CHIRAL CHEMICAL SYNTHESIS OF DNA CONTAINING (S)-9-(1,3-DIHYDROXY-2-PROPOXYMETHYL)GUANINE (DHPG) AND EFFECTS ON THERMAL-STABILITY, DUPLEX STRUCTURE, AND THERMODYNAMICS OF DUPLEX FORMATION

The antiviral compound 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG, Ganciclovir) is used clinically to treat cytomegaloviral infections i n immunocompromised hosts and more recently is being investigated as a chemotherapeutic agent to be used in combination with retroviral gene therapy. Structurally, DHPG, an analog of guanosine, lacks the 2'-deo xyribose carbon atom and is acyclic. It is therefore prochiral at the 4'-deoxyribose carbon, having both pro-R and pro-S isomers. This stere ochemistry is critical during biochemical conversions. DHPG retains th e equivalent of 3'-hydroxyl and 5'-hydroxyl moieties. These can potent ially support not only initial misincorporation of DHPG into DNA but a lso subsequent nucleotide addition. The mechanism of DHPG antiviral ac tion may thus not strictly be through chain terminations. To investiga te the structural and biochemical consequences of incorporation of DHP G into DNA, with particular attention to the relative contributions of the deoxyribose sugar to the overall structure and stability of DNA, we have developed a methodology for the chiral chemical synthesis of D NA oligomers containing DHPG. The stereochemistry of the DHPG phosphor amidite was established by a stereoselective acetyl transfer reaction catalyzed by porcine pancreatic lipase. The DNA resisted enzymatic dig estion at DHPG sites. Circular dichroism and copper phenanthroline cle avage studies indicated that the incorporation of DHPG into DNA does n ot significantly perturb the global B-conformation structure. Detailed thermodynamic investigations into DNA containing DHPG revealed reduce d thermal stability, as evidenced by a decrease in melting temperature , with significant alteration of the enthalpy, entropy, and free energ y of duplex formation. These data demonstrate that an intact deoxyribo se ring significantly contributes to the stability of a DNA duplex.