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Free fructose is conformationally locked.
Fructose has been examined under isolation conditions using a combination of UV ultrafast laser vaporization and Fourier-transform microwave (FT-MW) spectroscopy. The rotational spectra for the parent, all (six) monosubstituted (13)C species, and two single D species reveal unambiguously that the free hexoketose is conformationally locked in a single dominant β-pyranose structure. This six-membered-chair skeleton adopts a (2)C(5) configuration (equivalent to (1)C(4) in aldoses). The free-molecule structure sharply contrasts with the furanose form observed in biochemically relevant polysaccharides, like sucrose. The structure of free fructose has been determined experimentally using substitution and effective structures. The enhanced stability of the observed conformation is primarily attributed to a cooperative network of five intramolecular O-H···O hydrogen bonds and stabilization of both endo and exo anomeric effects. Breaking a single intramolecular hydrogen bond destabilizes the free molecule by more than 10 kJ mol(-1). The structural results are compared to ribose, recently examined with rotational resolution, where six different conformations coexist with similar conformational energies. In addition, several DFT and ab initio methods and basis sets are benchmarked with the experimental data.
Probing the limits of Q-tag bioconjugation of antibodies.
Site-selective labelling of antibodies (Abs) can circumvent problems from heterogeneity of conventional conjugation. Here, we evaluate the industrially-applied chemoenzymatic 'Q-tag' strategy based on transglutaminase-mediated (TGase) amide-bond formation in the generation of 89Zr-radiolabelled antibody conjugates. We show that, despite previously suggested high regioselectivity of TGases, in the anti-Her2 Ab Herceptin™ more precise native MS indicates only 70-80% functionalization at the target site (Q298H), in competition with modification at other sites, such as Q3H critically close to the CDR1 region.
Monitoring the Disassembly of Virus-like Particles by 19F-NMR.
Virus-like particles (VLPs) are stable protein cages derived from virus coats. They have been used extensively as biomolecular platforms, e.g., nanocarriers or vaccines, but a convenient in situ technique is lacking for tracking functional status. Here, we present a simple way to monitor disassembly of 19F-labeled VLPs derived from bacteriophage Qβ by 19F NMR. Analysis of resonances, under a range of conditions, allowed determination not only of the particle as fully assembled but also as disassembled, as well as detection of a degraded state upon digestion by cells. This in turn allowed mutational redesign of disassembly and testing in both bacterial and mammalian systems as a strategy for the creation of putative, targeted-VLP delivery systems.
Expanding the utility of proteases in synthesis: Broadening the substrate acceptance in non-coded amide bond formation using chemically modified mutants of subtilisin
The strategy of combined site directed mutagenesis and chemical modification creates chemically modified mutants (CMMs) with greatly broadened substrate specificities. We have previously reported that the CMMs of subtilisin Bacillus lentus (SBL) are efficient catalysts for the coupling of both L- and D-amino acids. We now report that these powerful catalysts also allow amide bond formation between a variety of non-coded carboxylic acids, including β-alanine and β-amino homologues of phenylalanine, with both L- and D-amino acid nucleophiles. As a guide to enzyme efficiency, a hydrolysis assay indicating pH change has been employed. CMMs selected by this screen furnished higher yields of coupling products compared to the wild-type enzyme (WT). Furthermore, both WT and CMM enzymes allow highly stereoselective aminolysis of a meso diester with an amino acid amine. These results highlight the utility of CMMs in the efficient formation of non-coded amides as potential peptide isosteres. © 2001 Published by Elsevier Science Ltd.
Carbohydrate-derived amino-alcohol ligands for asymmetric alkynylation of aldehydes.
[reaction: see text] Conformationally restricted amino alcohols based on carbohydrate scaffolds provide flexible and fine-tuneable libraries that greatly expand the range of ligands available in the Zn(OTf)(2)-mediated addition of alkynes to aldehydes, in some cases with very high stereoselectivities.
Glyco- and peptidomimetics from three-component Joullié-Ugi coupling show selective antiviral activity.
Chlorination-elimination chemistry coupled with three-component Joullié-Ugi reaction and facile deprotection allowed efficient access to an array of polyhydroxylated pyrrolidines through parallel synthesis that may be considered to be a library of imino (aza) sugars (glycomimetics) and/or dihydroxyprolyl peptides (peptidomimetics). The utility of generating such a library was illustrated by screening against 15 different targets that revealed potent and selective inhibition of the Gaucher's disease glycosyltransferase enzyme glucosylceramide synthase and of primary pathogen model for human hepatitis C virus (HCV) and bovine diarrhoeal virus (BVDV). An observed selectivity for this HCV model over hepatitis B virus and remarkably low toxicity suggest a novel mode of action.
Influence of preparation procedure on polymer composition: Synthesis and characterisation of polymethacrylates bearing β-D-glucopyranoside and β-D-galactopyranoside residues
Methacrylate derivatives bearing β-D-glucopyranoside and β-D-galactopyranoside residues are synthesised by glycosylation of 2-hydroxyethyl methacrylate (HEMA) with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide and 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide, respectively. β-Selectivity in the glycosylation reactions is ensured by neighbouring-group participation of acetyl groups at O-2 in the glycosyl donors. 2-(2′,3′,4′,6′-tetra-O-acetyl-β-D-glucosyloxy) ethyl methacrylate (AcGlcEMA, 1a) was obtained as a crystalline solid and its crystal structure was determined by single-crystal X-ray diffraction. Deprotected polymers are synthesised in two parallel ways; either polymerisation of the protected monomers and subsequent deacetylation of the resulting polymers, or polymerisation of the previously deprotected monomers. The number- and weight-average relative molecular masses of both the protected and deprotected polymers are determined by size exclusion chromatography (SEC). Absolute molecular masses are obtained using the previously estimated refractive-index increments, dn/dc. It is found that polymerisation of deprotected monomers leads to polymers of well-defined composition, in contrast to the deacetylation of protected polymers.
A convenient catalyst for aqueous and protein Suzuki-Miyaura cross-coupling.
A phosphine-free palladium catalyst for aqueous Suzuki-Miyaura cross-coupling is presented. The catalyst is active enough to mediate hindered, ortho-substituted biaryl couplings but mild enough for use on peptides and proteins. The Suzuki-Miyaura couplings on protein substrates are the first to proceed in useful conversions. Notably, hydrophobic aryl and vinyl groups can be transferred to the protein surface without the aid of organic solvent since the aryl- and vinylboronic acids used in the coupling are water-soluble as borate salts. The convenience and activity of this catalyst prompts use in both general synthesis and bioconjugation.
L-(+)-swainsonine and other pyrrolidine inhibitors of naringinase: Through an enzymic looking glass from D-mannosidase to L-rhamnosidase?
The synthesis and inhibitory properties towards naringinase (L-rhamnosidase) of L-(+)-swainsonine and of a number of more highly oxygenated analogues, and of some monocyclic equivalents, are reported. L-(+)-swainsonine and 1,4,6-trideoxy-1,4-imino-L-mannitol are powerful and specific inhibitors of naringinase.
Inhibition of naringinase (L-rhamnosidase) by piperidine analogues of L-rhamnose: Scaffolds for libraries incorporating trihydroxypipecolic acids
L-Deoxyrhamnojirimycin 1 does not inhibit naringinase significantly but 5-epi-L-deoxyrhamnojirimycin 2 is a potent inhibitor. Conversely, α-C-glycosides of 1 are good inhibitors of L-rhamnosidase whereas those of 2 are not. Intermediate azabicyclic lactones are likely to be of use for the incorporation of a number of trihydrocypipecolic acids into peptide libraries.
Enhanced aqueous Suzuki-Miyaura coupling allows site-specific polypeptide 18F-labeling.
The excesses of reagents used in protein chemistry are often incompatible with the reduced or even inverse stoichiometries used for efficient radiolabeling. Analysis and screening of aqueous Pd(0) ligand systems has revealed the importance of a guanidine core and the discovery of 1,1-dimethylguanidine as an enhanced ligand for aqueous Suzuki-Miyaura cross-coupling. This novel Pd catalyst system has now allowed the labeling of small molecules, peptides, and proteins with the fluorine-18 prosthetic [(18)F]4-fluorophenylboronic acid. These findings now enable site-specific protein (18)F-labeling under biologically compatible conditions using a metal-triggered reaction.
Towards an unprotected self-activating glycosyl donor system: Bromobutyl glycosides
Bromobutyl mannopyranosides have been successfully used as both protected and unprotected glycosyl donors both with and without the use of an external activator.
Ready display of antigenic peptides in a protein 'mimogen'.
Given the dependence of much modern biology upon the use of antibodies as tools and reagents, their variability and the often associated lack-of-detail about function and identity creates experimental errors. Here we describe the proof-of-principle for a potentially general, versatile method for the display of antigens in a soluble yet standard format on a lateral protein scaffold that mimics normal epitopes in a protein antigen (a 'mimogen') and confirm their utility in phosphorylation-dependent recognition by specific antibodies.
Control of phosphoryl migratory transesterifications allows regioselecive access to sugar phosphates.
Phosphate esters in polyhydroxylated systems are normally blighted by uncontrolled migration under a variety of reaction conditions. Cesium fluoride is demonstrated as a reagent to control migration of primary phosphates during transesterifications. This allows easy exchange of phosphoryl protecting groups enabling enhanced synthetic strategic flexibility and regioselective phosphate installation. Mechanistic analysis suggests that a fluoride-induced extended solvent sphere modulates steric bulk at phosphorus to favor the primary position.
Palladium-mediated cell-surface labeling.
Benign C-C bond formation at various sites in cell-surface channels has been achieved through Suzuki-Miyaura coupling of genetically positioned unnatural amino acids containing aryl halide side chains. This enabled site-selective cell surface manipulation of Escherichia coli ; the phosphine-free catalyst caused no cell death at required Pd loadings, suggesting future in vivo application of catalytic metal-mediated bond formation in more complex organisms.
Site-selective chemical protein glycosylation protects from autolysis and proteolytic degradation.
Glycosylation is often cited as having a stabilizing effect upon proteins with respect to proteolysis, thermolysis and other forms of degradation. We present here a model study on an autolytic protease that has been chemically glycosylated to produce single glycoforms. The resulting glycosylated enzymes are more stable with respect to their own autolytic degradation and that by other proteases. Kinetic parameters for protease activity with respect to the degradation of small-molecule amide substrate reveal no significant change in inherent activity thereby suggesting that reduced autolysis and proteolysis are a consequence of stabilization, perhaps by steric blockade of cleavage points or alteration of local unfolding kinetics. Variation in glycan identity suggests that greater glycan size leads to greater stabilization.
Hydrogen bonding and cooperativity in isolated and hydrated sugars: mannose, galactose, glucose, and lactose.
The conformation of phenyl-substituted monosaccharides (mannose, galactose, and glucose) and their singly hydrated complexes has been investigated in the gas phase by means of a combination of mass selected, conformer specific ultraviolet and infrared double resonance hole burning spectroscopy experiments, and ab initio quantum chemistry calculations. In each case, the water molecule inserts into the carbohydrate at a position where it can replace a weak intramolecular interaction by two stronger intermolecular hydrogen bonds. The insertion can produce significant changes in the conformational preferences of the carbohydrates, and there is a clear preference for structures where cooperative effects enhance the stability of the monosaccharide conformers to which the water molecule chooses to bind. The conclusions drawn from the study of monosaccharide-water complexes are extended to the disaccharide lactose and discussed in the light of the underlying mechanisms that may be involved in the binding of carbohydrate assemblies to proteins and the involvement, or not, of key structural water molecules.