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Applications of proton and Fluorine nuclear magnetic resonance spectroscopy to the study of large organic molecules Grant, Christopher William Maitland

Abstract

This thesis is divided into three chapters; each involving a different approach to the use of both proton (¹H) and fluorine (¹⁹F) nuclear magnetic resonance (n.m.r.) to study large organic molecules in solution. To some extent the three chapters represent an evolution in technique of the scientific world in general and of our laboratory in particular. Previous students in this laboratory have used high resolution n.m.r. spectroscopy (of ¹H, ³¹P and ¹⁹F nuclei) combined with double irradiation techniques to study progressively larger organic molecules. In the first chapter this work has been extended to "natural products" - molecules not previously very susceptible, because of their spectral complexity, to detailed n.m.r. studies. In particular ¹H and ¹⁹F n.m.r. have been employed in conjunction with heteronuclear noise modulated decoupling and ¹H-{¹H} INDOR. A series of steroids substituted with fluorine in the A, B or D rings have been examined as model systems: 2α-fluoro-cholestan-3-one, 6α- and 6β-fluoro-cholest-4-en-3-one and 16,16-difluoro-androst-5-en-3β-ol-17-one. In each case it has been possible to obtain coupling constants and chemical shifts for the nuclei in the area of the fluorine atom and hence to derive structural data from the n.m.r. spectra in spite of their complexity. The ¹H-{¹H} INDOR technique alone has been further applied to several problems posed by organic chemists involved with natural products. In each case this approach has been successful. Recently very large molecules such as enzymes have been studied by n.m.r. via their effects on the chemical shift and line width of smaller molecules with which they interact. As part of a programme to investigate the application of heteronuclear n.m.r. to problems of biological interest, we have used the above technique to study the interaction of various N-trifluoroacetylated monosaccharides with the enzyme, lysozyme. The ¹⁹F chemical shifts of N-trifluoroacetyl-D- glucosamine and its methyl glycosides have been studied as a function of enzyme concentration. The results suggest that the fluorine substituents affect the binding process to some extent but that such effects can be informative. The possibility of using n.m.r. to study the effects of substituents on enzyme-inhibitor interactions led to a study of the monosaccharides, N-acetyl-D-glucosamine-a-methyl glycoside and its C₆-iodo and C₅-methyl derivatives, all three of which gave similar results. This work is treated in Chapter II along with a brief discussion of the conformations of the inhibitors involved. Very recently organic chemists and biochemists have begun employing pulsed n.m.r. equipment in a variety of problems. We have become interested in the applications of relaxation time measurements to structural problems. In Chapter III pulsed ¹H n.m.r. experiments involving several model systems are reported: mixtures of the cis and trans isomers of 1,2-dichloro and 1,2-dibromoethylene and of the ethyl esters of maleic and fumaric acids have been studied. The results of these experiments are encouraging, indicating that, in this case at least, relaxation times are sensitive to structure and substituent in a consistent fashion. We have experimented almost exclusively with selective pulse techniques and have built and used a variety of equipment. In the Appendix are described two audiofrequency-pulse units which can be attached to a Varian HA-100 n.m.r. spectrometer and which were used for the experiments discussed in Chapter III. The same audiofrequency-pulse techniques have been applied to the measurement of nuclear relaxation times of individual protons in the alkaloid, vindoline, and in the sugar, 3,4,6-tri-0-acetyl-1-0-benzoyl-2-bromo-2- deoxy-β-D-glucopyranose and its 2-chloro analogue with less encouraging results. In addition we have reported the use of ¹⁹F pulsed n.m.r. to calculate the rate constants, k₋₁ and k₁, for the association of the α -anomer of N-trifluoroacetyl-D-glucosamine with lysozyme.

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