Two forms of the dinicotinate complex Cr(nic)2(H2O)3OH were formed which were yellow and blue, respectively. For the yellow form the nicotinic acid ligands were coordinated via the pyridine ring nitrogen atom but this complex was biologically inactive, while for the blue form nicotinic acid was coordinated via the carboxylate group and this compound was biologically active. Only Cr(III) formed a stable carboxylate coordinated dinicotinate complex. No stable complexes were formed with Fe(III) and Mn(III) due to significant olation, even at acidic pH's, and the complexes of nicotinic acid with Cr(II), Mn(II), Co(II) and Ni(II) were all pyridine nitrogen atom coordinated and biologically inactive. Several chromium (III) complexes with amino acids possessed biological activity also, and these included the α -carboxylate coordinated species Cr(gly)n(H2O)3+6-nand Cr(glu)n(H2O)3+6-n, the bidentate coordinated Cr(gln)2(H2O)2+ complex, and the NH4OH- eluted complexes obtained when Cr(gly)2(H2O)2+ and Cr(glu)2(H2O) 2+, but not Cr(cys) 2(H2O) 2+, were eluted from a DOWEX 50W-X12 cation-exchange column (loss of the α-amino coordination was postulated to have occurred). The biologically active mixed ligand complex postulated as Cr2(nic) 4(gly) 2(OH) 2
was prepared and found to be stable at neutral pH as a result of coordination of the glycine ligands. The activity of the chromium (III) complexes in the yeast fermentation assay suggested that similar effects would be found in mammalian systems. The yeast assay system was found to be a simple, quick and reproducible method of determining biological activity. All of the active chromium (III) complexes prepared were found to be similar, in structure, to the diguanide compound 1,4-diguanidinobutane which is known to lower blood sugar levels in mammals. This similarity in structure suggested a similar function might be possessed by the complexes reported in this thesi

Cooper, Juan Anton

English

Massey Research Online

Copyright is owned by the Author of the thesis.  Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only.  The thesis may not be reproduced elsewhere without the permission of the Author.  CHROMIUM (I I I) COMPLEXES AND THEIR RELATIONSHIP TO THE GLUCOSE TOLERANCE FACTOR A THESIS PRESENTED IN PARTIAL FULFILMEN T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN CHEMISTRY AT MASSEY UNIVERSITY NEW ZEALAND JUAN ANTON COOPER 1982 0fi!f·03 Cot:> MASSEY UNIVERSITY LIBRARY 88-04885 ii ABSTRACT To forms of the dinicotinate complex Cr(nic) 2 (H 2o) 3 0H were formed which were yellow and blue, respectively. For the yellow form the nicotinic acid ligands were coordinated via the pyridine ring nitrogen atom but this complex was biologically inactive, while for the blue form nicotinic acid was compound a stable No stable coordinated via the carboxylate was biologically active. Only group and this Cr(III) f ormed carboxylate coordinated dinicotinate complex. complexes were formed with Fe(III) and Mn(III) due to significant elation, even at acidic pH's, and the complexes of nicotinic acid with Cr (II), Mn (II) , Co ( I I) and Ni(II) were all pyridine nitrogen atom coordinated and biologically inactive. Several chromium (III) complexes with amino acids possessed biological activity also, and these included the 3+ a - carboxylate coordinated species Cr(gly)n (H 2 0) 6 _n and Cr(glu) n(H2 O)~~n, the bidentate coordinated Cr(gln) 2 + (H 2 o ) 2 complex, and the NH4 0H- eluted complexes obtained + . + when Cr(gly) 2 <H 2 o> 2 and Cr(glu) 2 (H2 o) 2 , but not Cr(cys ) 2 + (H 20 ) 2 , were eluted from a DOWEX 50W-X12 cation-exchange column (loss of the a-amino coordination was postulated to have occurred). The biologically active mixed ligand complex postulated as cr2 (nic) 4(gly) 2 (0H) 2 was prepared and found to b e stable at neutral pH as a result of coordination of the glycine ligands. The activity of the chromium (III) complexes in the yeast fermentation assay suggested that similar effects would be found in mammalian systems . The yeast assay system was found to be a simple, quick and reproducible method of determining biological activity. All of the active chromium (III) complexes prepared were found to be similar, in structure, to the diguanide compound iii 1 , 4-diguanidinobutane which is known to lower blood sugar levels in mammals. This similarity in structure suggested a similar function might be possessed by the complexe s reported in this thesis. iv ACKNOWLEDGEMENTS I wish to Dr Paul D. thank my supervisors Dr Len F. Blackwel 1 and Buckley for their invaluable advice and assist-ance and the encouragement offered throughout the course of this work. Thanks are also extended to all members of the Chemistry, Biochemistry and Biophysics Department for their help especially: Dr Steven J. Haylock, Mrs Rose Motion, Dr Alastair K.H. MacGibbon and Dr Eric W. Ainscough for their assistance and advice. Thanks also encouragement wife, Judith. to friends and family received during this for the support study, especiall y and my Finally, I would like to thank Mrs Veronica Fieldsend for the excellent job in typing this thesis. ABSTRACT ACKNOWLEDGEMENTS TABLE OF CONTENTS LIST OF FIGURES LIST OF TABLES TABLE OF CONTENTS SECTION ONE INTRODUCTION SECTION TWO NICOTINIC ACID COMPLEXES WITH CHROMIUM AND OTHER TRANSITION METALS 2. 1 Introduction 2.2 Transition Metal Chemistry 2.2.1 Introduction 2.2.2 Magnetic Properties 2.2.3 Electronic Structures and Spectral Properties 2.2.4 Charge Transfer Spectra 2.2.5 Electron Spin Resonance Spectra 2.3 Methods and Materials 2.3.1 Sources of Chemicals 2.3.2 Methods and Instrumentation 2.3.2.1 Micro analysis 2.3.2.2 Chromium determination 2.3.2.3 Electronic, infra-red and electron v Page ii iv v 1 5 5 6 6 7 8 1 5 1 5 1 6 1 6 1 6 16 1 6 spin resonance sp~ctroscopy 17 2.3.2.4 Magnetic susceptibility 17 2.3.2.5 X-ray diffractometry 17 2.3.2.6 Absorbance, pH and conductivity measurement 17 2.3.2.7 Ion-exchange resins 17 vi 2.3.2.8 Gel filtration resin 1 8 2.4 Results 1 8 2.4.1 The Yellow Chromium (III) Dinicotinate Complex 1 8 2.4.1.1 Preparation of yellow chromium (III) dinicotinate complex 1 8 2.4.1 .2 X-ray powder diffractometry 21 2.4.1 .3 Electronic spectra and magnetic properties 21 2.4.1.4 Electron spin resonance spectroscopy 25 2.4.1 .5 Infra-red spectra 2.4.2 Other Transition Metal Nicotinic Acid Complexes 25 28 2.4.2.1 Preparation of Co(nic) 2 (H 2o) 4 28 2.4.2.2 Preparation of Ni(nic) 2 (H 2o) 4 28 2.4.2.3 Preparation of Mn(nic) 2 (H 2o) 4 28 2.4.2.4 Preparation of Fe(nic) 2 (H 2o) 30H 29 2.4.2.5 Properties of the transition metal complexes 29 2.4.2.6 Electronic and magnetic properties 30 2.4.2.7 Infra-red spectra. 31 2.4.2.8 X-ray diffraction powder patterns 31 2.4.3 The Blue Chromium Dinicotinate Complex 35 2.4.3.1 Preparation of ·blue chromium dinicotinate complex 35 2.4.3.2 Electronic spectra and magnetic properties 35 2.4.3.3 Infra-red spectra 36 2.4.3.4 Electron spin resonance spectroscopy 39 2.4.3.5 X-ray powder diffractometry 3 9 2.4.4 Reaction of the Blue Chromium-Nicotinic Acid Complex with Acid 40 2.4.4.1 Electronic spectra 40 2.4.4.2 Ion-exchange and gel filtration chromatography 41 2.4.5 The Soluble Blue Chromium (III)-Mononicotin-ate Complex 41 2.4.5.1 Attempted preparation of a mono-nicotinate complex of chromium (III)41 vii 2.4.5.2 Electronic spectra 46 2.5 Discussion 46 2.5.1 Yellow Chromium (III)-Dinicotinate Complex 46 2.5.1 .1 Comparison with literature 53 2.5.2 Other Transition Metal Complexes with Nicotinic Acid 57 2.5.3 Blue Coloured Chromium (III)-Dinicotinate Complex 60 2.5.4 The Reaction of the Blue Cr(nic) 2 (H 2o) 30H Complex with Acid 2.5.5 The Soluble Blue Mononicotinate Complex 65 of Chromi~m (III) 6 9 2 .5.6 Compari~ons with Literature 7 5 2.6 Conclusion 78 SECTION THREE 82 AMINO ACID COMPLEXES OF CHROMIUM (II I ) 3. 1 Introduction 82 3.2 Methods and Materials 83 3.2.1 Sources of Chemicals 83 3.2.2 Experimental Methods and Instrumentation 83 3.2.2.1 Cation-exchange chromatography 83 3.2.2.2 Anion-exchange chromatography 84 3.2.2.3 Gel filtration chromatogr.aphy 84 3.2.2.4 Electronic spectroscopy 84 3.2.2.5 Absorbance, pH and conductivity measurement 84 3.2.2.6 Chromium determination 85 3.2.2.7 Micro analysis 85 3.3 Results 3.3.1 Chromium (III) Complexes with Glycine 85 85 viii 3.3.1 .1 Preparation of glycine complexes 85 3.3.1.2 Ion-exchange chromatography of glycine complexes 86 3.3.1 .3 Electronic Spectra 88 3.3.2 Chromium (III) Complexes with Cysteine 89 3.3.2.1 Preparation of cysteine complexes 89 3.3.2.2 Ion exchange chromatography of cysteine complexes 90 3.3.2.3 Electronic spectra of cysteine complexes 91 3.3.2.4 Titration of NaCr(cys) 2 .2H2o 93 3.3.2.5 Determination of cysteine by the DTNB reaction 98 3.3.3 Chromium (III) Complexes with Glutamic Acid 100 3.3.3.1 Preparation of glutamic acid complexes 1 00 3.3.3.2 Ion exchange of glutamic acid complexes 1 00 3.3.3.3 Electronic spectra 1 0 3 3.3.4 Chromium (III) Complexes with Glutamine 104 3.3.4.1 Preparation of [Ct(NH 3 ) 6 ] (N0 3 ) 3 104 3.3.4.2 Preparation of glutamine complexes 105 3.3.4.3 Ion exchange of the glutamine complexes 106 3.3.4.4 Electronic spectra 106 3.4 Discussion 1 07 3.4.1 Chromium (III) Complexes with Glycine 107 3.4.1 .1 The tris-glycine chromium (III) complex 107 3.4.1 .2 The dimer bis-glycine chromium (III) complex 109 3.4.1.3 Monodentate glycin3 complexes with c hromium (III) 111 3.4.1.4 The soluble bis-glycine chromium (III) complex + 3.4.1.5 The reaction of Cr(gly) 2 (H 2o> 2 NH 40H 1 1 4 with 1 1 8 ix 3.4.2 Chromium (III) Complexes with Cysteine 119 3.4.2.1 The tridentate cysteine complex with chromium (III) 119 3.4.2.2 The.reaction of NaCr(cys) 2 .2H 2o with acid 1 24 3.4.2.3 The reaction of NaCr(cys) 2 .2H 2o with base 1 28 3.4.2.5 The pH dependent equilibrium + between Cr(cys) 2 (H 2o) 2 and Cr(cys) 2- 1 29 3.4.3 Glutarnic Acid Complexes with Chromium (III) 130 3.4.3.1 The soluble pink glutamic acid complex with chromium (III) 130 3.4.3.2 The soluble purple glutamic acid complex with chromium (III) 133 3.4.3.3 The soluble blue monodentate glutamic acid complex with c hromium (III) 135 3.4.3.4 The effect of ammonia on the cationic glutamic acid complexes of chromium (JII) 136 3 .4.4 Glutamine Complex with Chromium (III) 1 36 3.4.4.1 The soluble red glutamine complex with chromium (III) 136 3.4.4.2 The soluble purple glutamine complex with chromium (III) 139 3.5 Conclusion 1 42 SECTION FOUR 1 46 MIXED LIGAND COMPLEXES WITH CHROMIUM (III) 4.1 Introduction 1 46 4.2 Methods and Materials 1 48 4.2.1 Sources of Chemicals 1 48 4.2.2 Methods and Instrumentation 1 48 4.2.2.1 Chromium determination 148 4.2.2.2 Electronic spectroscopy 148 4.2.2.3 Absorbance, pH and conductivity measurement 148 4.3 Results 4.2.2.4 Ion-exchange resins 4.2.2.5 Gel filtration resins 4.3.1 Preparation of a Mixed Solution of Chromium (III) Complexes with Nicotinic Acid and 1 48 1 48 1 49 x Glycine 149 4.3.1.1 Ion-exchange chromatography 149 4.3.1 .2 Gel filtration chromatography 4.3.1 .3 Electronic spectra 4.3.2 Preparation of a Mixed Solution of Chromium (III) Complexes with Nicotinic 1 50 1 50 Acid and Cysteine 150 4.3.2.1 Ion-exchange chromatography 4.3.2.2 Gel filtration chromatography 4.3.2.3 Electronic spectra 4.3.3 Preparation of Chromium (III)-glutathione 1 52 1 52 1 52 Complexes 153 4.3.3.1 Ion exchange chromatography and electronic spectra 153 4.3.4 Preparation of a Mixed Solution of Chromium (III) Complexes with Nicotinic Acid and Reduced Glutathione 153 4.3.4.1 Gel filtration chromatography and electronic sp~ctra 154 4.3.5 Preparation of a Mixed Solution of Chromium (III) Complexes with Nicotinic Acid, Glycine, Cysteine and Glutamic Acid 154 4.3.5.1 Ion-exchange separation 4.3.5.2 Gel filtration chromatography 4.3.5.3 Electronic spectra 4.3.5.4 Thin layer chromatography 4.4 Discussion 4.4.1 Mixed Ligand Complexes of Chromium (III) 1 55 1 57 1 58 1 61 164 with Nicotinic Acid and Glycine 164 4.4.2 Mixed Ligand Complexes of Chromium (III) with Nicotinic acid and Cysteine 168 4.4.3 Chromium (III) Complexes with Glutathione 171 xi 4.4.4 Mixed Ligand Complexes of Chromium (III) with Nicotinic Acid and Reduced Glutathione 173 4.4.5 Mixed Ligand Complexes of Chromium (III) with Nicotinic Acid, Glycine, Cysteine, and Glutamic Acid 174 4.5 Conclusion 177 SECTION FIVE 1 80 THE BIOLOGICAL ACTIVITY OF CHROMIUM (III) COMPLEXES 5. 1 Introduction 180 5.2 Methods and Materials 184 5.2.1 5.2.2 5.2.3 5.2.4 5.2.5 5.2.6 5.2.7 5.2.8 Yeast culture Pre-assay growth on plating medium Pre-assay growth in a liquid medium Assay growth in a defined medium Cell harvesting and concentration determ in -ation Standard assay technique Manometric calculations and data inter-pretation Optimum assay conditions 1 84 184 184 1 85 185 186 186 188 5.3 Results 189 5.3.1 Nicotinic Acid Complexes with Transition Metals 189 5.3.2 Chromium (III) Complexes with Various Amino Acids 192 5.3.3 Mixed Ligand Complexes of Chromium (III) 193 5.3.4 The Effect of Ammonium Hydroxide on Chromium (III) Complexes 195 5.4 Discussion 199 5.4.1 Nicotinic Acid Complexes with Transition Metals 199 5.4.2 Chromium (III) Complexes with Various Amino Acids 200 xii 5.4.3 Mixed Ligand Complexes with Chromium (III) 201 5.4.4 The Effect of Ammonium Hydroxide on Chromium (III) Complexes 202 APPENDIX REFERENCES SECTION SIX CONCLUSION LIST OF ABBREVIATIONS 205 211 212 xiii LIST OF FIGURES Figure Page 1.1 Hypothetical ternary complex of chromium at site of action 2. 1 2.2 2.3 2.4 2.5 Energy level diagram of an octahedral metal ion Ground state d-orbital configurations Tanabe-Sugano diagrams Apparatus for preparation and reaction of cr2+ Infra-red absorption spectra of (a) yellow Cr(nic) 2 (~ 2o) 30H , (b) Co(nic) 2 (H 2o) 4 , and (c) nicotinic acid 2.6(a) Electron spin resonance spectrum of yellow Cr(nic) 2 (H 2o) 30H 2.6(b) Electronspin resonance spectrum of blue Cr(nic) 2 (tt2o) 30H 2.7 Infra-red absorption spectrum of (a) Mn(nic) 2 (H 2o) 4 , (b) nicotinic acid, (c) Ni(nic) 2 (H 2o) 4 , (d) Co(nic) 2 (H 2o) 4 , and (e) yellow Cr(nic) 2 (H 2o) 30H 2.8 Infra-red absorption spectrum of (a) blue Cr(nic) 2 (H2o) 30H and (b) nicotinic acid 2.9 Elution profile of a blue coloured Cr(nic) 2 3+ (H 2o> 4 complex on a Sephadex G15 column 2.10 Elution profile of the blue coloured chromium (III)-nicotinic acid species on a Sephadex G15 column 3 1 1 1 1 1 3 1 9 20 26 26 32 37 42 45 xiv Figure Page 2.11 Molecular structure of Co(II)(nic) 2 (H 2o) 4 47 2.13 · Proposed structures of dinicotinate complex with bridging halogens 55 2.14 Structure of the blue Cr(nic) 2 (H 2o) 30H complex 66 2.15 Structure of the blue coloured Cr(nic) 2 (H 2o) 43+ species in acidic solution 2.16 Structure of soluble blue Cr(nic)(H2o) 52+ 2.17 Reaction scheme for dimeric chromium (III)-nicotinic acid complex 68 70 72 2.18 Predicted overlap of the elution profiles of the blue coloured Cr(nic) 2 (H 2o) 43+ and Cr(nic) (H 2o) 52+ species on a Sephadex G15 column 74 3.1 Electronic spectrum of NaCr(cys) 2 .2H 2o 92 3.2(a) Series of electronic spectra obtained when an aqueous solution of NaCr(cys) 2 .2H2o at pH 7.0 was titrated with dilute acid 94 3.2(b) Series of electronic spectra obtained when an aqueous solution of NaCr(cys) 2 .2H 2o at pH 7.0 was titrated with dilute base 3.3 Variation in intensity of the d-d bands in the visible spectrum of an aqueous solution of NaCr(cys) 2 .2H2o with pH 3.4(a) Titration curve for an aqueous solution of NaCr(cys) 2 .2H2o 95 96 97 Figure 3.4(b) Titration curve for an aqueous solution of cysteine HCl 3.5 Plot of absorbance at 412nm (for the DTNB anion) against time for the reaction of NaCr(cys) 2 .2H 2o at pH 8.0 with DTNB 3.6 3.7 3.8 3.9 3. 1 0 3. 11 3. 1 2 Guggenheim plot of ln(A-A') against time derived from the absorbance plot (Figure 3.5) Structure of [Cr ( gly) 2oH] 2 3+ Structure of postulated Cr(gly) 2 (H 2o) 4 Structure of NaCr(cys) 2 .2H 2o . + Structure of red coloured Cr(cys) 2 (H 2o) 2 3.13 Structure of Cr(glu) 2 (H 2o) 2 at pH 8.5 3.14 Reaction of chromium (III) with glutamic acid under acidic conditions 3. 1 5 3.16 4.1(a) Elution profile of the chromium (III)-nicotinate-glycine reaction mixture on a Sephadex G15 column 4.1(b) Elution profile of the dicysteine chromium (III)-nicotinic acid reaction mixture on a Sephadex G15 column xv Page 97 101 101 109 1 1 1 11 3 11 6 120 127 132 134 138 1 41 1 51 1 51 Figure 4.1(c) Elution profile of the chromium (III)-nicotinate-glutathione reaction mixture on a Sephadex G15 column 4.2(a) Elution profile of the cationic chromium (III) complexes prepared in Section 4.3.5 on a DOWEX xvi Page 1 51 50-X12 column 156 4.2(b) Elution profile of the cationic chromium (III) complexes prepared in Section 4.3.5 on a DOWEX 50-X12 column 156 4.3(a) Elution profile of fraction P on a Sephadex G10 column eluted with water 159 4.3(b) Elution profile of fraction P on a Sephadex G10 column eluted with 50 % EtOH 159 4.3(c) Elution profile of fraction G1 on a Sephadex G10 column eluted with water 4.4 pH titration of reaction mi~ture 4.5 Summary of the cation-exchange chromatography of the purple Cr(nic) 2 (H 2o) 43+-glycine reaction mixture 4.6 Postulated polymeric structure of [Cr(nic) 2 (OH)2]n 4.7 4.8 Summary of the cation-exchange chromatography of the purple Cr(cys) 2 (tt2o> 2+-nicotinic acid reaction mixture 1 59 1 60 164 166 167 168 Figure 4.9 Postulated structure of Cr(cys) 2 (nic) 2 5.1 Standard curves used in the determination of yeast cell concentration 5.2 Standard yeast assay showing carbon dioxide production 5.3 Activity of the NH 40H-eluted Cr(gly) 2 (H2o> 2+ species as a function of chromium (III) concentration 5.4 The structure of active monodentate chromium (III)-amirro acid complexes 6.1 Comparison of the structures of the active chromium (III) complexes xvii Page 170 172 187 190 197 204 209 Table 2. 1 2.2 LIST OF TABLES Electronic configuration of metal atoms "Spin only" magnetic moments for various numbers of unpaired electrons 2.3 Theoretical and experimental magnetic moments for various transition metal ions 2.4 2.5 2.6 2.7 2.8 Splitting of Russell-Saunders states Analytical data of nicotinic acid complexes X-ray diffraction powder patterns of yellow Cr(nic) 2 (tt 2o) 30H and Co(nic) 2 (H 2o) 4 Electronic absorption bands of nicotinic acid complexes Magnetic properties of nicotinic acid complexes 2.9 Infra-red spectrum of the yellow chromium-nicotinic acid complex 2.10 Infra-red spectra of nitrogen coordinated dinicotinic acid complexes 2.11 X-ray diffraction powder patterns of Ni(nic) 2 xviii Page 7 8 9 1 2 20 23 24 24 27 33 (H 2o) 4 and Mn(nic) 2 (H 2o) 4 34 2.12 Infra-red spectrum of blue chromium-nicotinic acid complex 3. 1 Analytical data for amino acid complexes 38 85 Table 3.2 Electronic spectra of glycine complexes 3.3 Electronic spectra of cysteine complexes 3.4 Reaction conditions for preparation of glycine complexes 3.5 Summary of amino acid complexes with Chromium (III) 4.1 Cationic fractions eluted from DOWEX 50-X12 column 4.2 Electronic spectra of cationic fractions 4.3 Thin layer chromatography of amino acids and nicotinic acid 4.4 Thin layer chromatography of non-chromatographed P fraction 4.5 Thin layer chromatography o~ G1 fraction 5.1 Activity of nicotinic acid complexes of transition metals 5.2 Activity of chromium (III) complexes with . various amino acids 5.3 Activity of amino acids 5.4(a) Activity of mixed ligand complexes of chromium (III) 5.4(b) Activity of mixed ligand complexes of chromium (III) xix Page 89 91 11 5 143 157 1 61 162 163 163 1 91 192 193 194 194 xx Table Page 5.5 Activity of Chromium (III) complexes after elution with NH 40H 5.6 Activity of Chromium (III) complexes after elution with NH 40H 196 1 9 8 

Chromium (III) complexes and their relationship to the glucose tolerance factor : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at Massey University

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Chromium (III) complexes and their relationship to the glucose tolerance factor : a thesis presented in partial fulfillment of the requirements for the degree of Master of Science in Chemistry at Massey University

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