Synthesis and conformational analysis of muramic acid delta-lactam structures and their 4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl) derivatives, characteristic of bacterial spore peptidoglycan.

1,6-Anhydro-4-O-benzyl-beta-muramic acid 1',2-lactam (2) was prepared by reduction of 1,6-anhydro-2-azido-4-O-benzyl-2-deoxy-3-O-[(R)-1- methoxycarbonylethyl]-beta-D-glucopyranose (1) followed by cyclisation. Debenzylation of 2 (-->3) and glycosylation of HO-4 with 3,4,6-tri-O-acetyl-2- deoxy-2-phthalimido-beta-D-glucopyranosyl chloride afforded 75% of a beta-(1-->4)-linked disaccharide derivative (7). Removal of the Phth group from 7, then acetylation, and O-deacetylation yielded 4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-amino-1,6-anhydro-3-O- [(R)- 1-carboxyethyl]-2-deoxy-beta-D-glucopyranose 1',2-lactam (10) Acetolysis of the 1,6-anhydro ring in the 4-acetate (4) of 3 and the 3',4',6'-triacetate (9) of 10, with saponification of the products 5 and 11, afforded 2-amino-3-O- [(R)-1-carboxyethyl]-2-deoxy-D-glucopyranose 1',2-lactam (6) and 4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-amino-3- O-[(R)-1-carboxyethyl]-2-deoxy-beta-D-glucopyranose 1',2-lactam (12), respectively. The structure of 12 corresponds to that of the disaccharide unit characteristic of the glycan chains of bacterial spore peptidoglycan. 1H NMR spectroscopy indicated that the beta-D-glucopyranose ring in the 1,6-anhydro 1',2-lactam derivatives adopts the BO,3 conformation. On cleavage of the 1,6-anhydro ring by acetolysis, the D-glucopyranose ring adopts the 4C1 conformation. X-ray analysis of 2, 4, and 5 confirmed the proposed structures. Molecular mechanics and molecular dynamics simulations were used to follow the transformation of the BO,3 conformation of the D-glucopyranose ring via transition states to the 4C1 form.

Synthesis and reactions of O-acetylated benzyl alpha-glycosides of 6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-N-acetylmuramoyl-L- alanyl-D-isoglutamine esters: the base-catalysed isoglutamine in equilibrium glutamine rearrangement in peptidoglycan-related structures.

Condensation of benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2- deoxy-3-O-[(R)-1-carboxyethyl]-alpha-D-glucopyranoside (2) and its 4-acetate (4) with L-alanyl-D-isoglutamine benzyl ester via the mixed anhydride method yielded N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-lacto yl)-L- alanyl-D-isoglutamine benzyl ester (5) and its 4-acetate (6), respectively. Condensation by the dicyclohexylcarbodi-imide-N-hydroxysuccinimide method converted 2 into benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl- 2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside 1',4-lactone (7). In the presence of activating agents, 7 underwent aminolysis with the dipeptide ester to give 5. Zemplén O-deacetylation of 5 and 6 led to transesterification and alpha----gamma transamidation of the isoglutaminyl residue to give N-(2-O-[benzyl 2-acetamido-6-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyr anosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (8) and -glutamine methyl ester (9). Treatment of 6 with MgO-methanol caused deacetylation at the GlcNAc residue to give a mixture of N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2- deoxy-beta-D-glucopyranosyl)-4-O-acetyl-2,3-dideoxy-alpha-D-glucopyra nosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (11) and -glutamine methyl ester (12). Benzyl or methyl ester-protection of peptidoglycan-related structures is not compatible with any of the reactions requiring alkaline media. Condensation of 2 with L-alanyl-D-isoglutamine tert-butyl ester gave N-(2-O-[benzyl 2-acetamido- 6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2,3-d ideoxy- alpha-D-glucopyranosid-3-yl]-(R)-lactoyl-L-alanyl-D-isoglutamine tert-butyl ester (16), deacetylation of which, under Zemplén conditions, proceeded without side-reactions to afford N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-la cotyl)-L- alanyl-D-isoglutamine tert-butyl ester (17).

A convenient synthetic route to the disaccharide repeating-unit of peptidoglycan.

Glycosylation of the readily accessible benzyl 2-acetamido-6-O-benzyl-2-deoxy-3-O-[(R)-1-(methoxycarbonyl)ethyl]-alpha- D- glucopyranoside with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl chloride (2), using the silver triflate method in the absence of a base, afforded 65-70% of the fully protected [beta-D-GlcNPhth-(1----4)-MurNAc] methyl ester derivative 4, the structure of which was ascertained on the basis of 500-MHz 1H-n.m.r. data. 2,2'-Dideoxy-2,2'-diphthalimido-beta,beta-trehalose hexa-acetate was a by-product. Removal of the Phth group from 4, followed by acetylation, yielded 90% of the acetylated 1,6-di-O-benzyl derivative 5, which, on saponification and catalytic hydrogenation, afforded 2-acetamido-4-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1- carboxyethyl]-2-deoxy-D-glucopyranose. Similarly, 5 was converted into the acetylated methyl ester derivative, which, on selective removal of the methyl ester group, gave benzyl 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-6-O-benzyl-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside. An alternative route for the preparation of 2 is described.

Synthesis, properties, and reactions of alpha- and beta-D-glucopyranosyl esters of some tripeptides.

The 2,3,4,6-tetra-O-benzyl-1-O-(N-benzyloxycarbonyltripeptidyl)-D-glucopyranoses ), 8, and 13 were synthesised from 2,3,4,6-tetra-O-benzyl-alpha-D-glucopyranose and the active esters of the appropriate N-protected tripeptides (Gly-Cly-Gly-, L-Phe-Gly-Gly-, and Gly-Gly-L-Phe-) in the presence of imidazole; the anomeric mixtures were resolved and the alpha and beta anomers characterised. The beta anomer of 13, containing the L and D enantiomers (ratio approximately 3:1) of Gly-Gly-Phe- as the aglycon, could be resolved by column chromatography into the pure isomeric forms. Catalytic hydrogenolysis of the beta anomers, in the presence and absence of a strong acid, yielded the free 1-esters 2 beta, 9 beta, and 14 beta, which were characterised as the monoxalate or trifluoroacetate salts and as free bases. Similarly, the alpha anomers afforded 2 alpha, 9 alpha, and 14 alpha, whereas omission of the strong acid led to accompanying 1 leads to 2 acyl migration, to give the 2-O-acyl derivatives. All of the compounds prepared were converted into the N-acetyl and/or peracetylated derivatives. The 1-esters 2 beta and 9 beta, both in the charged and uncharged form, and the trifluoroacetate salt of 14 beta, are susceptible to cleavage by beta-D-glucosidase; the enzyme had no effect on the uncharged form of 14 beta. This difference between 14 beta and its salt is discussed in conformational terms.

Isolation procedure and properties of monomer unit from lysozyme digest of peptidoglycan complex excreted into the medium by penicillin-treated Brevibacterium divaricatum mutant.

1. The peptidoglycan complex excreted in large amounts into the medium by the biotin-requiring mutant Brevibacterium divaricatum NRRL-2311 incubated in the presence of penicillin for 1 h has been investigated. A convenient isolation procedure with high yield for the pure monomeric unit from lysozyme digest of the accumulated polymer is described. 2. It is shown that the released peptidoglycan possesses the linear uncross-linked structure made of repeating disaccharide-pentapeptide unit [GlcNAc-MurNac-Ala-D-Glyn(meso-DAP-D-Ala-D-Ala)] which was isolated by stepwise gel filtration and fractionation of the digestion mixture in 10-mg quantities. Evidence that the minor digestion product of accumulated peptidoglycan possesses the glycan-linked dimer structure is given. Under conditions of beta-elimination, the monomeric unit yielded a lactylpentapeptide which was isolated in pure form by gel filtration. 3. The monomer unit originating from the cultures to which L-[U-14C]glutamic acid was added simultaneously with penicillin incorporated the label exclusively in the peptide chain, whereas that labeled from E11-14C]acetate as the precursor contained radioactivity in both the peptide chain (53%) and N-acetylamino groups (47%) of the glycan portion.

Synthesis and rearrangements of D-glucosyl esters of aspartic acid linked through the 1- or 4-carboxyl group.

Catalytic hydrogenation of 2,3,4,6-tetra-O-benzyl-1-O-[1-benzyl N-(benzyloxycarbonyl)-L-aspart-4-oyl]-alpha-D-glucopyranose (1alpha) in acetic acid-2-methoxyethanol gave 1-O-(L-beta-aspartyl)alpha-D-glucopyranose (2alpha) contaminated with 2-O-(L-alpha-aspartyl)-D-glucopyranose (8). Evidence that 8 was formed from the 1-oyl isomer of 1alpha, namely 2,3,4,6-tetra-O-benzyl-1-O-[4-benzyl N-(benzyloxycarbonyl)-L-aspart-1-oyl]-alpha-D-glucopyranose (7alpha), via 1 leads to 2 acyl migration, was obtained by submitting the deprotected D-glucosyl ester to successive N-acetylation, esterification, and O-acetylation; the final product was identified as a approximately 4:1 mixture of 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(acetyl)-L-aspart-4-oyl]-alpha-D-glucopyranose (4alpha) and 1,3,4,6-tetra-O-acetyl-2-O-[4-methyl N-(acetyl)-L-aspart-1-oyl]-D-glucopyranose (6) which were also prepared by definitive methods. On the other hand, deprotection of 1beta gave isomerically pure 2beta which was converted into the peracetylated ester derivative 4beta; an explanation for the differences in aglycon isomeric purity of 2alpha and 2beta is given. Hydrogenolysis of 7beta under the above conditions led to intermolecular transesterification with scission of the C-1 ester bond to give 1-(2-methoxyethyl) L-aspartic acid and D-glucose. Catalytic hydrogenation of 7alpha and 7beta, performed in the presence of trifluoroacetic acid, afforded 1-O-(L-alpha-aspartyl)-alpha- and -beta-D-glucopyranoside trifluoroacetate salts (11alpha and 11beta), respectively. The structure of 11beta was established by successive conversion into 2,3,4,6-tetra-O-acetyl-1-O-[4-methyl N-(acetyl)-L-aspart-1-oyl]-beta-D-glucopyranose (5beta) which was also prepared by definitive methods. Analogous treatment of 11alpha gave the N-acetyl derivative 12 which underwent 1 leads to 2 acyl migration during esterification with diazomethane to give the N-acetyl methyl ester derivative 10; acetylation of 10 afforded 6.

Synthesis and chemical behaviour of D-glucosyl esters of glutamic acid having the side-chain carboxyl group involved in the glycosidic linkage.

Simultaneous and stepwise deprotection of the fully benzylated D-glucosyl esters of 1-benzyl N-benzyloxycarbonyl- and N-tert-butyloxycarbonyl-L-glutamic acid (1 and 5, respectively) have been examined. Catalytic hydrogenation of 1 led to intramolecular aminolysis to give pyroglutamic acid and D-glucose, but similar treatment in the presence of trifluoroacetic acid afforded both anomers of 1-O-(L-gamma-glutamyl)-D-glucopyranose, which were characterized as trifluoroacetates (2alpha and 2beta) and converted into 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(acetyl)-L-glutam-5-oyl]-D-glucopyranose (4) which was also prepared by a definitive method. Hydrogenolysis of 5 gave both anomers of 1-O-[N-(tert-butyloxycarbonyl)-L-gamma-glutamyl]-D-glucopyranose (6), which, upon treatment with trifluoroacetic acid at - 10 degrees, afforded 2alpha and 2beta, respectively. The structure of 6beta was established by its conversion into 2,3,4,6-tetra-O-acetyl-1-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-beta-D-glucopyranose (7beta), whereas similar treatment of 6alpha gave a mixture of 1,3,4,6-tetra-O-acetyl-2-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-alpha-D-glucopyranose (9) and 7alpha. A 1 leads to 2 acyl migration occurred during esterification of the aglycon carboxyl group of 6alpha with diazomethane to give 2-O-[1-methyl N-(tert-butyloxycarbonyl)-L-glutam-5-oyl]-alpha-D-glucopyranose (8).

The kinetics of hydrolysis of synthetic glucuronic esters and glucuronic ethers by bovine liver and Escherichia coli beta-glucuronidase.

1. The relative rates of hydrolysis of synthetically prepared beta-d-glucuronic esters [aglycone: benzoic acid, veratroic (3,4-dimethoxybenzoic) acid, indol-3-ylacetic acid and ethylbutyric acid], and beta-d-glucuronic ethers (aglycone: phenolphthalein, p-nitrophenol, 3,4-dimethoxyphenol, 3,4-dimethoxybenzyl alcohol) by commercial preparations of beta-glucuronidase from bovine liver and Escherichia coli were investigated. The rates of hydrolysis of all compounds tested were followed by measuring the formation of glucuronic acid under conditions which do not affect the glycosidic ester bond. 2. The pH profiles of the substrates in reaction with the enzyme from both sources were determined, and substrate-saturation curves at the optimal pH for each substrate were constructed; double-reciprocal plots of activity against concentration were linear. 3. Comparison of kinetic data indicates that neither the type of sugar-aglycone linkage, nor the aglycone structure alone can explain the observed K(m) and V(max.) values. 4. alpha-d-Glucuronic esters of benzoic and veratroic acid resisted hydrolysis by beta-glucuronidase from both sources.

The chemical synthesis of 1-O-(indol-3'-ylacetyl)-beta-D-glucopyranose. The higher activity of the glucoside in comparison with exogenous indol-3-ylacetic acid in plant-section elongation tests.

1. The synthesis of 1-O-(indol-3'-ylacetyl)-beta-d-glucopyranose via the fully benzylated 1-O-(indol-3'-ylacetyl)-d-glucopyranose is described. The configuration of the free ester glucoside was confirmed by complete hydrolysis with beta-glucosidase and by the n.m.r. spectrum of the tetra-acetyl derivative. 2. The growth-promoting effect of the glucoside in Avena coleoptile- and pea stem-section tests distinctly exceeds the responses stimulated by equimolar amounts of indol-3-ylacetic acid or equimolar mixtures of indol-3-ylacetic acid and glucose at all concentrations investigated. Time-sequence experiments revealed that the sections stimulated by the glucoside exhibit a markedly greater rate of elongation than those promoted by indol-3-ylacetic acid. 3. 1-O-(Indol-3'-ylacetyl)-beta-d-glucopyranose was isolated from intact Avena coleoptiles. 4. According to the results, the conjugation of indol-3-ylacetic acid with glucose could not be considered merely as a detoxication mechanism for indol-3-ylacetic acid in plant tissues.