Phillip M. Rendle

Synthesis and utility of sulfated chromogenic carbohydrate model substrates for measuring activities of mucin-desulfating enzymes.

A chromogenic substrate, 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate was synthesized and used in combination with beta-N-acetylhexosaminidase for detection of the sulfatase, MdsA, by release of 4-nitrophenol. MdsA was originally isolated from the bacterium Prevotella strain RS2 and is believed to be involved in desulfation of sulfomucins, major components of the mucus barrier protecting the human colon surface. The exo nature of the MdsA sulfatase was indicated by its inability to de-esterify the disaccharide 4-nitrophenyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate. This latter compound was prepared from monosaccharide precursors by two different methods, the shorter requiring just six steps from 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and giving an overall yield of 26.4%. The syntheses of 4-nitrophenyl beta-D-galactopyranoside 3-triethylammonium sulfate and 6-triethylammonium sulfate and their use in combination with beta-galactosidase as chromogenic substrates for detecting Bacteroides fragilis sulfatases with different specificities was also demonstrated.

Mannosylated saponins based on oleanolic and glycyrrhizic acids. Towards synthetic colloidal antigen delivery systems.

Immunostimulatory saponin based colloidal antigen delivery systems show promise as adjuvants for subunit vaccines. For this reason, allyl oleanolate was glycosylated at the 3-position using trichloroacetimidate donors to give monodesmodic saponins following deprotection. Bisdesmodic saponins were synthesized by double glycosylation at the 3- and 28-positions of oleanolic acid. When formulated together with cholesterol and phospholipids, ring-like, helical and rod-like nanostructures were formed depending on the saponin concentrations used. As an indication of adjuvant activity, the ability of these formulations, and the saponins by themselves, to induce dendritic cell maturation was measured, but no significant activity was observed.

Glucofuranosylation with penta-O-propanoyl-β-d-glucofuranose

Abstract Readily available, crystalline penta- O -propanoyl-β- d -glucofuranose is shown to be a suitable glycosylating agent for the acid-catalysed, direct synthesis of O -, S - and N -glucofuranosyl compounds. β-Linked products are formed with good selectivity. Reaction with cyanotrimethylsilane gave the 1,2- O -(1-cyanopropylidene)acetal rather than the C -glycosyl cyanide. By selective acid-catalysed hydrolysis, the title compound was converted to the 1-hydroxy analogue from which the trichloroacetimidates were made as further potential glycosylating agents.

Recombinant, truncated B. circulans keratanase-II: Description and characterisation of a novel enzyme for use in measuring urinary keratan sulphate levels via LC-MS/MS in Morquio A syndrome.

OBJECTIVE Morquio A syndrome (mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficient N-acetylgalactosamine-6-sulphatase (GALNS) activity. Early and accurate diagnosis of this condition is critical for improved patient outcomes, particularly as enzyme replacement therapy has recently become available. An LC-MS/MS assay utilising keratan sulphate (KS) disaccharides derived from keratanase-II digestion provides a sensitive and specific means for quantitation of urinary KS, a screening biomarker for Morquio A (Oguma et al., 2007; Martell et al., 2011). To ensure a reliable supply of keratanase-II, we sought to produce a Bacillus circulans-derived enzyme via a recombinant approach in Escherichia coli. DESIGN AND METHODS Bioinformatics analysis of the B. circulans keratanase-II enzyme identified likely dispensable C-terminal domains amenable to enhancement via protein engineering. A truncated form of the enzyme was designed to remove the domains predicted to be unnecessary for catalytic activity and detrimental to recombinant expression in E. coli. RESULTS C-terminally truncated, recombinant B. circulans keratanase-II was purified to >98% homogeneity and extensively characterised, demonstrating desired activity, specificity and utility in LC-MS-based quantitation of urinary KS from Morquio A and control samples, and is functionally indistinguishable from full-length, native B. circulans-derived keratanase-II. CONCLUSIONS This novel, recombinant keratanase-II meets all performance requirements and can be produced in a rapid and reproducible manner. We speculate that other related bacterial enzymes of biomedical or industrial interest may be amenable to similar engineered enhancements.

Synthesis, Formulation, and Adjuvanticity of Monodesmosidic Saponins with Olenanolic Acid, Hederagenin and Gypsogenin Aglycones, and some C-28 Ester Derivatives†

Abstract In an attempt to discover a new synthetic vaccine adjuvant, the glycosylation of hederagenin, gypsogenin, and oleanolic acid acceptors with di‐ and trisaccharide donors to generate a range of mimics of natural product QS‐21 was carried out. The saponins were formulated with phosphatidylcholine and cholesterol, and the structures analyzed by transmission electron microscopy. 3‐O‐(Manp(1→3)Glcp)hederagenin was found to produce numerous ring‐like micelles when formulated, while C‐28 choline ester derivatives preferred self‐assembly and did not interact with the liposomes. When alone and in the presence of cholesterol and phospholipid, the choline ester derivatives produced nanocrystalline rods or helical micelles. The effects of modifying sugar stereochemistry and the aglycone on the immunostimulatory effects of the saponins was then evaluated using the activation markers MHC class II and CD86 in murine bone marrow dendritic cells. The most active saponin, 3‐O‐(Manp(1→3)Glcp)hederagenin, was stimulatory at high concentrations in cell culture, but this did not translate to strong responses in vivo.

Poly Ethoxy Ethyl Glycinamide (PEE‐G) Dendrimers: Dendrimers Specifically Designed for Pharmaceutical Applications

Poly ethoxy ethyl glycinamide (PEE‐G) dendrimers have been specifically designed and synthesized with the aim of providing a readily available dendrimer scaffold that can be used to make products that can meet the stringent requirements of pharmaceutical applications. The synthesis has been refined to produce dendrimers that are of high HPLC purity. The suitability of PEE‐G dendrimers for their designed use has been verified by subsequent measurements to demonstrate that they are of high stability, high aqueous solubility, low cytotoxicity, low immunogenicity and with low in vivo toxicity in an escalating‐dose rat study. PEE‐G dendrimers therefore provide a useful scaffold for researchers wanting to develop dendrimer‐based drug candidates.

The influence of boric acid on the acetylation of aldoses: ‘one-pot’ syntheses of penta-O-acetyl-β-D-glucofuranose and its crystalline propanoyl analogue

When glucose and boric acid are heated in acetic acid a soluble compound forms from which, with acetic anhydride and catalytic amounts of sulfuric acid, a mixture consisting of >90% of the glucofuranose per-acetates (α∶β ratio 1∶1.8) is obtained in high yield. In the absence of the sulfuric acid partial acetylation takes place and penta-O-acetyl-β-D-glucofuranose (α∶β ratio 1∶52) is obtainable in good yield by removal of boric acid and completion of the esterification by addition of acetic anhydride and pyridine. A new, crystalline glucofuranose, penta-O-propanoyl-β-D-glucofuranose, is obtained in 58% yield in a ‘one-pot’ procedure using boric acid. The effects of boric acid on the acid-catalysed acetylation of other aldo-hexoses and -pentoses suggest that the synthesis of furanosyl per-esters could be successful with xylose and idose as well as with glucose. p

Synthesis and biological activities of d-chiro-inositol analogues with insulin-like actions.

d-chiro-inositol (DCI, 1) evokes therapeutic actions in diabetes and insulin resistance but has sub-optimal pharmacokinetic profiles. To investigate what positions on the DCI cyclohexanol ring may be amenable to modification to improve pharmaceutical formulations, a series of analogues based on DCI were synthesised. These compounds were then evaluated for their ability to stimulate glucose transport using 3T3-L1 adipocytes as a model system. Positional analyses indicate that the hydroxyl group at position 1 is not essential for activity and can be modified without affecting glucose uptake. Removal of the hydroxyl at position 3 also had minimal effect on activity but this group is sensitive to modification. By comparison, the oxygen at position 2 is crucial to the potency of DCI, although this group can withstand modification without fundamentally affecting activity. These data reveal that positions 1 and 2 on the cyclohexanol ring of DCI offer further scope for modification to develop DCI analogues with desirable pharmacokinetic profiles for the potential treatment of metabolic disease.