J. Grant Buchanan

Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis

The incorporation of the specialized carbohydrate affinity ligand methacrylamido phenylboronic acid in polyacrylamide gels for SDS‐PAGE analysis has been successful for the separation of carbohydrates and has here been adapted for the analysis of post‐translationally modified proteins. While conventional SDS‐PAGE analysis cannot distinguish between glycated and unglycated proteins, methacrylamido phenylboronate acrylamide gel electrophoresis (mP‐AGE) in low loading shows dramatic retention of δ‐gluconolactone modified proteins, while the mobility of the unmodified proteins remains unchanged. With gels containing 1% methacrylamido phenylboronate, mP‐AGE analysis of gluconoylated recombinant protein Sbi results in the retention of the modified protein at a position expected for a protein that has quadrupled its expected molecular size. Subsequently, mP‐AGE was tested on HSA, a protein that is known to undergo glycation under physiological conditions. mP‐AGE could distinguish between various carbohydrate‐protein adducts, using in vitro glycated HSA, and discriminate early from late glycation states of the protein. Enzymatically glycosylated proteins show no altered retention in the phenylboronate‐incorporated gels, rendering this method highly selective for glycated proteins. We reveal that a trident interaction between phenylboronate and the Amadori cis 1,2 diol and amine group provides the molecular basis of this specificity. These results epitomize mP‐AGE as an important new proteomics tool for the detection, separation, visualization and identification of protein glycation. This method will aid the design of inhibitors of unwanted carbohydrate modifications in recombinant protein production, ageing, diabetes, cardiovascular diseases and Alzheimers disease.

Two epimerisations in the formation of oxetanes from l-rhamnose: towards oxetane-containing peptidomimetics

Abstract The methyl group in ( R )-3,5- O -benzylidene- l - rhamnono -1,4-lactone 2 , prepared from l -rhamnose 1 in 41% yield without need for chromatography, is axial and provides a good example of Mills’ rule that the ‘O-inside’ conformations are, in general, more stable than alternative ‘H-inside’ conformations. The lactone 2 may be converted in two steps in an overall 57% yield to the rhamnono -oxetane 3 , which should be useful in generating oxetane dipeptide isosteres and oxetane β-amino acids and in determining the value of the oxetane ring in inducing secondary structure in small peptidomimetics. Methyl 2,4-anhydro-( S )-3,5- O -benzylidene- l -rhamnonate 11 , in which both the phenyl and methyl groups are equatorial, is slightly more thermodynamically stable than 3 , providing a rare exception to Mills’ rule. X-ray crystal structures of 2 and 11 are reported.

Dyotropic rearrangement of alpha-lactone to beta-lactone: a computational study of small-ring halolactonisation

Transition structures have been optimised using the B3LYP/6-31+G* density functional level method, in vacuum and in implicit (PCM) and explicit (DFT/MM) aqueous solvation, for the degenerate rearrangement of the alpha-lactone derived by the formal addition of Cl(+) to acrylate anion and for the dyotropic rearrangement of this to the beta-lactone. Despite being lower in energy than the alpha-lactone, there is no direct pathway to the beta-lactone from the acrylate chloronium zwitterion, which is the transition structure for the degenerate rearrangement. This may be rationalised by consideration of the unfavorable angle of attack by the carboxylate nucleophile on the beta-position; attack on the alpha-position involves a less unfavorable angle. Formation of the beta-lactone may occur by means of a dyotropic rearrangement of the alpha-lactone. This involves a high energy barrier for the acrylate derived alpha-lactone, but dyotropic rearrangement of the beta,beta-dimethyl substituted alpha-lactone to the corresponding beta-lactone involves a much lower barrier, estimated at about 46 kJ mol(-1) in water, and is predicted to be a facile process.

Confirmation of the D configuration of the 2-substituted arabinitol 1-phosphate residue in the capsular polysaccharide from Streptococcus pneumoniae Type 17F.

The absolute configuration of the 2-substituted arabinitol 1-phosphate residue present in the repeating unit of the capsular polysaccharide (CPS) from Streptococcus pneumoniae Type 17F is confirmed as D, based on a comparison of proton and carbon chemical shifts in a synthetic oligosaccharide and in an oligosaccharide derived from the CPS by degradation.

Evidence for α-lactone intermediates in addition of aqueous bromine to disodium dimethyl-maleate and -fumarate

Crystallographic analysis of the bromo-β-lactones nobtained by addition of bromine to aqueous solutions of disodium n2,3-dimethylmaleate and 2,3-dimethylfumarate reveals stereochemistries nopposite to those originally assigned and suggests that the first-formed nintermediate in each case is an α-lactone.

The Walden cycle revisited: a computational study of competitive ring closure to α- and β-lactones

The text-book Walden cycle which interconverts the stereochemical configurations of chlorosuccinic and malic acids involves a β-lactone intermediate in preference to an α-lactone intermediate because the Onuc C Cl angle in the transition structure for the former (174°) is more favourable than that for the latter (139°), as determined by PCM(e = 78.4)/B3LYP/6-31+G* calculations; the smaller ring-strain energy of the β-lactone contributes little to the reactivity difference.

Carbohydrate Chemistry and Biochemistry, by Michael L. Sinnott

Carbohydrate Chemistry and Biochemistry represents a significant, single-author, synthesis of knowledge weighing in at 748 pages and containing only seven chapters. The author, Professor Michael Sinnott, has contributed many classic research papers in the field of the carbohydrate reactivity pertaining to the details of glycosyl hydrolase mechanism (1), the kinetic characterization of enzyme activity in evolving systems (2) and the preservation of knowledge in its physical forms (3). This text extends his prior wide-ranging reviews of the mechanisms of carbohydrate-active enzymes (4, 5), adopting a more tutorial approach. It represents, in the main, a detailed consideration of the literature till the end of 2006. Chapters 1 and 2 contain a conventional introduction to the structure and conformation of monosaccharides, with mutarotation being dealt with in detail. Chapter 3 focuses on the physical organic chemistry of nucleophilic substitution at the anomeric centre. It presages Chapter 5’s extensive treatment of enzyme-catalyzed glycosyl transfer. In between, Chapter 4 deals with primary structures and conformations of polysaccharides and includes a discussion of physical methods, including X-ray crystallography. Chapter 6 is a necessary collection of material, covering both synthetic and biologically-relevant heterolytic chemistry not involving the anomeric centre; in some topics (e.g. hydride transfer from NAD(P)H) discussed in this chapter, carbohydrates play only a very minor role. Chapter 7 deals with reactions involving homolytic chemistry. As noted in the author’s preface, this book was originally intended as a contribution to an RSC-sponsored series of volumes on physical organic chemistry but the untimely death of the series editor, Professor Andrew Williams, resulted in its publication as a stand-alone volume. This provenance has produced a book which extensively draws on the approaches of physical organic chemistry, applying them to the study of carbohydrate-active enzymecatalyzed reactions. The enzyme-catalyzed chemistry of the anomeric centre represents the author’s particular area of expertise, and Chapter 5 contains extensive, personal, valuable commentary on numerous studies in this area. The author provides a detailed, tutorial overview of the area, and so the systems chosen, though extensive, are not necessarily comprehensive. The historical overview of the enzymology of glycosyl hydrolases provided by this chapter is particulary interesting, highlighting several significant and perhaps under-appreciated papers. Sinnott writes with a fluid style throughout, and where he disagrees with the conclusions drawn by authors of the primary research papers, he makes clear why he does so. This encourages the reader to question the conclusions that can reasonably be drawn from experimental data and which experiments may be used to choose amongst competing mechanisms. The general approach adopted towards all enzyme-catalyzed glycosyl group transfers that occur with retention of chemistry should be readily approachable to those involved in the structural elucidation of stable enzyme-intermediate complexes. It assumes that if an