1068-1620/03/29032- $25.00 © 2003 MAIK “Nauka /Interperiodica” 0274 Russian Journal of Bioorganic Chemistry, Vol. 29, No. 3, 2003, pp. 274–280. Translated from Bioorganicheskaya Khimiya, Vol. 29, No. 3, 2003, pp. 303–309. Original Russian Text Copyright © 2003 by Kachalova, Tashlitsky, Stetsenko, Romanova, Gait, Oretskaya. 1 INTRODUCTION Conjugates of oligonucleotides with various mole- cules are currently extensively used for studying the regularities of protein and nucleic acid recognition and the problems of molecular biology. 2 Reporter mole- cules, intercalators, biologically active peptides, and enzymes have covalently been linked to fragments of nucleic acids [1–3]. At present, the methods of chemi- cal synthesis of oligonucleotide derivatives containing nucleophilic groups (amino [4] and mercapto groups [5]) and conjugates based on these oligonucleotide derivatives [6] have sufficiently been developed. On the other hand, the synthetic methods for the conjugates based on oligonucleotide derivatives with carboxyl or aldehyde function are few. The synthetic methods for these oligonucleotide derivatives described in [7, 8] imply the introduction of 3'-terminal carboxyl group in oligonucleotide through a modification of polymeric support by an additional nonnucleotide link. In [7], subsequent interactions with peptides and fluorescent labels were carried out in aqueous solution after removal of the oligonucleotide from the polymeric support. Hovinen, Guzaev, et al. [8] proposed an in situ method of synthesis of such conju- gates when oligonucleotide was removed from poly- meric support by the corresponding nucleophilic agent. Carboxyl group is introduced at 5'-terminus of an oligonucleotide either by attachment of a modified unit 1 Corresponding author; phone: +7 (095) 939-3148; fax: +7 (095) 939-3181; e-mail: oretskaya@belozersky.msu.ru 2 Abbreviations: HBTU, O-(1H-benzotriazole-1-yl)-N,N,N',N'-tet- ramethyluronium hexafluorophosphate; HOBt, N-hydroxybenzo- triazole; and rpHPLC, reversed-phase high performance liquid chromatography. during automated synthesis [9] or by formation of the functional group after termination of the elongation of oligonucleotide chain and complete deprotection of the oligonucleotide [10]. Subsequent interaction with the nucleophilic component was carried out in solution, which could result in low yields, an increased reaction time, and formation of by-products. In this connection, the method of solid phase synthesis of oligonucleotide derivatives appears to be more convenient due to sim- plicity of the reaction procedure and the removal of excessive reagents. For example, Guzaev et al. [11, 12] introduced into 5'-terminus of oligonucleotide chain a modified unit containing an active ester of carboxylic acid. Then nucleophilic components were covalently attached to the modified oligonucleotides before their removal from the polymeric support. The disadvan- tages of this method are a long time of conjugation (6– 12 h) [11], instability of the modified phosphoamidites on storage, and formation of by-products during conju- gation [12]. The goal of this study is the solid phase chemical synthesis of conjugates of oligonucleotides containing 5'-terminal carboxyl group with various amines and short peptides (Table 1). It was also of interest to find a correlation between the retention times of the resulting compounds in ion-pair rpHPLC and their physico- chemical characteristics. This would enable a rapid and reliable estimate of the efficiency of a modified oligo- nucleotide coupling with the corresponding nucleo- philic component and indirectly confirm the structure of the resulting compound. Solid Phase Synthesis and Chromatographic Characteristics of Nucleophilic Agents Conjugated with Oligonucleotides Containing 5'-Terminal Carboxyl Group A. V. Kachalova*, V. N. Tashlitsky*, D. A. Stetsenko**, E. A. Romanova*, M. J. Gait**, and T. S. Oretskaya** 1 *Faculty of Chemistry and Belozersky Institute of Physicochemical Biology, Moscow State University, Vorob’evy gory, Moscow, 119992 Russia **Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge, CB2 2QH, United Kingdom Received, March 6, 2002; in final form, June 7, 2002 Abstract—Conjugates of amines or short peptides with oligonucleotides containing 5'-terminal carboxyl group were prepared by solid phase chemical synthesis. A correlation between the physicochemical parameters and retention times of the synthesized conjugates was established by ion-pair reversed-phase HPLC. Key words: ion-pair reversed-phase HPLC, modified oligodeoxyribonucleotides, oligonucleotidopeptides