(Difluoromethylene)phosphates of Guanine Nucleosides as Probes of DNA Polymerases and G Proteins

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Biochemistry, ISSN: 1520-4995, Vol: 29, Issue: 29, Page: 6820-6826

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Arabshahi, Lili; Khan, Naseema N.; Butler, Michelle Marie; Noonan, Timothy; Brown, Neal C.; Wright, George E.
American Chemical Society (ACS)
Biochemistry, Genetics and Molecular Biology; Life Sciences; Medicine and Health Sciences
article description
5′-Polyphosphates of N-(p-n-butylphenyl)-2′-deoxyguanosine and -guanosine which contain a difluoromethylene group in place of a phosphoanhydride oxygen have been synthesized. 5′-[β,γ-(Difluoromethylene)triphosphates], including that of 2′-deoxyguanosine, were prepared by reaction of the corresponding 5′-phosphates, activated by l,l′-carbonyldiimidazole, with difluoromethanediphosphonate. The 5′-[(difluoromethylene)diphosphate] of N-(p-n-butylphenyl)guanosine was prepared by treatment of a protected 5′-tosyl nucleoside with difluoromethanediphosphonate, followed by deprotection. Condensation of this nucleotide, activated with l,l′-carbonyldiimidazole, with orthophosphate gave N-(p-n-butylphenyl)guanosine 5′-[(α,β-difluoromethylene)triphosphate]. Products were characterized byP and F NMR spectroscopy. The phosphonates were tested for their ability to displace [H]GDP from the GTP binding proteins cellular (EC) and oncogenic (Leu-61) Ha-ras p21, and for their ability to inhibit DNA polymerase α from Chinese hamster ovary cells. The p21s bound weakly to a triphosphonate when the CF group was in the β,γ position, but not when it was in the α,β position, and they did not bind to the corresponding (difluoromethylene)diphosphate. In contrast, the CF group had no effect on inhibition of DNA polymerase α by N-(p-n-butylphenyl)-2′-deoxyguanosine 5′-[(β,γ-difluoromethylene)triphosphate]. 2′-Deoxyguanosine 5′-[(β,γ-difluoromethylene)triphosphate] was found to be a bona fide substrate for several DNA polymerases and had a lower apparent K than dGTP with Bacillus subtilis DNA polymerase III. © 1990, American Chemical Society. All rights reserved.