Russian Journal of Bioorganic Chemistry, Vol. 30, No. 1, 2004, pp. 98–99. Translated from Bioorganicheskaya Khimiya, Vol. 30, No. 1, 2004, pp. 110–112. Original Russian Text Copyright © 2004 by Sinyakov, Feshchenko, Ryabinin.
LETTERS TO THE EDITOR
A Liquid-Phase Synthesis of DNA-Binding Polyamides Using Oligocarboxamide Blocks A. N. Sinyakov*, M. V. Feshchenko**, and V. A. Ryabinin*1 *Institute of Molecular Biology, Vector State Research Center of Virology and Biotechnology, pos. Kol’tsovo, Novosibirsk oblast, 630559 Russia **Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, pr. akademika Lavrent’eva 8, Novosibirsk, 630090 Russia Received August 13, 2003; in final form, August 18, 2003
Abstract—A method was developed for the synthesis of sequence-specific polyamides on the basis of 4-amino-1-methylpyrrole-2-carboxylic acid, 4-amino-1-methylimidazole-2-carboxylic acid, β-alanine, and γ-aminobutyric acid. Dimeric and trimeric oligocarboxamides were used as building blocks. Our synthetic scheme was applied for the synthesis of DNA minor groove binders containing up to twelve carboxamide units. Key words: distamycin, minor groove binder, liquid-phase synthesis, sequence-specific polyamides 1
Polyamides on the basis of N-methylpyrrole and Nmethylimidazole and structurally related to the natural compounds netropsin and distamycin are of a great interest for the design of ligands that are bound to the DNA minor groove in a sequence-specific manner (cf., e.g., [1]).2 At present, the solid-phase technique is the most effective for their synthesis [2–5]. Liquid-phase synthesis helps obtain ligands in larger quantities in comparison with the solid-phase techniques but it is more time-consuming and requires a chromatographic purification of intermediate oligocarboxamides. We have recently described a fast and efficient variant of the solid-phase synthesis of polyamides using di- and tricarboxamides as building blocks [5], which enabled a cutdown of the time of synthesis and an increase in yields and purity of products. However, all modifications of the solid-phase method require a large excess of activated component (no less than three equiv) to reach high yields at each cycle of the oligocarboxamide chain elongation. The usual removal of the synthesized oligocarboxamide from the solid support uses 1,1-dimethyl-1,3-diaminopropane, which prevents the subsequent ë-terminal modification of the ligand, whereas the postsynthetic alkaline treatment of the support releases the ligand in the form of carboxylic acid in a low yield. Moreover, the solid support with immobilized Boc-β-alanine (4-carbonylaminomethyl)benzyl ester commonly used in the solid phase synthesis [2–5] 1 Corresponding
author; phone: (3832) 30-4653; e-mail:
[email protected] 2 Abbreviations: HOBt, N-hydroxybenzotriazole; Im, 4-aminoimidazole-2-carboxylic acid residue; Py, 4-aminopyrrole-2-carboxylic acid residue; β, β-alanine residue; and γ, γ-aminobutyric residue.
determines the ligand structure: β-alanine residue is necessarily present at the C-terminus of the molecule. In this work, we studied a liquid-phase synthesis of oligocarboxamides using di- and tricarboxamides as building blocks. As in [5], we synthesized hairpin ligands composed of two tetrameric or pentameric fragments containing imidazole, pyrrole, and β-alanine residues linked with each other through γ-aminobutyric acid residues. In general, dimeric and trimeric compounds were used as building blocks. The blocks were chosen so that hydroxybenzotriazolide of pyrrolecarboxylic acid was not brought to the reaction with imidazole amino group, since this coupling reaction is not sufficiently rapid. Hydroxybenzotriazolides either obtained in the course of synthesis or preliminarily prepared [5] were used in the reaction of the oligocarboxamide chain elongation. The scheme of synthesis of a hairpin oligocarboxamide composed of two pentacarboxamide moieties joined by a linker (γ-aminobutyric acid), is given as an example. The starting monomer was an amino groupcontaining tricarboxamide. It was treated with an appropriate hydroxybenzotriazolide in DMF (1.2-fold excess) in the presence of triethylamine (4 equiv) for 2– 5 h at room temperature (TLC monitoring). The reaction mixture was diluted with water, and the precipitate was separated, dried, dissolved in chloroform, and applied onto a silica gel column. This was eluted with chloroform and 5% ethanol in chloroform to remove dicyclohexylurea and hydroxybenzotriazolide excess. The target product was eluted with 10–20% EtOH in CHCl3 in dependence on the length of oligocarboxamide chain. The eluate was evaporated, and the protective Boc group was removed by the treatment with 1 : 2 TFA–CH2Cl2 mixture for 30–40 min. The obtained
1068-1620/04/3001-0098 © 2004 MAIK “Nauka /Interperiodica”
A LIQUID-PHASE SYNTHESIS H N
99
H-β–ImPyOEt
Py = N
Boc-γ-PyPyOBt; (DMF, NEt3) TFA/CH2Cl2;
CO
H–γ–PyPy-β–ImPyOEt H N Im =
Boc-β-ImImOBt; (DMF, NEt3) TFA/CH2Cl2;
N N
β = –NH(CH2)3CO– γ = –NH(CH2)3CO–
HOBr =
H–β–ImIm–γ–PyPy-β–ImPyOEt
CO
Boc-γ-PyImOBt; (DMF, NEt3) NaOH/EtOH
Boc-γ–PyPy-β–ImIm-γ–PyPy-β–ImPy–OH
OH N N N
Scheme of synthesis of oligocarboxamide Boc-γ-PyPy-β-ImIm-γ-PyPy-β-ImPy-OH
amine was precipitated with diethyl ether, filtered, and dried. The chain elongation cycle was repeated as many times as required. When the last fragment was linked, the Boc group was not removed; the ethyl ester was instead saponified to the respective acid by an alkaline solution in aqueous alcohol according to [8]. In the above-described example, the oligocarboxamide was obtained after three elongation cycles instead of twelve cycles required in the conventional approach. A number of other ligands were synthesized according to the described scheme: Boc-γ-PyPy-β-PyIm-γPyPy-β-PyPy-OH, Boc-γ-PyPyPy-γ-PyPyPy-OH, Boc-γ-PyImImPy-γ-PyPyPyPy-OH, Boc-γ-ImPy-βImIm-γ-PyPy-β-ImPy-OH, Boc-γ-PyPyImIm-γ-PyPyPyIm-OH, and Boc-γ-PyPyPyIm-γ-PyPyPyPy-OH. They were used for obtaining derivatives with various ë-terminal substituents. In particular, Boc-γ-PyPy-βImIm-γ-PyPy-β-ImPy-OBt was treated with 1,1-dimethyl-1,3-diaminopropane with subsequent removal of the Boc group, and H-γ-ImPy-β-ImIm-γ-PyPy-βPyIm-NH(CH2)3N(CH3)2 was obtained (see the scheme). The yield per cycle of the chain elongation was 40–75%, and the total yield varied from 25 to 45%. Our method enables the obtaining of sufficiently large quantities of product; for example, we have synthesized nearly 1 g of the Boc-γ-PyPyPy-γ-PyPyPy-OH ligand. The structures of the resulting compounds were confirmed by NMR and mass spectra (data not shown). ACKNOWLEDGMENTS We are grateful to Jean-Paul Brouard and Lionel Dubost (Laboratoire de Chimie, Musée Nationale
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
d’Histoire Naturelle, Paris) for registration of MALDITOF mass spectra. This work was supported by INTAS (project no. 010638), the International Project Conjugates of Modified Oligonucleotides with the Minor Groove Ligands, Intercalators, and Topoisomerase Inhibitors: Stabilization of Triple Helix and Directed Modification of Double-Stranded DNA (project no. 04.70043-69), and Ministry of Foreign Affairs (France) (project EGIDE 04542ND). REFERENCES 1. Dervan, P.B. and Edelson, B.S., Curr. Opin. Struct. Biol., 2003, vol. 13, pp. 284–299. 2. Baird, E.E. and Dervan, P.B., J. Am. Chem. Soc., 1996, vol. 118, pp. 6141–6146. 3. Wurtz, N.R., Turner, J.M., Baird, E.E., and Dervan, P.B., Org. Lett., 2001, vol. 3, pp. 2101–2103. 4. Krutzik, P.O. and Chamberlin, R., Bioorg. Med. Chem. Lett., 2002, vol. 12, pp. 2129–2132. 5. Sinyakov, A.N., Feshchenko, M.V., and Ryabinin, V.A., Bioorg. Khim., 2003, vol. 29, pp. 449–451. 6. Grehn, L. and Ragnarsson, U., J. Org. Chem., 1981, vol. 46, pp. 3492–3497. 7. Vazquez, E., Caamano, A.M., Castedo, L., and Mascarenas, J.L., Tetrahedron Lett., 1999, vol. 40, pp. 3621– 3624. 8. Ryabinin, V.A. and Sinyakov, A.N., Bioorg. Khim., 1998, vol. 24, pp. 601–607.
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