Glycosylation of aromatic amines II: Kinetics and mechanisms of the hydrolytic reaction between kynurenine and glucose.

The kinetics of the weakly basic aromatic amine, kynurenine, with glucose were studied as model reactants aimed at mechanistic understanding of pharmaceutically relevant amine-aldehyde reactions. The reaction kinetics of the forward and reverse processes (glycosylamine formation and hydrolysis) were studied under first-order conditions in aqueous solutions at 40 degrees C in the pH range 1-6.5 in the presence of various buffers. The alpha-and beta-glycosylamines were reversibly formed via an acyclic imine that was not present in detectable quantities. Rate-limiting formation of the imine was complex and involved the addition of the amine and aldehyde to form the carbinolamine followed by the acid-catalyzed dehydration to the imine. The pH-rate profile was characterized by three kinetically distinguishable processes. At lower pH values, the profile was consistent with specific acid-catalyzed rate-determining addition of amine and aldehyde. In the pH range of 4-6 a downward bend was attributable to the change in rate determining step from addition to dehydration. In the pH region of 2-3 the rate law was described by specific acid catalysis and solvolysis of the zwitterionic form of kynurenine. Nonlinear buffer effects and Brönsted plots were shown to be consistent with this interpretation of the pH-rate profile.

Glycosylation of aromatic amines III: Mechanistic implications of the pH-dependent glycosylation of various aromatic amines (kynurenine, 2'-aminoacetophenone, daptomycin, and sulfamethoxzaole).

Glycosylation reaction kinetics of a series of aromatic amines (kynurenine, 2'-aminoacetophenone, daptomycin, and sulfamethoxazole) was compared to propose a unifying reaction mechanism. Kinetic studies were conducted in aqueous solutions containing glucose in the pH range 1-6.5 with 2'-aminoacetophenone and daptomycin. The resultant pH-rate profiles were compared to previously reported profiles for the reactions of glucose and kynurenine or sulfamethoxazole. Glycosylation of weakly basic aromatic amines involved the addition of the unprotonated amine to the aldehydic sugar leading to carbinolamine formation followed by specific acid catalyzed dehydration. All of the pH-rate profiles displayed characteristic downward bend at pH 4-5 due to a change from rate-determining addition to dehydration. In the pH-rate profile for kynurenine, a second downward bend was observed in the pH region 2-4. This feature was absent for the other substrates and was attributed to differences in reactivity of the two ionization states of the alpha carboxylic acid in kynurenine. This stabilization was not possible for the other amines studied.

Glycosylation of aromatic amines I: Characterization of reaction products and kinetic scheme.

The reactions of aliphatic and aromatic amines with reducing sugars are important in both drug stability and synthesis. The formation of glycosylamines in solution, the first step in the Maillard reaction, does not typically cause browning but results in decreased potency and is hence significant from the aspect of drug instability. The purpose of this research was to present (1) unreported ionic equilibria of model reactant (kynurenine), (2) the analytical methods used to characterize and measure reaction products, (3) the kinetic scheme used to measure reaction rates and (4) relevant properties of various reducing sugars that impact the reaction rate in solution. The methods used to identify the reversible formation of two products from the reaction of kynurenine and monosaccharides included LC mass spectrometry, UV spectroscopy, and 1-D and 2-D (1)H-(1)H COSY NMR spectroscopy. Kinetics was studied using a stability-indicating HPLC method. The results indicated the formation of alpha and beta glycosylamines by a pseudo first-order reversible reaction scheme in the pH range of 1-6. The forward reaction was a function of initial glucose concentration but not the reverse reaction. It was concluded that the reaction kinetics and equilibrium concentrations of the glycosylamines were pH-dependent and also a function of the acyclic content of the reacting glucose isomer.