RESUMEN
Fluorescent 11-[(9'-anthracenyl)methoxy]undecyl phosphate and P1-{11-[(9'-anthracenyl)methoxy]undecyl}-P2-(alpha-D-galactopyranosyl) diphosphate were chemically synthesized for the first time. The ability of the first compound to serve as substrate-acceptor ofgalactosyl phosphate residue and the second compound of mannosyl residue in enzymic reactions catalyzed by galactosylphosphotransferase and mannosyltransferase from Salmonella newport membrane preparation was demonstrated.
Asunto(s)
Galactósidos/síntesis química , Manosiltransferasas/metabolismo , Organofosfatos/síntesis química , Monosacáridos de Poliisoprenil Fosfato/síntesis química , Salmonella/enzimología , Galactósidos/química , Galactosiltransferasas/química , Galactosiltransferasas/metabolismo , Manosiltransferasas/química , Organofosfatos/química , Monosacáridos de Poliisoprenil Fosfato/química , Especificidad por SustratoRESUMEN
N-Acetylglucosaminyl(diphosphodolichol) N-acetylglucosaminyl transferase, also known as Enzyme II, is the second enzyme in the dolichol pathway. This pathway is responsible for the assembly of the tetradecasaccharide pyrophosphate dolichol, which is the substrate for oligosaccharyl transferase. In order to study the specificity of Enzyme II, four unnatural dolichol diphosphate monosaccharides were synthesized, with the C-2 acetamido group in the natural substrate Dol-PP-GlcNAc 1a replaced by fluoro, ethoxy, trifluoroacetamido, and amino functionalities. These analogues 1b-e were evaluated as glycosyl acceptors for Enzyme II, which catalyzes the formation of dolichol diphosphate chitobiose (Dol-PP-GlcNAc(2)) from UDP-GlcNAc and Dol-PP-GlcNAc. Enzyme II from pig liver was found to be highly specific for its glycosyl acceptor and the acetamido group shown to be a key functional determinant for this glycosylation reaction.
Asunto(s)
Dolicoles/metabolismo , N-Acetilglucosaminiltransferasas/química , N-Acetilglucosaminiltransferasas/metabolismo , Monosacáridos de Poliisoprenil Fosfato/síntesis química , Monosacáridos de Poliisoprenil Fosfato/metabolismo , Animales , Activación Enzimática/fisiología , Microsomas Hepáticos/metabolismo , Especificidad por Sustrato/fisiología , PorcinosRESUMEN
[reaction: see text] Tetrabutylammonium benzyl dihydrophytylphosphate was coupled to S-phenyl 2,3-di-O-benyl-4, 6-O-benzylidene-1-thio-alpha-D-mannopyranoside S-oxide on activation with triflic anhydride in toluene at -78 degrees C to give the corresponding beta-mannosyl phosphate in 56% yield with no detectable formation of the alpha-anomer. Treatment with sodium in liquid ammonia then afforded the unprotected beta-mannosyl phosphoisoprenoid.
Asunto(s)
Mycobacterium avium/química , Mycobacterium tuberculosis/química , Monosacáridos de Poliisoprenil Fosfato/síntesis química , Pared Celular/química , EstereoisomerismoRESUMEN
Bacterial Lipid I analogues containing different anomeric groups at the muramic acid moiety were synthesized and screened in MurG enzyme assays run in the presence and absence of cell wall membranes. The results obtained in this study help elucidate the role of the lipid diphosphate in the recognition of Lipid I by MurG.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/antagonistas & inhibidores , Glucolípidos/síntesis química , Glucolípidos/farmacología , Monosacáridos de Poliisoprenil Fosfato/síntesis química , Bacterias/química , Bacterias/enzimología , Bacterias/ultraestructura , Membrana Celular/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Datos de Secuencia Molecular , Ácidos Murámicos/síntesis química , Ácidos Murámicos/farmacología , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Peptidoglicano , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Efficient methods were developed in our group in recent years for chemical synthesis of polyprenyl phosphates, polyprenyl monophosphate sugars, and polyprenyl diphosphate sugars, which were known to serve as important intermediates in biosynthesis of complex carbohydrates. A simple procedure was developed involving the phosphorylation of aliphatic alcohols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile. Monophosphates of various natural and modified dolichols and polyprenols, as well as the derivatives of retinol, cholesterol, and nonacosanol, were prepared in high yields. First syntheses of dolichyl thiophosphate and dolichyl hydrogen phosphonate were developed, and these derivatives were of interest as analogs of dolichyl phosphate. Polyprenyl monophosphate sugars, including derivatives of alpha- and beta-anomers of D-glucopyranose, D-galactopyranose, D-mannopyranose, and 2-acetamido-2-deoxy-D-glucopyranose, were obtained smoothly from moraprenyl trichloroacetimidate and acylated glycosyl phosphates after deprotection. A method for the synthesis of polyprenyl diphosphate sugars from polyprenyl phosphoroimidazolidate and unprotected glycosyl phosphates was shown to be applicable for a wide range of the monosaccharide derivatives including hexoses, deoxyhexoses, 2-acetamido-2-deoxyhexoses, and uronic acids. A series of the oligosaccharide derivatives was also prepared by this method.
Asunto(s)
Monosacáridos de Poliisoprenil Fosfato/síntesis química , Oligosacáridos de Poliisoprenil Fosfato/síntesis química , Fosfatos de Poliisoprenilo/síntesis química , Secuencia de Carbohidratos , Fosfatos de Dolicol/síntesis química , Esterificación , Datos de Secuencia MolecularRESUMEN
The membrane fraction from a mutant of Salmonella anatum deficient in UDPgalactose-4-epimerase, utilized synthetic ficaprenyl alpha-D-galactosyl diphosphate as a substrate in the biosynthesis of the O-polysaccharide portion of lipopolysaccharide which has a mannosylrhamnosylgalactose repeating sequence. The galactosyl lipid was prepared by chemical synthesis from D-galactose and ficaprenol extracted from Ficus elastica. Membrane preparations catalyzed the transfer of rhamnose from TDP-rhamnose onto membrane-bound ficaprenyl galactosyl diphosphate forming rhamnosylgalactosyl ficaprenyl diphosphate; the reaction was dependent on the prior insertion of the synthetic glycosyl-lipid into the membrane, and was proportional to incubation time up to 4 min at 29 degrees C. When both TDP-rhamnose and GDP-mannose were added, the product formed was O-polysaccharide. These results indicate that the chemically synthesized ficaprenyl galactosyl diphosphate can be an active substrate for the in vitro synthesis of the Salmonella O-polysaccharide.