Microwave-Mediated Synthesis of Labeled Nucleotides with Utility in the Synthesis of DNA Probes

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-aminodeoxycyticline 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.