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PRELIMINARY RESULTS ON THE SYNTHESIS OF METAL COMPLEX-PNA CONJUGATES Felix Zelder, Andriy A. Mokhir, Roland Krämer, Igor Fritsky Ruprecht-Karls-Universität Heidelberg, Anorganisch-Chemisches Institut Im Neuenheimer Feld 270, D-69120 Heidelberg. E-mail: [email protected] RESULTS ABSTRACT Nonenzymatic hydrolysis of the phosphodiester backbone of nucleic acids is an attractive research aim in molecular biology, but the sequence specific hydrolysis of ssDNA was achieved only in one case with a conjugate consisting of a Ce(IV)-complex attached to a DNA-oligonucleotide.[1] The nonenzymatic sequence specific hydrolysis of dsDNA is not reported. Since peptide nucleic acids show interesting binding properties we are focussing on the design of metal-complex-PNAs in a way that allows the generation of combinatorial libraries. Their synthesis and characterization will be discussed. INTRODUCTION Restriction enzymes that hydrolyse the phosphodiester backbone of DNA play an important role in biological processes and in biotechnology. These enzymes recognize specific sequences of 4-8 base pairs. n+ M Here we report on the synthesis of some conjugates of PNA (peptide nucleic acids) (blue) with a varity of polydentate metal- binding fragments (yellow) , which can hydrolyse the phosphodiester bonds of target oligonucleotides (black). n+ M Coupling of carboxylic acids onto 5’-terminus of PNA was accomplished using an activation mixture of HBTU, HOBT, and DIEA and protected carboxylic acids. Yields were consistently higher then 90 %. After treatment with different transition metal ions the chemical stability of the modified PNA was determined by MALDI- TOF MS. Metal-complex PNA conjugates were analysed by MALDI- TOF MS. POLYDENTATE LIGANDS ATTACHED TO 5´- TERMINUS OF PNA Fig. 1: Mechanism of sequence specific cleavage of polynucleotides with artificial nucleases. n+ M Although artificial restriction enzymes offer unlimited sequence specifity and better chemical stability, only one case is reported in which sequence specific hydrolysis of ssDNA was achieved by a conjugate consisting of a Ce(IV)complex attached to a DNA- oligonucleotide.[1] NAE n+ M N n+ n+ M M N N O N N N N O O n+ M O O O N Tetradentate ligands Bidentate ligands SOLID PHASE SYNTHESIS OF POTENTIAL SEQUENCE SPECIFIC NUCLEASES R: PNA (peptide nucleic acids) T T O NH C O O N N H2N N O N N NH N O NH O O HN N O N O NH O O HN N O N N HN T T C T T C T T T C NH2 R O T TBhoc C T T Bhoc C T T T CBhoc T TBhoc C T T CBhoc T T T CBhoc N HN 1 2 R T T C T T C T T T C N OH O HO N HO O Fig. 2: 1; RCOOH, HBTU, HOBT, DIEA, DMF. 2; TFA/ m-Cresol. MALDI- TOF MS OF A CONJUGATE OF PNA AND METAL COMPLEX ACKNOWLEDGEMENTS This work is supported by Ruprecht-KarlsUniversität Heidelberg. Authors thank Dr. Gross (Ruprecht-KarlsUniversität Heidelberg) for his kind support with mass spectrometric measurements. O O HO N TTCTTCTTTC H N HO O ZrIV O O O O Fig. 3: MALDI- TOF MS spectra of crude NAE-TTCTTCTTTC [M+H]th+ = 2807,5. and after treatment with Zr(IV) [M+Zr(IV)-3H th] = 2896,3. Uncertainty of m/ z = 0. 1%. O Zr N H N TTCTTCTTTC 2+ LITERATURE [1]M. Komiyama, J. Biochem. 1995, 118, 665- 670.