Development of novel 19F NMR pH indicators: synthesis and evaluation of a series of fluorinated vitamin B6 analogues.

We have synthesized a series of novel fluorinated vitamin B6 analogues (6-fluoropyridoxol derivatives) as potential 19F NMR pH indicators for use in vivo. Modifications included addition of aldehyde, carboxyl or aminomethyl groups at the 4- or 5-ring position, and examination of a trifluoromethyl moiety as an internal chemical shift standard. The variation in chemical shift with respect to acid-base titration showed pKa values in the range 7.05-9.5 with a chemical shift sensitivity in the range 7.4-12 ppm. Several of the molecules readily cross cell membranes providing estimates of both intra- and extra-cellular pH in whole blood. 6-Fluoropyridoxamine (6-FPAM) exhibits a pKa = 7.05, which is closer to normal physiological pH than the parent molecule 6-fluoropyridoxol (6-FPOL) (pKa = 8.2), and should thus, be useful for precise and accurate measurements of pH in vivo. Enhanced spectral resolution for 6-FPAM over 6-FPOL is demonstrated in whole blood and the perfused rat heart.

Identification of determinants for inhibitor binding within the RNA active site of human telomerase using PNA scanning.

Telomerase is a ribonucleoprotein that participates in the maintenance of telomere length. Its activity is up-regulated in many tumor types, suggesting that it may be a novel target for chemotherapy. The RNA component of telomerase contains an active site that plays at least two roles&sbd;binding telomere ends and templating their replication [Greider, C. W., & Blackburn, E. H. (1989) Nature 337, 331-337]. The accessibility of RNA nucleotides for inhibitor binding cannot be assumed because of the potential for RNA secondary structure and RNA-protein interactions. Here we use high-affinity recognition by overlapping peptide nucleic acids (PNAs) [Nielsen, P. E., et al. (1991) Science 254, 1497-1500] to identify nucleotides within the RNA active site of telomerase that are determinants for inhibitor recognition. The IC50 for inhibition decreases from 30 microM to 10 nM as cytidines 50-52 (C50-52) at the boundary between the alignment and elongation domains are recognized by PNAs overlapping from the 5' direction. As C50-52 are uncovered in the 3' direction, IC50 increases from 10 nM to 300 nM. As cytidine 56 at the extreme 3' end of the active site is uncovered, IC50 values increase from 0.5 microM to 10 microM. This analysis demonstrates that C50-C52 and C56 are important for PNA recognition and are physically accessible for inhibitor binding. We use identification of these key determinants to minimize the size of PNA inhibitors, and knowledge of these determinants should facilitate design of other small molecules capable of targeting telomerase. The striking differences in IC50 values for inhibition of telomerase activity by related PNAs emphasize the potential of PNAs to be sensitive probes for mapping complex nucleic acids. We also find that PNA hybridization is sensitive to nearest-neighbor interactions, and that consecutive guanine bases within a PNA strand increase binding to complementary DNA and RNA sequences.