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 in 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'-deoxyribose carbon atom and is acyclic. It is therefore prochiral at the 4'-deoxyribose carbon, having both pro-R and pro-S isomers. This stereochemistry is critical during biochemical conversions. DHPG retains the equivalent of 3'-hydroxyl and 5'-hydroxyl moieties. These can potentially support not only initial misincorporation of DHPG into DNA but also subsequent nucleotide addition. The mechanism of DHPG antiviral action may thus not strictly be through chain terminations. To investigate the structural and biochemical consequences of incorporation of DHPG 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 DNA oligomers containing DHPG. The stereochemistry of the DHPG phosphoramidite was established by a stereoselective acetyl transfer reaction catalyzed by porcine pancreatic lipase. The DNA resisted enzymatic digestion at DHPG sites. Circular dichroism and copper phenanthroline cleavage studies indicated that the incorporation of DHPG into DNA does not significantly perturb the global B-conformation structure. Detailed thermodynamic investigations into DNA containing DHPG revealed reduced thermal stability, as evidenced by a decrease in melting temperature, with significant alteration of the enthalpy, entropy, and free energy of duplex formation. These data demonstrate that an intact deoxyribose ring significantly contributes to the stability of a DNA duplex.
Marshalko
S J
SJ
Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Schweitzer
B I
BI
Beardsley
G P
GP
D001665:Binding Sites; D004247:DNA / Q000138:chemical synthesis / Q000737:chemistry*; D018736:DNA Adducts / Q000138:chemical synthesis / Q000737:chemistry; D004355:Drug Stability; D015774:Ganciclovir / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D013237:Stereoisomerism; D013816:Thermodynamics
D018736:DNA Adducts; D004247:DNA; D015774:Ganciclovir