Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 43-65).

Thesis (M. Med. Sc.)--University of Hong Kong, 2001. / Includes bibliographical references.

Thesis (Ph. D.)--University of Hong Kong, 2001. / Includes bibliographical references (leaves 128-151).

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

The reaction of nitric oxide with secondary amines to form diazeniumdiolate anions with the structure R2NN2O2 - has been studied in order to investigate the stereochemistry and mechanism of this reaction. The stereochemical preference of the cis (Z) isomer for these compounds was proved using vibrational spectroscopy, namely infrared spectroscopy (IR) and Raman. Theoretical (B3LYP/aug-cc-pVDZ) rotation barriers for the simple diazeniumdiolate anion, Me2NN(O)=NO- are calculated in the gas phase to be 26.2 kcal/mol with the cis isomer predicted to be stabilized by only 0.5 kcal/mol compared to the trans isomer, therefore stereochemical preference for the cis is due to kinetic rather than thermodynamic control. Using vibrational spectroscopy to characterize the anion, it was determined that if the trans (E) isomer is present it represents <0.002% of the total composition. / The mechanism of nitric oxide's reaction with secondary amines was elucidated using both synthetic and kinetic methods. Attempts at preparing diazeniumdiolates (NONOates) by reacting nitric oxide directly to various radical anions proved to be unsuccessful. The result of these reactions is the formation of many reaction products, many of which could not be conclusively identified. / The rate of these NO-amine condensation reactions was found to be dependent upon the solvent in which the reaction was conducted, with modest rate enhancements obtained in heterocyclic organic bases. In addition to differences in rate, the overall yield of diazeniumdiolate recovered was found to be highly dependent on solvent choice as well as amine structure. / Kinetic measurements for the formation of these compounds indicate that the rate law is multi-term and the order of reaction with respect to nitric oxide depends greatly on the concentration of nitric oxide. At low nitric oxide concentrations the dependency on nitric oxide is first order, however as the NO concentration increases the order of the reaction becomes second order in [NO]. / The decomposition profile of amine derived diazeniumdiolates in aqueous media is significantly different than in organic solutions. In alkaline aqueous solutions the rate of decomposition is slow, with no significant decomposition occurring after several hours. This decomposition can be accelerated by lowering the pH. In non-aqueous solutions, the rate decomposition of the anion was determined to be comparable to the rate in neutral aqueous solutions. / Finally the equilibrium constant for the rate determining step for the reaction of nitric oxide with the secondary amine, pyrrolidine in pyridine was elucidated. The equilibrium constant for the rate limiting step was found to be 1.1 M-1.

A study of the reactions of various S-nitrosothiols, particularly S-nitroso-N- acetylpenicillamine (SNAP), was undertaken. These compounds were known to produce nitric oxide (NO) when decomposing, which has important and diverse biological roles. An example of their use in physiological research was demonstrated. The Griess method was used to determine the stoichiometry of nitrite production from S-nitrosothiol decomposition in various buffer solutions. In all cases the production was found to be almost quantitative. The kinetic measurement of SNAP decomposition in a variety of buffers and pH was undertaken. The results were complex and often erratic, conforming to first order but also half order kinetics in many cases. There was some indication that decomposition products and light could affect the reaction. The presence of disulphide (dimer) as a major reaction product was confirmed in the case of SNAP. Free-radical traps were used to probe the decomposition mechanism, as were hemin and haemoglobin as NO detectors to determine decomposition kinetics. The true agent of S-nitrosothiol decomposition was found to be intrinsic copper in the water supply and buffer salts. S-nitrosothiols were found to be stoichiometrically decomposed by Hg(^2+) ions, but catalytically decomposed by Cu(^2+) ions. Kinetic measurement confirmed the complex nature of the catalysis. The importance of SNO and NH(_2), and SNO and COO- as binding sites was demonstrated. Some explanation was found for the differing structure/reactivity relationships observed. It was shown that transnitrosation of a thiol could occur, involving thiolate anion attack upon the S-nitrosothiol. However, the reaction appeared to be very slow at physiological pH. The nitrosation of N-methylaniline by S-nitrosothiols was found to occur only in the presence of oxygen - direct transfer of NO did not occur, nitrosation being mediated after SNAP decomposition.

A detailed study concerning the aqueous decomposition characteristics of S-nitrosothiols in both the presence and absence of cupric ions was undertaken. Spectrophotometric measurements established that the true catalytic species generating nitric oxide from S-nitrosothiols is Cu(^+), formed by the reduction of copper(II) ions by thiolate, which is present as an impurity in solution. Introduction of the specific cuprous ion chelator neocuproine inhibited reaction, with the concentration of thiol in situ having a significant influence on the absorbance/time traces obtained. Under certain conditions thiolate ions clearly promoted S-nitrosothiol decomposition, whereas at times an opposite effect was noted. These results have been correlated with the reductive ability and chelation properties towards Cu(^2+) of each thiol in question. Structure/reactivity studies were extended further to include a range of S-nitrosated aromatic and heterocyclic thiols which generated the corresponding disulfides in distilled water yet reformed the appropriate thione at pH 7.4, along with nitric oxide in both media. A mechanism has been proposed which accounts for these observations. The reaction of S-nitrosothiols with cupric ions bound to biologically significant molecules such as amino acids, peptides and proteins was followed. Despite Cu(^2+) being chelated in this manner, S-nitrosothiol decomposition was apparent, albeit at a slower rate than that seen when copper(II) sulfate pentahydrate was utilised. Thiolate ions were capable of reducing Cu(^2+) Cu(^+) which was bound to such molecules suggesting a possible mechanism for nitric oxide formation from S-nitrosothiols in vivo. The blue copper protein ceruloplasmin also promoted NO generation under physiological conditions. A brief investigation into the direct reaction of thiolate ion with its corresponding S-nitrosothiol was also carried out. It was discovered that the major reaction product in this instance is ammonia and not nitric oxide, suggesting that a different copper-ion independent process is occurring involving direct interaction between the two species.

Thesis (D. Med. Sci.)--University of Tartu, 2004. / Vita. Includes bibliographical references.