RESUMEN
A universal, photochemically cleavable DNA base analogue would add desirable versatility to a number of methods in molecular biology. A novel C-nucleoside, nitropiperonyl deoxyriboside (NPdR, P), has been investigated for this purpose. NPdR can be converted to its 5'-DMTr-3'-CE-phosphoramidite and was incorporated into pentacosanucleotides by conventional synthesis techniques. The destabilizing effect on hybrid formation with a complementary strand when this P base opposes A, T, and G was found to be 3-5 kcal/mol, but 9 kcal/mol when it opposes C. Brief irradiation (lambda > 360 nm, 20 min) of DNA containing the P base and piperidine treatment causes strand cleavage giving the 3'- and 5'-phosphates. Two significant recent interests, universal/non-hydrogen-bonding base analogues and photochemical backbone cleavage, have thus been combined in a single molecule that serves as a light-based DNA scissors.
Asunto(s)
ADN/química , Nucleósidos/química , Secuencia de Bases , Hidrólisis , Resonancia Magnética Nuclear Biomolecular , Hibridación de Ácido Nucleico , Fotoquímica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TermodinámicaRESUMEN
Juvenile hormone (JH) undergoes metabolic degradation by two major pathways involving JH esterase and JH epoxide hydrolase (EH). While considerable effort has been focussed on the study of JH esterase and the development of inhibitors for this enzyme, much less has been reported on the study of JH-EH. In this work, the asymmetric synthesis of two classes of inhibitors of recombinant JH-EH from Trichoplusia ni, a glycidol-ester series and an epoxy-ester series is reported. The most effective glycidol-ester inhibitor, compound 1, exhibited an I(50) of 1.2x10(-8) M, and the most effective epoxy-ester inhibitor, compound 11, exhibited an I(50) of 9.4x10(-8) M. The potency of the inhibitors was found to be dependent on the absolute configuration of the epoxide. In both series of inhibitors, the C-10 R-configuration was found to be significantly more potent that the corresponding C-10 S-configuration. A mechanism for epoxide hydration catalyzed by insect EH is also presented.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Epóxido Hidrolasas/antagonistas & inhibidores , Compuestos Epoxi/síntesis química , Mariposas Nocturnas/enzimología , Propanoles/síntesis química , Animales , Línea Celular , Estructura Molecular , Spodoptera/citologíaRESUMEN
The majority of the JH III epoxide hydrolase activity in last stadium day 3 (gate 1) wandering Trichoplusia ni was membrane bound with approximately 9% of the activity found in the cytosol. Both the microsomal and cytosolic JH epoxide hydrolases were stable, retaining 30% of their original activity after incubation at 4 degrees C for 15 days. 18O-labeled water underwent enzyme catalyzed regioselective addition to the least substituted C10 position of JH III. In multiple turnover reactions with JH epoxide hydrolase in 97.9% 18O-labeled water, only 91.3% 18O incorporation was observed. This is consistent with an SN2 reaction likely involving a carboxylate in the active site of JH epoxide hydrolase. The DNA amplification cloning of a fragment of a putative T. ni epoxide hydrolase is reported. The deduced amino acid sequence shares 67% similarity to the rat microsomal epoxide hydrolase.