Caiping Li

Preparation and anticoagulant activity of the phosphosulfomannan PI-88

A yeast-derived phosphomannan mixture was chemically sulfonated and the composition and structure of the product mixture was studied. This phosphosulfomannan mixture, PI-88, is currently under clinical evaluation as an anti-cancer agent. Analysis using capillary electrophoresis demonstrated that PI-88 was a multi-component mixture. Gel permeation chromatography provided four fractions of PI-88 that contained components which differed in size from disaccharide to hexasaccharide, and by degree of sulfation. These fractions were characterised by spectroscopic and chromatographic methods and the structure of PI-88 is that expected based on the structure of the phosphomannan starting material. The anticoagulant activity of these fractions was evaluated and the structural requirements for activity are described.

Large-scale preparation of the oligosaccharide phosphate fraction of Pichia holstii NRRL Y-2448 phosphomannan for use in the manufacture of PI-88

Mild acid-catalysed hydrolysis of the extracellular phosphomannan of the yeast Pichia holstii NRRL Y-2448 produces a high-molecular-weight phosphomannan core, a low-molecular-weight oligosaccharide phosphate fraction, and a neutral oligosaccharide fraction. A method was developed for the large-scale preparation of the oligosaccharide phosphate fraction, consisting predominantly of the pentasaccharide phosphate, 6-O-PO3H2-alpha-D-Man-(1 --> 3)-alpha-D-Man-(1 --> 3)-alpha-D-Man-(1 C 3)-alpha-D-Man-(1 --> 2)-D-Man, for use in the manufacture of the promising new anti-cancer agent, PI-88. Further insights were also gained into the structure of the phosphomannan by HPLC analysis of the time course of the hydrolysis reaction.

Determination of the composition of the oligosaccharide phosphate fraction of Pichia (Hansenula) holstii NRRL Y-2448 phosphomannan by capillary electrophoresis and HPLC

The promising new anticancer agent, PI-88, is prepared by the sulfonation of the oligosaccharide phosphate fraction of the extracellular phosphomannan produced by the yeast Pichia (Hansenula) holstii NRRL Y-2448. The composition of the oligosaccharide phosphate fraction was determined by capillary electrophoresis (CE) with indirect UV detection using 6 mM potassium sorbate at pH 10.3 as the background electrolyte. Further confirmation of the composition was obtained by HPLC analysis of a sample dephosphorylated by treatment with alkaline phosphatase. The structure of the hexasaccharide component has been determined by isolation and NMR spectroscopic analysis of its dephosphorylated derivative. Additionally, the structure of a second, previously undetected tetrasaccharide component (a hexosamine) has been determined by isolation and NMR spectroscopic analysis of the acetate of its dephosphorylated derivative. It is demonstrated that CE is an ideal method for the quality control of the oligosaccharide phosphate fraction for use in the production of PI-88.

Synthesis of a heparan sulfate mimetic library targeting FGF and VEGF via click chemistry on a monosaccharide template

A disulfated methyl 6‐azido‐6‐deoxy‐α‐D‐mannopyranoside template was used as a core structure for binding to the angiogenic growth factors FGF‐1, FGF‐2, and VEGF. The core structure was diversified in a rapid, parallel manner by employing the CuI‐catalyzed Huisgen azide–alkyne cycloaddition (“click”) reaction. The diversity was further extended by incorporating a Swern oxidation–Wittig reaction sequence on a click adduct of propargyl alcohol. Thus, the sulfated core was linked by various spacers to selected hydrophobic or polar motifs, which were designed to probe the protein surface surrounding the cationic heparan sulfate binding sites of the growth factors in order to improve affinity and selectivity. The affinities of the compounds for the growth factors were measured by surface plasmon resonance solution affinity assays. A lead compound was identified with micromolar binding affinity toward both FGF‐1 and VEGF (Kd=84 and 49 μM, respectively) and good selectivity over FGF‐2 (29‐ and 51‐fold, respectively).

A focused sulfated glycoconjugate Ugi library for probing heparan sulfate-binding angiogenic growth factors.

A library of small molecule heparan sulfate (HS) mimetics was synthesized by employing the Ugi four-component condensation of d-mannopyranoside-derived isocyanides with formaldehyde as the carbonyl component and a selection of carboxylic acids and amines, followed by sulfonation. The library was used to probe the subtle differences surrounding the ionic binding sites of three HS-binding angiogenic growth factors (FGF-1, FGF-2 and VEGF). Each compound features 3 or 4 sulfo groups which serve to anchor the ligand to the HS-binding site of the protein, with a diverse array of functionality in place extending from C-1 or C-6 to probe for adjacent favorable binding interactions. Selectivity of binding to these proteins was clearly observed and supported by molecular docking calculations.