1,2-Diol and hydrazide phosphoramidites for solid-phase synthesis and chemoselective ligation of 2′-modified oligonucleotides

Article abstract of DOI:10.1081/NCN-120022969

The preparation of two novel 2′-O-alkyl phosphoramidites bearing 1,2-diol and hydrazide functions for a chemoselective ligation is described. The former amidite was used to obtain 2′-modified oligodeoxyribonucleotides, which can be later oxidised by NaIO<

Full text of DOI:10.1081/NCN-120022969

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1,2-Diol and Hydrazide Phosphoramidites for Solid-
Phase Synthesis and Chemoselective Ligation of 2′-
Modified Oligonucleotides
Eugeny M. Zubin a , Dmitry A. Stetsenko b c , Tatiana S. Oretskaya a & Michael J. Gait b
a Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology , M.V.
Lomonosov Moscow State University , Moscow, Russia
b Medical Research Council Laboratory of Molecular Biology , Cambridge, UK
c Laboratory of Molecular Biology , Medical Research Council , Hills Road, CB2 2QH,
Cambridge, UK
Published online: 31 Aug 2006.
To cite this article: Eugeny M. Zubin , Dmitry A. Stetsenko , Tatiana S. Oretskaya & Michael J. Gait (2003) 1,2-Diol and
Hydrazide Phosphoramidites for Solid-Phase Synthesis and Chemoselective Ligation of 2′-Modified Oligonucleotides,
Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1375-1378, DOI: 10.1081/NCN-120022969
To link to this article: http://dx.doi.org/10.1081/NCN-120022969
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1375–1378, 2003
1,2-Diol and Hydrazide Phosphoramidites for Solid-Phase
Synthesis and Chemoselective Ligation of
20-Modified Oligonucleotides
1
Eugeny M. Zubin,1 Dmitry A. Stetsenko,2, Tatiana S. Oretskaya,
*
and Michael J. Gait2
1Chemistry Department and A.N. Belozersky Institute of Physico-Chemical
Biology, M.V. Lomonosov Moscow State University, Moscow, Russia
2Medical Research Council Laboratory of Molecular Biology,
Cambridge, UK
ABSTRACT
The preparation of two novel 20-O-alkyl phosphoramidites bearing 1,2-diol and
hydrazide functions for a chemoselective ligation is described. The former amidite
was used to obtain 20-modified oligodeoxyribonucleotides, which can be later oxi-
dised by NaIO4 to generate 20-aldehyde oligonucleotides. These were successfully
conjugated to acceptor molecules. The latter amidite also showed good coupling
yields, but the hydrazide function was demonstrated to be labile under basic
deprotection conditions.
Key Words: Oligonucleotide; Peptide; Conjugate; Aldehyde; Hydrazide.
*Correspondence: Dmitry A. Stetsenko, Laboratory of Molecular Biology, Medical Research
Council, Hills Road, CB2 2QH Cambridge, UK; Fax: +44 1223 412178; E-mail: ds@mrc-
lmb.cam.ac.uk.
1375
DOI: 10.1081/NCN-120022969
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1376
Zubin et al.
20-O-Alkyloligoribonucleotides have attracted particular attention as steric
block inhibitors of gene expression due to their high duplex stability with RNA
and improved nuclease resistance. Conjugation of an oligonucleotide analogue to
certain peptide sequences may facilitate its cellular uptake and=or affect intracellular
localisation.[1] To link multiple peptides covalently to a single oligonucleotide
sequence, the 20-sugar position may offer less disturbance of duplex stability. One
general method of preparation of peptide-oligonucleotide conjugates is total stepwise
solid-phase synthesis.[2] An alternative way is a separate solid-phase assembly of oli-
gonucleotide and peptide fragments, deprotection and purification followed by a
chemoselective ligation mediated by mutually reactive groups introduced into each
component during solid-phase assembly or post-synthetically.[2]
We described recently an efficient method for introduction of electrophilic
groups into the 20-position of an oligonucleotide.[3] Furthermore, we demonstrated
Scheme 1.

1,2-Diol and Hydrazide Phosphoramidites
1377
the possibility of a multiple peptide 20-conjugation via oxime=thiazolidine=hydrazine
chemistry.[4] Now we would like to report on the synthesis of two new 20-O-alkyl
phosphoramidite reagents for introduction of reactive groups for chemoselective
ligation.
Initially, we used 20-O-(2,3-dihydroxypropyl)uridine to introduce 20-aldehyde
function into oligonucleotides.[3] However, to maintain satisfactory duplex stabi-
lity and to be able to modulate linker length, another approach to 20-electrophilic
nucleosides proved to be successful. Thus N3-pivaloyloxymethyl-50-O-(4,40-di-
methoxytrityl)-20-O-[N-(2,3-diacetoxypropyl)aminocarbonyl]methyluridine 30-phospho-
ramidite was obtained from N3,30,50-O-protected uridine in six steps (see Sch. 1).
This synthetic route was shorter and more straightforward and high-yielding than
that described in.[3] The coupling efficiency of the monomer in a DNA synthesis
was > 97%. Singly- and doubly-modified 20-deoxyoligonucleotides were isolated,
their integrity and purity assessed by HPLC and MALDI-TOF analysis. These oli-
gonucleotides were then treated with NaIO4 to generate 20-aldehyde function as
described in.[4] New 20-aldehyde oligonucleotides were successfully conjugated to
9-hydrazinoacridine and H-Cys-Gly-OH dipeptide via hydrazone and thiazolidine
linkages, respectively.
We also attempted to prepare modified oligonucleotides containing a 20-hydra-
zide group which can react with aldehydes e.g. peptide C- or N-terminal aldehydes.
In the main step, N3,30,50-O-protected 20-O-(benzyloxycarbonyl)methyluridine was
treated with N2H4=EtOH to give the corresponding hydrazide derivative, which
was further N-protected by a Fmoc group (Sch. 1). 20-Hydrazide phosphoramidite
showed good reactivity in solid-phase coupling ( > 96% stepwise yield). However,
deprotection revealed an unexpected lability of the hydrazide function. Cleavage
and deprotection carried out with conc. aq. NH3 at 55ꢀC overnight or at room tem-
perature for 8–10 h and 0.4 M NaOH=MeOH did not lead to a product with the
correct molecular mass.
In conclusion, we described the preparation of two novel 20-O-alkyl phosphor-
amidites bearing 1,2-diol and hydrazide groups for a chemoselective ligation. The
former phosphoramidite was used to prepare 20-modified oligodeoxyribonucleotides,
which can be later oxidised by NaIO4 to generate a 20-aldehyde group. These 20-alde-
hyde oligonucleotides can be conjugated successfully to acceptor molecules. The
latter phosphoramidite showed good coupling yields, but the hydrazide function
was found to be labile under basic deprotection conditions.
ACKNOWLEDGMENTS
The authors thank Anna Kachalova and Andrey Malakhov for their help with
HPLC.
REFERENCES
1. Stetsenko, D.A.; Arzumanov, A.A.; Korshun, V.A.; Gait, M.J. Peptide conju-
gates of oligonucleotides as enhanced antisense agents. Mol. Biol. (Russ.)
2000, 34 (6), 852–859.

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Zubin et al.
2. Zubin, E.M.; Romanova, E.A.; Oretskaya, T.S. Modern methods for the
synthesis of peptide-oligonucleotide conjugates. Russ. Chem. Rev. 2002, 71 (3),
239–264.
3. Kachalova, A.V.; Zatsepin, T.S.; Romanova, E.A.; Stetsenko, D.A.; Gait, M.J.;
Oretskaya, T.S. Synthesis of modified nucleotide building blocks containing
electrophilic groups in the 20-position. Nucleosides Nucleotides Nucleic Acids
2000, 19 (10–12), 1693–1707.
4. Zatsepin, T.S.; Stetsenko, D.A.; Arzumanov, A.A.; Romanova, E.A.; Gait,
M.J.; Oretskaya, T.S. Synthesis of peptide-oligonucleotide conjugates with
single and multiple peptides attached to 20-aldehydes through thiazolidine,
oxime, and hydrazine linkages. Bioconjug. Chem. 2002, 13 (4), 822–830.