Tobacco smoke chemistry 3. Aromatic acids of cigarette smoke condensate.

A fraction containing mainly aromatic acids has been isolated from cigarette smoke condensate. Gas chromatographic and mass spectral analysis of the corresponding methyl esters and comparison with reference compounds, many of which were synthesized for this purpose, made possible the identification of 27 constituents (Table 1). Eighteen of these have not been detected in tobacco smoke condensate before.

Tobacco smoke chemistry. 2. Alkyl and alkenyl substituted guaiacols found in cigarette smoke condensate.

A series of alkyl and alkenyl substituted guaiacols, which comprise a group of biologically and organoleptically active compounds, have been synthesized. Mass spectra and GC retention times for these have been recorded and compared with those obtained for constituents of a weakly acidic fraction of smoke condensate derived from American blend type cigarettes. On the basis of these results, 25 guaiacols have been identified, 18 of which have not been detected in tobacco smoke condensate previously.

Tobacco smoke chemistry. 1. A chemical and mass spectrometric study of tobacco smoke alkyl 2-hydroxy-2-cyclopentenones.

A series of alkyl 2-hydroxy-2-cyclopentenones, which comprise biologically and organoleptically active compounds, have been synthesized and subjected to high resolution mass spectrometric studies to clarify structurally significant fragmentation pathways. On the basis of these results, 26 alkyl 2-hydroxy-2-cyclopentenones were identified in the weakly acidic fraction of smoke condensate from American blend type cigarettes, eighteen of which had not been detected in tobacco smoke previously. The utility for identification purposes of the corresponding quinoxaline derivatives, obtained through condensation with o-phenylenediamine, is discussed.

Carbohydrate binding studies on the lectin from Datura stramonium seeds.

The carbohydrate-binding properties of the Datura stramonium seed lectin were studied by equilibrium dialysis, quantitative precipitation of natural and synthetic glycoproteins, and hapten inhibition of precipitation. The dimeric lectin (Mr = 86,000) possesses two carbohydrate-binding sites for N,N'N",N"'- tetraacetylchitotetritol /mol protein, with an apparent Ka = 8.7 X 10(3) M-1 at 4 degrees C. Whereas fetuin and orosomucoid reacted poorly with the Datura lectin, the asialo derivatives of these glycoproteins gave strong precipitation with the lectin. Carcinoembryonic antigen, type 14 pneumococcal capsular polysaccharide, and bovine serum albumin, highly substituted with N,N'- diacetylchitobiose units, also precipitated the lectin. Of the homologous series of chitin oligosaccharides tested, N,N',N"'- triacetylchitotriose was over 6-fold more potent than the disaccharide (N,N'- diacetylchitobiose ) which, in turn, was 90 times more reactive than N-acetyl-D-glucosamine. N-Acetyllactosamine [beta-D-Gal-(1----4)-D-GlcNAc] was also a potent inhibitor of Datura lectin being equivalent to N,N'- diacetylchitobiose . The requirement for an N-acetyl-D-glucosaminyl unit linked at the C-4 position was established. The biantennary pentasaccharide (penta-2,6) was a 500-fold more potent inhibitor than N-acetyllactosamine, suggesting that it might interact with both saccharide-binding sites of the Datura lectin simultaneously.

Binding of synthetic oligosaccharides to the hepatic Gal/GalNAc lectin. Dependence on fine structural features.

A series of synthetic oligosaccharides, resembling natural N-acetyllactosamine type glycans, were tested for their ability to inhibit the binding of labeled ligand to the mammalian hepatic lectin on rabbit hepatocytes at 2 degrees C. A dramatic hierarchy of inhibitory potency (tetraantennary greater than triantennary much greater than biantennary much greater than monoantennary) could be demonstrated. The range of concentration required for 50% inhibition of labeled ligand binding extended from approximately 1 mM, for the monoantennary oligosaccharides, to approximately 1 nM for triantennary oligosaccharides, even though the absolute Gal concentration increased only 3-fold. It was found that the number of Gal residues/cluster and their branching mode are major determinants of binding affinity of ligands to the hepatic lectin on the surface of hepatocytes.

Synthesis of oligosaccharides that form parts of the complex type of carbohydrate moieties of glycoproteins. Three glycosides prepared for conjugation to proteins.

Condensation of 3,6-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-2-deoxy-2- phthalimido-beta-D-glucopyranosyl bromide with p-nitrophenyl 3-O-benzoyl-4,6-di-O-benzylidene-alpha-D-mannopyranoside, p-nitrophenyl 3,6-di-O-benzyl-alpha-D-mannopyranoside and p-nitrophenyl 3,4-di-O-benzyl-alpha-D-mannopyranoside gave protected tri- and pentasaccharides. The oligosaccharide glycosides 1, 2 and 3 were obtained after de-protection of these condensation products. These oligosaccharides will, after suitable conversions, be conjugated to proteins for biological studies.

Mitogenic leukoagglutinin from Phaseolus vulgaris binds to a pentasaccharide unit in N-acetyllactosamine-type glycoprotein glycans.

The carbohydrate binding specificity of leukoagglutinin (La; Phaseolus vulgaris isolectin L4) was studied by using quantitative precipitation and precipitation-inhibition. A series of purified glycopeptides and synthetic oligosaccharides were used as inhibitors. The minimal structural unit required for La binding was the disaccharide GlcNac(1 leads to beta 2)Man. Additions for this basic unit of different sugar residues gave a positive or negative contribution to binding. The most complementary structure was the pentasaccharide (formula: see text). This pentasaccharide units occurs in tetraantennary N-acetyllactosamine-type glycoprotein glycans. Glycoproteins containing such structures were accordingly precipitated by La. Selected glycopeptides and oligosaccharides were also tested as inhibitors of La-induced DNA synthesis in human lymphocytes. The pattern of inhibition was essentially the same as that obtained by precipitation-inhibition, indicating that binding to lymphocytes via the carbohydrate binding site of the lectin is an essential step in the activation process.

Synthesis of three oligosaccharides that form part of the complex type of carbohydrate moiety of glycoproteins.

Silver trifluoromethanesulfonate-promoted condensation of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl bromide with benzyl 3,6-di-O-benzyl-alpha-D-mannopyranoside and benzyl 3,4-di-O-benzyl-alpha-D-mannopyranoside gave the protected 2,4- and 2,6-linked trisaccharides in yields of 54 and 32%, respectively. After exchanging the 2-deoxy-2-phthalimido groups for 2-acetamido-2-deoxy groups and de-blocking, the trisaccharides 2,4-di-O-(2-deoxy-beta-D-glucopyranosyl)-D-mannose and 2,6-di-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-D-mannose were obtained. Similar condensation of 3,6-di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-beta-D-glucopyranosyl bromide with benzyl 3,4-di-O-benzyl-alpha-D-mannopyranoside gave a pentasaccharide derivative in 52% yield. After transformations analogous to those applied to the trisaccharides, 2,6-di-O-[beta-D-galactopyranosyl-(1 leads to 4)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)]-D-mannose was obtained.