Structural studies of the Vibrio cholerae o-antigen

The dominant part of the O-antigen of Vibrio cholerae is a homopolysaccharide composed of (1→2)-linked 4-amino-4,6-dideoxy-α-d-mannopyranosyl (perosaminyl) residues, the amino groups of which are acylated by 3-deoxy-l-glycero-tetronic acid. Most of the amino sugar is decomposed during acid hydrolysis. Treatment of the polymer with anhydrous hydrogen fluoride, which cleaves the glycosidic linkages but does not cause N-deacylation, followed by acid hydrolysis under mild conditions, produced the monomer in good yield. Treatment of the N-deacylated polysaccharide with nitrous acid caused deamination with concomitant rearrangements, typical of 4-amino-4-deoxyhexopyranosyl residues in which the amino group occupies an equatorial position.     

A glycopeptide resistant to exoglycosidases

The carboxyl group of the terminal N-acetylneuraminic acid residue of the glycopeptide, prepared from α₁-acid glycoprotein by protease digestion, was esterified with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and then reduced with sodium borohydride. The reduced glycopeptide, thus prepared, containing the reduced N-acetylneuraminic acid, was resistant to hydrolysis by neuraminidase, and consequently to other exoglycosidases. The penultimate β-d-galactosyl residue of the oligosaccharide chain of the reduced glycopeptide was hydrolyzed by β-d-galactosidase only after the removal of the terminal, reduced, sialic acid by mild hydrolysis with acid. The reduced glycopeptide should be a useful substrate for the assay of endoglycosidases in the presence of exoenzymes. It should also find use as a carbon source in the growth of endoglycosidase-elaborating bacteria.     

Immunological studies in ulcerative colitis: extract, fractionation, and immunological characterization of germ-free rat colon antigen

Intestinal mucins from germ-free rats contained antigens reactive with sera from patients with ulcerative colitis, in addition to human blood group A- and H-like antigens. A crude antigen extract was obtained by phenol-water extraction at 65 °C. Two intestinal glycoproteins were purified from the extract by fractionated ethanol precipitation, ion-exchange chromatography, and gel filtration. The two glycoproteins (2aI and 4aIIb) were homogeneous in regard to electrical charge and molecular size. Both were glycoproteins of the blood group substance type. Component 2aI was very rich in N-acetylgalactosamine and threonine and low in N-acetylglucosamine and sialic acid(s). It had strong blood group A-like activity, weak blood group H activity, and no colon antigen activity as defined by patients' sera. Component 4aIIb was rich in sialic acid(s). About 40% of the sera from patients with ulcerative colitis reacted with this component. No blood group A- or H-like activity could be demonstrated. Colon antigen activity was sensitive to periodate oxidation, but resistant to boiling at neutral pH. It was very sensitive to acid hydrolysis. In fact, colon antigen activity was significantly reduced when subjected to weak acid hydrolysis under conditions which only appeared to release sialic acids.     

Decomposition of phosphoserine and phosphothreonine during acid hydrolysis

The rate of decomposition of phosphoserine and phosphothreonine, both as free O-phosphoamino acids and in peptides, was studied under conditions of acid hydrolysis, using 1, 2, and 6 n HCl at 110°C. For the free O-phosphoamino acids, the decomposition follows first-order kinetics and is two- to fourfold faster for phosphoserine than phosphothreonine. The rate of destruction of these O-phosphoamino acids during hydrolysis of peptides is dependent on the neighboring amino acid residues, and thus the hydrolysis of a free O-phosphoamino acid generally is not a good model for the hydrolysis of that O-phosphoamino acid in a peptide. For the three peptides studied, maximal recoveries of O-phosphoamino acids are obtained after hydrolysis in 6 n HCl for 2 to 4 hr.     

Pharmacokinetic studies of naproxen amides of some amino acid esters with promising colorectal cancer chemopreventive activity

Naproxen (nap) is belonging to Non-steriodal anti-inflammatory drugs (NSAIDs) group of drugs that characterized by their free carboxylic group. The therapeutic activity of nap is usually accompanied by GI untoward side effects. Recently synthesized naproxen amides of some amino acid esters prodrugs to mask the free carboxylic group were reported. Those prodrugs showed a promising colorectal cancer chemopreventive activity. The current study aims to investigate the fate and hydrolysis of the prodrugs kinetically in different pH conditions, simulated gastric and intestinal fluids with pHs of 1.2, 5.5 and 7.4 in vitro at 37 °C. The effect of enzymes on the hydrolysis of prodrugs was also studied through incubation of these prodrugs at 37 °C in human plasma and rat liver homogenates. The pharmacokinetic parameters of selected prodrugs and the liberated nap were studied after oral and intraperitoneal administration in male wistar rats. The results showed the hydrolysis of naproxen amides of amino acid esters to nap through two steps first by degradation of the ester moiety to form the amide of nap with amino acid and the second was through the degradation of the amide link to liberate nap. The two reactions were followed and studied kinetically where K₁ and K₂ (rate constants of degradation) is reported. The hydrolysis of prodrugs was faster in liver homogenates than in plasma. The relative bioavailability of the liberated nap in vivo was higher in case of prodrug containing ethyl glycinate moiety than that occupied l-valine ethyl ester moiety. Each of nap. prodrugs containing ethyl glycinate and l-valine ethyl ester moieties appears promising in liberating nap, decreasing direct GI side effect and consequently their colorectal cancer chemopreventive activity.     

Cloning of a Gene Cluster from Cellvibrio mixtus which Codes for Cellulase, Chitinase, Amylase, and Pectinase

The soil isolate Cellvibrio mixtus UQM2294 degraded a variety of polysaccharides including microcrystalline cellulose. Among 6,000 cosmid clones carrying C. mixtus DNA, constructed in Escherichia coli with pHC79, 50 expressed the ability to degrade one or more of the following substrates: carboxymethyl cellulose, chitin, pectin (polygalacturonic acid), cellobiose, and starch. These degradative genes are encoded in a single 94.1-kilobase segment of the C. mixtus genome; a preliminary order of the genes is starch hydrolysis, esculin hydrolysis, cellobiose utilization, chitin hydrolysis, carboxymethyl cellulose hydrolysis, and polygalacturonic acid hydrolysis. A restriction endonuclease cleavage map was constructed, and the genes for starch, carboxymethyl cellulose, cellobiose, chitin, and pectin hydrolysis were subcloned.     

The linkage of 4-O-methyl-L-galactose in the sulphated polysaccharide of Aeodes ulvoidea

Methyl 2-acetamido-5,6-di-O-benzyl-2-deoxy-β-d-glucofuranoside (11) was obtained in six steps from the known methyl 3-O-allyl-2-benzamido-2-deoxy-5,6-O-isopropylidene-β-d-glucofuranoside. Mild acid hydrolysis, followed by benzylation gave the 5,6-dibenzyl ether. The benzamido group was exchanged for an acetamido group by strong alkaline hydrolysis, followed by N-acetylation, and the allyl group was isomerized into a 1-propenyl group that was hydrolyzed with mercuric chloride. Treatment of 11 with l-α-chloropropionic acid and with diazomethabe gave methyl 2-acetamido-5,6-di-O-benzyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-β-d-glucofuranoside which formed on mercaptolysis the internal ester 16, further converted into 2-acetamido-4-O-acetyl-5,6-di-O-benzyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-d-glucose diethyl dithioacetal (18) by alkaline treatment followed by esterification with diazomethane and acetylation. Attempts to remove the O-acetyl group of the corresponding dimethyl acetal 20 with sodium methoxide in mild conditions were not successful.     

Identification of 2-amino-6-O-(2-amino-2-deoxy-beta-D-glucopyranosyl)-2-deoxy-D-glucose as a major constituent of the hydrophobic region of the Bordetella pertussis endotoxin

2-Amino-6-O-(2-amino-2-deoxy-β- d-glucopyranosyl)-2-deoxy- d-glucose substituted on the amino group of the reducing 2-amino-2-deoxy- d-glucose unit by a 3-hydroxytetradecanoyl group was shown to be a major constituent of the “Lipid A” fragment obtained by acid hydrolysis of the Bordetella pertussis endotoxin.     

Multifunctional hydrolytic catalysis: VI. Catalytic hydrolysis of p-nitrophenyl acetate by N-(4-imidazolylmethyl)benzohydroxamic acid

A bifunctional catalyst, N-(4-imidazolylmethyl)benzohydroxamic acid, was synthesized from benzohydroxamic acid and chloromethylimidazole, and used for the hydrolysis of p-nitrophenyl acetate. The reaction proceeded via the formation of the acetyl hydroxamate and its subsequent decomposition. The deacylation step was shown to be general base-catalyzed by the intramolecular imidazole group on the basis of the deuterium solvent kinetic isotope effect of 2.0. The efficiency of water attack on the acetyl hydroxamate was enhanced 130-fold by the imidazole group. The catalytic process is compared with the reactions of related monofunctional compounds, and finally its significance as a model of the charge relay system is discussed.     

Analysis of adipimidate-crosslinked lysine

The chromatographic conditions for separation of N,N′-bislysyl(?-N)adipamidine and N-lysyl(?-N)adipamidinic acid, which were the products of acid hydrolysis of proteins treated with adipimidate esters, from other amino acids on an amino acid analyzer were established including their ninhydrin color values. Kinetics of decomposition of these lysine derivatives under the conditions of total acid hydrolysis of protein are also reported.     

The preparation of matrix-bound proteases and their use in the hydrolysis of proteins

Four proteases, crude acid protease from Aspergillus, pronase, amino-peptidase M, and prolidase, have been covalently attached to activated agarose and to amino propyl glass beads. The matrix-bound enzymes have been tested as catalysts for the complete hydrolysis of protein substrates, with the primary goal to isolate unstable amino acid derivatives present in the substrate protein. Under conditions used in the present work, the total amino acid release from the protease-catalyzed hydrolysis of four substrate proteins (pancreatic ribonuclease, egg white lysozyme, yeast enolase, and bovine insulin) was 95–103% of that observed in standard acid hydrolysis. Recovery of individual amino acids showed greater deviation from the theoretical values, but cystine was the only amino acid recovered in low yields (42–77%) from all four proteins. Derivatized amino acids, such as methionine sulfoxide, O-(butylcarbamoyl)-serine, and N-glycosyl asparagine have been obtained from chemically modified proteins or from unmodified glycoprotein in good yield, and normal amino acid constituents of proteins which cannot be quantified after acid hydrolysis (tryptophan, asparagine, and glutamine) have also been determined either directly after proteolysis or after proteolysis in conjunction with acid hydrolysis.     

The linkage between the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei

1. The linkage between the polysaccharide and mucopeptide components of the cell wall of Lactobacillus casei is rapidly hydrolysed under mild acid-hydrolysis conditions. 2. The release of the polysaccharide is accompanied by the hydrolysis of an N-acetylhexosaminide linkage. The N-acetylhexosamine residue readily forms chromogen and it is concluded that it is substituted on C₍₃₎ by the adjacent sugar. 3. Continued heating of the polysaccharide in acid results in a slower release of reactive N-acetylhexosamine due to the hydrolysis of glycosidic linkages within the polysaccharide. 4. After the linkage between the polysaccharide and mucopeptide has been hydrolysed, acid phosphatase will release approx. 40% of the total phosphorus as inorganic phosphate. 5. It is concluded that the polysaccharide component of the cell wall is joined through its reducing end group to a phosphate grouping in the mucopeptide.     

Influence of the location of substituents in substituted celluloses on solution hydrolysis of the d-glucosidic linkages

Sensitivity of the d-glucosidic linkages in cellulose to hydrolysis in homogeneous acidic media was found to be directly related to the location of a substituent in the d-glucoyranosyl unit. The 2-diethylaminoethyl (DEAE) substituent caused sensitivity toward hydrolysis to decrease in the order d-glucose > 3-O >6-O- 2-O-DEAE-d-glucopyranosyl-unit in hydrolyses beginning in 72% sulfuric acid and in 100% trifluoroacetic acid (TFA). Differences in the substituent effects were larger in TFA than in sulfuric acid. The effects reported for acid-catalyzed hydrolyses in homogeneous media are discussed relative to enzymic hydrolysis of a water-soluble, O-substituted cellulose.     

Characterisation of two ornithine-containing lipids from Erwina aroideae

An apparently pure ornithine-containing lipid (OCL) was isolated from Erwina aroideae by solvent extraction and thin-layer chromatography (TLC). However, selective hydrolysis of the lipid under acidic and basic conditions and analysis of hydrolysates by gas chromatography-mass spectrometry (GCMS) showed that two structurally similar OCL were in fact present. These lipids both contained a 3-hydroxyhexadecanoic acid moiety which was linked to ornithine by an amide group formed between the 2-amino group of ornithine and the carboxyl group of the acid. The two lipids, however, differ in the nature of the fatty acid bound through an ester linkage to the hydroxyl group of the 3-hydroxyhexadecanoic acid moiety. One lipid is the ester of hexadecanoic acid whereas the other lipid is the ester of octadecenoic acid. These lipids are present in approximately equal amounts.     

Hydrolysis of novel diacylglycerol and phorbol diesters by serum lipase

Rat serum, active in the hydrolysis of the tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was examined with regard to lipid interferences of [³H]TPA hydrolysis and enzyme substrate specificity. The enzymatic hydrolysis of TPA could be enhanced 8-fold, ever crude serum, by using a lipid-free acetone powder of rat serum. Addition of lipid to the lipid-free acetone powder produced potent inhibition of TPA hydrolysis. The inclusion of multilamallar liposomes resulted in similar inhibition, and isolation of liposomes by high-speed centrifugation showed that 95% of the radiolabeled TPA was associated with the fatty pellet. Substrate specificity studies demonstrated that the serum activity hydrolyzes the long-chain ester of TPA and the long-chain primary acyl group of diacylglycerols. TPA was hydrolyzed at approximately twice the rate of dioleoylglycerol; however, the most reactive substrates were those synthetic analogs of diacylglycerol containing a short-chain ester group at the sn-2 position. Palmitic acid was liberated from [1-¹⁴C]palmitoyl-2-acetyl-sn-glycerol and [1-¹⁴C]palmitoyl-2-butyryl-sn-glycerol at 120- and 33-tinies the rate of TPA hydrolysis, respectively. Lipase resistant 1-hexadecyl-2-[³H]acetylglycerol was also used as substrate, but the sn-2 ester moiety showed poor lability. The diacylglycerol analogs are new lipase substrates and, in view of their similarities to the fatty acyl portion of TPA, it is thought that these compounds could serve as protein kinase C activators.     

The hydrolytic susceptibility of prochelator BSIH in aqueous solutions

The prochelator BSIH ((E)-N′-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzylidene)isonicotinohydrazide) contains a boronate group that prevents metal coordination until reaction with peroxide releases the iron chelator SIH ((E)-N′-(2-hydroxybenzylidene)isonicotinohydrazide). BSIH exists in aqueous buffer and cell culture media in equilibrium with its hydrolysis products isoniazid and (2-formylphenyl)boronic acid (FBA). The relative concentrations of these species limit the yield of intact SIH available for targeted iron chelation. While the hydrolysis fragments are nontoxic to retinal pigment epithelial cells, these results suggest that modifications to BSIH that improve its hydrolytic stability yet maintain its low inherent cytotoxicity are desirable for creating more efficient prochelators for protection against cellular oxidative damage.     

Candida rugosa lipase mediated multigram synthesis of acid part of S(+)-atliprofen,a new NSAID and molecular modeling studies aimed at predicting selectivity of the enzyme

An efficient procedure to prepare S-4-(3-thienyl)phenyl-α-methylacetic acid, an intermediate of a recently approved non-steroidal anti-inflammatory cyclooxygenase inhibitor atliprofen by enantioselective hydrolysis of the corresponding esters in presence of candida rugosa lipase is reported. The methyl and butyl esters of the racemic acid 2 were synthesized and subjected to enantioselective hydrolysis by the lipase to give S-4-(3-thienyl)phenyl-α-methylacetic acid upto 97.86% ee. The observed enantioselectivity during the hydrolysis of the substrate by the lipase was rationalized by molecular modeling studies. The methyl esters of both R and S-enantiomers of 4-(3-thienyl)phenyl-α-methylacetic acid, naproxen and ketoprofen were taken for the modeling studies. The results of the modeling studies are in conformity with the experimental observations.     

Asymmetric acetalation of α,α-trehalose: Synthesis of α-d-galactopyranosyl α-d-glucopyranoside and 6-deoxy-6-fluoro-α-d-glucopyranosyl α-d-glucopyranoside

Selective acetalation of α,α-trehalose with ethyl or methyl isopropenyl ether and toluene-p-sulphonic acid in N,N-dimethylformamide gave the 4,6-isopropylidene acetal as the major product, isolated as its hexa-acetate 1 (38%). The gluco-galacto analogue 6 of α,α-trehalose was synthesized from 1 by the sequence: hydrolysis of the isopropylidene group with trifluoroacetic acid, mesylation of the resulting diol, benzoate displacement, and saponification of the product. Deacetylation of 1 followed by benzylation and hydrolysis of the acetal group furnished a hexa-O-benzyl derivative 9. Tosylation of the primary hydroxyl group in 9, treatment of the product with tetrabutylammonium fluoride in acetonitrile, and subsequent catalytic hydrogenolysis of the benzyl groups gave 6-deoxy-6-fluoro-α,α-trehalose (12). Compounds 6 and 12 and 6-deoxy-6-iodo-α,α-trehalose are substrates for cockchafer trehalase, but have very low V_max values.     

THE ARGININE AND PREARGININE GROUPS IN EDESTIN

The author corroborates the data of Schmidt showing that the dissociation index of the third group of arginine is pK₃'' = 12.5. New titration data of edestin have been obtained in very alkaline solutions and show that there is a corresponding group with a titration index of pG'' = 12.0, but present in much less quantity than can account for the arginine found on hydrolysis. The data support the theory that the combination of strong base or strong acid with proteins is produced by the formation of salts with the "extra groups" of those trivalent amino acids which can be isolated from the protein, with the exception of arginine. Arginine contributes to the titration curve in much smaller amount than is found on hydrolysis. This deficiency in the arginine group may be accounted for by the basic group in proteins having a titration index of pG'' = 3.8 to 4.6 (depending on the protein), which apparently yields arginine on hydrolysis, and may properly be called prearginine.     

Glucuronyl glycine,a novel N-terminus in a glycoprotein

Treatment of cutinase, an extracellular glycoprotein produced by Fusarium solani f. pisi, with NaB³H₄ at pH 7.0 generated labeled enzyme. Acid hydrolysis showed that all of the label was in an acidic carbohydrate which was identified as gulonic acid. The N-terminal amino group of the enzyme is blocked; the precursor of gulonic acid has a free reducing group and it is attached via a linkage resistant to β-elimination. Furthermore, pronase digestion of NaB³H₄-treated cutinase gave rise to a ninhydrin negative compound which contained the bulk of the ³H and this compound was identified as N-gulonyl glycine. These results strongly suggest that the amino group of glycine, the N-terminal amino acid of this enzyme, is in amide linkage with glucuronic acid.