Z-DNA is the only DNA conformation that has a left-handed helical twist. Although Z-DNA has been implicated in both carcinogenesis and mutagenesis, its specific biological role remains uncertain. We have demonstrated that the formation of C8-arylguanine DNA adducts, derived from arylhydrazines, shifts the B/Z-DNA equilibrium toward the Z-DNA conformation in d(CG)5 sequences. However, our previous work examined the effect of two adducts in the duplex, and it was unclear whether the two base modifications were working together to cause the equilibrium shift toward the Z-DNA conformation. Here we report the synthesis and characterization of a hairpin oligonucleotide sequence (d(CG)5T4(CG)5) containing only one C8-arylguanine modified base. The unmodified hairpin and the previously studied unmodified double-stranded oligonucleotide were conformationally similar, and each required ∼3 M NaCl to yield a B-/Z-DNA ratio of 1:1. The introduction of a single C8-arylguanine modification significantly reduced the NaCl concentration needed to produce a 1:1 B-/Z-DNA ratio in the hairpin. Further, the addition of MgCl2 and spermine to the C8-arylguanine-modified hairpin shifts the B/Z-DNA equilibrium such that the Z form predominated under physiological conditions. NMR and molecular modeling indicated the conformational effects produced by the C8-arylguanine modification occurred locally at the site of modification while CD data demonstrated that the C8-arylguanine-modified base destabilized the B form. Additionally, our data show that adopting the Z-DNA conformation is preferred over denaturation to the single-stranded form. Finally, the conformational effects of the C8-arylguanine modifications were not additive and the introduction of any such modifications drive Z-DNA formation under physiological conditions, which may provide a novel carcinogenesis mechanism where DNA adducts confer their carcinogenicity through a Z-DNA-mediated mechanism.

DNA has many different conformations however only Z‐DNA adopts a left‐handed helix. The zigzag phosphate backbone of Z‐DNA brings the negatively charged phosphate residues closer together rendering Z‐DNA a higher energy form of DNA. Z‐DNA has been implicated in carcinogenesis from its role in gene expression and mutagenesis because Z‐DNA formation can stimulate large scale gene deletions, translocations, and rearrangements in vivo. The inability to generate a stable Z‐DNA structure under physiological conditions has made investigation of B‐ to Z‐DNA interconversion difficult since Z‐DNA requires external stimuli to stabilize its formation like chemical modifications or specific proteins known to stabilize Z‐DNA, Z‐DNA binding proteins (ZBP). We have demonstrated that C8‐arylguanine DNA adducts, derived from carcinogenic arylhydrazines, drive Z‐DNA formation and the addition of MgCl2 stabilizes the Z form under physiological conditions. The conformational effects of the C8‐arylguanine mutation were determined by nuclear magnetic resonance (NMR) and circular dichroism (CD). In addition, preliminary results using in silico molecular dynamics and CD have demonstrated that ZBPs prefer to bind to the C8‐arylguanine modified DNA. C8‐Arylguanine can be used as a tool to clarify the mechanism of ZBP mediated B‐ to Z‐DNA transition. The current view fails to explain the ZBP selectivity for the Z form and leads to a contradictory conclusion that ZBPs cannot bind Z‐DNA. C8‐Arylguanine modified oligonucleotides can be used to fully elucidate the binding of ZBPs to DNA, as both the B and Z forms can be generated under physiological conditions, using surface plasmon resonance (SPR). This may reveal the underlying factors that control ZBP‐DNA interactions which could become a potential therapeutic target. (Supported by a fellowship to BCT (HEPC.dsr.09013) and (NSF EPS‐1003907)

The 1H and 13C NMR spectra of 10‐deoxymethynolide (1), 8.9‐dihydro‐10‐deoxymethynolide (2) and its glycosylated derivatives (3–9) were analyzed using gradient‐selected NMR techniques, including 1D TOCSY, gCOSY, 1D NOESY (DPFGSENOE), NOESY, gHMBC, gHSQC and gHSQC‐TOCSY. The NMR spectral parameters (chemical shifts and coupling constants) of 1–9 were determined by iterative analysis. For the first time, complete and unambiguous assignment of the 1H NMR spectrum of 10‐deoxymethynolide (1) has been achieved in CDCl3, CD3OD and C6D6 solvents. The 1H NMR spectrum of 8,9‐dihydro‐10‐deoxymethynolide (2) was recorded in CDCl3, (CD3)2CO and CD3OD solutions to determine the conformation. NMR‐based conformational analysis of 1 and 2 in conjugation with molecular modeling concluded that the 12‐membered ring of the macrolactones may predominantly exist in a single stable conformation in all solvents examined. In all cases, a change in solvent caused only small changes in chemical shifts and coupling constants, suggesting that all glycosylated methymycin analogs exist with similar conformations of the aglycone ring in solution. Copyright © 2013 John Wiley & Sons, Ltd.

A novel class of N-substituted tetrahydropyridine derivatives was found to have multiple kinetic mechanisms of monoamine oxidase A inhibition. Eleven structurally similar tetrahydropyridine derivatives were synthesized and evaluated as inhibitors of MAO-A and MAO-B. The most potent MAO-A inhibitor in the series, 2,4-dichlorophenoxypropyl analog 12, displayed time-dependent mixed noncompetitive inhibition. The inhibition was reversed by dialysis, indicating reversible enzyme inhibition. Evidence that the slow-binding inhibition of MAO-A with 12 involves a covalent bond was gained from stabilizing a covalent reversible intermediate product by reduction with sodium borohydride. The reduced enzyme complex was not reversible by dialysis. The results are consistent with slowly reversible, mechanism-based inhibition. Two tetrahydropyridine analogs that selectively inhibited MAO-A were characterized by kinetic mechanisms differing from the kinetic mechanism of 12. As reversible inhibitors of MAO-A, tetrahydropyridine analogs are at low risk of having an adverse effect of tyramine-induced hypertension.

The B form of DNA exists in equilibrium with the Z form and is mainly affected by sequence, electrostatic interactions, and steric effects. C8-purine substitution shifts the equilibrium toward the Z form though how this interaction overcomes the unfavorable electrostatic interactions and decrease in stacking in the Z form has not been determined. Here, a series of C8-arylguanine derivatives, bearing a para-substituent were prepared and the B/Z equilibrium determined. B/Z ratios were measured by CD and conformational effects of the aryl substitution determined by NMR spectroscopy and molecular modeling. The para-substituent was found to have a significant effect on the B/Z DNA equilibrium caused by altering base-pair stacking of the B form and modifying the hydration/ion shell of the B form. A unique melting temperature versus salt concentration was observed and provides evidence relevant to the mechanism of B/Z conformational interconversion.