Carbohydrate Research 337 (2002) 139 – 146 www.elsevier.com/locate/carres Determination of the composition of the oligosaccharide phosphate fraction of Pichia (Hansenula ) holstii NRRL Y-2448 phosphomannan by capillary electrophoresis and HPLC Vito Ferro, a, * Caiping Li, a Kym Fewings, a Maria C. Palermo, a Robert J. Linhardt, b Toshihiko Toida c a Department of Research & Deelopment, Progen Industries Ltd, PO Box 28, Richlands BC, Qld 4077, Australia b Department of Chemistry, Diision of Medicinal and Natural Products Chemistry, Department of Chemical and Biochemical Engineering, Uniersity of Iowa, Iowa City, IA 52242, USA c School of Pharmaceutical Sciences, Chiba Uniersity, 1 -33 Yayoi, Inage, Chiba 263 -8522, Japan Received 3 August 2001; accepted 6 November 2001 Abstract 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. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Pichia (Hansenula ) holstii ; Phosphomannan; Oligosaccharide phosphate fraction; Capillary electrophoresis; PI-88 1. Introduction The novel sulfated oligosaccharide agent known as PI-88 has recently been identified as a promising in- hibitor of tumour growth and metastasis 1 and is cur- rently undergoing clinical evaluation in cancer patients. PI-88 is an inhibitor of angiogenesis by virtue of its ability to block heparan sulfate binding of angiogene- sis-inducing growth factors. PI-88 also blocks metasta- sis by inhibiting heparanase, 2 a key enzyme involved in the degradation of the extracellular matrix surrounding tumour cells, thus preventing their spread to other sites via entry into blood vessels and lymphatics. In addition to its anticancer activities, PI-88 shows promise as a potential anticoagulant/antithrombotic agent with a novel mode of action. 3–5 It is a specific ligand for heparin cofactor II, enhancing its ability to inhibit thrombin (factor IIa). However, it does not interact with antithrombin-III and thus shows no anti-Xa or AT-III mediated ant-IIa activity. PI-88 is prepared 6 by the sulfonation of the oligosac- charide phosphate fraction (OPF) of the extracellular phosphomannan produced by the yeast Pichia (Hansenula ) holstii NRRL Y-2448. 7,8 Early structural studies of the OPF found that the principal component is the pentasaccharide phosphate 1 9 and that smaller amounts of tetrasaccharide 9,10 and hexasaccharide 10 were also present. More recently, NMR and mass spectral studies have demonstrated that it contains * Corresponding author. Tel.: +61-7-32739100; fax: +61- 7-33751168. E -mail address: vito.ferro@progen.com.au (V. Ferro). 0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII:S0008-6215(01)00300-7