Use of an aminooxy linker for the functionalization of oligodeoxyribonucleotides

Article abstract of DOI:10.1016/S0960-894X(01)00108-1

We describe the preparation of oligonucleotides containing a 5′-linker bearing an aminooxy group. Use of the trityl protecting group for the aminooxy moiety allows purification of the modified oligonucleotide by reverse phase HPLC and cleavage in mild aci

Full text of DOI:10.1016/S0960-894X(01)00108-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 931–933
Use of an Aminooxy Linker for the Functionalization of
Oligodeoxyribonucleotides
Eric Defrancq* and Jean Lhomme
´
LEDSS, Chimie Bioorganique, UMR CNRS 5616, Universite Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France
Received 8 November 2000; revised 25 January 2001; accepted 14 February 2001
Abstract—We describe the preparation of oligonucleotides containing a 50-linker bearing an aminooxy group. Use of the trityl
protecting group for the aminooxy moiety allows purification of the modified oligonucleotide by reverse phase HPLC and cleavage
in mild acidic conditions. Derivatization with an aldehydic reporter group is efficient and rapid. # 2001 Elsevier Science Ltd. All
rights reserved.
Introduction
by transamination6 or by use of a phosphoramidite
bearing a phthaloyl protected aminooxy group.7 In the
latter case, the phthaloyl group was cleaved during the
ammonia treatment required for deprotection of the
exocyclic amines of the nucleobases. The intermediate
aminooxy-modified oligonucleotide was not isolated
and the crude deprotected oligonucleotide had to be
used without purification as a consequence of the high
reactivity of the aminooxy group. We thus considered
the use of a phosphoramidite bearing the aminooxy
moiety protected by a group that could be selectively
removed. The trityl protection was selected as it is stable
to the basic deprotection conditions and the hydro-
phobicity of the trityl group allows the use of reverse
phase HPLC for purification of the oligonucleotide.
Modified oligonucleotides represent new tools in molec-
ular biology for use as linkers, probes, primers, etc., and
in therapy for gene expression inhibition. Their potential
use for analytical and therapeutic applications has
strongly spurred the development of new methods for
linking covalently reporter groups on oligonucleotides.
For these purposes, conjugation of oligonucleotides
with fluorescent, lipophilic, intercalating, crosslinking,
alkylating or DNA cleaving entities has been per-
formed.1 Most strategies involve generation of an ali-
phatic amino group at the 50-terminus of the
oligonucleotide and subsequent reaction with an elec-
trophilic reporter molecule.1ꢀ3 However, this approach
generally requires large excess of the electrophilic
reporter molecule (activated ester, isothiocyanate, etc.)
to achieve complete conjugation.
In this paper, we report on the preparation of the
phosphoramidite 1 (Fig. 1) and its incorporation into
oligodeoxyribonucleotides. We describe the efficient
conjugation of such modified oligonucleotides with the
aldehydic fluorescein derivative 2 taken as an example.
In recentpapers, 4,5 we described an efficientmethod for
derivatization of oligonucleotides by use of the amino-
oxy–aldehyde coupling reaction. The oligonucleotide
was functionalized with an aldehydic group located
either on a base at any preselected position inside the
0
sequence or athte 5 extremity, and the reporter mol-
ecule carried an aminooxy group. We then considered the
‘reverse strategy’ in which the aminooxy moiety is sup-
ported by the oligonucleotide at the 50-end. Aminooxy
linkers have already been anchored on oligonucleotides
*Corresponding author. Fax: +33-476-514-382; e-mail: eric.
defrancq@ ujf-grenoble.fr
Figure 1.
0960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00108-1

932
E. Defrancq, J. Lhomme / Bioorg. Med. Chem. Lett. 11 (2001) 931–933
Scheme 1. (a) N-Hydroxy-5-norbornene-2,3-dicarboximide, K2CO3, DMF; (b) N2H4, EtOH; (c) Tr–Cl, pyridine; (d) N,N,N0,N0-tetraisopropyl-2-
cyanoethylphosphorodiamidite, N,N0-diisopropylammonium tetrazolide, CH2Cl2.
Scheme 2. (a) 80% aqueous AcOH; (b) acetone; (c) 2, DMF.
Results and Discussion
aminooxy derivative that proved to be highly unstable.
The more stable trityl protection was thus introduced by
reacting 4 with trityl chloride in dry pyridine. Phosphi-
tylation with N,N,N0,N0-tetraisopropyl-2-cyanoethyl-
phosphorodiamidite afforded the phosphoramidite
synthon 1.9
Preparation of the phosphoramidite 1 (Scheme 1)
Preparation of the phosphoramidite 1 was accomplished
by the route depicted in Scheme 1. The aminooxy
moiety was introduced using N-hydroxy-5-norbornene-
2,3-dicarboximide as a precursor instead of the usual
N-hydroxyphthalimide as the yields of coupling and
Oligonucleotides synthesis (Scheme 2)
8
subsequentcleavage were higher. The desired O-pro-
tected oxyamine 3 was obtained in 80% yield by heating
commercial 6-bromohexanol with the dicarboximide
derivative at 50 ꢁC in DMF in the presence of potassium
carbonate. The aminooxy group was then deprotected
using hydrazine in EtOH to afford the linker 4 in 92%
yield. We then protected selectively the aminooxy func-
tion in 4 with the trityl group. Reaction of 4 with
monomethoxytrityl chloride gave the corresponding
Two oligonucleotides containing the 50-modification
were synthesized according to standard b-cyanoethyl-
phosphoramidite chemistry: the homopyrimidine
d(XTTTTTTTT) and the undecamer d(XCGCACA-
CACGC) in which X represents the 50-aminooxy linker.
After deprotection with concentrated ammonia for 24 h
at50 ꢁC, the oligonucleotides 6 and 7 containing the
trityl group at the 50 extremity were purified by reverse
Figure 2. HPLC profiles of (A) crude mixture of undecamer 7; (B) purified undecamer 7; (C) crude detritylation mixture of 7.

E. Defrancq, J. Lhomme / Bioorg. Med. Chem. Lett. 11 (2001) 931–933
933
phase HPLC. Figure 2 shows the HPLC profile of
Acknowledgements
the crude (A) and purified (B) oligonucleotide 7.
Both oligonucleotides 6 and 7 were characterized by
ES-MS.10 Cleavage of the trityl protection in the
homopyrimidine oligonucleotide 6 was performed in
80% aqueous AcOH at room temperature for 6 h lead-
ing to selective formation of the deprotected oligonu-
cleotide 8. The same conditions were used for the
purines containing oligonucleotide 7. Even in this case
no side reaction (depurination) occurred during the
acidic treatment (Fig. 2C). Taking advantage of the
high reactivity of the aminooxy function with carbonyl
derivatives, the structures of 8 and 9 were confirmed by
formation of the corresponding oxime ethers 10 and 11
by reacting the aminooxy oligonucleotides with ace-
tone.10
The authors wish to thank Dr. V. Ducros and D. Ruffieux
(Biochimie C, CHU Grenoble) for ES-MS measurements.
We are grateful to Dr. P. Cros and Dr. A. Laayoun from
bioMerieux S.A. for their interest in this work.
References and Notes
1. (a) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 1925.
(b) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 10441.
(c) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1992, 48, 2223.
2. Markiewicz, W. T.; Groger, G.; Rosch, R.; Zebrowska, A.;
Markiewicz, M.; Klotz, M.; Hinz, M.; Godzina, P.; Seliger, H.
Nucleic. Acids Res. 1997, 25, 3672.
3. Goodchild, J. Bioconjugate Chem. 1990, 1, 165.
4. Trevisiol, E.; Renard, A.; Defrancq, E.; Lhomme, J. Tetra-
hedron Lett. 1997, 38, 8687.
5. Trevisiol, E.; Renard, A.; Defrancq, E.; Lhomme, J.
Nucleosides, Nucleotides Nucleic Acids 2000, 19, 1427.
6. Adarichev, V. A.; Kalachikov, S. M.; Kiseliova, A. V.;
Dymshits, G. M. Bioconjugate Chem. 1998, 9, 671.
7. Salo, H.; Virta, P.; Hakala, H.; Prakash, T. P.; Kawasaki,
A. M.; Manoharan, M.; Lonnberg, H. Bioconjugate Chem.
1999, 10, 815.
The coupling reaction of the aminooxy-derivatized oli-
gonucleotides with the fluorescein derivative 2 taken as
an example of an aldehydic reporter group was exam-
ined. Reaction of 2 with oligonucleotides 8 and 9 was
carried out at room temperature in water at pH 5 for
5 h using a 2-fold excess of the aldehydic fluorophore.
In both cases the reaction was highly selective affording
the corresponding oxime ethers 12 and 13. The structure
of the conjugated oligonucleotides 12 and 13 was
confirmed by ES-MS.10
8. Rougny, A.; Daudon, M. Bull. Soc. Chim. Fr. 1976, 5–6,
833.
9. Phosphoramidite 1 was obtained as an oil; 1H NMR
(CDCl3) d 7.33–7.26 (15H, m, H–Ar trityl), 6.26 (1H, s, NH),
3.82 (2H, m, CH2O), 3.68–3.58 (4H, m, CH2O), 2.62 (2H, t,
CH2CN), 1.60–1.44 (5H, m, 2CH2 and CH), 1.21–1.17 (17H,
m, 4CH3, 2CH2 and CH). 31P NMR (CDCl3) d 145.4. MS
(FAB, NBA matrix) m/z 575 [M+.].
In conclusion, introduction of an aminooxy moiety at
the 50-end of oligonucleotides using the phosphor-
amidite 1 proved very efficient. This approach has the
major advantage of an easy purification of well-charac-
terized intermediates. Mild acidic cleavage of the trityl
protection affords the aminooxy oligonucleotide with-
out by-product formation. Derivatization with an alde-
hydic reporter group is rapid and quantitative as
previously reported for that kind of aminooxy–carbonyl
coupling reaction.4,5,11,12
10. ES-MS: calcd for 6: 2808.6, found 2808.0; calcd for 7:
3708.2, found 3710.9; calcd for 10: 2606.6, found 2605.7; calcd
for 11: 3506.6, found 3505.7; calcd for 12: 3024.6, found
3023.6; calcd for 13: 3924.2, found 3925.7.
11. Trevisiol, E.; Defrancq, E.; Lhomme, J.; Laayoun, A.;
Cros, P. Eur. J. Org. Chem. 2000, 1, 211.
12. Trevisiol, E.; Defrancq, E.; Lhomme, J.; Laayoun, A.;
Cros, P. Tetrahedron 2000, 56, 6501.