Solid-Phase Synthesis of Deoxynucleic Guanidine (DNG) Oligomers and Melting Point and Circular Dichroism Analysis of Binding Fidelity of Octameric Thymidyl Oligomers with DNA Oligomers

Abstract

A practical solid-phase synthesis of deoxynucleic guanidine (DNG), a positively charged DNA backbone analogue, is reported. The nucleoside coupling step in the solid-phase synthesis of DNG involves the attack of a terminal 3‘-amine upon an electronically activated 5‘-carbodiimide to create a protected guanidinium internucleoside linkage. The activated carbodiimide is synthesized in situ by the mercury(II) abstraction of sulfur from an unsymmetrically substituted thiourea in which one substituent is an electron-withdrawing protecting group and the other is the 5‘-nucleoside monomer. This produces, in addition to the carbodiimide, a mercury sulfide precipitate which accumulates in the pores of the solid support, restricting solvent and reagent access and reducing the coupling yields with each successive cycle. This obstacle is overcome by a simple washing step involving a thiophenol solution which readily removes the mercury salt. The addition of this step to the cycle enables DNG oligomers to be synthesized using standard macroporous SPS supports. Coupling yields of 98% were estimated from the HPLC analysis of the product mixtures. An octameric thymidyl oligomer (II) was synthesized and the fidelity of binding to octameric adenyl DNA oligomers containing cytidyl mismatches was determined. Binding was studied by thermal denaturation (Tm), Job plots, and circular dichroism spectrophotometry. The DNG oligomer (II) formed a 2:1 complex with octameric adenyl DNA (III) with a melting temperature of 63 °C. Each cytidyl mismatch induced a penalty of 4 to 5 °C in the observed melting temperatures. DNA sequences with four or more mismatches showed no base pairing in the presence of II. No association was observed between II and octameric cytidyl DNA. These observations demonstrate that DNG oligomers of moderate length are able to discriminate between complementary and mismatched DNA oligomers.