RESUMO
Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5'-diphospho-N-acetyl-D-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts D-fructose-6-phosphate (Fru-6P) and L-glutamine (Gln) into D-glucosamine-6-phosphate (GlcN-6P) and L-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme.
Assuntos
Ensaios Enzimáticos , Glutamina-Frutose-6-Fosfato Transaminase (Isomerizante)/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frutosefosfatos/análise , Frutosefosfatos/metabolismo , Glucosamina/análogos & derivados , Glucosamina/análise , Glucosamina/metabolismo , Glucose-6-Fosfato/análogos & derivados , Glucose-6-Fosfato/análise , Glucose-6-Fosfato/metabolismo , Ácido Glutâmico/análise , Ácido Glutâmico/metabolismo , Glutamina/análise , Glutamina/metabolismo , Cinética , Especificidade por SubstratoRESUMO
The parallel synthesis of O-aryloxyamines remains an unfulfilled need in the field of medicinal chemistry and fragment-based approaches. To fill this gap a solution-phase two-step process based on (1) a copper-catalyzed cross-coupling of aryl boronic acids with a fluorous tagged N-hydroxyphthalimide, and (2) a supported aminolysis was designed and optimized using Taguchi's method. A library of O-aryloxyamines was synthesized in high yields with high purity and diversity.