ABSTRACT
Novel synthetic analogue of immunomodulatory peptidoglycan monomer 1 (PGM), (adamant-1-yl)-CH2CO-PGM (2), was prepared by acylation of epsilon-amino group of diaminopimelic acid with symmetrical (adamant-1-yl)-acetic acid anhydride in the presence of triethylamine. The product was isolated by gel filtration on Sephadex G-25, followed by ion exchange chromatography on SP-Sephadex C-25. The susceptibility of (adamant-1-yl)-CH2CO-PGM to hydrolysis with N-acetylmuramyl-L-alanine amidase was demonstrated, and the product of hydrolysis, (adamant-1-yl) CH2CO-pentapeptide 3, was characterized. Both 2 and 3 are water soluble and non-pyrogenic compounds. Immunomodulatory activity of PGM (adamant-1-yl)-CH2CO-PGM and structurally related derivative Boc-Tyr-PGM was compared in experiments in vivo, in mice, using ovalbumin (OVA) as an antigen. All three tested compounds exhibited comparable immunostimulating effects with respect to the induction of anti-ovalbumin immunoglobulin G. The results of evaluation of biological activity show that the substitution of free amino group in the parent peptidoglycan molecule with bulky lipophilic substituents did not affect the susceptibility to hydrolysis with N-acetylmuramyl-L-alanine amidase and did not alter markedly the immunostimulating activity. The results also indicate that the free amino group in the peptide chain is not a necessary requirement in the mechanism of immunostimulation of tested immunomodulators.
Subject(s)
Adamantane/analogs & derivatives , Adamantane/chemical synthesis , Adjuvants, Immunologic/pharmacology , Peptidoglycan/metabolism , Adamantane/analysis , Amidohydrolases/metabolism , Animals , Chromatography, Thin Layer , Enzyme-Linked Immunosorbent Assay , Immunization , Lipids/chemistry , Mice , Mice, Inbred C57BL , Molecular StructureABSTRACT
Peptidoglycan monomer, the disaccharide pentapeptide beta-D-Glcp-N-Ac-(1-->4)-D-Murp-N-Ac-L-Ala-D-mesoA2pm- (epsilonN H2)-D-Ala-D-Ala (PGM) is an immunomodulator. PGM and/or its derivative N-tert-butyloxycarbonyl-L-tyrosyl peptidoglycan monomer (Boc-Tyr-PGM) were coupled to two polysaccharides: the glucuronoxylomannan (GXM) from Cryptococcus neoformans, type B, solubilized by ultrasonic irradiation (MW 12-400 kDa) and to the dextran FP 70 (MW 70 kDa). Both polysaccharides were activated by CNBr. Initially, unprotected PGM was coupled via its amino group to GXM. The reactions yielded 42%-52% of the conjugate, containing only 0.18%-0.31% of PGM. In another approach Boc-Tyr-PGM (having its amino group blocked) was reacted via its free carboxyl group. Both CNBr-activated polysaccharides were first coupled to adipic acid dihydrazide (ADH) and then subsequently coupled to Boc-Tyr-PGM. The dextran conjugate (approximately 80% yield ) contained 6.3% of Boc-Tyr-PGM. The isolation of GXM conjugate required several modifications and it was obtained in lower yield (approximately 30%) but contained 13.7% of Boc-Tyr-PGM. Both conjugates were water soluble and apyrogenic and suitable for further testing of biological activity.
Subject(s)
Adjuvants, Immunologic/chemistry , Peptidoglycan/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Cryptococcus neoformans/chemistry , Molecular Sequence Data , Polysaccharides/chemistryABSTRACT
Selective pivaloylations of methyl beta-D-xylopyranoside have been studied under various reaction conditions. Partially pivaloylated products were submitted to additional acetylations. The structures were established by 1H NMR spectroscopy. Representatives of acylated methyl beta-D-xylopyranosides (acyl being pivaloyl, acetyl, or a combination of both) were submitted to hydrolysis catalyzed by rabbit serum and esterases isolated from rabbit serum.
Subject(s)
Esterases/metabolism , Methylglycosides/chemical synthesis , Acylation , Animals , Esterases/blood , Hydrolysis , Magnetic Resonance Spectroscopy , Methylglycosides/metabolism , Pentanoic Acids/chemistry , Pentanoic Acids/metabolism , RabbitsABSTRACT
The preparation and characterization of the essential components to be used in the radioimmunoassay of peptidoglycan monomer (PGM) is described. In order to raise the anti-peptidoglycan monomer antibodies 14C-labelled peptidoglycan monomer-bovine serum albumin conjugate was prepared by the coupling of 14C-peptidoglycan monomer to bovine serum albumin in the presence of glutaraldehyde in 0.1 M NaHCO3 at pH 8.3. The prepared conjugate elicited anti-PGM response in rabbits. A synthetic analog of peptidoglycan monomer, Boc-L-tyrosyl-peptidoglycan monomer was prepared by condensation of unprotected peptidoglycan monomer and N-hydroxysuccinimidester of Boc-L-tyrosine in the presence of triethylamine and the obtained disaccharide-hexapeptide was labelled with Na125I. This compound exhibited the ability of binding to anti-peptidoglycan monomer antibodies. The prepared compounds, namely anti-PGM antibodies and 125I-labelled Boc-L-tyrosyl-peptidoglycan monomer, were used as essential components in competitive radioimmunoassay for peptidoglycan monomer determination in mammalian and human sera and plasma, respectively.
Subject(s)
Peptidoglycan/analysis , Amino Acid Sequence , Antibodies , Brevibacterium , Carbohydrate Sequence , Carbon Radioisotopes , Chromatography, Gel/methods , Chromatography, Ion Exchange/methods , Chromatography, Thin Layer/methods , Electrophoresis, Paper/methods , Molecular Sequence Data , Peptidoglycan/isolation & purification , Radioimmunoassay/methodsABSTRACT
14C-Labelled methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside was used as a substrate for the detection of esterase activity in the isolation of esterase II from rabbit serum. On treatment of methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside with rabbit serum and esterase II, the 6-O-pivaloyl group was removed selectively. Likewise, the 6-O-pivaloyl group was removed selectively from methyl 2-acetamido-2-deoxy-3,4,6-tri-O-pivaloyl-alpha-D-glucopyranoside.
Subject(s)
Carbohydrate Metabolism , Esterases/isolation & purification , Animals , Esterases/blood , Molecular Structure , Pentanoic Acids/metabolism , RabbitsABSTRACT
Selective pivaloylation of 2-acetamido-2-deoxy-D-glucose, its methyl alpha- and beta-glycosides, and the methyl alpha-glycoside of N-acetyl-D-muramic acid under various conditions has been studied. The structures of the products were established by 1H-n.m.r. spectroscopy and acetylation. The orders of acylation, HO-6 greater than HO-3 greater than HO-1 greater than HO-4 for 2-acetamido-2-deoxy-D-glucose and HO-6 greater than HO-3 greater than HO-4 for its methyl glycosides, were established. Methyl 2-acetamido-2-deoxy-3,6-di-O-pivaloyl-alpha- and -beta-D-glucopyranosides and 2-acetamido-2-deoxy-1,3,4,6-tetra-O-pivaloyl-D-glucopyranose were hydrolysed by rabbit serum esterases.
Subject(s)
Acetylglucosamine/analogs & derivatives , Acetylglucosamine/metabolism , Glucosamine/analogs & derivatives , Methylglycosides/metabolism , Muramic Acids/metabolism , Pentanoic Acids/metabolism , Sugar Acids/metabolism , Valerates/metabolism , Acylation , Animals , RabbitsABSTRACT
The effects of immunomodulating peptidoglycans, peptidoglycan monomer (PGM) and muramyl dipeptide (MDP), on hepatic microsomal UDP-glucuronyltransferase (uridine diphosphoglucuronate glucuronosyl transferase, EC 2.4.1.17) and beta-glucuronidase (beta-D-glucuronide glucuronohydrolase, EC 3.2.1.31) were tested in female C57Bl mice. 4-Methylumbelliferone and p-nitrophenol were used as representative substrates for one functional form of UDP-glucuronyltransferase (GT1) and testosterone for the second functional form (GT2) of the enzyme. Both PGM and MDP were found to transiently inhibit the activity of UDP-glucuronyltransferase. There was no significant difference in the magnitude of inhibition of the two functionally different enzyme forms. The activity of microsomal beta-glucuronidase was tested using 4-methylumbelliferyl glucuronide and p-nitrophenyl glucuronide as substrates. Time dependent transient inhibition of beta-glucuronidase activity was observed with both peptidoglycans. In addition, the effect of MDP on cytochrome P-450 was tested, since we have shown previously that PGM affected this system. MDP decreased the content of cytochrome P-450 and inhibited the activity of related enzymes.
