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
Sj. Marshalko et al., 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, Biochemistry, 34(28), 1995, pp. 9235-9248
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.