Syntheses of linear tetra-, hexa-, and octa-saccharide fragments of the i-blood group active poly-(N-acetyl-lactosamine) series. Blockwise methods for the synthesis of repetitive oligosaccharide sequences

N-Phthaloylation of lactosamine gave various glycosyl donors (beta-chloride, beta-trichloroacetimidate) and glycosyl acceptors (3',4'-diol). Coupling of the chloride with a methyl beta-D-glycoside led to the tetrasaccharide fragment, beta-D-Galp-(1----4)-beta-D-GlcpNac-(1----3)-beta-D-Galp-(1----4)- beta-D-GlcpNAcOMe. Acetolysis of the protected tetrasaccharide, followed by treatment with hydrogen chloride, gave a tetrasaccharide chloride which was coupled with the methyl beta-glycoside of lactosamine. A hexasaccharide fragment, [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)]2-beta-D-Galp-(1----4)-bet a- D-GlcpNAcOMe, was thus obtained by this ("n + 1") method. A more efficient ("n + n") method was applied for the synthesis of an octasaccharide fragment, [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)]3-beta-D-Galp- (1----4)-beta-D-GlcpNAcOMe (38), where di- and tetra-saccharide intermediates having a 3,4-O-isopropylidene-beta-D-galactopyranosyl nonreducing terminal group and a benzyl beta-D-glycoside group were precursors, either as glycosyl donors (beta-trichloroacetimidates) or glycosyl acceptors (3,4-diols as nonreducing terminal groups). Thus, doubling the length of the repetitive oligosaccharide sequence could be efficiently accomplished at each glycosylation step.     

Synthesis of a trisaccharide of 3-deoxy-D-manno-2-octulopyranosylonic acid (KDO) residues related to the genus-specific lipopolysaccharide epitope of Chlamydia

The disaccharides, O-(sodium 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosylonate)-(2----8)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate, were prepared via glycosylation of methyl (allyl 4,5,7-tri-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosid)onat e with methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-D-manno-2-octulopyranosyl bromide)onate under Helferich and Koenigs-Knorr conditions, respectively. Based on g.l.c.-m.s. data of the alpha- and beta-(2----8)-linked disaccharide derivatives, obtained after carbonyl- and carboxyl-group reduction, followed by methylation, the alpha-anomeric configuration was assigned to the terminal KDO-residue in the KDO-region of Chlamydial lipopolysaccharide. The trisaccharide O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----8)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----4)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate was obtained via block synthesis using an alpha-(2----8)-linked disaccharide bromide derivative as the glycosyl donor. Copolymerization of the allyl glycosides with acrylamide gave water-soluble macromolecular antigens, suitable for defining epitope specificities of monoclonal antibodies directed against Chlamydial LPS.     

Synthesis of a branched pentasaccharide sequence of "bisected" structure of N-glycoproteins

For the synthesis of the threefold-branched pentasaccharide, O-alpha-D-mannopyranosyl-(1----3)-O-[(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----4)]-O-[alpha-D-mannopyranosyl-(1----6)]-O-beta-D- mannopyranosyl-(1----4)-2-acetamido-2-deoxy-D-glucopyranose (20), which is a part of the structure of the N-glycoproteins, the disaccharide 4-O-(4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl) -1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranose was synthesized as a key compound by use of the silver silicate-catalyst procedure. After elimination of the 4-O-acetyl group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group was attached according to the phthalimido method. Further elimination of the allyl groups allowed the linkage of two alpha-D-mannopyranosyl groups in the presence of mercury salt. A deblocking sequence consisting of four steps gave 20.     

Identification of N-glycolylneuraminic acid-containing gangliosides of cat and sheep erythrocytes. 252Cf fission fragment ionization mass spectrometry in the analysis of glycosphingolipids

A number of gangliosides were isolated from cat and sheep erythrocytes for use in analyzing the specificity of a panel of human anti-heterophile monoclonal antibodies. The structures of these compounds were determined by a combination of different procedures, including sugar analysis, glycosidase treatment, periodate oxidation, TLC immunostaining, methylation analysis, and mass spectrometry. These methods identified the cat erythrocytes gangliosides (C1 and C2) as N-glycolylneuraminic acid (NeuGc)-containing hematosides; C1 was shown to be NeuGc alpha 2----8NeuGc alpha 2----3Gal beta I----4Glc-Cer [NeuGc)2GD3) and C2 to be NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)GD3). The two sheep gangliosides (S1 and S2) were found to be novel glycolipids based on the paragloboside sequence; S1 was identified as NeuGc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuGc)2-disialylparagloboside) and S2 as NeuAc alpha 2----8NeuGc alpha 2----3Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4Glc-Cer [NeuAc-NeuGc-)-disialylparagloboside). Structural analysis of these compounds was aided by the use of 252Cf fission fragment ionization time-of-flight mass spectrometry. This method provided easily interpretable spectra on methylated derivatives which were particularly useful in determining the sialic acid composition of the gangliosides and the sequence of their disialosyl side chains.     

A new type of carbohydrate-containing synthetic antigen: synthesis of carbohydrate-containing polyacrylamide copolymers having the specificity of O:3 and O:4 factors of Salmonella

The synthesis of a new type of synthetic antigen that contains no protein is described. Two linear polyacrylamide copolymers with carbohydrate branches were obtained via radical copolymerisation of the allyl glycosides of the oligosaccharide determinants O-beta-D-mannopyranosyl-(1----4)-O-alpha-L-rhamnopyranosyl-(1----3 )-beta-D- galactopyranose and O-3,6-dideoxy-alpha-D-xylo-hexopyranosyl-(1----3)-alpha-D-mannopyranose with acrylamide. These copolymers, which contained 30% of carbohydrate and had molecular masses exceeding 100 kilodaltons, had the group specificity E and B of Salmonella.     

Synthesis of 2-acrylamidoethylglycosides of 3-O-(beta-D- glucopyranosyluronic acid)-alpha-L-rhamnose and 3-O-(alpha-L- rhamnopyranosyl)-beta-D-glucopyranuronic acid and copolymeric artificial antigens based on them

The synthesis of disaccharide repeating units, D-GlcA-(beta 1----3)-L-Rha (fragment A) and L-Rha-(alpha 1----3)-D-GlcA (fragment B), of the K54-antigenic polysaccharide from uropathogenic Escherichia coli 06:K54:H10 is described. Essential stages of the synthesis of fragment A involved the glycosylation of methyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside followed by acetolysis of the methyl bioside obtained and further transformation into 2-(benzyloxycarbonylamino)ethyl glycoside; deprotection and, finally, conversion into 2-(acrylamido)ethyl glycoside. Selective opening of lactone ring in 2-azidoethyl 2,4-di-O-acetyl-beta-D-glucopyranoside-6,3-lactone was used for deprotection of 3-OH group in the synthesis of fragment B. Rhamnosylation of the glucuronic acid derivative thus obtained followed by transformation into 2-(acrylamido)ethyl glycoside and deprotection gave fragment B. Both fragments A and B were converted into artificial antigens of copolymer type.     

An efficient synthesis of ganglioside GM3: highly stereocontrolled glycosylations by use of auxiliaries

An efficiently stereocontrolled total synthesis of GM3 alpha-D-Neup5Ac-(2----3)-beta-D-Galp-(1----4)-beta-D-Glcp-(1----1) -Cer was achieved by employing both methyl 5-acetamido-4,7,8,9-tetra-O-benzyl-2-bromo-2,3,5-trideoxy-3- phenylthio-D-erythro-beta-L-gluco-2-nonulopyranosonate for the key sialylation step, and O-[methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha -D-galacto-2-nonulopyranosyl)onate]-(2----3)-O-(2,4,6-tri-O- acetyl-beta-D-galactopyranosyl-(1----4)-3,6-di-O-acetyl-2-O-pivaloyl- alpha-D-glucopyranosyl trichloroacetimidate and fluoride for the key coupling step with a ceramide derivative. These two steps were significantly altered and improved in comparison with our previous synthesis that had been executed without use of stereocontrolling auxiliaries. GM3 was obtained in 4.5% overall yield in 19 steps starting from allyl O-(2,6-di-O-acetyl-3,4-O-isopropylidene-beta-D-galactopyranosyl)-(1----4 )-2,3,6-tri-O-acetyl-beta-D-glucopyranoside.     

Synthesis of an appropriately protected core glycotetraoside, a key intermediate for the synthesis of "bisected" complex-type glycans of a glycoprotein

A stereocontrolled synthetic route to a glycotetraoside, allyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-3, 6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O- benzyl- 2-deoxy-6-O-p-methoxy-phenyl-2-phthalimido-beta-D-glucopyranoside, an important intermediate for the synthesis of "bisected" complex type glycans of glycoproteins has been established by employing two glycosyl donors, 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate and 4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-alpha-D-mannopyranosyl bromide, and a glycosyl acceptor, allyl O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4) -3-O- benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D-glucopyranoside.     

A nuclear magnetic resonance spectroscopic investigation of Kdo-containing oligosaccharides related to the genus-specific epitope of Chlamydia lipopolysaccharides.

The 1H- and 13C-NMR parameters, chemical shifts and coupling constants, for the pentasaccharide of the genus-specific epitope of Chlamydia lipopolysaccharide and related di-, tri-, and tetra-saccharides have been measured and assigned completely using 1D and 2D techniques, and their structures have been confirmed. NOE experiments indicated the preferred conformation of the pentasaccharide and the component oligosaccharides. The 3JH,H demonstrate a change in conformation by rotation of the C-6-C-7 bond of the side chain of the (2----8)-linked Kdo (unit b) in alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcN-(1--- -6)- GlcNol, alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1- ---O)- allyl, and alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl relative to that preferred in alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, alpha-Kdo-(2----8)-alpha-Kdo-(2----O)-allyl, alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl, and alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, irrespective of the size of the aglycon, e.g., allyl or beta-D-GlcN residues. The conformational results have been substantiated by computer calculations using the HSEA approach.     

Synthesis of a selectively protected trisaccharide building block of the capsular polysaccharide of Streptococcus pneumoniae types 6A and 6B

4-Methoxybenzyl 2,4-di-O-benzyl-3-O-[2,4,6-tri-O-benzyl-3-O-(3,4,6-tri-O-benzyl-alpha-D- galactopyranosyl)-alpha-D-glucopyranosyl]-alpha-L-rhamnopyranoside (22), a building block for the alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap fragment of the capsular polysaccharides of Streptococcus pneumoniae types 6A and 6B [----2)-alpha-D-Galp-(1----3)-alpha-D-Glcp-(1----3)-alpha-L-Rhap-( 1----X)-D- RibOH-(5-P----]n (6A, X = 3; 6B, X = 4) has been synthesised. Ethyl 3-O-allyl-2,4,6-tri-O-benzyl-1-thio-beta-D-glucopyranoside was coupled with 4-methoxybenzyl 2,4-di-O-benzyl-alpha-L-rhamnopyranoside in ether, using methyl triflate as promoter. The resulting alpha-D-Glcp-(1----3)-alpha-L-Rhap derivative was deallylated with KOBut in N,N-dimethylformamide followed by 0.1M HCl in 9:1 acetone-water. The product was coupled with 3,4,6-tri-O-acetyl-2-O-allyl-alpha,beta-D-galactopyranosyl trichloroacetimidate in ether, using trimethylsilyl triflate, to yield 19. Deacetylation, benzylation, and deallylation then gave 22.     

Examination of aglycone-binding site of human salivary alpha-amylase by means of transglycosylation reactions

The active site of human salivary alpha-amylase is composed of tandem subsites (S3, S2, S1, S1',S2', etc.) geometrically complementary to several glucose residues, and the glycosidic linkage of the substrate is split between S1 and S1'. As a matter of convenience, the subsites to which the non-reducing-end part (glycone) and the reducing-end part (aglycone) of the substrate being hydrolyzed are bound are named the glycone-binding site (S3, S2, S1) and the aglycone-binding site (S1', S2'), respectively. The features of the aglycone-binding site of human salivary alpha-amylase were examined by means of transglycosylation reaction using phenyl alpha-maltoside (GG phi: G-G-phi) and its derivatives (GAG phi: G-AG-phi, GCG phi: G-CG-phi, AGG phi: AG-G-phi, and CGG phi: CG-G-phi) in which one of the glucose residues (G) has been converted to 6-amino-6-deoxy-glucose (AG) or glucuronic acid (CG) residue as the acceptor. A fluorogenic derivative of maltotetraose, p-nitrophenyl O-6-deoxy-6-[(2-pyridyl)amino]-alpha-D-glucopyranosyl-(1----4)-O-alpha-D -glucopyranosyl-(1----4)-O-alpha-D-glucopyranosyl-(1----4)-alpha-D- glucopyranosyl-(1----4)-alpha-D-glucopyranoside (FG4P, FG-G-G-G-P), was used as the substrate. HSA catalyzed both hydrolysis of FG4P to FG3 (FG-G-G) and p-nitrophenyl alpha-glucoside (G-P) and transfer of the FG3 residue of FG4P to the acceptors. Transfer to GAG phi occurred more effectively than to GG phi. Transfers to GCG phi and CGG phi were less than to GG phi and very little transfer to AGG phi occurred.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of the tetrasaccharide core region of antigenic lipo-oligosaccharides characteristic of Mycobacterium kansasii

The oligosaccharide core region, beta-D-Glcp-(1----3)-beta-D-Glcp-(1----4)-alpha-D-Glcp- 1----1)-alpha-D-Glcp (1), of the lipo-oligosaccharide-type antigens isolated from M. kansasii has been synthesised from 2,3,2',3',4',6'-hexa-O-benzyl-6-O-(1-phenylethyl)-alpha, alpha-trehalose (4). Compound 4 was obtained by LiAlH4-AlCl3-type hydrogenolysis of 2,3,2',3',4',6'-hexa-O-benzyl-4,6-O-(S)-(1-phenylethylidene)-alpha , alpha-trehalose. The beta-laminaribiosyl part of the molecule was built-up by sequential glycosylation steps using 2,4,6-tri-O-acetyl-3-O-allyl-alpha-D-glucopyranosyl bromide in the presence of HgBr2 and methyl 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranoside promoted by methyl triflate. The complete a priori 13C-n.m.r. spectrum assignment of 1 was achieved by applying 2D methods.     

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural elucidation of two capsular polysaccharides from one strain of Bacteroides fragilis using high-resolution NMR spectroscopy.

The capsule of Bacteroides fragilis is unusual in that it consists of two distinct capsular polysaccharides. Using a combination of high-resolution NMR spectroscopy, theoretical calculations, and as few chemical procedures as required, the structure of both polysaccharide antigens (polysaccharides A and B) was elucidated. Using the above procedures, it was possible to obtain the complete structures using minimal quantities of polysaccharides A and B (8 and 5 mg, respectively). Only small amounts of each subjected to chemical analysis were not recoverable. Polysaccharide A is composed of the following repeating unit: [----3)alpha-D-AATp(1----4)[beta-D-Galf(1----3)]alpha-D- GalpNAc(1----3)beta-D-Galp(1----], where AAT is 2-acetamido-4-amino-2,4,6-trideoxygalactose. A pyruvate substituent having the R configuration spans O-4 and O-6 of the beta-D-galactopyranosyl residue. Polysaccharide B is composed of the following repeating unit: [----4)alpha-L-QuipNAc(1----3)beta-D-QuipNAc(1----4)[alpha-L - Fucp(1----2)beta-D-GalpA(1----3)beta-D-GlcpNAc(1----3)]alpha -D-Galp(1----]. A 2-aminoethylphosphonate substituent is situated on O-4 of the N-acetyl-beta-D-glucopyranosyl residue.     

Phenylpropanoid and iridoid glycosides from Pedicularis striata.

A new phenylpropanoid glycoside, pedicularioside A, and five known glycosides, acteoside, isoacteoside, decaffeoylacteoside, echinacoside and 8-acetylharpagide, were isolated from whole plants of Pedicularis striata. On the basis of spectral and chemical evidence, pedicularioside A was shown to be 1'-O-beta-D-(3,4-dihydroxy-beta-phenyl)-ethyl-4'-O-caffeoyl-beta-D -apiosyl-(1----3')-alpha-L-rhamnosyl-(1----6')-glucopyranosi de.     

Synthesis of oligosaccharide fragments of the repeating unit of Salmonella kentucky O-specific polysaccharide and conversion of the oligosaccharides into the glycosyl phosphates

alpha-D-Man-(1----2)-alpha-D-Man-(1----3)-D-Gal, a structural fragment of the main chain of Salmonella serogroups C2 and C3 O-specific polysaccharides, and the isomer with the central residue beta have been synthesised, as have some oligosaccharides related to the structure of the O-specific polysaccharide of S. kentucky (serogroup C3), namely, alpha-D-Glc-(1----4)-D-Gal, alpha-D-Man-(1----3)-[alpha-D-Glc-(1----4)]-D-Gal, and alpha-D-Man-(1----2)-alpha-D-Man-(1----3)-[alpha-D-Glc-(1----4)]-D-Gal, and the isomers with the D-Glc unit beta. Each oligosaccharide was converted into the alpha-glycosyl phosphate.     

Synthesis of oligosaccharides with group specificity for blood types A, B and H (Type 3)

Chemical synthesis of A, B, and H (type 3) human blood group determinant oligosaccharides (as R-glycosides, R = OCH2CH2CH2NHCOCF3) and their polymeric derivatives are reported. 4,6; 4',6'-Di-O-benzylidene derivative of Gal beta 1----3GalNAc alpha 1----R was chloroacetylated selectively at 3'-OH, the chloroacetate was alpha-fucosylated and dechloroacetylated to give protected H (type 3) trisaccharide bearing free 3'-OH. alpha-Glycosylation of the trisaccharide with 2-azido-3,4,6-tri-O-benzyl-beta-D-galactopyranosyl chloride and 2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl bromide gave rise to protected A and B tetrasaccharides, respectively. Deprotected R-glycosides were converted to OCH2CH2CH2NH2 derivatives. Their reaction with poly(4-nitrophenylacrylate) affords polyacrylamide-coupled conjugates with A, B, and H (type 3) specificity.     

Three saponins from Oxytropis species.

Three flavonoids and three saponins have been isolated from Oxytropis species. Their structures were determined as isorhamnetin-3-O-beta-D-glucoside, rhamnetin-3-O-beta-D-galactoside, apigenin, 3-O-[alpha-L-rhamnopyranosyl (1----2)-beta-D-glucopyranosyl(1----4)-beta-D-glucuronopyranosyl]+ ++soyasapogenol B, 3-O-[beta-D-glucopyranosyl(1----2)-beta-D-glucuronopyranosyl] azukisapogenol and a new saponin 3-O-[beta-D-glucopyranosyl(1----2)-beta-D-glucopyranosyl]-25-O-alpha-L- rhamnopyranosyl-(20S,24S)-3 beta,16 beta, 20,24,25-pentahydroxy-9,19-cycloanostane.     

Selective high-efficiency cross-linking of mammalian ribosomal proteins with cleavable thiol-directed heterobifunctional reagents: identification and binding directions of major protein complexes

Rat liver and mouse ascitic tumour ribosomal proteins are cross-linked selectively in good yield with the newly developed cleavable heterobifunctional reagents 2-(4-hydroxy-2-maleimidophenylazo)benzoic acid N-hydroxysuccinimide ester (reagent A) and 4-(4-hydroxy-3-maleimidophenylazo)[carboxyl-14C]benzoic acid N-hydroxysuccinimide ester (reagent B). The primary function of the reagents, an N-aroylated maleimide, binds quantitatively at low pH to accessible cysteine groups. After eliminating the free reagent, the pH is increased to make the secondary function, a juxtanuclear aroyl ester, reactive against neighboring amino groups, essentially lysine. The spacer, 4-phenylazophenol, is readily cleaved by reduction with dithionite. The ranges of cross-linking of the two reagents are approx. 8 and 12 A, respectively. Using the radiolabelled reagent B the secondarily attached protein (and its contact sequence) is made recognizable even in trace amounts. The order of binding of the interacting proteins is thereby established. The two reagents produce similar, but not identical, patterns of selective cross-linking. The following protein complexes are readily observed after conventional staining. With reagent A: S8-S11, L4-L14, L4-L18, L6-L29 and L21-L18a. With the radioactive, longer-range reagent B: L4 ---- L13a, L4 ---- L18, L4 ---- L18a, L4 ---- L26, L6 ---- L29, L14 ---- L13a, L21 ---- L18a and L27 ---- L30 (arrows indicating the direction of binding). Ternary and quaternary complexes are also obtained, especially of the large protein L4. With both reagents a protein designated L6' is cross-linked to L23. The predominant cross-linked complexes can be obtained on a preparative scale for isolation and characterization of contact sequences by optional fragmentation and fractionation methods.     

Novel blood group H glycolipid antigens exclusively expressed in blood group A and AB erythrocytes (type 3 chain H). I. Isolation and chemical characterization

A series of blood group H antigens reacting with monoclonal antibody MBrl has been found in human blood group A and AB erythrocytes, but not in O or B erythrocytes. These H antigens are clearly different from the globo-H structure (Fuc alpha 1----2Gal beta 1----3GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer), which was previously isolated from O erythrocytes and is also reactive with the MBrl antibody. The new series of H antigens associated with blood group A has been characterized as having TLC mobilities which approximately coincide with those of H2, H3, and H4 glycolipids. One of these A-associated H antigens, having a similar TLC mobility as the H2 glycolipid, was isolated from A erythrocytes and was characterized by 1H NMR spectroscopy, methylation analysis, and enzymatic degradation as having the structure shown below: (formula, see text). The structure represents a precursor of the repetitive A epitope attached to type 2 chain, previously called type 3 chain A (Clausen, H., Levery, S. B., Nudelman, E., Tsuchiya, S., and Hakomori, S. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1199-1203). This A-associated H structure is hereby called type 3 chain H.