Vanadate and Peroxovanadate Complexes of Biomedical Relevance – A speciation approach with focus on diabetes András Gorzsás Department of Chemistry Inorganic Chemistry Umeå University Umeå, Sweden Akademisk avhandling som med tillstånd av rektorsämbetet vid Umeå Universitet för erhållande av Filosofie Doktorsexamen framlägges till offentlig granskning vid Kemiska institutionen, sal KB3A9, KBC–huset, fredagen den 22 april 2005, kl 13.00. Fakultetsopponent: Professor Carlos F. G. C. Geraldes, Department of Biochemistry, Faculty of Science and Technology, University of Coimbra, P. O. Box 3126, P – 3001 – 401 Coimbra, Portugal i TITLE Vanadate and Peroxovanadate Complexes of Biomedical Relevance – A speciation approach with focus on diabetes AUTHOR András Gorzsás ADDRESS Department of Chemistry, Inorganic Chemistry, Umeå University, SE – 901 87 Umeå, Sweden ABSTRACT Diabetes mellitus is one of the most threatening epidemics of modern times with rapidly increasing incidence. Vanadium and peroxovanadium compounds have been shown to exert insulin–like actions and, in contrast to insulin, are orally applicable. However, problems with side–effects and toxicity remain. The exact mechanism(s) by which these compounds act are not yet fully known. Thus, a better understanding of the aqueous chemistry of vanadates and peroxovanadates in the presence of various (bio)ligands is needed. The present thesis summarises six papers dealing mainly with aqueous speciation in different vanadate – and peroxovanadate – ligand systems of biological and medical relevance. Altogether, five ligands have been studied, including important blood constituents (lactate, citrate and phosphate), a potential drug candidate (picolinic acid), and a dipeptide (alanyl serine) to model the interaction of (peroxo)vanadate in the active site of enzymes. Since all five ligands have been studied both with vanadates and peroxovanadates, the number of systems described in the present work is eleven, including the vanadate – citrate – lactate mixed ligand system. The pH–independent formation constants have been determined for 33 ternary vanadate – ligand, 41 quaternary peroxovanadate – ligand and two vanadate – mixed ligand species in addition to the pKa values of all five ligands. These constants have been used to model physiological conditions, and the biomedical relevance of the different species is discussed. The studies have been performed at 25 ºC in the physiological medium of 0.150 M Na(Cl), i.e. the ionic strength of human blood. No buffers have been used, and wide pH–ranges have usually been covered. The applied experimental techniques comprise mostly 51V NMR and potentiometry, but 31P, 13C, 1H and 14N NMR as well as EPR and ESI–MS have also been used to gain additional information. Multimethod data have been treated by the least–squares program LAKE and modelling has been carried out by the software package WinSGW. Whenever possible, solution structures of the species have been proposed. In addition, simple biological tests have been carried out to determine the stability of the formed peroxovanadate complexes in the presence of human catalase. A brief comparison is given of the different vanadate – ligand and peroxovanadate – ligand systems with emphasis on observed trends and general features. KEYWORDS: vanadium, hydrogen peroxide, lactate, citrate, phosphate, alanyl serine, picolinic acid, speciation, formation constants, equilibrium, solution structure, potentiometry, NMR (51V, 31P, 13C, 1H) ISBN 91–7305–854–8 74 pages and 6 papers ii “et dixit amen dico vobis nisi conversi fueritis et efficiamini sicut parvuli non intrabitis in regnum cælorum” Biblia Sacra Vulgata, Matthæus 18:3 Babának iii Printed by: Arkitektkopia AB, Umeå, Sweden, 2005 ISBN 91–7305–854–8 iv Vanadate and Peroxovanadate Complexes of Biomedical Relevance – A speciation approach with focus on diabetes András Gorzsás Department of Chemistry, Inorganic Chemistry Umeå University SE – 901 87 Umeå SWEDEN This thesis contains a summary and discussion of the following papers, referred to in the text by their roman numbers I–VI. + – I. Speciation in the aqueous H /H2VO4 /H2O2/picolinate system relevant to diabetes research Ingegärd Andersson, András Gorzsás, Lage Pettersson Dalton Transactions, 2004, 421. + – II. A speciation study of the aqueous H /H2VO4 /H2O2/L–α–alanyl–L–serine system. András Gorzsás, Ingegärd Andersson, Hauke Schmidt, Dieter Rehder, Lage Pettersson Dalton Transactions, 2003, 1161. + – III. Speciation in the aqueous H /H2VO4 /H2O2/L–(+)–lactate system András Gorzsás, Ingegärd Andersson, Lage Pettersson Dalton Transactions, 2003, 2503. + – IV. Speciation in the aqueous H /H2VO4 /H2O2/citrate system of biomedical interest András Gorzsás, Kendra Getty, Ingegärd Andersson, Lage Pettersson Dalton Transactions, 2004, 2873. + – V. Speciation in the aqueous H /H2VO4 /H2O2/phosphate system Ingegärd Andersson, András Gorzsás, Csaba Kerezsi, Imre Tóth, Lage Pettersson Manuscript VI. Speciation in peroxovanadate systems Lage Pettersson, Ingegärd Andersson, András Gorzsás Coordination Chemistry Reviews, 2003, 237, 77. Publications of interest but not included in the thesis: • A simple PCR–heteroduplex screening method for detection of a common mutation of the catalase gene in Hungary László Góth, András Gorzsás, Tibor Kálmán Clinical Chemistry, 2000, 46, 1199. • Synthesis of new cyclitol compounds that influence the activity of phosphatidylinositol 4–kinase isoform, PI4K230 István F. Pelyvás, Zoltán G. Tóth, György Vereb, András Balla, Elza Kovács, András Gorzsás, Ferenc Sztaricskai, Pál Gergely Journal of Medical Chemistry, 2001, 44, 627. v Table of Contents ABBREVIATIONS ............................................................................................................................................... 1 POPULÄRVETENSKAPLIG SAMMANFATTNING ..................................................................................... 2 1. INTRODUCTION............................................................................................................................................. 4 1.1 AQUEOUS BIOINORGANIC CHEMISTRY OF VANADIUM.................................................................................... 4 1.1.1 Vanadium(III)....................................................................................................................................... 4 1.1.2 Vanadium(IV) ....................................................................................................................................... 5 1.1.3 Vanadium(V)......................................................................................................................................... 5 1.2 AQUEOUS BIOINORGANIC CHEMISTRY OF PEROXOVANADATES ..................................................................... 8 1.3 PHYSIOLOGICAL EFFECTS AND TOXICOLOGY OF VANADIUM ......................................................................... 9 1.4 INSULIN–ENHANCING EFFECTS OF VANADIUM COMPOUNDS ........................................................................ 10 1.5 AIM OF THE PRESENT WORK ........................................................................................................................ 13 2. EXPERIMENTAL AND TECHNIQUES..................................................................................................... 14 2.1 IONIC MEDIUM............................................................................................................................................. 14 2.2 POTENTIOMETRY......................................................................................................................................... 14 2.2.1 Potentiometric titrations..................................................................................................................... 14 2.2.2 Separate pH measurements ................................................................................................................ 15 2.3 NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY........................................................................ 15 51 2.3.1 V NMR.............................................................................................................................................. 18 31 2.3.2 P NMR.............................................................................................................................................. 19 1 13 14 2.3.3 H, C and N NMR.......................................................................................................................... 19 2.4 ELECTRON PARAMAGNETIC RESONANCE (EPR) SPECTROSCOPY................................................................ 20 2.5 ELECTROSPRAY IONISATION MASS SPECTROMETRY (ESI–MS).................................................................. 21 2.6 CRYSTALLISATION EXPERIMENTS................................................................................................................ 22 2.7 SIMPLE BLOOD TESTS .................................................................................................................................. 22 3. TREATMENT OF EQUILIBRIUM DATA ................................................................................................. 24 3.1 EQUILIBRIA AND MASS BALANCES............................................................................................................... 24 3.2 EVALUATION OF EQUILIBRIA ......................................................................................................................