title: Microwave-mediated synthesis of labeled nucleotides with utility in the synthesis of DNA probes.
abstract: A novel method of linking haptens to deoxycytidine 5'-triphosphate via microwave-mediated bisulfate-catalyzed transamination with hydrazine has been developed. This method enables the tethering of small molecule haptens to dCTP via a discrete polyethylene glycol (PEG) spacer, yielding N(4)-aminodeoxycytidine 5'-triphosphate-dPEG-haptens. This synthetic approach employs microwave-catalyzed hydrazinolysis that enables the attachment of spacers via hydrazine linkages. The microwave-mediated introduction of this hydrazine handle provides a significant improvement in yield over those of published thermal methods. The microwave reaction was shown to be scalable, and the final product was amenable to labeling with a wide variety of haptens. The resulting nucleotide triphosphates, N(4)-aminodeoxycytidine 5'-triphosphate-dPEG-haptens, can serve as unique substrates for the enzyme-mediated labeling of DNA probes. The efficacy of incorporation of one such novel nucleotide, N(4)-aminodeoxycytidine 5'-triphosphate-dPEG(4)-DNP, has been demonstrated in nick translation labeling of HER2 and HPV probes. The labeled probes have been shown to be effective in visualizing their target genes in tissue.
authors.affiliation: Ventana Medical Systems, Inc., Tucson, Arizona 85755, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Lefever
authors.forename: Jerome W
authors.initials: JW
authors.lastname: Kosmeder
authors.forename: Michael
authors.initials: M
authors.lastname: Farrell
authors.forename: Christopher
authors.initials: C
authors.lastname: Bieniarz
mesh_terms: D015342:DNA Probes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000138:chemical synthesis* / Q000737:chemistry*; D006241:Haptens / Q000737:chemistry; D006801:Humans; D008872:Microwaves; D027383:Papillomaviridae / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D012995:Solubility; D013194:Staining and Labeling; D014867:Water / Q000737:chemistry
chemical_list: D015342:DNA Probes; D003845:Deoxycytosine Nucleotides; D006241:Haptens; D011092:Polyethylene Glycols; D018719:Erb-b2 Receptor Tyrosine Kinases; D014867:Water; C024107:2'-deoxycytidine 5'-triphosphate