RESUMEN
Reactions between formaldehyde and guanosine in the range pH 4-10 gave N2-hydroxymethylguanosine 1, bis-N2-guanosinylmethane 2 and 7-[7-(beta-D-ribofuranosyl)-purin-6(7H)- on-2-yl]-3-(beta-D-ribofuranosyl)-5,6,7,8-tetrahydro-sym-triazino[ 1,2-alpha] purin-10(3H)-one 3a. The latter is a new adduct, the formation of which occurs in neutral solution, is favoured at higher pH and can be rationalised by a sequence of condensations involving two molecules each of guanosine and formaldehyde.
Asunto(s)
Aductos de ADN/síntesis química , Formaldehído/química , Guanosina/química , Formaldehído/toxicidad , Estructura Molecular , Mutagénesis/fisiologíaRESUMEN
Convenient synthesis of rac-glycidaldehyde from rac-but-3-ene-1,2-diol and (R)-glycidaldehyde from D-mannitol are described. (R)-Glycidaldehyde (1) reacts with guanosine in water (pH 4-11, faster reaction at higher pH) to give initially 6(S)-hydroxy-7(S)-(hydroxymethyl)-3-(beta-D-ribofuranosyl)-5,6,7- trihydroimidazo[1,2-alpha]purin-9(3H)-one (7a) and 6(S),7(R)-dihydroxy-3-(beta-D-ribofuranosyl)-5,6,7,8- tetrahydropyrimido[1,2- alpha]purin-10(3H)-one (8a). The former decomposes to 7-(hydroxymethyl)-5,9-dihydro-9-oxo-3-(beta-D-ribofuranosyl)imidazo[1,2- alpha]purine (3a), 5,9-dihydro-9-oxo-3-(beta-D-ribofuranosyl)imidazo[1,2-alpha]purine (5a, 1,N2-ethenoguanosine), and formaldehyde, while the latter adduct is relatively stable. The position of the hydroxymethyl group on the imidazo ring of 7-(hydroxymethyl)-5,9-dihydro-9-oxo-3-(beta-D-ribofuranosyl)imidazo-[1,2 - alpha]purine was proved by 13C NMR analysis of adducts derived from [1-15N]guanosine and [amino-15N]guanosine. At longer reaction times, the adduct 7,7'-methylenebis[5,9-dihydro-9-oxo-3-(beta-D-ribofuranosyl)imidazo[1,2- alpha]purine (4a) is formed from guanosine and glycidaldehyde. The structure analysis of this adduct was also aided by 13C NMR analysis of the 15N-labeled adduct derived from [1-15N]guanosine. Analogous adducts were obtained from the reaction between glycidaldehyde and deoxyguanosine. Mechanisms of formation of the adducts from glycidaldehyde and guanosine/deoxyguanosine are proposed and supported by model studies with simple amines. The formaldehyde produced in the reactions described reacts with guanosine to give the known adduct N2-(hydroxymethyl)guanosine (9).
Asunto(s)
Aldehídos/síntesis química , Aductos de ADN/biosíntesis , Desoxiguanosina/química , Compuestos Epoxi/síntesis química , Guanosina/química , Aldehídos/química , Aminas/química , Carcinógenos/química , Reactivos de Enlaces Cruzados , Aductos de ADN/química , Compuestos Epoxi/química , Espectroscopía de Resonancia Magnética , Relación Estructura-Actividad , Agua/químicaRESUMEN
(R)-(2-dodecanamidoisohexyl)phosphocholine (DAHPC), labelled with 13C at the amide carbonyl group, has been synthesized and its binding to bovine pancreatic phospholipase A2 (PLA2) studied by n.m.r. and i.r. spectroscopy. Two-dimensional 1H-n.m.r. spectra show that, in the presence of Ca2+, DAHPC binds to the active site of the enzyme in a similar manner to other phospholipid amide substrate analogues. The environment of the labelled carbonyl group has been investigated by a combination of 13C n.m.r. and difference-Fourier-transform i.r. spectroscopy. The carbonyl resonance shifts 3 p.p.m. downfield on the binding of DAHPC to PLA2. The carbonyl absorption frequency decreases by 14-18 cm-1, accompanied by a marked sharpening of the absorption band. These results indicate that the carbonyl bond undergoes significant polarization in the enzyme-ligand complex, facilitated by the enzyme-bound Ca2+ ion. This suggests that ground-state strain is likely to promote catalysis in the case of substrate binding. Simple calculations, based on the i.r. data, indicate that the carbonyl bond is weakened by 5-9 kJ.mol-1. This is the first report of observation of the amide vibration of a bound ligand against the strong background of protein amide vibrations.
Asunto(s)
Espectroscopía de Resonancia Magnética , Fosfolipasas A/metabolismo , Fosforilcolina/metabolismo , Animales , Sitios de Unión , Isótopos de Carbono , Bovinos , Fenómenos Químicos , Química Física , Análisis de Fourier , Enlace de Hidrógeno , Estructura Molecular , Páncreas/enzimología , Fosfolipasas A/química , Fosfolipasas A2 , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Espectrofotometría Infrarroja , TermodinámicaRESUMEN
A series of substrate analogue inhibitors of pancreatic phospholipase A2 has been designed and synthesized. The compounds were tested in a novel dual-screening system based on parallel assays with monomeric and micellar substrates. Intermolecular nuclear Overhauser effects between vinylic protons on one inhibitor and identified active site residues on the bovine pancreatic enzyme have been observed in solution NMR studies of the enzyme-inhibitor complex. It can be deduced from both the biochemical results and the NMR data that the mode of interaction between this type of inhibitor and the active site of phospholipase A2 is essentially the same, irrespective of the presence or absence of an aggregated phospholipid surface. A model of the binding between the enzyme and inhibitor which incorporates the two-dimensional NMR data has been developed. The model can account for the activity of modified inhibitor structures and can be extrapolated to an assessment of the mode of binding of the natural substrate itself.
Asunto(s)
Fosfolipasas A/antagonistas & inhibidores , Animales , Bovinos , Simulación por Computador , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Espectroscopía de Resonancia Magnética , Micelas , Modelos Moleculares , Páncreas/enzimología , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Relación Estructura-ActividadRESUMEN
The structure and the mechanism of formation of the 1:1 adduct from the reaction of guanosine with glycidaldehyde have been investigated. From this reaction, a 2:2 adduct has also been isolated, and can be rationalized as a product of self-condensation of the 1:1 adduct, with extrusion of formaldehyde.