The chemical synthesis of glucosaminylphosphatidylglycerol. Comparison with a new phospholipid isolated from Bacillus megaterium.
ABSTRACT: 1. We describe the synthesis of a glucosamine derivative of phosphatidylglycerol having the same structure as that of the natural compound isolated from Bacillus megaterium. 2. 2-O-(3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-d-glucopyranosyl)-3-O-benzyl-1-iodo-sn-glycerol was prepared by a Königs-Knorr condensation between 3-O-benzyl-1-toluene-p-sulphonyl-sn-glycerol and 3,4,6-tri-O-acetyl-1-bromo-2-deoxy-2-phthalimido-d-glucopyranose followed by replacement of the toluene-p-sulphonyl group with iodine. The iodide was treated with the silver salt of 2-isolauroyl-1-oleoyl-sn-glycerol 3-(monobenzyl hydrogen phosphate) to form the fully protected phosphoglycolipid. 3. Removal of benzyl protecting groups by catalytic hydrogenolysis, phthaloyl group with hydrazine and acetyl groups with pH10 buffer furnished 2-O-(2-amino-2-deoxy-d-glucopyranosyl)-1-(2-isolauroyl-1-stearoyl-sn-glycero-3-phosphoryl)-sn-glycerol. 4. The synthetic and natural compounds appeared identical when compared by chromatography and by identification of hydrolysis products from chemical and enzymic degradations.
Project description:Dimethylthexylsilyl 2-acetamido-3-O-allyl-2-deoxy-6-O-(4-methoxybenzyl)-beta-D-glucopyranoside was prepared by reduction of the corresponding 4,6-O-(4-methoxybenzylidene) acetal with sodium cyanoborohydride and trifluoroacetic acid. This alcohol was coupled to 2-O-benzoyl-3,4,6-tri-O-benzyl-alpha-D-glucopyranosyl trichloroacetimidate to give a beta-glucoside that was converted to dimethylthexylsilyl 3,4,6-tri-O-benzyl-beta-D-mannopyranosyl-(1-->4)-2-acetamido-3-O-allyl-2-deoxy-6-O-(4-methoxybenzyl)-beta-D-glucopyranoside by saponification, Dess-Martin oxidation, and sodium borohydride reduction. Sulfonylation then gave dimethylthexylsilyl 2-O-(benzylsulfonyl)-3,4,6-tri-O-benzyl-beta-D-mannopyranosyl-(1-->4)-2-acetamido-3-O-allyl-2-deoxy-6-O-(4-methoxybenzyl)-beta-D-glucopyranoside.
Project description:1. The isolation, characterization and properties of two by-products in the preparation of 2-acetamido-3,4,6-tri-O- acetyl-2-deoxy-beta-d-glucopyranosylamine are described. They are bis(2-acetamido-2-deoxy-d-glucopyranosyl)amines. 2. An independent synthesis of the bis-glycopyranosylamines is reported and conditions are given for their preparation in high yield. 3. Further improvements are given for the synthesis of 2-acetamido-1-N-(beta-l- aspartyl)-2-deoxy-beta-d-glucopyranosylamine and the alpha-l-aspartyl isomer. 4. The synthesis of 2-acetamido-1-N-acetyl-2-deoxy-beta-d-glucopyranosylamine is described.
Project description:1. The lipids of Bacillus megaterium were extracted and three lipids containing glucosamine were identified. One of these is not a phospholipid, but the other two, which differ in their chromatographic behaviour, contain phosphorus, glycerol, fatty acid and d-glucosamine in the molar proportions 1:2:2:1. 2. In both phosphoglycolipids, the fatty acids are bound in ester linkage, and both yield 2,5-anhydromannose and 3-sn-phosphatidyl-1'-sn-glycerol on treatment with sodium nitrite. 3. Both phosphoglycolipids were N-acetylated and, after removal of fatty acids by mild alkaline hydrolysis, in both cases N-acetylglucosamine was quantitatively released by beta-N-acetylhexosaminidase. 4. The glucosaminylglycerols derived from the two phosphoglycolipids by partial acid hydrolysis differ in their behaviour towards periodate. In one case 1 mole of periodate is rapidly consumed/mole of glucosaminylglycerol, but in the other case under identical conditions the consumption of periodate is negligible. 5. The phosphoglycolipids were identified as 1'-(1,2-diacyl-sn-glycero-3-phosphoryl)-3'-O-beta-(2-amino-2-deoxy-d-glucopyranosyl)-sn-glycerol and as 1'-(1,2-diacyl-sn-glycero-3-phosphoryl)-2'-O-beta-(2-amino-2-deoxy-d-glucopyranosyl)-sn-glycerol. 6. Both phosphoglycolipids are good substrates for phospholipase A: neither is a substrate for phospholipase C from Clostridium perfringens, and only the 3'-glucosaminide is a substrate for phospholipase D.
Project description:Regioselective modification of d-glucosamine (2-amino-2-deoxy-d-glucopyranose, GA) through C-1 and C-2 positions to synthesized thermo-responsive D-Glucosamine-poly(N-iso-propylacrylamide) (PNIPAM) via atom transfer radical polymerization (ATRP) was investigated for the first time. Two different schemes of the synthesis for GA derivatives (GA-PNIPAM (i) and (ii)) with well-defined structures using 3,4,6-tri-o-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranose and 1,3,4,6-tetra-o-acetyl-2-amino-2-deoxy-β-d-glucopyranose intermediates were examined. The GA-PNIPAM (ii) had an amino at C-2 position, while there was a hydroxyl in GA-PNIPAM (i) at this position. Both the resulting oligomers (i) and (ii) had a narrow dispersity, and no significant cytotoxic response of copolymers (i) and (ii) was observed in the cell line over the concentration range from 0.1 μg/mL to 1000 μg/mL at any of the exposure times. In addition, it was discovered that GA-PNIPAM (i) and (ii) inhibited the proliferation of Human Hepatocellular Carcinoma Cells HepG2 as the concentration and the time changed, and the inhibitory activity of polymer (ii) was higher than that of he (i). The results suggest that the GA-PNIPAM polymers show excellent biocompatibility in vitro.
Project description:1. The phosphatidylglucose structure proposed previously (Smith & Henrikson, 1965) for the glucose-containing phospholipid from Acholeplasma laidlawii is incorrect. 2. The structure now proposed is 3-(sn-glycerol-3-phosphoryl-6'-[O-alpha-d-glucopyranosyl-(1-->2)-O-alpha-d-glucopyranosyl])- sn-1,2-diglyceride, a new type of bacterial lipid. 3. Deacylation of the lipid gave a single water-soluble phosphate ester which could be distinguished on chromatography from synthetic samples of glucosylphosphorylglycerols. 4. Hydrolysis of the lipid with alkali gave a mixture of fatty acids, glycerol 2-phosphate, sn-glycerol 3-phosphate and O-alpha-d-glucopyranosyl-(1-->2)-O-alpha- d-glucopyranosyl-(1-->1)-d-glycerol. 5. The lipid was unaffected on incubation with phospholipases A, C and D. 6. Diglucosyl diglyceride was isolated after treatment of the lipid with 60% HF, establishing the location of the fatty acid residues. 7. Periodate oxidation studies showed that the sn-glycerol 3-phosphate was esterified to the 6-hydroxyl group of one of the glucose residues in diglucosyl diglyceride.
Project description:Four glycoconjugate building blocks for the construction of combinatorial PNA like glycopeptide libraries were prepared in 75-79% yield by condensing tert-butyl N-[2-(N-9-fluorenylmethoxycarbonylamino)ethyl]glycinate (AEG) 5 with 3-oxo-3-(2,3,4,6-tetra-O-acetyl-?-D-glucopyranosylamino)- (6a), 3-oxo-3-(?-D-galactopyranosylamino)- (6b), 3-oxo-3-(2-acetamido-2-deoxy-3,4,6-tetra-O-acetyl-?-D-glucopyranosylamino)- (6c) and 3-oxo-3-(2-acetamido-2-deoxy-3,4,6-tetra-O-acetyl-?-D-galactopyranosylamino)propanoic acid (6d), respectively. The resulting AEG glycoconjugates 1a-d were converted into the corresponding free acids 2a-d in 97-98% yield by treatment with aqueous formic acid. The Fmoc group of compound 1c was removed and the intermediate amine 9 was condensed with 2a to afford the corresponding glycosylated AEG dipeptide 4 in 58% yield. All glycoconjugate building blocks showed the presence of cis and trans rotamers. Compounds 1a, 1b and 4 were subjected to temperature dependent 1H NMR spectroscopy in order to determine the coalescence temperature which resulted in calculated rotation barriers of 17.9-18.3 kcal/mol for the rotamers.
Project description:Several esters of the alpha-N-toluene-p-sulphonyl and alpha-N-benzoyl derivatives of S-(3-aminopropyl)-l-cysteine and the methyl ester of S-(4-aminobutyl)-N-toluene-p-sulphonyl-l-cysteine were synthesized. The kinetics of hydrolysis of these and esters of the alpha-N-toluene-p-sulphonyl and alpha-N-benzoyl derivatives of l-arginine, l-lysine, S-(2-aminoethyl)-l-cysteine and esters of gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyric acid and alpha-N-toluene-p-sulphonyl-l-homoarginine by alpha- and beta-trypsin were compared. On the basis of values of the specificity constants (k(cat.)/K(m)), the two enzymes display similar catalytic efficiency towards some substrates. In other cases alpha-trypsin is less efficient than beta-trypsin. It is possible that alpha-trypsin possesses greater molecular flexibility than beta-trypsin.
Project description:1. Esters of gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyric acid (alpha-N-toluene-p-sulphonyl-l-norarginine) have been synthesized and shown to be hydrolysed by bovine trypsin and thrombin. As substrates for these enzymes, they were better than esters of alpha-N-toluene-p-sulphonyl-l-homoarginine or of alpha-N-toluene-p-sulphonyl-l-ornithine but not as good as esters of alpha-N-toluene-p-sulphonyl-l-arginine. 2. With trypsin as catalyst, the methyl and propyl esters are hydrolysed at the same rate at high substrate concentrations and hence deacylation of the acyl-enzyme appears to be rate-determining. In the presence of thrombin, however, the methyl ester is hydrolysed much faster than the n-propyl ester. 3. The variation of k(0) with pH indicates that groups with pK((app.)) values of 7.05+/-0.02 and 6.53+/-0.02 must be dissociated in trypsin and thrombin respectively for hydrolysis to proceed. 4. Activation constants have been determined for the trypsin-catalysed hydrolysis of methyl gamma-guanidino-l-alpha-toluene-p-sulphonamidobutyrate and have been compared with the corresponding constants for the hydrolysis of homologous substrates. 5. Cholate increases k(0) and decreases K(m); the effects are more pronounced with thrombin than with trypsin.
Project description:The first total synthesis of 5'-O-?-d-glucopyranosyl tubercidin was successfully developed. It is a structurally unique disaccharide 7-deazapurine nucleoside exhibiting fungicidal activity, and was isolated from blue-green algae. The total synthesis was accomplished in eight steps with 27% overall yield from commercially available 1-O-acetyl-2,3,5-tri-O-benzoyl-?-d-ribose. The key step involves stereoselective ?-O-glycosylation of the corresponding 7-bromo-6-chloro-2',3'-O-isopropylidene-?-d-tubercidin with 2,3,4,6-tetra-O-benzyl-glucopyranosyl trichloroacetimidate. All spectra are in accordance with the reported data for natural 5'-O-?-d-glucopyranosyl tubercidin. Meanwhile, 5'-O-?-d-glucopyranosyl tubercidin was also prepared using the same strategy.
Project description:Glycerol kinase catalyses the phosphorylation of the symmetrical substrate, 2-dexoy-2-flurooglycerol, by ATP to an asymmetric product, 2-deoxy-2-fluoro-sn-glycerol 3-phosphate. The stereospecificity of the enzymic reaction was extablished by unambiguous chemical synthesis of 2-deoxy-2-fluoro-sn-glycerol labelled with 2H at C-1, followed by glycerol kinase-catalysed phosphorylation and isolation of the labelled phosphate. The configuration of the 2H-labelled phosphate was determined by n.m.r. spectroscopy. This enzymic phosphorylation of 2-dexoy-2-fluoroglycerol is absolutely stereospecific in the same sence as that of glycerol, with fluorine replacing the C-2 hydroxy group. The behaviour of fluorine as a hydroxy analogue in directing the stereospecific course of the enzyme reaction is relevant to the use of the fluorine atom of fluoro analogues of substrate as a reporter group for hydroxy-binding sites of enzymes.