Journal of the American Chemical Society

Squaryl group as a new mimic of phosphate group in modified oligodeoxynucleotides: synthesis and properties of new oligodeoxynucleotide analogues containing an internucleotidic squaryldiamide linkage.

PMID 12392419


This paper describes the synthesis and properties of a new type of modified oligodeoxynucleotide containing a neutral but highly polarized squaryl group as a novel mimic of the phosphate group. A modified thymidine dimer derivative (TsqT) having a squaryldiamide linkage was synthesized in almost quantitative yield by a two-step substitution of diethyl squarate with 3'-amino-5'-O-(4,4'-dimethoxytrityl)-3'-deoxythymidine and 5'-amino-5'-deoxythymidine. The CD and UV studies of TsqT suggest that this dimer has basically a structure similar to that of TpT. The NMR studies of TsqT show a unique property, namely, that the squaryl group of TsqT is influenced by Mg2+ concentration. The ab initio calculations of TsqT showed a highly polarized structure resembling that of a phosphate group. This dimer structural motif was finally incorporated into oligodeoxynucleotides by use of the phosphoramidite approach. The hybridization affinity of these modified oligodeoxynucleotides for the complementary and mismatched oligodeoxynucleotides was studied in detail by using Tm experiments. Consequently, it turned out that in a matched duplex of 5'-d(CGCATsqTAGCC)-3'/5'-d(GGCTAATGCG)-3' the A-T base pairs at the modified site can be preserved, but instead thermal destabilization of the overall structure was observed. To estimate the structure of the duplex, two kinds of fluorescein chromophores (fluorescein (FL) and cyanine 3 (Cy3)) were introduced into the 5'-terminal site of 5'-d(GACGCATsqTAGCCGAT)-3' and 5'-d(ATCGGCTAATGCGTC)-3', respectively. The fluorescence resonance energy transfer experiments using these functionalized oligodeoxynucleotides suggest that the matched duplexes have a bent structure at the modified site. This conclusion was also strongly supported by computational MM and MD simulations.

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