Showing papers by "Daniel M. Brown published in 2000"

Synthesis and Incorporation of Pyrrole Carboxamide Nucleoside Triphosphates by DNA Polymerases

Abstract: We have synthesised and examined the enzymatic incorporation properties of the 5′-triphosphates of 2′-deoxyribosyl pyrrole 3-monocarboxamide (dMTP) and 2′-deoxyribosyl pyrrole 3,4-dicarboxamide (dDTP). These analogues we had hoped would behave as ambivalent base analogues in that they can present two alternative hydrogen-bonding faces either by rotation about the carboxamide group or about the glycosidic bond. The two pyrrole derivatives, dMTP and dDTP, exhibit a preference for incorporation with Klenow polymerase. They are preferentially incorporated as either A or C.

The Synthesis of Bicyclic N4‐Amino‐2′‐deoxycytidine Derivatives.

Abstract: Nucleosides which have ambivalent tautomeric properties have value in a variety of nucleic acid hybridization applications, and as mutagenic agents. We describe here synthetic studies directed to stable derivatives of this kind of nucleoside based on N4-aminocytosine. Treatment of the 4-(1H-1,2,4-triazol-1-yl)-5-(chloroethyl)pyrimidinone nucleoside derivative 5 with hydrazine leads to formation of the 6,6-bicyclic pyrimido-pyridazin-7-one 3, and with methylhydrazine to the corresponding fixed tautomeric 1-methyl derivative 7 (Scheme 1). If these cyclization reactions are carried out in the presence of a base, the 6-ring bicyclic derivatives undergo rearrangement to their corresponding 5-ring pyrrolo-pyrimidin-2-one analogues 8 (Scheme 2). In the reaction of the triazolyl derivative 5 with 1-[(benzyloxy)carbonyl]-1-methylhydrazine, spontaneous cyclization gives the 5-ring derivative 13 related to 8 rather than the open-chain product 12 (Scheme 4). Reaction of an acetylated analogue of triazolyl derivative 5 with 1,1-dimethylhydrazine gives rise to some of the open-chain product 9, but it too cyclizes to a product that we have assigned the structure of the 6,6-ring quaternary ammonium salt 11 (Scheme 3).

Random Mutagenesis Using 2‐Amino‐9‐(2‐deoxy‐β‐D‐ribofuranosyl)purine‐5′‐triphosphate and the Polymerase Chain Reaction.

Abstract: Base analogues offer an attractive method for mutagenising DNA in combination with the polymerase chain reaction (PCR). We have synthesised the 5′-triphosphate-2′-deoxyribosyl derivative of 2-aminopurine (dAPTP), one of the first base analogues to be used for mutagenesis, and examined its utility in PCRs. An E. coli amber suppressor gene, supF, was used as a template for mutagenesis. The analogue induced exclusively transition mutations, but at a low frequency, consistent with its weak mutagenicity in vivo.