Dennis M. Whitfield

Functional characterization of dehydratase/aminotransferase pairs from Helicobacter and Campylobacter: enzymes distinguishing the pseudaminic acid and bacillosamine biosynthetic pathways.

Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-α-d-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His6-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-β-l-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-α-d-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-α-d-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose.

Targeted metabolomics analysis of Campylobacter coli VC167 reveals legionaminic acid derivatives as novel flagellar glycans.

Glycosylation of Campylobacter flagellin is required for the biogenesis of a functional flagella filament. Recently, we used a targeted metabolomics approach using mass spectrometry and NMR to identify changes in the metabolic profile of wild type and mutants in the flagellar glycosylation locus, characterize novel metabolites, and assign function to genes to define the pseudaminic acid biosynthetic pathway in Campylobacter jejuni 81-176 (McNally, D. J., Hui, J. P., Aubry, A. J., Mui, K. K., Guerry, P., Brisson, J. R., Logan, S. M., and Soo, E. C. (2006) J. Biol. Chem. 281, 18489-18498). In this study, we use a similar approach to further define the glycome and metabolomic complement of nucleotide-activated sugars in Campylobacter coli VC167. Herein we demonstrate that, in addition to CMP-pseudaminic acid, C. coli VC167 also produces two structurally distinct nucleotide-activated nonulosonate sugars that were observed as negative ions at m/z 637 and m/z 651 (CMP-315 and CMP-329). Hydrophilic interaction liquid chromatography-mass spectrometry yielded suitable amounts of the pure sugar nucleotides for NMR spectroscopy using a cold probe. Structural analysis in conjunction with molecular modeling identified the sugar moieties as acetamidino and N-methylacetimidoyl derivatives of legionaminic acid (Leg5Am7Ac and Leg5AmNMe7Ac). Targeted metabolomic analyses of isogenic mutants established a role for the ptmA-F genes and defined two new ptm genes in this locus as legionaminic acid biosynthetic enzymes. This is the first report of legionaminic acid in Campylobacter sp. and the first report of legionaminic acid derivatives as modifications on a protein.

The CMP-legionaminic acid pathway in Campylobacter: Biosynthesis involving novel GDP-linked precursors

The sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-nonulosonic acid, or legion-aminic acid, is found as a virulence-associated cell-surface glycoconjugate in the Gram-negative bacteria Legionella pneumophila and Campylobacter coli. L. pneumophila serogroup 1 strains, causative agents of Legionnaires disease, contain an alpha2,4-linked homopolymer of legionaminic acid within their lipopolysaccharide O-chains, whereas the gastrointestinal pathogen C. coli modifies its flagellin with this monosaccharide via O-linkage. In this work, we have purified and biochemically characterized 11 candidate biosynthetic enzymes from Campylobacter jejuni, thereby fully reconstituting the biosynthesis of legionaminic acid and its CMP-activated form, starting from fructose-6-P. This pathway involves unique GDP-linked intermediates, likely providing a cellular mechanism for differentiating between this and similar UDP-linked pathways, such as UDP-2,4-diacetamido-bacillosamine biosynthesis involved in N-linked protein glycosylation. Importantly, these findings provide a facile method for efficient large-scale synthesis of legionaminic acid, and since legionaminic acid and sialic acid share the same D-glycero-D-galacto absolute configuration, this sugar may now be evaluated for its potential as a sialic acid mimic.

Can the stereochemical outcome of glycosylation reactions be controlled by the conformational preferences of the glycosyl donor

Previous static and dynamical density functional theory studies of the 2,6-di-O-acetyl-3,4-O-isopropylidene-D-galactopyranosyl cations and their methanol adducts has led to an hypothesis that these cations exist in two families of conformers characterized as (2)S(O) and B(2,5), respectively. These families differ by ring inversion, each with its own reactivity. New calculations on the 2,6-di-O-acetyl-3,4-di-O-methyl-D-galactopyranosyl cation confirmed these trends. Removing the isopropylidene group allows more flexibility, but two families of conformers can be discerned with the monocyclic oxocarbenium ions in the E(3) conformation and the bicyclic dioxolenium ions in the (4)H(5) conformation. Attack on the beta-face of these monocyclic cations is favored by hydrogen bonding and the anomeric effect. The experimentally observed high beta-stereoselectivity of mannopyranosyl donors and high alpha-stereoselectivity of glucopyranosyl donors with the 4,6-O-benzylidene protecting groups can be rationalized assuming that the trans-fused 1,3-dioxane ring allows population of only one family of conformers. The combination of hydrogen bonding and conformational changes of the pyranose ring in response to the C-5[bond]O-5[bond]C-1[bond]C-2 torsion angle changes are identified as key factors in stereoselectivity. Based on these observations a strategy to design face discriminated glycosyl donors that exist predominantly in only one family of conformers is proposed.

Quantitative description of six-membered ring conformations following the IUPAC conformational nomenclature

Abstract Although several methods of quantitative conformational characterization exist in the literature, all these methods use a spherical polar coordinate representation which is in contrast to the qualitative description based on the IUPAC nomenclature. To bridge this gap this paper introduces a method to characterize six-membered ring conformations as a linear combination of ideal basic conformations. The linear combination coefficients are derived by projection of the vector of torsion angles onto those of ideal basic conformations. As the IUPAC nomenclature uses subscripts and superscripts to indicate atoms below and above the reference plane, the linear combination coefficients combined with the IUPAC name provide an instant visual image of the conformation. The method introduced here is based on endocyclic dihedral angles and requires only three dihedral angles for a full characterization, which is often available by NMR measurements for rigid conformations. We provide a table of equations to determine the missing dihedral angles based on redundancy conditions. The relationship between linear combinations and spherical representation similar to the well-known Cremer–Pople parameters is presented. In deriving the spherical conformational parameters we solved an inconsistency of previous definitions for spherical representation, namely that none of previous definitions place the intermediate halfchair or twistboat conformations exactly halfway between the pole (chair) and the equator (boat and twistboat) of the sphere as expected based on intuitive stereochemistry. To make our method generally available we provide an interface on the Internet that carries out all calculations described in the paper and allows the user to visualize, rotate and manipulate the ring (http://www.nrc.ca/ibs/6ring.html). By simplifying both the concepts and the access to carry out the calculations more experimental chemists can benefit from the description of ring conformation.

Low-temperature approach to highly emissive copper indium sulfide colloidal nanocrystals and their bioimaging applications.

We report our newly developed low-temperature synthesis of colloidal photoluminescent (PL) CuInS2 nanocrystals (NCs) and their in vitro and in vivo imaging applications. With diphenylphosphine sulphide (SDPP) as a S precursor made from elemental S and diphenylphosphine, this is a noninjection based approach in 1-dodecanethiol (DDT) with excellent synthetic reproducibility and large-scale capability. For a typical synthesis with copper iodide (CuI) as a Cu source and indium acetate (In(OAc)3) as an In source, the growth temperature was as low as 160 °C and the feed molar ratios were 1Cu-to-1In-to-4S. Amazingly, the resulting CuInS2 NCs in toluene exhibit quantum yield (QY) of ~23% with photoemission peaking at ~760 nm and full width at half maximum (FWHM) of ~140 nm. With a mean size of ~3.4 nm (measured from the vertices to the bases of the pyramids), they are pyramidal in shape with a crystal structure of tetragonal chalcopyrite. In situ (31)P NMR (monitored from 30 °C to 100 °C) and in situ absorption at 80 °C suggested that the Cu precursor should be less reactive toward SDPP than the In precursor. For our in vitro and in vivo imaging applications, CuInS2/ZnS core-shell QDs were synthesized; afterwards, dihydrolipoic acid (DHLA) or 11-mercaptoundecanoic acid (MUA) were used for ligand exchange and then bio-conjugation was performed. Two single-domain antibodies (sdAbs) were used. One was 2A3 for in vitro imaging of BxPC3 pancreatic cancer cells. The other was EG2 for in vivo imaging of a Glioblastoma U87MG brain tumour model. The bioimaging data illustrate that the CuInS2 NCs from our SDPP-based low-temperature noninjection approach are good quality.

Chapter 4 Computational Studies of the Role of Glycopyranosyl Oxacarbenium Ions in Glycobiology and Glycochemistry

Publisher Summary This chapter discusses the computational studies about the role of glycopyranosyl oxacarbenium ions in glycobiology and glycochemistry. An understanding of the formation and breaking of glycosidic linkages is at the core of both glycobiology and glycochemistry. With only a few exceptions, both the formation and breaking of glycosidic linkages involve processes that proceed through species having a high degree of oxacarbenium ion character. Two current reports have studied model systems for glycopyranosides— namely, 2-methoxytetrahydropyran and 2-ethoxytetrahydropyran, with special emphasis on determining precisely the anomeric effect. To achieve these goals, a so-called complete basis set (CBS) calculation was performed in the gas phase at 0 K. To reach agreement with the experiment, the standard corrections of including the zero point energy, a temperature correction to room temperature, and estimating the entropy were added. One of the many factors that affect the reactivity and stability of the glycopyranosyl oxacarbenium ion is the orientation and the conformation of the side chains attached to C-2, C-3, C-4, and C-5. In this study the implications for reactivity of the different stereochemistry, and hence orientations at C- 4 of galacto-versus gluco-configured ions are one of the main considerations. The C-5–C -6 conformation has also been extensively studied in neutral oligosaccharides.

SYNTHESIS, NMR, AND CONFORMATIONAL STUDIES OF FUCOIDAN FRAGMENTS. III.[1] EFFECT OF BENZOYL GROUP AT O-3 ON STEREOSELECTIVITY OF GLYCOSYLATION BY 3-O- AND 3,4-DI-O-BENZOYLATED 2-O-BENZYLFUCOSYL BROMIDES

The effect of a benzoyl group at O-3 on stereoselectivity of glycosylation by 3-O- and 3,4-di-O-benzoylated 2-O-benzyl-L-fucopyranosyl bromides was studied by direct chemical experiments and computational chemistry. The influence of a benzoyl group at O-3 of the fucosyl donors was shown to have a larger effect on the efficiency of α-fucosylation than a benzoyl group at O-4. It is hypothesized that this is a result of the ability of a benzoyl group at O-3 to participate in glycosyl cation stabilization.

Adjuvant potential of archaeal synthetic glycolipid mimetics critically depends on the glyco head group structure

Subunit vaccines capable of providing protective immunity against the intracellular pathogens and cancers that kill millions of people annually require an adjuvant capable of directing a sufficiently potent cytotoxic T lymphocyte response to purified antigens, without toxicity issues. Archaeosome lipid vesicles, prepared from isoprenoid lipids extracted from archaea, are one such adjuvant in development. Here, the stability of an archaeal core lipid 2,3-di-O-phytanyl-sn-glycerol (archaeol) is used to advantage to synthesize a series of disaccharide archaeols and show that subtle variations in the carbohydrate head group alters the type and potency of immune responses mounted in a mammal. Critically, a glycosylarchaeol was required to elicit high cytotoxic CD8(+) T cell activity, with highest responses to the antigen entrapped in archaeosomes containing disaccharides of glucose in beta- or alpha1-6 linkage (beta-gentiobiose, beta-isomaltose), or of beta-lactose. This first study on synthetic archaeal lipid adjuvants reveals potential for this class of regulatory friendly, easily scalable, inexpensive, and potent glyco-adjuvant.

Polymer-supported and chemoenzymatic synthesis of the Neisseria meningitidis pentasaccharide: a methodological comparison

Neisseria meningitidis trisaccharide [GlcNAc[(1-->3)Galbeta(1-->4)Glc-R], tetrasaccharide [Galbeta(1-->4)GlcNAcbeta(1--> 3)Galbeta(1-->4)Glc-R], and a pentasaccharide [Neu5Acalpha(2-->3)Galbeta(1-->4)GlcNAcbeta(1-->3)G albeta(1-->4)Glc-SPh] were prepared via conventional chemical synthesis, polymer-supported synthesis, and chemoenzymatic methods, starting from D-lactose. The polymer polyethyleneglycol monomethylether (MPEG) and the linker dioxyxylene (DOX) were used with a lactose-bound acceptor to improve the purification process. Several enzymes (LgtA, GalE-LgtB fusion, and CMP-Neu5Ac synthetase/sialyltransferase fusion) were used for syntheses of these oligosaccharides. Excellent stereo- and regioselectivities as well as high yield (> 90% from Gal(1-->4)Glc-SPh) of the pentasaccharide were obtained. Both of the convenient processes are suitable for efficient preparation of target oligosaccharides.