functionalized peptide and oligonucleotide fragments
followed by a coupling reaction in the solution phase is
therefore preferred. To date, however, only a limited
number of such reactions are known. For instance,
formation of a disulfide bond,7 reaction of cysteine peptide
with maleimido oligonucleotide,8 reaction of bromoacetyl
peptide with thiol-functionalized oligonucleotide,9 and
formation of amide bonds10 have been reported. These
established routes still suffer from certain disadvantages.
Earlier work from our laboratory11 and others’ labo-
ratories12 has focused on developing the use of chemo-
selective oxime, thiazolidine, or hydrazone linkages for
efficient preparation of oligonucleotide conjugates. Herein,
the oligonucleotides functionalized with an aldehyde
group are reacted with the peptides containing nucleo-
philic groups such as aminooxy, aminothiol, or hydrazine.
The conjugation reactions are efficient but suffer from
the fact that oligonucleotide aldehyde has poor stability
and a known propensity to react with the purine bases.
In a search for an alternative strategy to prepare
POCs, we decided to explore the glyoxylic aldehyde
functionality. The advantage of using the glyoxylic alde-
hyde is that it is highly stable to air oxidation and does
not react with lysine side chains or R-amino groups
during ligation or storage, unlike the oligodeoxynucleo-
tide (ODN) aldehydes. These are extensively used in
protein engineering.13 However, there is only one report
on the use of oligonucleotide functionalized with glyoxylic
aldehyde for conjugation through a hydrazone linkage.12c
Unfortunately, no data on the hydrolytic stability has
been reported. Herein, we describe a new, convenient,
and straightforward method to prepare oligonucleotides
functionalized with glyoxylic aldehyde at the 5′ end. The
efficiency of the method is illustrated by coupling the
ODN-glyoxylic aldehyde to a nuclear localizing signal
(NLS) peptide sequence functionalized with an aminooxy
function. Also, the hydrolytic stability of the glyoxylic
New Method To Prepare
Peptide-Oligonucleotide Conjugates
through Glyoxylic Oxime Formation
Yashveer Singh, Eric Defrancq,* and Pascal Dumy
LEDSS, UMR CNRS 5616, ICMG FR2607, Universite´
Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France
Received August 24, 2004
Abstract: A new method to prepare peptide-oligonucleo-
tide conjugates through chemoselective glyoxylic oxime
linkage is reported. A novel phosphoramidite reagent,
readily accessible from serine, was prepared and used in
automated DNA synthesis to prepare oligonucleotides car-
rying a glyoxylic aldehyde functionality at the 5′ terminus.
This was efficiently coupled to a peptide functionalized with
an aminooxy group. The method could be of general use to
prepare a broad range of oligonucleotide conjugates.
Oligonucleotides and their analogues constitute an
important class of therapeutic agents that are being
investigated for their use as specific inhibitors of gene
expression.1 The efficacy of oligonucleotides in cell culture
is, however, often limited by poor cellular uptake.2
Certain peptide carriers have been reported to enhance
the cell delivery of the oligonucleotides.3 These peptides
have been either used as additives or covalently conju-
gated to the oligonucleotide to achieve an improved cell
delivery.4 In this context, the synthesis of peptide-
oligonucleotide conjugates (POCs) has attracted consider-
able interest, and various synthetic approaches have been
developed for POC synthesis.4c The total stepwise solid-
phase synthesis that involves complete synthesis of the
two fragments on the same or different support5 as well
as fragment coupling on solid support has been proposed.6
These methods, however, do not find much favor on
account of poor compatibility between the peptide and
oligonucleotide chemistries. A postassembly conjugation
strategy (the fragment-coupling approach) that involves
separate synthesis and deprotection of appropriately
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(b) Antopolsky, M.; Azhayeva, E.; Tengvall, U.; Auriola, S.; Jaaskel-
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tides: Process and Applications; Langel, U¨ ., Ed.; CRC Press: Boca
Raton, FL, 2002; pp 347-363.
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M. Bioconjugate Chem. 1998, 9, 260-267. (b) Morris, M. C.; Vidal, P.;
Chaloin, L.; Heitz, F.; Divita, G. Nucleic Acids Res. 1997, 25, 2730-
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Eur. J. 2004, in press. (b) Edupuganti, O. P.; Renaudet, O.; Defrancq,
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10.1021/jo0485177 CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/02/2004
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J. Org. Chem. 2004, 69, 8544-8546