A parallel synthesis scheme for generating libraries of DNA polymerase substrates and inhibitors.
We report a combinatorial approach aimed at producing in a single step a large family of nucleoside triphosphate derivatives that could be tested for their ability to be substrates for DNA polymerases. We propose as a unique triphosphate building block a nucleotide with a hydrazine function anchored to an imidazole ring. Condensation between the 5'-triphosphate derivative of 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide (dY(NH(2))TP) and any aldehyde or ketone, followed by reduction of the intermediate hydrazones dXmTP, resulted in the corresponding hydrazides (dXnTP). Following this scheme, a series of aldehydes having various aromatic parts yielded a number of adducts dY(NHR)TP. Vent (exo-) DNA polymerase is found to be able to catalyse the single incorporation of these bulky triphosphate derivatives. Subsequent extensions of the modified pairs with canonical triphosphates resulted mainly in abortive elongations at primer+2, except after the incorporation of dY(NHben)TP and, to a lesser extent, dY(NHphe)TP opposite C. These results illustrate the potential of this parallel synthetic scheme for generating new substrates or inhibitors of replication in a single step.
Strobel
Heike
H
Institut Pasteur, Unité de Chimie Organique (URA CNRS 2128), 28 rue du Docteur Roux, 75724 Paris cedex 15, France.
Dugué
Laurence
L
Marlière
Philippe
P
Pochet
Sylvie
S
D017931:DNA Primers / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015723:Gene Library; D006834:Hydrazines; D019384:Nucleic Acid Synthesis Inhibitors; D009711:Nucleotides / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D013379:Substrate Specificity
D017931:DNA Primers; D006834:Hydrazines; D019384:Nucleic Acid Synthesis Inhibitors; D009711:Nucleotides; C070331:Tli polymerase; D004259:DNA-Directed DNA Polymerase