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 N4-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, N4-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, N4-aminodeoxycytidine 5′-triphosphate−dPEG4−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

Structural Requirements for CNS Active Opioid Glycopeptides

Glycopeptides related to β-endorphin penetrate the blood–brain barrier (BBB) of mice to produce antinociception. Two series of glycopeptides were assessed for opioid receptor binding affinity. Attempts to alter the mu-selectivity of [d-Ala²,<i>N</i>-MePhe⁴,Gly-ol⁵]­enkephalin (DAMGO)-related glycopeptides by altering the charged residues of the amphipathic helical address were unsuccessful. A series of pan-agonists was evaluated for antinociceptive activity (55 °C tail flick) in mice. A flexible linker was required to maintain antinociceptive activity. Circular dichroism (CD) in H₂O, trifluoroethanol (TFE), and SDS micelles confirmed the importance of the amphipathic helices (<b>11s</b> → <b>11sG</b> → <b>11</b>) for antinociception. The glycosylated analogues showed only nascent helices and random coil conformations in H₂O. Chemical shift indices (CSI) and nuclear Overhauser effects (NOE) with 600 MHz NMR and CD confirmed helical structures in micelles, which were rationalized by molecular dynamics calculations. Antinociceptive studies with mice confirm that these glycosylated endorphin analogues are potential drug candidates that penetrate the BBB to produce potent central effects

Discovery of 4-Morpholino-6-aryl-1<i>H</i>-pyrazolo[3,4-<i>d</i>]pyrimidines as Highly Potent and Selective ATP-Competitive Inhibitors of the Mammalian Target of Rapamycin (mTOR): Optimization of the 6-Aryl Substituent

Design and synthesis of a series of 4-morpholino-6-aryl-1H-pyrazolo[3,4-d]pyrimidines as potent and selective inhibitors of the mammalian target of rapamycin (mTOR) are described. Optimization of the 6-aryl substituent led to the discovery of inhibitors carrying 6-ureidophenyl groups, the first reported active site inhibitors of mTOR with subnanomolar inhibitory concentrations. The data presented in this paper show that 6-arylureidophenyl substituents led to potent mixed inhibitors of mTOR and phosphatidylinositol 3-kinase α (PI3K-α), whereas 6-alkylureidophenyl appendages gave highly selective mTOR inhibitors. Combination of 6-alkylureidophenyl groups with 1-carbamoylpiperidine substitution resulted in compounds with subnanomolar IC50 against mTOR and greater than 1000-fold selectivity over PI3K-α. In addition, structure based drug design resulted in the preparation of several 6-arylureidophenyl-1H-pyrazolo[3,4-d]pyrimidines, substituted in the 4-position of the arylureido moiety with water solubilizing groups. These compounds combined potent mTOR inhibition (IC50 50 < 1 nM)