First and Stereoselective Synthesis of an α‑(2→5)-Linked Disaccharide of 3‑Deoxy‑d-<i>manno</i>-oct-2-ulosonic Acid (Kdo)

Resistance of bacterial pathogens toward antibiotics has revived interest in lipopolysaccharide (LPS) motifs as potential therapeutic targets. The LPS of several pathogenic <i>Acinetobacter</i> strains comprises a 4,5-branched Kdo trisaccharide containing an uncommon (2→5)-linkage. In this contribution the first stereoselective glycosylation method for obtaining an α-Kdo-(2→5)-α-Kdo disaccharide in good yield is highlighted. The synthetic approach used for accessing this linkage type will allow for future studies of the immunoreactivity associated with this unique bacterial Kdo inner core structure

Synthesis of Zwitterionic 1,1′-Glycosylphosphodiester: A Partial Structure of Galactosamine-Modified <i>Francisella</i> Lipid A

Synthesis of a “double glycosidic” phosphodiester comprising anomeric centers of two 2-amino-2-deoxy-sugars is reported. The carbohydrate epitope of <i>Francisella</i> lipid A modified with α-d-galactosamine at the anomerically linked phosphate has been stereoselectively prepared and coupled to maleimide-activated bovine serum albumin via an amide-linked thiol-terminated spacer group. H-Phosphonate and phosphoramidite approaches have been explored for the coupling of 4,6-DTBS-2-azido-protected GalN lactol and peracetylated spacer-equipped reducing βGlcN(1→6)­GlcN disaccharide via phosphodiester linkage. Deprotection conditions preserving the integrity of the labile glycosidic zwitterionic phosphodiester were elaborated

Stereoselective Synthesis of α- and β‑l‑Ara4N Glycosyl H‑Phosphonates and a Neoglycoconjugate Comprising Glycosyl Phosphodiester Linked β‑l‑Ara4N

Stereoselective synthesis of variably protected α- and β-l-Ara4N glycosyl H-phosphonates as key intermediates in the syntheses of β-l-Ara4N-modified LPS structures and α-l-Ara4N-containing biosynthetic precursors is reported. A facile one-pot approach toward β-l-Ara4N glycosyl H-phosphonates includes anomeric deallylation of protected 4-azido β-l-Ara4N via terminal olefin isomerization followed by ozonolysis and methanolysis of formyl groups to furnish exclusively β-configured lactols that are phosphitylated with retention of configuration. The carbohydrate epitope of β-l-Ara4N-modified Lipid A, βGlcN(1→6)­αGlcN­(1→P←1)­β-l-Ara4N, was stereoselectively synthesized and linked to maleimide-activated bovine serum albumin

Convergent Synthesis of 4‑<i>O</i>‑Phosphorylated l-<i>glycero</i>-d-<i>manno</i>-Heptosyl Lipopolysaccharide Core Oligosaccharides Based on Regioselective Cleavage of a 6,7‑<i>O</i>‑Tetraisopropyldisiloxane-1,3-diyl Protecting Group

The structurally conserved lipopolysaccharide core region of many Gram-negative bacteria is composed of trisaccharides containing 4-<i>O</i>-phosphorylated l-<i>glycero</i>-d-<i>manno</i>-heptose (l,d-Hep) units, which act as ligands for antibodies and lectins. The disaccharides Glc-(1→3)-Hep4P Hep-(1→3)-Hep4P and Hep-(1→7)-Hep4P and the branched trisaccharide Glc-(1→3)-[Hep-(1→7)]-Hep4P, respectively, have been synthesized from a methyl heptopyranoside acceptor in less than 10 steps. The synthetic strategy was based on the early introduction of a phosphotriester at position 4 of heptose followed by a regioselective opening of a 6,7-<i>O</i>-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl) group allowing for a straightforward access to glycosylation at position 7. Perbenzylated <i>N</i>-phenyl trifluoroacetimidate glucosyl and heptosyl derivatives served as α-selective glycosyl donors

Theoretical Foundation for the Presence of Oxacarbenium Ions in Chemical Glycoside Synthesis

Glycoside formation in organic synthesis is believed to occur along a reaction path involving an activated glycosyl donor with a covalent bond between the glycosyl moiety and the leaving group, followed by formation of contact ion pairs with the glycosyl moiety loosely bound to the leaving group, and eventually solvent-separated ion pairs with the glycosyl moiety and the leaving group being separated by solvent molecules. However, these ion pairs have never been experimentally observed. This study investigates the formation of the ion pairs from a covalent intermediate, 2,3,4,6-tetra-<i>O</i>-methyl-α-d-glucopyranosyl triflate, by means of computational chemistry. Geometry optimization of the ion pairs without solvent molecules resulted in re-formation of the covalent α- and β-triflates but was successful when four solvent (dichloromethane) molecules were taken into account. The DFT­(M06-2X) computations indicated interconversion between the α- and β-covalent intermediates via the α- and β-contact ion pairs and the solvent-separated ion pairs. The calculated activation Gibbs energy of this interconversion was quite small (10.4–13.5 kcal/mol). Conformational analyses of the ion pairs indicated that the oxacarbenium ion adopts ⁴H₃, ²H₃/E₃, ²H₃/²S₀, E₃, ^2,5B, and B_2,5 pyranosyl ring conformations, with the stability of the conformers being strongly dependent on the relative location of the counteranion

2,4,5-Trihydroxy-3-methylacetophenone: A Cellulosic Chromophore as a Case Study of Aromaticity

The title compound (2,4,5-trihydroxy-3-methylacetophenone, <b>1</b>) was isolated as chromophore from aged cellulosic pulps. The peculiar feature of the compound is its weak aromatic system that can be converted into nonaromatic (quinoid or cyclic aliphatic) tautomers, depending on the conditions and reaction partners. In alkaline media, the participation of quinoid canonic forms weakens aromaticity, whereas in neutral and acidic media, the strong hydrogen bond between the 2-hydroxyl group and the acetyl moiety plays an important role in favoring quinoid tautomers. As a result, compound <b>1</b>, with quinoid contributions being already “preset”, is relatively stable toward oxidation and hardly undergoes alkylation or nitration at CH-6, whereas the 2,4,5-trimethoxyderivative, being “properly” aromatic and even more sterically hindered, is readily alkylated or nitrated. The lability of the aromatic system is best demonstrated by the unusual reaction of <b>1</b> with hydroxylamine, producing a tetroxime that is derived from its 2,4,5-triketo tautomer. The high oxidative stability and low reactivity of the compound hinder oxidative bleaching of this chromophore in cellulosic pulps and detection reactions for analytical purposes

Transferase Activity of Lactobacillal and Bifidobacterial β‑Galactosidases with Various Sugars as Galactosyl Acceptors

The β-galactosidases from Lactobacillus reuteri L103 (<i>Lreu</i>βgal), Lactobacillus delbrueckii subsp. <i>bulgaricus</i> DSM 20081 (<i>Lbul</i>βgal), and Bifidobacterium breve DSM 20281 (<i>Bbre</i>βgal-I and <i>Bbre</i>βgal-II) were investigated in detail with respect to their propensity to transfer galactosyl moieties onto lactose, its hydrolysis products d-glucose and d-galactose, and certain sugar acceptors such as <i>N</i>-acetyl-d-glucosamine (GlcNAc), <i>N</i>-acetyl-d-galactosamine (GalNAc), and l-fucose (Fuc) under defined, initial velocity conditions. The rate constants or partitioning ratios (<i>k</i>_Nu/<i>k</i>_water) determined for these different acceptors (termed nucleophiles, Nu) were used as a measure for the ability of a certain substance to act as a galactosyl acceptor of these β-galactosidases. When using <i>Lbul</i>βgal or <i>Bbre</i>βgal-II, the galactosyl transfer to GlcNAc was 6 and 10 times higher than that to lactose, respectively. With lactose and GlcNAc used in equimolar substrate concentrations, <i>Lbul</i>βgal and <i>Bbre</i>βgal-II catalyzed the formation of <i>N</i>-acetyl-allolactosamine with the highest yields of 41 and 24%, respectively, as calculated from the initial GlcNAc concentration

Synthesis of a Pentasaccharide Fragment Related to the Inner Core Region of Rhizobial and Agrobacterial Lipopolysaccharides

The pentasaccharide fragment α-d-Man-(1 → 5)-[α-d-Kdo-(2 → 4)-]­α-d-Kdo-(2 → 6)-β-d-GlcNAc-(1 → 6)-α-d-GlcNAc equipped with a 3-aminopropyl spacer moiety was prepared by a sequential assembly of monosaccharide building blocks. The glucosamine disaccharideas a backbone surrogate of the bacterial lipid A regionwas synthesized using an 1,3-oxazoline donor, which was followed by coupling with an isopropylidene-protected Kdo-fluoride donor to afford a protected tetrasaccharide intermediate. Eventually, an orthogonally protected <i>manno</i>-configured trichloroacetimidate donor was used to achieve the sterically demanding glycosylation of the 5-OH group of Kdo in good yield. The resulting pentasaccharide is suitably protected for further chain elongation at positions 3, 4, and 6 of the terminal mannose. Global deprotection afforded the target pentasaccharide to be used for the conversion into neoglycoconjugates and “clickable” ligands

Complete Structural Elucidation of an Oxidized Polysialic Acid Drug Intermediate by Nuclear Magnetic Resonance Spectroscopy

Polysialic acid (PSA) is a high molecular weight glycan composed of repeat units of α(2→8) linked 5-<i>N</i>-acetyl-neuraminic acid. Mild periodate oxidation of PSA selectively targets the end sialic acid ring containing three adjacent alcohols generating a putative aldehyde, which can be used for terminal attachment of PSA to therapeutic proteins. The work presented here permitted complete NMR peak assignments of not only the repeat units, but also the two terminal units at each end of oxidized PSA, an intermediate, which can be used to improve drug performance. The assignments were made using a variety of NMR techniques on oligomers of sialic acid as well as oxidized PSA with molecular masses of 4 and 20 kDa. This enabled structure elucidation that showed the actual moiety formed was not the expected aldehyde or its hydrate, but is a hemiacetal between the oxidation site on the terminal sialic acid ring and the penultimate ring. The existence of a hemiacetal structure has major implications on stability, reactivity, and conjugation chemistry of oxidized PSA. The assignment process also revealed deuterium exchange of the axial hydrogen at the 3- (methylene) position of the ring, which was in agreement with the literature

Reaction of Oxidized Polysialic Acid and a Diaminooxy Linker: Characterization and Process Optimization Using Nuclear Magnetic Resonance Spectroscopy

Native polysialic acid (natPSA) is a high-molecular-weight glycan composed of repeat units of α-(2 → 8) linked <i>N</i>-acetylneuraminic acid (Neu5Ac). Mild periodate oxidation of PSA selectively targets the end sialic acid ring containing three adjacent alcohols generating a putative aldehyde, which can be used, after attachment of a linker molecule, for terminal attachment of PSA to protein. Previously, we showed that the oxidized PSA (oxoPSA) contained a hemiacetal at the oxidation site and can react with a linker containing an aminooxy group in a conjugation reaction to form a stable oxime linkage. Thus, reagents containing an aminooxy group may be prepared for conjugation of PSA to the carbohydrate moiety of therapeutic proteins, thereby increasing their half-life. These aminooxy–PSA reagents can selectively react with aldehyde groups generated by mild NaIO₄ oxidation of glycans on the surface of the target protein. To comprehend the conjugation, unoxidized tetrasialic acid and Neu5Ac were reacted in model reactions with a diaminooxy linker to define the nuclear magnetic resonance (NMR) chemical shifts. Based on these data, we were able to show that, in the case of PSA, the reaction with the linker occurs not only at the expected oxidized end to form an aldoxime but also at the end distal to the oxidation to form a ketoxime. We determined that, in aged solutions, both oxoPSA and PSA aldoxime were hydrolyzed. PSA aldoxime was also shown to disproportionate to form a dimer (PSA-linker-PSA), which then could react further with the released linker at one of its PSA termini. Furthermore, NMR was used to monitor the effects of deliberate process changes so that conditions could be optimized for attachment of linker at the desired end of the PSA chain, which led to a well-defined product