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 electronwithdrawing 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.

Backbone Modification of Nucleic Acids: Synthesis, Structure and Therapeutic Applications

“…Kinetic studies have also revealed that the rate of triple helix formation between DNG (T 5 ) with short oligodeoxyadenylates, is an order of magnitude faster than the formation of DNA triplexes at similar salt concentrations [146]. Also, investigations with DNG (T 5 and T 8 ), show that T m 's are significantly reduced by base pair mismatches [147,148]. This suggest that sequence specific base pairing is the predominant mode of binding between DNG and nucleic acid targets, although it has still to be established whether or not this is accompanied by any non-specific binding, via salt bridges, between the oppositely charged backbones.…”

“…Such studies appear close at hand with the development of an efficient solid phase synthetic method for the synthesis of DNG (Fig. 18) [148]. The synthesis involves the coupling of a 3´-amino nucleoside 58, immobilised on a polystyrene resin, with a 5´-carbodiimide intermediate which is derived from the thiourea 59, upon treatment with HgCl 2 .…”

Recent Developments in the Chemistry and in the Biological Applications of Amidoximes

“…From the plethora of modifications reported hitherto, oligonucleotides with the amide linkage 385 (Scheme 62) have shown very promising antisense properties by displaying higher binding affinity and very good resistance towards nucleases [339,340]. Oligomers with positively charged guanidine [341][342][343][344][345][346] linker 386 lead in the development of new antisense therapeutics, since the attractive forces between them and the negatively charged DNA or RNA contribute significantly to the stability of heteroduplex and triplex structures formed between these species. In addition, they are stable to enzymatic hydrolysis due to the lack of a phosphodiester linkage.…”

Solid‐Phase Synthesis of Guanidinium Derivatives from Thiourea and Isothiourea Functionalities

“…[71Ϫ73] Replacing the phosphate backbone with positively charged guanidinium groups [74] increases the binding of these analogs and makes them resistant to nucleases. [76] Bruice and co-workers have reported the synthesis of deoxynucleotide guanidine oligomers (Scheme 6). [76] Bruice and co-workers have reported the synthesis of deoxynucleotide guanidine oligomers (Scheme 6).…”

“…[75] The positively charged guanidinium might also give rise to cell membrane permeability by electrostatic attraction of the oligonucleoside to the negatively charged phosphate groups on the cell surface. [76] At first, the resin 6A was loaded with protected and modified nucleotide 6B in the presence of HgCl 2 in DMF to create guanidine 6C. [76] At first, the resin 6A was loaded with protected and modified nucleotide 6B in the presence of HgCl 2 in DMF to create guanidine 6C.…”