ISSN 0012-5008, Doklady Chemistry, 2008, Vol. 419, Part 2, pp. 108–110. © Pleiades Publishing, Ltd., 2008.Original Russian Text © T.S. Zatsepin, Le Thi Hien, T.S. Oretskaya, 2008, published in Doklady Akademii Nauk, 2008, Vol. 419, No. 6, pp. 767–769.

108

Oligonucleotides, modified ones including, arewidely used in different fields of molecular biology.They are used as primers for sequencing and amplify-ing DNA and determining the DNA primary structureby the hybridization method and are promising thera-peutic agents [1]. One of the fields of application ofmodified oligonucleotides is the preparation of theirconjugates with various compounds such as peptides,oligosaccharides, and dyes for studying the behavior ofnucleic acids in vitro or in vivo [2]. The attachment ofdyes or electrochemically active compounds to oligo-nucleotides provides an opportunity to use these deriv-atives for the design of systems of DNA detection bythe hybridization method [3]. The presence of markergroups considerably increases the sensitivity of DNAdetermination. The conjugates of oligonucleotides withpeptides, carbohydrates, and lipophilic compoundsoften better penetrate through cell membranes as com-pared with natural nucleic acids, which can be used inantisense biotechnology and RNA interference [4]. Atpresent, oligonucleotides bearing aldehyde groups are

widely used for preparing different conjugates with flu-orophores, peptides, and proteins [5].

In this work, we propose a new method allowing theintroduction of the aldehyde function at the internucle-otide phosphate group (Scheme 1). This is possibleupon the insertion of the H-phosphonate nucleotideunit followed by its oxidation with 3-aminopropanediolin carbon tetrachloride. The resulting diol derivative isa precursor for aldehyde formation. In spite of the factthat the H-phosphonate method is scarcely ever used atpresent for oligonucleotide synthesis, it is often appliedfor the introduction of different modified units into anoligonucleotide chain [6].

We performed the synthesis of an oligonucleotidechain on an automated synthesizer using the ami-dophosphite method. At the site of expected modifica-tion, a nucleoside 3'-H-phosphonate derivative wasattached and the H-phosphonate group was oxidizedwith 3-aminopropanediol to a phosphamide fragment;the 1,2-diol group and unreacted 5'-hydroxy groupswere simultaneously blocked with acetate protectivegroups, and oligonucleotide synthesis was continued

New Approach to the Synthesis of Modified Oligonucleotides Bearing an Aldehyde Group

T. S. Zatsepin, Le Thi Hien, and T. S. Oretskaya

Presented by Academician A.A. Bogdanov September 3, 2007

Received September 24, 2007

DOI:

10.1134/S0012500808040071

Moscow State University, Vorob’evy gory, Moscow, 119992 Russia

CHEMISTRY

Sequences of synthesized oligonucleotides, the yields of conjugates, and the data of MALDI TOF mass spectrometry of modi-fied oligonucleotides and conjugates based on them

Oligonucleotide (5'

→

3')

Mass, [M + H]

+

, calculated/found Yield of conjugate,

%initial oligonucleotide

with 1,2-diololigonucleotide conju-

gate with peptide

ctatggcgaggcgattaagttgggcaacgtp*cagggtcttccgaacgac 14948.8/14980 15833.7/15850 45

ctatggcgaggcgattaagttgggp*caacgtcagggtcttccgaacgac 14948.8/14997 15833.7/15871 60

ctatggcgaggcgattaagttgggcaacgtcagggtp*cttccgaacgac – – 58

ctatggcgaggcgattaap*gttgggcaacgtcagggtcttccgaacgac – – 54

ctatggcgaggcp*gattaagttgggcaacgtcagggtcttccgaacgac – – 18

Note: p* is the modified internucleotide phosphate group.

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NEW APPROACH TO THE SYNTHESIS OF MODIFIED OLIGONUCLEOTIDES 109

on an automated synthesizer (see Scheme 1). Amongthe advantages of this method are that the synthesis isfast and simple and can use only commercial chemicalsand that an aldehyde group can be introduced into anyprescribed position of the oligonucleotide chain. Theremoval of the products of oligonucleotide synthesisfrom the polymer support and the deprotection of oligo-nucleotides were carried out by treatment with concen-trated aqueous ammonia at 55

°

C. The structures of oligo-nucleotides

I

–

IV

are presented in the table. The purity ofthe products was determined by reversed-phase HPLCand matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry.

To produce an aldehyde group, we used a commonmethod, the periodate oxidation of a 1,2-diol group inthe corresponding precursor [7]. Oxidation productshad the same retention time as initial oligonucleotideswhen the reaction mixture was analyzed by reversed-phase HPLC. To show the reactivity of the aldehydegroup, we obtained model conjugates with

O

-benzylhy-droxylamine and

N

-aminooxyacetylpeptide (Scheme 1).The resulting oligonucleotide conjugates were isolatedby reversed-phase HPLC and analyzed by ion-pairreversed-phase HPLC and MALDI TOF mass spec-trometry.

B

O

ODMTrO

Cm

B

O

ODMTrO

PO H

OR1

Cm

H2N OH

OH

B

O

ODMTrO

PHN O

OR1

Cm

AcO

AcO

B

O

OR2O

PHN O

OR3

O

B

O

OR2O

PHN O

OR3

NOR4

(1) Standard amidophosphite

(2) Attachment of a hydrophosphoryl

(1) Standard amidophosphite

(2) Deprotection and purification(1)

(3) Sodium periodate

B is a heterocyclic base;Cm is a glass with a given pore size;

R1 is a fragment of the protected oligonucleotide chain;R2 and R3 are fragments of the oligonucleotide chain;

R4 = Bn, CH2CO–DPGYIGSR–NH2;

1, 2, and 3 are reaction stages.

Scheme. Synthesis of oligonucleotides containing the aldehyde function

oligonucleotide synthesis

synthon

(2) Ac2O, DMAP, pyridine

oligonucleotide synthesis

of the oligonucleotide

R4ONH2

at the internucleotide phosphate group.

Thus, we have suggested a new approach to the syn-thesis of oligonucleotides bearing aldehyde groups.Two new types of modified oligonucleotides containinga 1,2-diol or aldehyde group attached to the internucle-

otide phosphate were obtained. The reactivity of thealdehyde group within the oligonucleotides was dem-onstrated. We plan to use these derivatives for the affin-ity modification of DNA-binding proteins.

110

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ZATSEPIN et al.

EXPERIMENTAL

Oligodeoxyribonucleotides were obtained on anApplied Biosystems 394A automated DNA synthesizeraccording to a standard procedure using commercialchemicals and solvents. The polymer support used wasa glass with a definite pore size (CPG-500) containingimmobilized nucleoside. The specific load of the poly-mer by the first monomeric unit was 25–30

µ

mol/g.3'-Amidophosphites of natural 2'-deoxyribonucleo-sides were used as 0.1 M solutions in dry acetonitrile.First, the synthesis of the 3'-terminal fragment of theoligonucleotide was carried out in the “Trityl OFF”mode. Then, the column was removed from the synthe-sizer and the attachment of a modified unit was per-formed manually using 1-mL disposable syringes con-nected to both sides of the column (for the use of thistechnique, see [8]). To attach the nucleotide unit, a solu-tion of nucleoside H-phosphonate (10 mg in 500

µ

L ofa pyridine–acetonitrile (1 : 1, v/v) mixture) and pivaloylchloride (10 mg in 500

µ

L of the same solvent) wasmixed immediately before application to the columnand the reaction took place for 2 min. The resulting H-phosphonate was further oxidized with a solution of 3-aminopropanediol (50 mg in 1.5 mL of carbon tetra-chloride) for 10 min. After the 1,2-diol group and theunreacted 5'-hydroxy groups were protected for 30 swith 1 mL of a mixture containing 5% acetic anhydride,5% pyridine, and 4%

N

-methylimidazole in tetrahydro-furan, the synthesis of an oligomeric chain was contin-ued on the automated synthesizer. The conversion atthis stage was slightly lower than for 3'-amidophosphi-tes of natural 2'-deoxyribonucleosides, about 90%. Oli-godeoxyribonucleotides were deprotected and removedfrom the polymeric support with a concentrated aque-ous ammonia solution at 55

°

C for 18 h, oligonucleotidesolutions were concentrated in vacuum, and the targetcompounds were isolated by reversed-phase HPLC.

The formation of the aldehyde group, the prepara-tion of conjugates, the purification of oligonucleotidesand conjugates, and the recording of mass spectra werecarried out as described in [9].

ACKNOWLEDGMENTSWe are grateful to Prof. O.I. Lavrik (Institute of

Chemical Biology and Fundamental Medicine, Sibe-rian Branch, RAS). This work was supported by theRussian Foundation for Basic Research (project no.06–04–49196a) and the Council for Grants of the Pres-ident of the Russian Federation for Support of YoungRussian Scientists (project no. MK–5631.2006.4).

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