Part One. Structural refinement methodology by using three-dimensional NMR data. Part Two. Chemical nuclease design, synthesis, activity and mechanistic study

Jiyan Chen, Purdue University

Abstract

Part one. A hybrid-hybrid matrix method is described that quantitatively analyzes 3D NOE-NOE NMR data. Experimental 3D data are deconvoluted into a 2D hybrid NOESY spectrum. The deconvoluted 2D hybrid NOESY spectrum can then be merged with experimental 2D NOESY data along with additional simulated 2D data as necessary to create a hybrid-hybrid 2D NOE volume matrix. This hybrid-hybrid volume matrix is then used with the complete relaxation program, MORASS, to calculate a rate matrix, and the resulting distances taken from the off-diagonal cross-relaxation rates can then be utilized in a distance geometry or restrained molecular-dynamics refinement of the structure. Part two. Bipyridine was covalently linked from its 4-position to C5 of 2$\sp\prime$-deoxyuridine through the tether, -SCH$\rm\sb2CH\sb2NH$(CO)-, to give 5- (2-(4$\sp \prime$-methyl-2,2$\sp \prime$-dipyrid-4-yl-carboxamido)ethylthio) -2$\sp\prime$-deoxyuridine. This nucleoside was incorporated into oligonucleotides of different sequences via standard phosphoramidite chemistry. In the presence of 0.01 mM Fe(II) and 0.4 mM H$\rm\sb2O\sb2,$ a complementary substrate DNA can be cleaved by the bipyridine tethered targeting oligonucleotide. When the bipyridine is positioned close to the 3$\sp\prime$-end of the targeting strand, the opposing strand can be cleaved consistently in 80% yield over a three nucleotide span regardless of DNA duplex sequences. No targeting strand self-fragmentation is observed during the cleavage reaction. The cleavage yield is very sensitive to salt concentration, temperature and certain structure modifications. The cleavage can not be quenched by DMSO at 0.24 mM concentration. Fe(III) shows substantial nuclease activity in the presence of H$\rm\sb2O\sb2$ and the absence of reductants. On the other hand, Fe(II) does not show nuclease activity in the presence of reductants as co-reactants. It was postulated that a fundamental step in the cleavage reaction includes abstraction of a 3$\sp\prime$ hydrogen atom from deoxyribose on the opposing sequence. However, no isotope effect was observed in the cleavage reaction when C3$\sp\prime$ was labeled with $\sp2$H.

Degree

Ph.D.

Advisors

Bergstrom, Purdue University.

Subject Area

Organic chemistry

COinS