmol). The reaction mixture was stirred at
room temperature for 1 h (monitored by HPLC) and conjugate 4
(0.17 mg, 41%) was obtained after RP-HPLC purification.
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