Synthesis and Hybridization Properties of DNA±PNA Chimeras Carrying 5-Bromouracil and 5-Methylcytosine Elisenda Ferrer, Anna Shevchenko and Ramon Eritja* European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117 Heidelberg. Germany Received 22 June 1999; accepted 8 September 1999 AbstractÐThe preparation of 5-bromouracil and 5-methylcytosine peptide nucleic acid (PNA) monomers is described. These PNA monomers were used for the preparation of several DNA±PNA chimeras and their hybridization properties are described. The substitution of cytosine by 5-methylcytosine in DNA±PNA chimeras increased duplex stability while substitution of thymine by 5- bromouracil maintained it. Moreover, binding of DNA±PNA chimeras to double-stranded DNA to form triple helices was studied. In contrast to DNA, the presence of 5-methylcytosine and 5-bromouracil in DNA±PNA chimeras destabilized triple helix. # 2000 Elsevier Science Ltd. All rights reserved. Introduction Peptide nucleic acids (PNA) are oligonucleotide ana- logues in which the sugar-phosphate backbone has been replaced by N-(2-aminoethyl)glycine units. 1 In spite of the change in the backbone structure, PNA molecules bind strongly to complementary DNA and RNA sequences, 2 and they are achiral and uncharged. For these reasons PNA oligomers have a strong potential as therapeutic agents, diagnostic tools and probes in molecular biology. 3 Although PNA oligomers have excellent binding prop- erties, they show poor solubility and a tendency to self- aggregation. 4 Some of these problems have been solved by preparing DNA±PNA chimeras. These chimeras are more water soluble, maintain the ability of DNA to activate RNase H and retain some of the enhanced binding and nuclease resistance properties of PNA. 5±12 The preparation of DNA±PNA chimeras has been described by several authors. 5±12 The most common strategy is to use the MMT group for the temporary protection of the backbone amino function, and acyl groups for the exocyclic amino functions of the nucleo- bases. 5±8 The MMT is removed under mild conditions (3% TCA in DCM), and the nucleobase-protecting groups are removed using concentrated aqueous ammonia. These conditions are similar to those used in DNA synthesis allowing the preparation of PNA±DNA chimeras. 5±12 We report the preparation of DNA±PNA chimeras carrying 5-bromouracil and 5-methylcytosine. Substitu- tion of cytosine by 5-methylcytosine in oligonucleotides increases the stability of both double 13,14 and triple 15,16 helices. Oligonucleotides carrying 5-bromouracil are used in photo-cross-linking experiments, 17 in X-ray dif- fraction studies, 18 as non-radioactive labels 19 and for triple-helix stabilization. 16,20 The eect of the incorpora- tion of 5-bromouracil and 5-methylcytosine in PNA oli- gomers or DNA±PNA chimeras on these properties remains to be determined. A PNA oligomer carrying 5- bromouracil has been prepared 21 using Boc-chemistry, which is, however, incompatible with the synthesis of DNA±PNA chimeras. Recently, a new strategy has been developed for the preparation of MMT-protected PNA monomers which 0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0968-0896(99)00308-9 Bioorganic & Medicinal Chemistry 8 (2000) 291±297 Keywords: 5-bromouracil; duplex stability; 5-methylcytosine; PNA; PNA±DNA chimeras. Abbreviations: ACN: acetonitrile; AcOEt: ethyl acetate; AcOH: acetic acid; Boc: tert-butoxycarbonyl; DBU: 1,8-diazabicyclo[5.4.0]undec-7- ene; DCM: dichloromethane; DIP: diisopropylcarbodiimide; DIPEA: diisopropylethylamine; DMAP: 4-N,N-(dimethylamino)pyridine; DMF: N,N-dimethylformamide; DMT: dimethoxytrityl; Et 3 N: trie- thylamine; HATU: N-[(dimethylamino)-1H; 1,2,3-triazolo-[4,5-b]pyr- idin-1-yl-methylene]-N-methylmethanaminium hexa¯uorophosphate N-oxide; HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine; MMT: monomethoxytrityl; MeOH: methanol; PEG-PS: poly- ethyleneglycol-polystyrene; PNA: peptide nucleic acid; TCA: tri- chloroacetic acid; TFA: tri¯uoroacetic acid; THF: tetrahydrofuran; . *Corresponding author at present address: Centro de Investigacio n y Desarrollo, C.S.I.C., Jordi Girona 18-26, E-08034 Barcelona, Spain. Tel.: +34-93-4006145; fax: +34-93-2045904; e-mail: recgma@ cid.csic.es