Branched montbretin A mimics allow derivatisation and potent amylase inhibition.

Mimics of the complex flavonol glycoside montbretin A in which a flavonol moiety is coupled to a caffeic acid via partially peptidic linkers have proved to be potent inhibitors of human pancreatic alpha-amylase with potential as therapeutics for control of blood glucose levels. After exploring optimal linker length, a synthetic route to a version with a branched linker was devised based on the structure of the enzyme/inhibitor complex. The resultant branched inhibitors were shown to retain nanomolar potency even when decorated with polymers as a means of modifying solubility. Similar improvements, along with nanomolar affinity, could also be achieved through conjugation to cyclodextrins which have the potential to bind to starch binding sites found on the surface of human amylase. Incorporation of a conjugatable branch into this unusual pharmacophore thereby affords considerable flexibility for further modifications to improve pharmacokinetic behaviour or as a site for attachment of capture tags or fluorophores.

Identification of a second glycoform of the clinically prevalent O1 antigen from Klebsiella pneumoniae.

Carbapenemase and extended ß-lactamase-producing Klebsiella pneumoniae isolates represent a major health threat, stimulating increasing interest in immunotherapeutic approaches for combating Klebsiella infections. Lipopolysaccharide O antigen polysaccharides offer viable targets for immunotherapeutic development, and several studies have described protection with O-specific antibodies in animal models of infection. O1 antigen is produced by almost half of clinical Klebsiella isolates. The O1 polysaccharide backbone structure is known, but monoclonal antibodies raised against the O1 antigen showed varying reactivity against different isolates that could not be explained by the known structure. Reinvestigation of the structure by NMR spectroscopy revealed the presence of the reported polysaccharide backbone (glycoform O1a), as well as a previously unknown O1b glycoform composed of the O1a backbone modified with a terminal pyruvate group. The activity of the responsible pyruvyltransferase (WbbZ) was confirmed by western immunoblotting and in vitro chemoenzymatic synthesis of the O1b terminus. Bioinformatic data indicate that almost all O1 isolates possess genes required to produce both glycoforms. We describe the presence of O1ab-biosynthesis genes in other bacterial species and report a functional O1 locus on a bacteriophage genome. Homologs of wbbZ are widespread in genetic loci for the assembly of unrelated glycostructures in bacteria and yeast. In K. pneumoniae, simultaneous production of both O1 glycoforms is enabled by the lack of specificity of the ABC transporter that exports the nascent glycan, and the data reported here provide mechanistic understanding of the capacity for evolution of antigenic diversity within an important class of biomolecules produced by many bacteria.

Vinyl Halide-Modified Unsaturated Cyclitols are Mechanism-Based Glycosidase Inhibitors.

Suitably configured allyl ethers of unsaturated cyclitols act as substrates of ß-glycosidases, reacting via allylic cation transition states. Incorporation of halogens at the vinylic position of these carbasugars, along with an activated leaving group, generates potent inactivators of ß-glycosidases. Enzymatic turnover of these halogenated cyclitols (F, Cl, Br) displayed a counter-intuitive trend wherein the most electronegative substituents yielded the most labile pseudo-glycosidic linkages. Structures of complexes with the Sulfolobus ß-glucosidase revealed similar enzyme-ligand interactions to those seen in complexes with a 2-fluorosugar inhibitor, the lone exception being displacement of tyrosine 322 from the active site by the halogen. Mutation of Y322 to Y322F largely abolished glycosidase activity, consistent with lost interactions at O5, but minimally affected (7-fold) rates of carbasugar hydrolysis, yielding a more selective enzyme for unsaturated cyclitol ether hydrolysis.

A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family.

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-ß-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.

Development of an active site titration reagent for α-amylases.

α-Amylases are among the most widely used classes of enzymes in industry and considerable effort has gone into optimising their activities. Efforts to find better amylase mutants, such as through high-throughput screening, would be greatly aided by access to precise and robust active site titrating agents for quantitation of active mutants in crude cell lysates. While active site titration reagents designed for retaining ß-glycosidases quantify these enzymes down to nanomolar levels, convenient titrants for α-glycosidases are not available. We designed such a reagent by incorporating a highly reactive fluorogenic leaving group onto unsaturated cyclitol ethers, which have been recently shown to act as slow substrates for retaining glycosidases that operate via a covalent 'glycosyl'-enzyme intermediate. By appending this warhead onto the appropriate oligosaccharide, we developed efficient active site titration reagents for α-amylases that effect quantitation down to low nanomolar levels.

New insights into lipopolysaccharide assembly and export.

Lipopolysaccharide is an important immunomodulatory and structural component found in the outer membrane of Gram-negative bacteria. The biosynthesis of this glycoconjugate proceeds by a highly conserved pathway and, as such, is an attractive target for antibiotic action. We highlight here recent work focused on understanding this pathway with an emphasis on new insights related to chain length control and transport across the inner and outer cell membranes.

Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification.

A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-d-Galp-(1→3)-ß-d-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat-unit structure to the nonreducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-d-Galp-(1→3)-ß-d-Galp-(1→] or O2c [→3)-ß-d-GlcpNAc-(1→5)-ß-d-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1 antigen polymerization and forming a complex with the O2a glycosyltransferase WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp-dependent GT-A galactosyltransferase adding ß-(1→3)-linked d-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)-linked d-Galp These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc transferase and noted that WbmW represents a UDP-Galf-dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf. Our results provide mechanistic and conceptual insights into an important strategy for polysaccharide antigen diversification in bacteria.

A Route to Polyprenol Pyrophosphate-Based Probes of O-Polysaccharide Biosynthesis in Klebsiella pneumoniae O2a.

An approach for the assembly of polyprenol pyrophosphate-based probes of O-polysaccharide biosynthesis in Klebsiella pneumoniae serotype O2a is described. This convergent route features high-yielding, diastereoselective glycosylations and the late-stage installation of the polyprenol pyrophosphate moiety. Although applied to the synthesis of a nonasaccharide bearing a farnesyl group (1), the modular nature of the route makes it amenable to the synthesis of additional derivatives containing either larger glycans or different lipid domains.

The LPG1x family from Leishmania major is constituted of rare eukaryotic galactofuranosyltransferases with unprecedented catalytic properties.

Galactofuranosyltransferases are poorly described enzymes despite their crucial role in the virulence and the pathogenicity of numerous microorganisms. These enzymes are considered as potential targets for therapeutic action. In addition to the only well-characterised prokaryotic GlfT2 from Mycobacterium tuberculosis, four putative genes in Leishmania major were previously described as potential galactofuranosyltransferases. In this study, we have cloned, over-expressed, purified and fully determined the kinetic parameters of these four eukaryotic enzymes, thus demonstrating their unique potency in catalysing the transfer of the galactofuranosyl moiety into acceptors. Their individual promiscuity revealed to be different, as some of them could efficiently use NDP-pyranoses as donor substrates in addition to the natural UDP-galactofuranose. Such results pave the way for the development of chemoenzymatic synthesis of furanosyl-containing glycoconjugates as well as the design of improved drugs against leishmaniasis.

Rearranged T Cell Receptor Sequences in the Germline Genome of Channel Catfish Are Preferentially Expressed in Response to Infection.

Rearranged V(D)J genes coding for T cell receptor α and ß chains are integrated into the germline genome of channel catfish. Previous analysis of expressed TCR Vß2 repertoires demonstrated that channel catfish express multiple public clonotypes, which were shared among all the fish, following infection with a common protozoan parasite. In each case a single DNA sequence was predominately used to code for a public clonotype. We show here that the rearranged VDJ genes coding for these expressed public Vß2 clonotypes can be amplified by PCR from germline DNA isolated from oocytes and erythrocytes. Sequencing of the Vß2 PCR products confirmed that these expressed public Vß2 clonotypes are integrated into the germline. Moreover, sequencing of PCR products confirmed that all five Vß gene families and Vα1 have rearranged V(D)J genes with diverse CDR3 sequences integrated into the germline. Germline rearranged Vß2 and Vß4 genes retain the intron between the leader and Vß sequence. This suggests that the germline rearranged TCR Vß genes arose through VDJ rearrangement in T cells, and subsequently moved into the germline through DNA transposon mediated transposition. These results reveal a new dimension to the adaptive immune system of vertebrates, namely: the expression of evolutionarily conserved, rearranged V(D)J genes from the germline.

Synthesis of the Campylobacter jejuni 81-176 Strain Capsular Polysaccharide Repeating Unit Reveals the Absolute Configuration of its O-Methyl Phosphoramidate Motif.

The O-methyl phosphoramidate (MeOPN) motif is a non-stoichiometric modification of capsular polysaccharides (CPS) in ≈70 % of all Campylobacter jejuni strains. Infections by C. jejuni lead to food-borne illnesses and the CPS they produce are key virulence factors. The MeOPN phosphorus atom in these CPS is stereogenic and is found as a single stereoisomer. However, to date, the absolute stereochemistry at this atom has been undefined. We report the synthesis of the three repeating units found in C. jejuni 81-176 CPS; one of these possesses a MeOPN group. In the course of these studies we established that the stereochemistry of the phosphorus atom in this MeOPN group is R. These studies represent the first unequivocal proof of stereochemistry of this group in any C. jejuni CPS. The compounds produced are anticipated to be useful tools in investigations targeting the function and biosynthesis of this structurally-interesting modification, which so far has only been identified in campylobacter.

Stereocontrolled Synthesis of 2-Deoxy-galactopyranosides via Isopropylidene-Protected 6- O-Silylated Donors.

The stereocontrolled synthesis of 2-deoxy-d- arabino-hexopyranosides ("galactopyranosides") using 3,4- O-isopropylidene-6- O- tert-butyldiphenylsilyl-protected glycosyl donors is reported. 2-Deoxy-thioglycoside 3e gives excellent α-selectivity, while galactal 9 leads to, in a two-step protocol, 2-deoxy-ß-glycosides in high stereoselectivity. The selectivity of both reagents is believed to arise from the combination of the isopropylidene acetal spanning O-3 and O-4 together with the sterically demanding silyl group on O-6. The utility of the method was demonstrated through the synthesis of a trisaccharide that contains both 2-deoxy α- and ß-d-galactopyranosyl residues.