Retrostructural model to predict biomass formulations for barrier performance.

Barrier performance and retrostructural modeling of the macromolecular components demonstrate new design principles for film formulations based on renewable wood hydrolysates. Hardwood hydrolysates, which contain a fair share of lignin coexisting with poly- and oligosaccharides, offer excellent oxygen-barrier performance. A Hansen solubility parameter (HSP) model has been developed to convert the complex hydrolysate structural compositions into relevant matrix oxygen-permeability data allowing a systematic prediction of how the biomass should be formulated to generate an efficient barrier. HSP modeling suggests that the molecular packing ability plays a key role in the barrier performance. The actual size and distribution of free volume holes in the matrices were quantified in the subnanometer scale with Positron annihilation lifetime spectroscopy (PALS) verifying the affinity-driven assembly of macromolecular segments in a densely packed morphology and regulating the diffusion of small permeants through the matrix. The model is general and can be adapted to determine the macromolecular affinities of any hydrolysate biomass based on chemical composition.

Efficient chemical synthesis of a dodecasaccharidyl lipomannan component of mycobacterial lipoarabinomannan.

Lipomannan (LM) is one of the domains of lipoarabinomannan (LAM) glycolipids, the latter being one of several cell surface organic molecules that fortify mycobacterial species against external attack. Some members of mycobacterial families are pathogenic, most notably Mycobacterium tuberculosis and Mycobacterium leprae, while others are nonpathogenic, and used in the clinic, such as Mycobacterium smegmatis. Additional biological significance arises from the fact that LM has been implicated in several health disorders outside of those associated with mycobacterial pathogens, notably for treatment of bladder cancer. LM is comprised of a heavily lipidated phosphoinositide dimannoside headgroup, from which a mannan array, of varied complexity, extends. The latter consists of a 1,6-alpha-linked backbone flanked at position O2, not necessarily regularly, with alpha-linked mannosides. This paper gives an example of lipomannan synthesis in which all of the sugar components, whether functioning as donors or acceptors, are obtained from n-pentenyl orthoesters, themselves in turn prepared in three easy steps from D-mannose. Assembly of the mannan array is facilitated by the exquisite regioselectivity occasioned by the use of ytterbium triflate/N-iodosuccinimide as the trigger for reaction of n-pentenyl orthoesters.

Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guerin. Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains.

The attenuated strain of Mycobacterium bovis Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy, is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. Mycobacterium tuberculosis and other mycobacterial species produce a glycogen-like alpha-glucan composed of repeating units of 4-linked glucosyl residues substituted at some 6 positions by short oligoglucosyl units that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of alpha-glucans purified from the extracellular materials and boiling water extracts of three vaccine substrains. The polysaccharides were purified, and their structural features were established by mono- and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like alpha-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted alpha-glucans in mycobacteria.

Characterization of the epitope of anti-lipoarabinomannan antibodies as the terminal hexaarabinofuranosyl motif of mycobacterial arabinans.

mAb CS-35 is representative of a large group of antibodies with similar binding specificities that were generated against the Mycobacterium leprae lipopolysaccharide, lipoarabinomannan (LAM), and which cross-reacted extensively with LAMs from Mycobacterium tuberculosis and other mycobacteria. That this antibody also cross-reacts with the arabinogalactan (AG) of the mycobacterial cell wall, suggesting that it recognizes a common arabinofuranosyl (Araf)-containing sequence in AG and LAM, is demonstrated. The antibody reacted more avidly with 'AraLAM' (LAM with naked Araf termini) compared to 'ManLAM' (in which many Araf termini are capped with mannose residues) and mycolylarabinogalactan-peptidoglycan complex (in which the terminal Araf units are substituted with mycolic acids). Neither did the antibody bind to AG from emb knock-out mutants deficient in the branched hexa-Araf termini of AG. These results indicate that the terminal Araf residues of mycobacterial arabinan are essential for binding. Competitive ELISA using synthetic oligosaccharides showed that the branched hexa-Araf methyl glycoside [beta-D-Araf-(1-->2)-alpha-D-Araf-(1-)(2)-(3 and 5)-alpha-D-Araf-(1-->5)-alpha-D-Araf-OCH(3)] was the best competitor among those tested. The related linear methyl glycoside, beta-D-Araf-(1-->2)-alpha-D-Araf-(1-->5)-alpha-D-Araf-(1-->5)-alpha-D-Araf-OCH(3), representing one linear segment of the branched hexa-Araf, was less effective and the other linear tetrasaccharide, beta-D-Araf-(1-->2)-alpha-D-Araf-(1-->3)-alpha-D-Araf-(1-->5)-alpha-D-Araf-OCH(3), was ineffective. The combined results suggest that the minimal epitope recognized by antibody CS-35 encompasses the beta-D-Araf-(1-->2)-alpha-D-Araf-(1-->5)-alpha-D-Araf-(1-->5)-alpha-D-Araf within the branched hexa-Araf motif of mycobacterial arabinans, whether present in LAM or AG.

CD1-restricted T cell recognition of microbial lipoglycan antigens.

It has long been the paradigm that T cells recognize peptide antigens presented by major histocompatibility complex (MHC) molecules. However, nonpeptide antigens can be presented to T cells by human CD1b molecules, which are not encoded by the MHC. A major class of microbial antigens associated with pathogenicity are lipoglycans. It is shown here that human CD1b presents the defined mycobacterial lipoglycan lipoarabinomannan (LAM) to alpha beta T cell receptor-bearing lymphocytes. Presentation of these lipoglycan antigens required internalization and endosomal acidification. The T cell recognition required mannosides with alpha(1-->2) linkages and a phosphotidylinositol unit. T cells activated by LAM produced interferon gamma and were cytolytic. Thus, an important class of microbial molecules, the lipoglycans, is a part of the universe of foreign antigens recognized by human T cells.

Structural definition of acylated phosphatidylinositol mannosides from Mycobacterium tuberculosis: definition of a common anchor for lipomannan and lipoarabinomannan.

Based on chemical analysis, we have previously concluded that the biologically important lipoarabinomannan (LAM) and lipomannan (LM) from Mycobacterium are multiglycosylated forms of the phosphatidylinositol mannosides (PIMs), the characteristic cell envelope mannophosphoinositides of mycobacteria. Using definitive analytical techniques, we have now re-examined the reported multiacylated nature of PIMs in order to gain a better insight into their possible roles as biosynthetic precursors of LM and LAM. High-sensitivity fast atom bombardment-mass spectrometry analyses of the perdeuteroacetyl and permethyl derivatives of PIMs from Mycobacterium tuberculosis and Mycobacterium leprae enabled us to define the exact fatty acyl compositions of the multiacylated, heterogeneous PIM families, notably the dimannoside (PIM2) and the hexamannoside (PIM6). Specifically, in conjunction with other chemical and gas chromatography-mass spectrometry (GC-MS) analyses, the additional C16 fatty acyl substituent on PIM2 and its lyso form were defined as attached to the C6 position of mannose. We also present evidence for triacylated mannophosphoinositide as a common lipid anchor for both LM and LAM, and further postulate that acylation of PIM2 may constitute a key regulatory step in their biosynthesis.

Lipoarabinomannan. Multiglycosylated form of the mycobacterial mannosylphosphatidylinositols.

The lipopolysaccharides of mycobacteria, lipoarabinomannan (LAM) and lipomannan (LM), of key importance in host-pathogen interaction, were recently shown to contain a phosphatidylinositol "anchoring domain." We now have established that LAM and LM are based on the phosphatidylinositol mannosides, the characteristic glycophospholipids of mycobacteria. Digestion of the arabinose-free LM with an endo-alpha 1----6-mannosidase yielded evidence for the presence of the 1-(sn-glycerol-3-phospho)-D-myo-inositol-2,6-bis-alpha-D-mannopyranoside unit, indistinguishable from that derived from phosphatidylinositol dimannoside. This same inositol substitution pattern was shown to be present in LAM by methylation analysis before and after dephosphorylation. Positions C-2 and C-6 of the inositol unit of LAM are occupied by mannosyl residues and C-1 by a phosphoryl group. Partial acid hydrolysis of per-O-methylated LAM and comparison by gas chromatography-mass spectrometry of the resulting derivatized oligosaccharides with like products from phosphatidylinositol hexamannoside demonstrated that the C-6 of inositol is the point of attachment of the mannan core of LAM, which consists of an alpha 1----6-linked backbone with considerable alpha-1----2 side chains. Thus, a structural and presumably biosynthetic relationship is established between some of the membranous mannosylphosphatidylinositols described some 25 years ago and the newly emerging, biologically active lipopolysaccharides of mycobacteria.

Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobacterium tuberculosis.

The recent availability (Hunter, S.W., Gaylord, H., and Brennan, P.J. (1986) J. Biol. Chem. 261, 12345-12351) of the well known arabinomannan of Mycobacterium leprae and Mycobacterium tuberculosis as the pure native lipoarabinomannan has resulted in its implication in key aspects of the immunopathogenesis of leprosy and tuberculosis. We had indicated that the lipid moiety of lipoarabinomannan is probably based on a diacylglycerol unit in that glycerol and the two fatty acids, hexadecanoate and 10-methyloctadecanoate, were identified. In addition, lipoarabinomannan was also shown to contain myo-inositol 1-phosphate. Evidence is now presented, based on selective radiolabeling and analysis of various cleavage fragments, that the inositol phosphate exists as both an alkalilable phosphodiester and as part of a phosphatidylinositol "membrane anchor." The mannan of M. tuberculosis was also isolated as the native lipomannan. It also apparently contains a phosphatidylinositol unit but is devoid of the alkali-labile inositol phosphate residues. These lipopolysaccharides are apparently multiglycosylated versions of the well known myocobacterial mannosyl phosphatidylinositols and are prokaryotic versions of the growing list of phosphatidylinositol-anchored macromolecules. Immunogold labeling demonstrates that lipoarabinomannan is a true antigenic capsular or extracellular product of M. tuberculosis. The presence of a phosphatidylinositol residue on lipoarabinomannan may explain its interaction with macrophage membranes and role in mycobacterial pathogenesis.

An n.m.r. and conformational analysis of the terminal trisaccharide from the serologically active glycolipid of Mycobacterium leprae in different solvents.

The 1H- and 13C-n.m.r. spectra of allyl 2-O-[4-O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-2,3-di-O-methyl-alpha-L -rhamnopyranosyl]-3-O-methyl-alpha-L-rhamnopyranoside (3), a glycoside of the terminal trisaccharide found in the phenolic glycolipid I from Mycobacterium leprae, and those of the two component disaccharides, allyl 4-O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-2,3-di-O- methyl-alpha-L-rhamnopyranoside (1) and allyl 2-O-(2,3-di-O-methyl-alpha-L-rhamnopyranosyl)-3-O-methyl-alpha-L- rhamnopyranoside (2) have been assigned completely by 1D and 2D techniques. The preferred conformations, determined by chemical shift and n.O.e. studies, were different in D2O, CD3OD, and CDCl3. The preferred conformation of 3 accorded with the results of hard-sphere exo-anomeric (HSEA) calculations.

Synthesis of galactofuranose disaccharides of biological significance.

Methyl beta-D-galactofuranoside was readily obtained by tin(IV) chloride-catalyzed glycosylation of penta-O-benzoyl-alpha,beta-D-galactofuranose, followed by debenzoylation with sodium methoxide. Glycosylation of 1 with 2,3,5-tri-O-benzoyl-D-galactono-1,4-lactone or with the 6-O-trityl-lactone derivative 5 gave the benzoylated beta-D-galactofuranosyl-(1----6)-D-galactono-1,4-lactone 6 in excellent yield. The structure of disaccharide 6 was confirmed by borohydride reduction to the glycosyl-alditol 7. A byproduct of the condensation reaction of 1 with 4 or 5 was identified as the benzoylated (1----1)-beta,beta'-D-galactofuranosyl disaccharide 8. Compound 8 was readily prepared (88% yield) by controlled addition of water to 1, in the presence of stannic chloride. O-Debenzoylation of 8 afforded crystalline beta'-D-galactofuranosyl-(1----1)-beta-D-galactofuranoside. The glycosyl-lactone 6 constitutes a key intermediate for the synthesis of a disaccharide derivative having both units in the furanoid form. Thus, diisoamylborane reduction of the lactone function of 6 led to the disaccharide derivative 10, from which the methyl glycoside 12 was prepared. O-Debenzoylation of 12 gave the corresponding methyl beta-D-galactofuranosyl-(1----6)-beta-D-galactofuranoside. The free disaccharide beta-D-Galf-(1----6)-D-Galp and its acetylated derivative were also synthesized from 10.

A transient rise in agalactosyl IgG correlating with free interleukin 2 receptors, during episodes of erythema nodosum leprosum.

The proportion of oligosaccharide chains on the Fc fragment of IgG which terminate with N-acetylglucosamine and not galactose (%GO) has previously been shown to be raised in rheumatoid arthritis (RA), Crohn's disease (CD) and tuberculosis (Tb), but to be normal in sarcoidosis (SA), and in both lepromatous and tuberculoid leprosy. However we have now studied %GO in sequential serum samples collected from lepromatous leprosy patients undergoing episodes of erythema nodosum leprosum (ENL). During ENL %GO is transiently raised, and this rise parallels an increase in circulating interleukin 2 receptors (IL-2R). These findings confirm that changes in T cell function occur during ENL. Moreover it appears that %GO rises when there is, simultaneously, T-cell-mediated tissue damage and an acute phase response (RA, CD, Tb, ENL), but not when there is an acute phase response without major T cell involvement, or chronic T cell activity alone (SA, and tuberculoid leprosy). We suggest therefore that %GO is an indicator of a type of T cell activity with broad immunopathological implications.

Chemical synthesis of an artificial antigen containing the trisaccharide hapten of Mycobacterium leprae.

The trisaccharide allyl O-(3,4-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl-al pha-L- rhamnopyranosyl)-(1----2)-3-O-methyl-alpha-L-rhamnopyranoside was synthesized from partially methylated monosaccharide derivatives. Condensation of 1,4-di-O-acetyl-2,3-di-O-methyl-alpha-L-rhamnopyranose promoted by boron trifluoride etherate with the appropriate alcohol proceeded stereoselectively and with very high yields. Selective deacetylation and glycosylation with 2,4-di-O-acetyl-3,6-di-O-methyl-alpha-D-glucopyranosyl bromide led to a trisaccharide. The acrylamide copolymers of mono-, di-, and tri-saccharide were compared in their ability to specifically bind antibodies from leprosy patients.

Synthesis and immunoreactivity of neoglycoproteins containing the trisaccharide unit of phenolic glycolipid I of Mycobacterium leprae.

The trisaccharide segment, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, of the Mycobacterium leprae-specific phenolic glycolipid I has been synthesized as its 8-(methoxycarbonyl)octyl glycoside and coupled to a carrier protein, to produce a leprosy-specific neoglycoprotein, the so-called natural trisaccharide-octyl-bovine serum albumin (NT-O-BSA). Special features of the synthetic strategy were the use of silver trifluoromethanesulfonate (triflate) to promote glycosylation, resulting in the rhamnobiose in high yield and absolute stereospecificity. The terminal 3,6-di-O-methyl-D-glucopyranosyl group was introduced after O-deallylation of the rhamnobiose. Removal of protecting groups yielded the trisaccharide hapten suitable for coupling to carrier protein. Poly(acrylamide)-gel electrophoresis of the neoglycoprotein demonstrated its purity, and subsequent immunoblotting with a monoclonal antibody directed to the terminal 3,6-di-O-methyl-beta-D-glucopyranosyl epitope of the native glycolipid demonstrated its antigenicity. Comparative serological testing in enzyme-linked immunosorbent assays of NT-O-BSA, the corresponding disaccharide-containing products, and another trisaccharide-containing neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O- methyl-alpha-L-rhamnopyranosyl)-(1----2)-(3-O-methyl-alpha-L-rhamnopy ran osyl)- (1----4')-oxy-(3-phenylpropanoyl)-BSA (NT-P-BSA) [Fujiwara et al., Agric. Biol. Chem., 51 (1987) 2539-2547] against sera from leprosy patients and control populations showed concordance; the presence of the innermost sugar did not contribute significantly to sensitivity or specificity. The di- and tri-saccharide-containing neoantigens, on account of ready availability and solubility, provide greater flexibility than the native glycolipid for the serodiagnosis of leprosy.

Demonstration that the galactosyl and arabinosyl residues in the cell-wall arabinogalactan of Mycobacterium leprae and Mycobacterium tuberculosis are furanoid.

By a complex process involving methylation, partial hydrolysis with acid, reduction with sodium borodeuteride, ethylation, further hydrolysis and reduction, and subsequent capillary gas-liquid chromatography-mass spectrometry of the derived alditol acetates, it was established that the arabinogalactans of Mycobacterium leprae and Mycobacterium tuberculosis contain arabinofuranyosyl and galactofuranosyl residues exclusively. Thus, the covalently bound, highly immunogenic arabinogalactan of mycobacteria, and presumably of other actinomycetes, is highly unusual, in that all of the glycosyl residues are in the furanoid form. Furthermore, by establishing that the galactofuranosyl residues are either 5-, 6-, or 5,6-linked, their linkage pattern was established, and the literature is corrected on this point.

Structure of the major triglycosyl phenol-phthiocerol of Mycobacterium tuberculosis (strain Canetti).

Phenol-phthiocerol glycolipids have been found previously in Mycobacterium leprae, M. kansasii, M. bovis and M. marinum, but not in M. tuberculosis. A search for glycolipids in this latter species showed that the Canetti strains of M. tuberculosis synthesize a major triglycosyl phenol-phthiocerol, accompanied by minor amounts of other glycolipids with a similar aglycone moiety. The triglycoside moiety has the following structure: 2,3,4-tri-O-methyl L-fucopyranosyl(alpha 1----3)L-rhamnopyranosyl(alpha 1----3)2-O-methyl L-rhamnopyranosyl(alpha 1-. The aglycone moiety consists in phenol-phthiocerol (two homologs). Its two secondary alcohol functions are esterified by mycocerosic acids (homologs with 26-32 carbon atoms and with 2-4 methyl branches). The proposed structure differs on several points from the M. leprae glycolipids, but presents some analogy with the major glycolipid of M. kansasii. A minor monoglycosyl phenol-phthiocerol was also studied. Its overall structure is very similar to that of M. bovis, with 2-O-methyl rhamnose as sugar moiety.

Chemical synthesis of the trisaccharide unit of the species-specific phenolic glycolipid from Mycobacterium leprae.

O-(3,6-Di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, the haptenic trisaccharide of the Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) antigen, and related trisaccharides, were synthesized by allylation of O-2 of benzyl 4-O-benzyl-alpha-L-rhamnopyranoside using phase-transfer catalysis, methylation of the product, deallylation, and coupling to O-(2,4-di-O-acetyl-3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3- di-O-methyl-L-rhamnopyranosyl bromide or related disaccharides. Anomeric mixtures of the trisaccharide derivatives were separated by preparative t.l.c., deacetylated, and hydrogenolyzed, to give the pure trisaccharides. It had already been demonstrated that only those trisaccharides containing an intact, terminal 3,6-di-O-methyl-beta-D-glucopyranosyl unit are effective in inhibiting the specific binding between PGL-I and anti-PGL-I immunoglobulin M antibodies in human lepromatous leprosy sera.

Chemical synthesis and serology of disaccharides and trisaccharides of phenolic glycolipid antigens from the leprosy bacillus and preparation of a disaccharide protein conjugate for serodiagnosis of leprosy.

We examined the structural requirements within the species-specific 3,6-di-O-methyl-beta-D-glucopyranosyl-(1 leads to 4)-2,3-di-O-methyl- alpha-L-rhamnopyranosyl-(1 leads to 2)-3-O-methyl-alpha-L-rhamnopyranose unit of the phenolic glycolipid I antigen of Mycobacterium leprae for binding to anti-glycolipid immunoglobulin M from human leprosy sera. We used chemically defined, partially deglycosylated fragments of phenolic glycolipid I, two other minor M. leprae-specific phenolic glycolipids (those containing 6-O-methyl-beta-D-glucopyranosyl-(1 leads to 4)-2,3-di-O-methyl-alpha- L-rhamnopyranosyl-(1 leads to 2)-3-O-methyl-alpha-L-rhamnopyranose and 3,6-di-O-methyl-beta-D-glucopyranosyl-(1 leads to 4)-3-O-methyl-alpha- L-rhamnopyranosyl-(1 leads to 2)-3-O-methyl-alpha-rhamnopyranose units), and phenolic glycolipids from other mycobacteria. Additionally, the trisaccharide of phenolic glycolipid I, the 3,6-di-O-methyl-beta-D-glucopyranosyl-(1 leads to 4)-2, 3-di-O-methyl-alpha-L-rhamnopyranose, the 6-O-methyl-beta-D-glucopyranosyl-(1 leads to 4)-2,3-di-O-methyl-alpha- L-rhamnopyranose, and the beta-D-glucopyranosyl-(1 leads to 4)-2,3-di-O-methyl-alpha- L-rhamnopyranose disaccharides were synthesized and characterized, and their activities were examined. Only the phenolic glycolipids containing 3,6-di-O-methyl-beta-D-glucopyranosyl at the nonreducing terminus were efficient in binding the anti-glycolipid immunoglobulin M, and the 3,6-di-O-methyl-beta-D-glucopyranosyl-containing di- and trisaccharides were the most effective in inhibiting this binding. Thus, the 3,6-di-O-methyl-beta-D-glucopyranosyl substituent was recognized as the primary antigen determinant in phenolic glycolipid I. With this information, bovine serum albumin containing reductively aminated 3,6-di-O-methyl-beta-D-glucopyranosyl-(1 leads to 4)-2,3-di-O-methyl- L-rhamnose was prepared and shown to be highly active in the serodiagnosis of leprosy.