Synthesis of oligopeptides consisting of L-leucyl-L-leucyl-L-ananyl and L-methionyl-L-methionyl-L-leucyl repeating units with an improved preparation of Nps-amino acids

Two series of peptides with hydrophobic side chains, Nps-(L -Leu-L -Leu-L -Ala)_n-OEt and Nps-(L -Met-L -Met-L Leu)_n-OEt (n = 1–6), were synthesized by the fragment condensation method using dicyclohexylcarbodiimide in the presence of N-hydroxysuccinimide. The tripeptide fragments were prepared stepwise by dicyclohexylcarbodiimide-mediated reaction of Nps-amino acids, which were synthesized by an improved rapid procedure.     

Sequential oligopeptides. Conformational studies of the oligopeptides and a polypeptide with the repeating sequence L-norvalyl-glycyl-L-proline

Conformational studies of a series of oligopeptides (from the tripeptide to the octadecapeptide) with the repeating sequence L -norvalyl-glycyl-L -proline and a polytripeptide with this sequence are reported. By means of chiroptical techniques, unordered conformations are found for all oligopeptides in water, trifluoroethanol, and ethylene glycol and for the water-insoluble polymer in trifluoroethanol. In ethylene glycol the polymer assumes a collagen-like structure. Infrared studies indicate that all the oligomers, in contrast to the polymer, are unordered in the solid state.     

Sequential oligopeptides. Synthesis and characterization of the oligopeptides and a polypeptide with the repeating sequence L-norvalyl-glycyl-L-proline

The synthesis and characterization of a series of oligopeptides (from the tripeptide to the octadecapeptide) with the repeating sequence L -norvalyl-glycyl-L -proline and a polytripeptide with this sequence are reported. The oligomers were synthesized step by step using the mixed anhydride method. All the products were chemically and optically pure. The polymer was prepared by the active ester method, using the p-nitrophenyl ester as the polymerizable tripeptide derivative. Good yield of relatively high average molecular-weight polymer was obtained. In the accompanying paper conformational investigations, both in solution and in the solid state, on the oligomers and the polymer are described.     

Synthesis of the repeating units of Schizophyllan

The tetrasaccharides β-d-Glcp-(1→3)-β-d-Glcp-(1→3)-[β-d-Glcp-(1→6)]-d-Glcp, β-d-Glcp-(1→3)-[β-d-Glcp-(1→6)]-β-d-Glcp-(1→3)-d-Glcp, and β-d-Glcp-(1→6)-β-d-Glcp-(1→3)-β-d-Glcp-(1→3)-d-Glcp, corresponding to the three possible repeating-units of Schizophyllan, have been synthesised by silver trifluoromethanesulfonate-promoted Koenigs-Knorr type condensations, using 2,4,6-tri-O-acetyl-3-O-allyl-α-d-glucopyranosyl bromide as the key intermediate.     

Synthesis and conformational analysis of the repeating units of bacterial spore peptidoglycan

Deprotection of the fully blocked disacharide allyl O-(2-amino-4,6-O-benzylidene-3-O-[(R)-1-carboxyethyl]-2-deoxy-beta-D-glucopyranosyl-1',2-lactam)-(1-->4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside by selective de-O-allylation and parallel removal of the benzylidene and O-benzyl groups is described. The resulting beta-muramyl lactam-(1-->4)-GlcNAc disaccharide is characterised as the per-O-acetylated derivative by 1H and 13C NMR spectroscopy and X-ray structure analysis. Conformational analysis about glycosidic bond of repeating units of bacterial spore cortex is based on experimental data and molecular modelling.     

Clostridium difficile cell-surface polysaccharides composed of pentaglycosyl and hexaglycosyl phosphate repeating units

Clostridium difficile is a Gram-positive bacterium that is known to be a cause of enteric diseases in humans. It is the leading cause of antibiotic-associated diarrhea and pseudomembranous colitis. Recently, large outbreaks of C. difficile-associated diarrhea have been reported internationally, and there have been reports of increases in severe disease, mortality and relapse rates. At the moment, there is no vaccine against C. difficile, and the medical prevention of C. difficile infection is mostly based on the prophylactic use of antibiotics; however, this has led to an increase in the incidence of the disease. Here, we describe the chemical structure of C. difficile cell-surface polysaccharides. The polysaccharides of three C. difficile strains were structurally analyzed; ribotype 027 (North American pulsotype 1) strain was observed to express two polysaccharides, one was composed of a branched pentaglycosyl phosphate repeating unit: [-->4)-alpha-l-Rhap-(1-->3)-beta-D-Glcp-(1-->4)-[alpha-l-Rhap-(1-->3]-alpha-D-Glcp-(1-->2)-alpha-D-Glcp-(1-->P] and the other was composed of a hexaglycosyl phosphate repeating unit: [-->6)-beta-D-Glcp-(1-->3)-beta-D-GalpNAc-(1-->4)-alpha-D-Glcp-(1-->4)-[beta-D-Glcp-(1-->]-beta-D-GalpNAc-(1-->3)-alpha-D-Manp-(1-->P]. The latter polysaccharide was also observed to be produced by strains MOH900 and MOH718. The results described here represent the first literature report describing the covalent chemical structures of C. difficile cell-surface polysaccharides, of which PS-II appears to be a regular C. difficile antigen. These C. difficile teichoic-acid-like polysaccharides will be tested as immunogens in vaccine preparations in a rat and horse model.     

Conformation of the hexasaccharide repeating subunit from the Vibrio cholerae O139 capsular polysaccharide

In the past decade, several outbreaks of cholera have been reported to be caused by Vibrio cholerae O139, a strain which differs from the more common O1 strain in that the former is encapsulated. The hexasaccharide repeating subunit has been isolated from the V. cholerae O139 capsular polysaccharide by digestion with a recently discovered polysaccharide lyase derived from a bacteriophage specific for this serogroup. It specifically cleaves at a single position of the 4-linked galacturonic acid producing an unsaturated sugar product in quantities for conformational studies by (1)H and (13)C NMR spectroscopy. We report conformational studies on this oligosaccharide by molecular modeling and NMR spectroscopy including nuclear Overhauser effects and residual dipolar coupling of a sample weakly oriented in liquid crystalline solution. The structure contains a tetrasaccharide epitope homologous to the human Lewis(b) blood group antigen, which adopts a relatively well-defined single conformation. Comparison of these results with those of a previously published study of the intact capsular polysaccharide indicates that the conformations of the epitope in the two cases are identical or at least closely similar. Thus, this epitope, which may be essential for the pathogenicity of this V. cholerae strain, is not a "conformational epitope" requiring a certain critical size for antigenicity as has been reported for several other bacterial capsular antigens.     

Potential of mean force for separation of the repeating units in cellulose and hemicellulose

As a first step toward understanding the energetics of removal of cello-oligomers from the cellulose surface, we have performed umbrella sampling calculations to determine the free energy required for separation of repeating units of cellulose and hemicellulose from each other. Molecular dynamics (MD) simulations were performed for both the stacked and non-stacked arrangements of the cellobiose pair system and the xylobiose pair system. These stacked and non-stacked arrangements were taken as representative systems for the crystalline and amorphous domains in cellulose and hemicellulose. In addition, similar calculations were also carried out to determine the energetics involved in the separation of the cellobiose–xylobiose molecule pair in the non-stacked arrangement. The potential of mean force profiles exhibit a single minimum in all cases and are qualitatively similar. Our results show that the location of the minimum as well as the depth of the well can be correlated with the size of the disaccharide molecules.     

Structural heterogeneity in the lipopolysaccharides of Pseudomonas syringae with O-polysaccharide chains having different repeating units.

Studies by sugar and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy revealed a structural heterogeneity in the O-polysaccharides of Pseudomonas syringae pvs. coronafaciens IMV 9030 and atrofaciens IMV 8281 owing to the presence of different types of repeating units. In strain IMV 9030, the major repeating units are a linear alpha-L-rhamnose trisaccharide and a tetrasaccharide (A, n=0 or 1). A minor repeating unit is a branched pentasaccharide with an alpha-L-rhamnose main chain and a lateral 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) residue (B, X=2, n=1). In strain IMV 8281, all repeating units are branched and differ in size and position of substitution of one of the alpha-L-rhamnose residues (tetrasaccharide, B, X=3, n=0; pentasaccharides, B, X=2 or 3, n=1). [structure--see text] Reinvestigation of the structure of the branched O-polysaccharide of P. syringae pv. tomato IPGR 140 showed that, together with the major tetrasaccharide repeating unit (B, X=3, n=0) [Knirel, Y. A., et al. Carbohydr. Res. 1993, 243, 199-204], it has a minor pentasaccharide repeating unit (B, X=3, n=1).     

CD studies of double-stranded polydeoxynucleotides composed of repeating units of contiguous homopurine residues

Double-stranded synthetic polydeoxynucleotides of the general form poly[d(G_nC_n)] · poly[d(G_nC_n)], poly[d(G_nC)] · poly[d(GC_n)], and poly[d(A_nT_n)] · poly[d(A_nT_n)] have been synthesized. When n = 4 or larger, the CD spectra of polymers of the form poly[d(G_nC_n)] · poly[d(G_nC_n)] or poly[d(G_nC)] · poly[d(GC_n)] closely resemble the spectrum of poly[dG] · poly[dC], suggesting that a string of four continguous guanosine residues is sufficient to induce a conformation resembling that of the polypurine · polypyrimidine. With polymers of the form poly[d(A_nT_n)] · poly[d(A_nT_n)], however, the CD spectrum only gradually approaches that of poly[dA] · poly[dT].     

Distribution of a family of Haemophilus influenzae genes containing CCAA nucleotide repeating units

A family of genes containing lengths of CCAA nucleotide repeating units directly following the sequence encoding the leader peptide has been identified in Haemophilus influenzae. The length of the CCAA repeats ranges from 6 to 43 and all of the identified genes encode proteins or predicted proteins with a significant homology to bacterial iron- or heme-related outer membrane proteins. We have previously shown that two of these gene products, HgpA and HgpB, bind hemoglobin and the hemoglobin-haptoglobin complex. Studies were performed to define the species distribution of the five identified genes and the CCAA repeats. We show that both the CCAA motif and the structural genes for hemoglobin and hemoglobin-haptoglobin binding are widely distributed among H. influenzae strains.     

The synthesis of two repeating units of Haemophilus influenzae type a capsular antigen

The repeating units 2-O-beta-D-glucopyranosyl-L-ribitol 4'- and 1-phosphate of Haemophilus influenzae type a capsular antigen have been synthesised by condensation of an alpha-D-glucopyranosyl bromide derivative with 5-O-allyl-1,2,3-tri-O-benzyl-D-ribitol followed by selective deprotection of HO-4' or HO-1, phosphorylation, and removal of the blocking groups.     

Multigram syntheses of the disaccharide repeating units of chondroitin 4- and 6-sulfates.

The multigram syntheses of beta-D-glucopyranosyluronic acid-(1-->3)-2-acetamido-2-deoxy-4- and 6-O-sulfo-D-galactopyranose disodium salt, the disaccharide repeating units of chondroitin 4- and 6-sulfates, are described. The disaccharide benzyl methyl 2,3,4-tri-O-benzoyl-beta-D-glucopyranosyluronate- (1-->3)-2-acetamido-2-deoxy-alpha-D-galactopyranoside was used as a common intermediate. Selective benzoylation at O-6 followed by O-sulfonation at C-4 of the aminosugar moiety, saponification and catalytic hydrogenation afforded the 4-O-sulfo derivative, whereas selective O-sulfonation at C-6 followed by similar deprotection steps provided the 6-O-sulfo derivative in high yield.     

The structure of the gene for mouse filaggrin and a comparison of the repeating units

Filaggrins are an important class of intermediate filament-associated proteins that are involved in the organization of keratin filaments in the terminal stages of mammalian epidermal differentiation. Filaggrins are initially synthesized as very large polyprotein precursors consisting of many tandemly arranged repeats that are later liberated by proteolytic processes to yield many copies of the functional protein. We have recently characterized a cDNA clone to mouse filaggrin (Rothnagel, J. A., Mehrel. T., Idler, W. W., Roop, D. R., and Steinert, P. M. (1987) J. Biol. Chem. 262, 15643-15648) which encodes a 750-base pair (250-amino acid) repeating element having properties consistent with a filaggrin molecule. Southern blot analysis of total mouse DNA and the mouse gene isolated from a cosmid library (cosmid clone cFM6.1A2) has also revealed a repeat length of about 750 base pairs. The cosmid clone contains most of the mouse filaggrin gene, but it is missing the 5'-noncoding sequences and possibly some coding sequences as well. We report here that cosmid clone cFM6.1A2 contains 20 filaggrin repeats and 15,213 base pairs of coding sequences. Sequence analysis of this clone has revealed at least two different types of repeating element. Type B has a repeat length of 750 base pairs (250 amino acids), whereas type A is 765 base pairs (255 amino acids) long and contains an additional five amino acids inserted next to an acidic sequence that delineates the amino and carboxyl termini of the filaggrin repeats. It is supposed that these additional five amino acids may alter the proteolytic sensitivity of the acidic linker sequence, thereby affecting the processing of the precursor. The random distribution of the two types of repeats in the precursor indicates that the mouse filaggrin gene arose by a complicated series of duplications and/or rearrangements.