RESUMEN
The functional mechanism of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) raises fundamental questions since the transfer of cations must differ from the better-known principles of rhodopsin-based proton pumps. Addressing these questions must involve a better understanding of its photointermediates. Here, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance spectroscopy on cryo-trapped photointermediates shows that the K-state with 13-cis retinal directly interconverts into the subsequent L-state with distinct retinal carbon chemical shift differences and an increased out-of-plane twist around the C14-C15 bond. The retinal converts back into an all-trans conformation in the O-intermediate, which is the key state for sodium transport. However, retinal carbon and Schiff base nitrogen chemical shifts differ from those observed in the KR2 dark state all-trans conformation, indicating a perturbation through the nearby bound sodium ion. Our findings are supplemented by optical and infrared spectroscopy and are discussed in the context of known three-dimensional structures.
Asunto(s)
Rodopsina , ATPasa Intercambiadora de Sodio-Potasio , Carbono/metabolismo , Flavobacteriaceae , Iones/metabolismo , Espectroscopía de Resonancia Magnética , Rodopsina/química , Sodio/química , ATPasa Intercambiadora de Sodio-Potasio/químicaRESUMEN
Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.
Asunto(s)
Reactivadores de la Colinesterasa/química , Reactivadores de la Colinesterasa/farmacología , Agentes Nerviosos/toxicidad , Relación Estructura-Actividad , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Animales , Dominio Catalítico , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos/métodos , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Agentes Nerviosos/química , Cloruro de Obidoxima/farmacología , Compuestos Organofosforados/química , Compuestos Organofosforados/toxicidadRESUMEN
An asymmetric total synthesis of (+)-cis-sylvaticin is described. Key steps include the use of permanganate-mediated oxidative cyclization of 1,5-dienes to synthesize the two major fragments 2 and 3 and a catalytically efficient tethered RCM to unite these THF-containing fragments. In addition, t-BuP 4 base was found to reliably promote rapid alkylation of the butenolide precursor fragment 4.