Chemical Synthesis and Immunological Evaluation of Helicobacter pylori Serotype O6 Tridecasaccharide O-Antigen Containing a dd-Heptoglycan

The development of glycoconjugate vaccines against Helicobacter pylori is challenging. An exact epitope of the H. pylori lipo-polysaccharide (LPS) O-antigens that contain Lewis determinant oligosaccharides and unique dd -heptoglycans has not yet been identified. Reported here is the first total synthesis of H. pylori serotype O6 tridecasaccharide O-antigen containing a terminal Le^y tetrasaccharide, a unique α-(1→3)-, α-(1→6)-, and α-(1→2)-linked heptoglycan, and a β-d -galactose connector, by an [(2×1)+(3+8)] assembly sequence. Seven oligosaccharides covering different portions of the entire O-antigen were prepared for immunological investigations with a particular focus on elucidation of the roles of the dd -heptoglycan and Le^y tetrasaccharide. Glycan microarray analysis of sera from rabbits immunized with isolated serotype O6 LPS revealed a humoral immune response to the α-(1→3)-linked heptoglycan, a key motif for designing glycoconjugate vaccines for H. pylori serotype O6.     

Synthesis of 8-Methoxycarbonyloctylβ-Glycosides of Tri-and Tetrasaccharides Related to Schizophyllan and Neoglycoproteins Therefrom

Abstract

The 8-methoxycarbonyloctyl β-glycosides of the trisaccharides O-β-d-Glcp-(1 → 6)- O-β-d-Glcp-(1 → 3)-d-Glcp and O-β-d-Glcp-(1 → 3)-O-[β-d -Glcp-(1 → 6)]-d-Glcp and of the tetrasaccharide O-β-d-Glcp-(1 → 3)-O-[β-d-Glcp-(1 → 6)]-O-β-d-Glcp-(1 → 3)-d-Glcp, corresponding to the fragments of schizophyllan, have been synthesized by using mono- to tetrasaccharide 1-thioglycosides as glycosyl donors, each bearing a participating benzoyl group in the 2-position, and N-iodosuccinimide and silver triflate as promoter. Saponification of the tri- and tetrasaccharide β-glycosides, followed by attachment to bovine serum albumin of the resulting sugar derivatives having a carboxyl group at the aglycon terminal, provided neoglycoproteins for immunological studies of the polysaccharide.     

Block Synthesis of Streptococcus pneumoniae Type 14 Capsular Polysaccharide Structures*

Synthesis of a thioglycoside tetrasaccharide block, β‐D‐Galp‐(1→4)‐β‐DGlcp‐(1→6)‐[β‐D‐Galp‐(1→4)]‐β‐D‐GlcNPhthp‐(1→SEt, corresponding to the repeating unit of Streptococcus pneumoniae serotype 14 CPS is described. Coupling of this block with a spacer followed by removal of an isopropylidene acetal yielded an acceptor, which was elongated with the donor block to give a protected dimer of the repeating unit. Iteration of this methodology yielded the trimer. Deprotection then produced an octa and a dodecasaccharide derivative ready for conjugation to proteins to afford immunoactive glycoconjugates.     

Chemical Synthesis Elucidates the Immunological Importance of a Pyruvate Modification in the Capsular Polysaccharide of Streptococcus pneumoniae Serotype 4

Carbohydrate modifications are believed to strongly affect the immunogenicity of glycans. Capsular polysaccharides (CPS) from bacterial pathogens are frequently equipped with a pyruvate that can be placed across the 4,6‐, 3,4‐, or 2,3‐positions. A trans‐2,3‐linked pyruvate is present on the CPS of the Gram‐positive bacterium Streptococcus pneumoniae serotype 4 (ST4), a pathogen responsible for pneumococcal infections. To assess the immunological importance of this modification within the CPS repeating unit, the first total synthesis of the glycan was carried out. Glycan microarrays containing a series of synthetic antigens demonstrated how antibodies raised against natural ST4 CPS specifically recognize the pyruvate within the context of the tetrasaccharide repeating unit. The pyruvate modification is a key motif for designing minimal synthetic carbohydrate vaccines for ST4.     

Chemical Synthesis and Immunological Evaluation of Helicobacter pylori Serotype O6 Tridecasaccharide O‐Antigen Containing a dd‐Heptoglycan

The development of glycoconjugate vaccines against Helicobacter pylori is challenging. An exact epitope of the H. pylori lipo‐polysaccharide (LPS) O‐antigens that contain Lewis determinant oligosaccharides and unique dd ‐heptoglycans has not yet been identified. Reported here is the first total synthesis of H. pylori serotype O6 tridecasaccharide O‐antigen containing a terminal Le^y tetrasaccharide, a unique α‐(1→3)‐, α‐(1→6)‐, and α‐(1→2)‐linked heptoglycan, and a β‐d ‐galactose connector, by an [(2×1)+(3+8)] assembly sequence. Seven oligosaccharides covering different portions of the entire O‐antigen were prepared for immunological investigations with a particular focus on elucidation of the roles of the dd ‐heptoglycan and Le^y tetrasaccharide. Glycan microarray analysis of sera from rabbits immunized with isolated serotype O6 LPS revealed a humoral immune response to the α‐(1→3)‐linked heptoglycan, a key motif for designing glycoconjugate vaccines for H. pylori serotype O6.     

The Synthesis of Four Dideoxygenated Analogues of β-Maltosyl-(1→4)-Trehalose

Abstract

Four derivatives of β-maltosyl-(1→4)-trehalose were prepared, each with two deoxy functions in one of the constitutive disaccharide building blocks. 2,3-Di-O-acetyl-4,6-dideoxy-4,6-diiodo-α-D-galactopyranosyl- (1→4) ?1,2,3,6-tetra-O-acetyl-D-glucopyranose (3) was employed as a precursor for the 4?,6?-dideoxygenated tetrasaccharide 9: coupling of 3 with 2,3,6-tri-O-benzyl-α-D-glucopyranosyl 2,3,6-tri-O-benzylidene-α-D-glucopyranoside (4) furnished the tetrasaccharide 5 which was deiodinated and deprotected to yield the target tetrasaccharide 9. Secondly, the dideoxygenated maltose derivative 3-deoxy-4,6-O-isopropylidene-2-O-pivaloyl-β-D-glucopyranosyl- (1→4) ?1,6-anhydro-3-deoxy-2-O-pivaloyl-β-D-glucopyranose (10) was ring-opened to the anomeric acetate 11. A [2+2] block synthesis with 4 in TMS triflate mediated glycosylation gave a tetrasaccharide which was deprotected to the 3″,3?-dideoxygenated analogue of β-maltosyl-(1→4)-trehalose. For the third tetrasaccharide, 2,3,2″,3′-tetra-O-benzyl-α,α-trehalose was iodinated at the primary positions and deiodinated in the presence of palladium-on-carbon, then this acceptor was selectively glycosylated with hepta-O-acetyl-maltosyl bromide (20). Removal of protective groups furnished the maltosyl trehalose tetrasaccharide deoxygenated at positions C-6 and C-6′. to prepare a 3,3′-dideoxygenated trehalose, the free hydroxyl groups of 2-O-benzyl-4,6-O-(R)-benzylidene-α-D-glucopyranosyl 2-O-benzyl-4,6-O-(R)-benzylidene-α-D-glucopyranoside (25) were reduced by Barton-McCombie deoxygenation. One of the benzylidene groups was opened reductively with sodium cyanoborohydride. The resulting free hydroxyl group at the 4′-position was glycosylated in a Koenigs-Knorr reaction with 20 to yield the 3,3′-dideoxygenated tetrasaccharide 32, the fourth target oligosaccharide, after deprotection.     

Convergent synthesis of the tetrasaccharide repeating unit related to the O-antigenic polysaccharide of Escherichia coli 78

A convergent synthesis of the tetrasaccharide repeating unit of the O-antigenic cell wall polysaccharide of Escherichia coli 78, as the corresponding methyl glycoside (I), is being reported. It involved stereoselective glycosidation of a β-linked mannodisaccharide acceptor with a β-linked glucosamine based disaccharide thioglycoside donor, which were prepared from the corresponding functionalised monosaccharide based glycosyl donors and acceptors. The resulting tetrasaccharide derivative was finally converted to (I) by selective deprotection and also by global protection and deprotection techniques.     

On the Effect of the Aglycon Structure of Three Aryl β-D-Galactosides on the Yield and the Regioselectivity of the Transglycolytic Synthesis of N-Acetyllactosamine

ABSTRACT

We examined the effect as donors of three aryl β-D-galactosides (i.e. p-nitrophenyl β-D-galactopyranoside, o-nitrophenyl β-D-galactopyranoside and phenyl β-D-galacto-pyranoside) on the regioselectivity and the yield of the synthesis of N-acetyllactosamine obtained from the transglycosylation reaction catalyzed by a crude preparation of β-D-galactosidase from Bacillus circulans at 25 °C, 37 °C and 55 °C, respectively. Using p-nitrophenyl β-D-galactopyranoside the reaction results were fully regiospecific at all the temperatures considered: the maximum molar yield (74%) was obtained at an incubation temperature of 55 °C. Using o-nitrophenyl β-D-galactopyranoside as the donor the reaction was still highly regioselective and the maximum molar yield (50%) was achieved at an incubation temperature also of 55 °C. Using phenyl β-D-galactopyranoside transglycolytic products appear only at an incubation temperature of 55 °C but at very low molar yield (about 14%) and lower regioselectivity.     

Investigation by NMR Spectroscopy and Molecular Modeling of the Conformations of Some Modified Disaccharide Antigens for Shigella Dysenteriae Type 1

Abstract

The O-polysaccharide of Shigella dysenteriae type 1 is made up of multiple repeats of the linear tetrasaccharide 3)-α-L-Rhap-(1→2)-α-D-Galp-(1→3)-α-D-GlcpNAc-(1→3)-α-L-Rhap-(1→, for which the antigenic determinant for a murine monoclonal IgM antibody is the disaccharide α-L-Rhap-(1→2)-α-D-Galp. This disaccharide and various analogs have been studied by 2D NOESY, ROESY, and TOCSY NMR spectroscopy, in conjunction with proton spin-lattice relaxation rate measurements, restrained molecular mechanics, and restrained molecular dynamics with simulated annealing. It has been found that replacement of any single hydroxyl group in the determinant by a hydrogen atom, or replacement of any single hydroxyl group in the Gal residue by a fluorine atom has little if any influence on the conformation of the resulting derivatives.

     

Copolymerization of aryldiazomethanes

Several aryldiazoalkanes (M₂) have been copolymerized with phenyldiazomethane (M₁) in toluene-methanol solution at 40°C, namely, the p-chloro-, p-methoxy-, p-mesyl-o and -p-methyl-, 2,4- and 3,4-dichlorophenyldiazoalkanes, and the α- and β- naphthyl-diazoalkanes. The copolymerization parameters r₁ and r₂ have been evaluated. By plotting 1/r₁ against the Hammett σ values a negative ρ values was found equal to ?0.88. From cationic copolymerizations carried out at ?78°C in the presence of boron trifluoride-diethyl ether as catalyst a similar plot of 1/r₁ against σ gives a value of ρ equal to ?0.82. The negative sign and the agreement between these ρ values demonstrates the cationie mechanism of the methanol polymerization and copolymerization of aryldiazoalkanes.     

The mass spectra of six fluorinated β-diketones and their monothio analogues

The mass spectra of the fluorinated β-diketones RCOCH₂COCF₃ (R = Ph, p-FC₆H₄, p-ClC₆H₄, p-BrC₆H₄, p-MeC₆H₄ and 2-thienyl) and their monothio analogues RC(SH)?CHCOCF₃ have been obtained. The replacement of one oxygen by sulphur in the β-diketones brings about a greater complexity in the mass spectra. The β-diketones fragment by first losing a ·CF₃ radical and then successively lose CH₂?C?O and CO to yield \documentclass{article}\pagestyle{empty}\begin{document}${\rm [R}\mathop {\rm C}\limits^{\rm + } {\rm = O]} $\end{document} and [R]⁺. The monothio-β-diketones also fragment by an analogous reaction pathway: viz. the initial loss of ·CF₃ is followed by the successive loss of CH₂?C?O and CS to yield \documentclass{article}\pagestyle{empty}\begin{document}${\rm [R}\mathop {\rm C}\limits^{\rm + } {\rm = S]} $\end{document} and [R]⁺. However, they also fragment by two other pathways involving the initial loss of ·H and ·X (X=F, Cl, Br, Me), the latter occurring only with those monothio-β-diketones having R=p-XC₆H₄.     

A study of benzobisoxazole and benzobisthiazole compounds and polymers under hydrolytic conditions

The stability of benzobisoxazole and benzobisthiazole compounds and polymers under hydrolytic conditions was studied. 2,6-Bis(4-tert-butylphenyl)benzo[1,2-d;4,5-d′]bisoxazole (1) dissolved in acetonitrile containing sulfuric acid and water at 80°C is stable. A suspension of 2,6-bis[4-(2-benzoxazoyl)phenyl]benzo[1,2-d;5,4-d′]bisoxazole (2) in 0.2 N H₂SO₄ or 0.2 N NaOH solution at 100°C for 21 days is stable. The intrinsic viscosity of a poly(p-phenylene)benzobisoxazole (PBO) fiber sample soaked in 0.2 N H₂SO₄, water with 1 wt % polyphosphoric acid (PPA), or 0.2 N NaOH remained the same. Under very severe hydrolytic conditions such as dissolution of compound 2 or PBO in PPA or methanesulfonic acid with residual water followed by coagulation in water, benzobisoxazole underwent bond cleavage to generate carboxylic acid and o-aminophenol functional groups. This is in contrast to an earlier hypothesis that the decrease in intrinsic viscosity under these conditions was due to chain association. Poly(p-phenylene)benzobisthiazole (PBT) also underwent bond cleavage under these very severe conditions, which are unlikely to be encountered in normal applications. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 2637–2643, 1999     

The Synthesis of the 2′′- and 2′′′-Monodeoxygenated Analogues of β-Maltosyl-(1→4)-Trehalose

Abstract

Two derivatives of β-maltosyl-(1→4)-trehalose monodeoxygenated at C-2′′ or C-2′′′ have been synthesized in [2+2] block syntheses. O-(2,3,4,6-Tetra-O-benzyl-α-D-glucopyranosyl)-(1→4)-3,6-di-O-benzyl-1,2-di-O-acetyl-β-D-glucopyranose (6), prepared from the respective orthoester, was coupled to the glycosyl acceptor 2,3-di-O-benzyl-4,6-O-benzylidene-α-D-glucopyranosyl 2,3,6-tri-O-benzyl-α-D-glucopyranoside. In the resulting tetrasaccharide 8, the only ester group was removed and replaced by a xanthate which was reduced in a Barton-McCombie reaction to afford the 2′′-deoxygenated tetrasaccharide 12. For the synthesis of a 2′′′-deoxygenated derivative, a maltose building block was assembled from two monosaccharides. The key building block was ethyl 2,3,6-tri-O-benzyl-1-thio-β-D-glucopyranoside (14) which was used i) as a glycosyl acceptor in a phenylselenyl chloride mediated coupling reaction with tri-O-benzyl-glucal and ii) after the first coupling as a glycosyl donor to react with glycosyl acceptor 7 to give tetrasaccharide 18. The phenylselenyl group was reduced with tributyltin hydride on the disaccharide level. Deprotection of 18 furnished the 2′′′-deoxy-maltosyl-(1→4)-trehalose 20.     

Anomeric 5-Aza-7-deaza-2′-deoxyguanosines in Silver-Ion-Mediated Homo and Hybrid Base Pairs: Impact of Mismatch Structure,Helical Environment,and Nucleobase Substituents on DNA Stability

Nucleoside configuration (α-d vs. β-d ), nucleobase substituents, and the helical DNA environment of silver-mediated 5-aza-7-deazaguanine-cytosine base pairs have a strong impact on DNA stability. This has been demonstrated by investigations on oligonucleotide duplexes with silver-mediated base pairs of α-d and β-d anomeric 5-aza-7-deaza-2′-deoxyguanosines and anomeric 2′-deoxycytidines incorporated in 12-mer duplexes. To this end, a new synthetic protocol has been developed to access the pure anomers of 5-aza-7-deaza-2′-deoxyguanosine by glycosylation of either the protected nucleobase or its salt followed by separation of the glycosylation products by crystallization and chromatography. Thermal stability measurements were performed on duplexes with α-d /α-d and β-d /β-d homo base pairs or α-d /β-d and β-d /α-d hybrid pairs within two sequence environments, positions 6 or 7, of oligonucleotide duplexes. The respective T_m stability increases observed after silver ion addition differ significantly. Homo base pairs with β-d /β-d or α-d /α-d nucleoside combinations are more stable than α-d /β-d hybrid base pairs. The positional switch of silver-ion-mediated base pairs has a significant impact on stability. Nucleobase substituents introduced at the 5-position of the dC site of silver-mediated base pairs affect base pair stability to a minor extent. Our investigation might lead to applications in the construction of bioinspired nanodevices, in DNA diagnostics, or metal-DNA hybrid materials.     

Use of aromatic dialdehydes in the preparation of poly(p-benzoquinono)diimidazoles

Poly(p-benzoquinono)diimidazoles have been synthesized under mild conditions (100 or 150°C) by one-stage polycondensation in N,N-dimethylacetamide (DMAc) or in polyphosphoric acid of 2,3,5,6-tetraamino-p-benzoquinone (TABQ) with terephthalaldehyde and isophthalaldehyde. The model compound 2,6-diphenyl-1,5-dihydro-1′,4′-benzoquinono[2′,3′-d:5′,6′-d′]diimidazole was also prepared by condensation of TABQ with benzaldehyde in DMAc at 100°C. The prepared polymers were completely insoluble in organic solvents but slightly soluble (0.5%) in strong acids with viscosities (η_inh) in the range of 0.17–0.38 dL/g (c = 0.2% in formic acid). Their thermal stability was studied by TG analysis.     

Synthesis of a Tri- and Tetrasaccharide Fragment Specific for the Shigella flexneri Serotype 5a O-Antigen. A Reinvestigation

ABSTRACT

Stereocontrolled, stepwise synthesis of methyl α-L-rhamnopyranosyl-(1→2)-[α-D-glucopyranosyl-(1→3)]-α-L-rhamnopyranoside (A(E)B, 1) and methyl 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-L-rhamnopyranosyl-(1→2)-[α-D-glucopyranosyl-(1→3)]-α-L-rhamnopyranoside (DA(E)B, 2) is described; these constitute the methyl glycosides of fragments of the O-specific polysaccharide of Shigella flexneri serotype 5a. Two routes to trisaccharide 1 were considered. Route 1 involved the coupling of a precursor to residue A and a disaccharide EB, whereas route 2 was based on the condensation of a precursor to residue E and a disaccharide AB. Rather surprisingly, the latter afforded the β-anomer of 1, namely methyl α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→3)]-α-L-rhamnopyranoside as the major product. Route 1 was preferred. Overall, several observations made during this study suggested that, for the construction of higher fragments, a suitable precursor to rhamnose A would require protecting groups of low bulkiness at position 3 and 4. Therefore, the 2-O-acetyl-3,4-di-O-allyl-α-L-rhamnopyranosyl trichloroacetimidate (35) was the precursor of choice to residue A in the synthesis of the tetrasaccharide 2. The condensation product of 35 and methyl 2,3,4,6-tetra-O-benzyl-α-D-glucopyranosyl-4-O-benzyl-α-L-rhamnopyranoside was selectively deacylated and condensed to 2-trichloroacetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl trichloroacetimidate to afford the corresponding fully protected tetrasaccharide 45. Controlled stepwise deprotection of the latter proceeded smoothly to afford the target 2. It should be emphasised that the preparation of 45 was not straightforward, several donors and coupling conditions that were tested resulted only in the complete recovery of the acceptor. Distortion of several signals in the ¹³C NMR spectra of the fully or partially protected tetrasaccharide intermediates suggested that steric hindrance, added to the known low reactivity of HO-2 of rhamnosyl acceptors, probably played a major role in the outcome of the glycosidation attempts.     

Studies towards a Conjugate Vaccine for Anthrax: Synthesis of the Tetrasaccharide Side Chain of the Bacillus anthracis Exosporium

The first synthesis of β‐L ‐glycoside 17 of the tetrasaccharide β‐Ant‐(1 → 3)‐α‐L ‐Rhap‐(1 → 3)‐α‐L ‐Rhap‐(1 → 2)‐L ‐Rhap is described (Schemes 1–3). Its spacer can be functionalized to make it amenable to conjugation to proteins by different conjugation methods. The synthesis was performed in a stepwise manner starting from the aglycon‐bearing terminal saccharide with thioglycosides as glycosyl donors. To attach the upstream terminal anthrose residue, the assembled linker‐equipped trisaccharide was glycosylated with ethyl 4‐azido‐3‐O‐benzyl‐2‐O‐(bromoacetyl)‐4,6‐dideoxy‐1‐thio‐β‐D ‐glucopyranoside ( 11 ). Further functionalization of the tetrasaccharide thus obtained, followed by deprotection gave the target substance 17 . Synthesis of substructures of 17 equipped with the same spacer, namely β‐L ‐Rhap‐1‐O‐(CH₂)₅COOMe ( 21 ), α‐L ‐Rhap‐(1 → 2)‐β‐L ‐Rhap‐1‐O‐(CH₂)₅COOMe ( 22 ), and α‐L ‐Rhap‐(1 → 3)‐α‐L ‐Rhap‐(1 → 2)‐β‐L ‐Rhap‐1‐O‐(CH₂)₅COOMe ( 23 ), is also described (Scheme 4).     

The spin delocalization substituent parameters σ: 3. The spin-delocalizing abilities of the acetyl and trimethylsilyl groups. Synthesis of four substituted trifluorostyrenes

Four substituted α, β, β-trifluorostyrenes (TFSs) namely, ptrimethylsilyltrifluorostyrene (1), p-acetyltrifluorostyrene ethylene ketal (2), p-acetyltrifluorostyrene (3) and p-phenoxytrifluorostyrene (4), have been synthesized. The rate constants (k₂) of the thermal cyclodimerization of 1 and 3 have been measured in the temperature range 120—160°C. The polar parameters _σP of the four TFSs calculated from ¹⁹F NMR chemical shifts are: for p-trimethylsilyl-1, 0.21; 1-ethylenedioxy-ethyl-, —0.01; p-acetyl-, 0.61 and p-phenoxy-,—0.47. The spin de-localization substituent parameters σ?_T of p-trimethylsilyl and p-acetyl groups are 0.29 and 0.52 respectively. Thus both the p-trimethylsilyl and the p-acetyl groups act as electron-attracting groups which can also effectively stabilize a spin.