title: Two novel dATP analogs for DNA photoaffinity labeling.
abstract: Two new photoreactive dATP analogs, N(6)-[4-azidobenzoyl-(2-aminoethyl)]-2'-deoxyadenosine-5'-triphospha+ ++ te (AB-dATP) and N(6)-[4-[3-(trifluoromethyl)-diazirin-3-yl]benzoyl-(2-aminoethyl) ]-2 '-deoxyadenosine-5'-triphosphate (DB-dATP), were synthesized from 2'-deoxyadenosine-5'-monophosphate in a six step procedure. Synthesis starts with aminoethylation of dAMP and continues with rearrangement of N(1)-(2-aminoethyl)-2'-deoxyadenosine-5'-monophosphate to N(6)-(2-aminoethyl)-2'-deoxyadenosine-5'-monophosphate (N(6)-dAMP). Next, N(6)-dAMP is converted into the triphosphate form by first protecting the N-6 primary amino group before coupling the pyrophosphate. After pyrophosphorylation, the material is deprotected to yield N(6)-(2-aminoethyl)-2'-deoxyadenosine-5'-triphosphate (N(6)-dATP). The N-6 amino group is subsequently used to attach either a phenylazide or phenyldiazirine and the photoreactive nucleotide is then enzymatically incorporated into DNA. N(6)-dATP and its photoreactive analogs AB-dATP and DB-dATP were successfully incorporated into DNA using the exonuclease-free Klenow fragment of DNA polymerase I in a primer extension reaction. UV irradiation of the primer extension reaction with AB-dATP or DB-dATP showed specific photocrosslinking of DNA polymerase I to DNA.
authors.affiliation: Department of Biochemistry and Molecular Biology, Southern Illinois University School of Medicine, Room 229C, Neckers Building, 1245 Lincoln Drive, Carbondale, IL 62901-4413, USA.
authors.forename: M
authors.initials: M
authors.lastname: Zofall
authors.forename: B
authors.initials: B
authors.lastname: Bartholomew
mesh_terms: D000225:Adenine / Q000737:chemistry / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D001483:Base Sequence; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism; D011756:Diphosphates / Q000378:metabolism; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001386:Azides; D004268:DNA-Binding Proteins; D003838:Deoxyadenine Nucleotides; D003839:Deoxyadenosines; D011756:Diphosphates; D007202:Indicators and Reagents; D019920:Photoaffinity Labels; D000249:Adenosine Monophosphate; C014747:phenylazide; D004247:DNA; D004256:DNA Polymerase I; D000225:Adenine; C026600:2'-deoxyadenosine triphosphate