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Porphyrin chemistry pertaining to the design of anticancer agents Meng, Grant G. 1993

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PORPHYRIN CHEMISTRY PERTAINING TO THE DESIGN OF ANTICANCER AGENTS by GRANT GUANGZHEN MENG B . Sc., Peking University, Beijing, 1983 M.Sc., Peking University, Beijing, 1986 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULT OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA SEPTEMBER 1993 ©Grant Guangzhen Meng, 1993  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Department of  ^CA &IP/ .,5Z-.^  The University of British Columbia Vancouver, Canada  Date OCt.^  DE-6 (2/88)  3  Abstract A limitation of radiotherapy is the lack of selectivity (in term of cell killing) toward tumor cells, especially toward hypoxic tumor cells. Porphyrin drugs used in photodynatnic therapy (PDT) have good selectivity (in term of drug accumulation) toward tumors, but the therapy has a limitation due to poor penetration of light through tissues. The initial objective of this thesis work was to design, synthesize and test for potential porphyrin radiosensitizers with improved tumor selectivity. The rationale was that the radio sensitization abilities of the porphyrins might be improved by introduction of certain functional groups (including nitro groups and metal ions), while retaining or improving (by balancing lipophilicity and hydrophilicity) selective properties of porphyrins. Such a drug would first selectively accumulate in tumors, then sensitize the effects of X-rays, which penetrate tissues well, to kill cells, thus overcoming some of the limitations of both radiotherapy and PDT. If such compounds can be synthesized, they may also be used in other aspects in cancer treatments and diagnosis, including PDT, chemotherapy, boron neutron capture therapy, and magnetic resonance imaging diagnosis. The potential of such compounds to be bioreductive drugs by targeting the hypoxic cells in tumors is also of interest in this project. This thesis work focuses mainly on the synthesis and characterization of some designed porphyrins and metalloporphyrins. Some preliminary in vitro studies of these compounds are also presented. A method of combining pyrrole condensation with aldehydes and subsequent modification is developed and used to synthesize a large variety of porphyrins. Among 40 porphyrins synthesized, 22 are new and 15 are water-soluble. A new class of porphyrins with three different meso substituents is synthesized (e.g., cis-(NPh)PyB(SPh)P, the structure of which is shown below). Substituents which are presumed to improve the properties of radiosensitization (NO2), DNA association (NH2, pyridyl), and watersolubility (methylpyridinium and sulfonatophenyl) are introduced into the porphyrin  structures. The Ili NMR spectra of these porphyrins are presented with each of the signals assigned. The aggregation of the porphyrin free-bases in aqueous solutions is reported. All the water-soluble porphyrin free-bases aggregate in aqueous solutions, but to different degrees. Different models for the aggregation are suggested, including a new "slide-over" model for the aggregation of tris-ionic porphyrins. Equilibrium constants (K) for an assumed monomer dimer process for 6 porphyrins have been calculated, and the K values for anionic porphyrins are in the order: Tet(SPh)P < PyT(SPh)P (APh)T(SPh)P <<trans-BPyB(SPh)P (structures are shown below). 10  15  20 cis-(NPh)PyB(SPh)P = 5-(4-nitropheny1)-10-(4-pyridy1)-15,20-(4-sulfonatophenyl)porphyrin Tet(SPh)P = 5,10,15,20-tetralcis(4-sulfonatophenypporphyrin PyT(SPh)P = 5-(4-pyridy1)-10,15,20-tris(4-sulfonatophenyl)porphyrin (APh)T(SPh)P = 5-(4-aminopheny1)-10,15,20-tris(4-sulfonatophenyl)porphyrin trans-BPyB(SPh)P = 5,15-bis(4-pyridy1)-10,15,20-bis(4-sulfonatophenyl)porphyrin Tet(MPy)P = 5,10,15,20-tetralcis(4-methylpyridinium)porphyrin T(MPy)PhP = 5,10,15-tris(4-methylpyridinium)-20-(4-phenyl)porphyrin T(MPy)(NPh) = 5,10,15-tris(4-methylpyridinium)-20-(4-nitrophenyl)porphyrin cis-B(MPy)B(NPh) = 5,10-tris(4-methylpyridinium)-15,20-bis(4-nitrophenyl)porphyrin  Cobalt complexes of the water-soluble porphyrins Tet(MPy)P and Tet(SPh)P have been reported in the literature in the investigation of several chemical and biological systems. However, questions remain in the literature regarding the synthesis and characterization of these compounds, especially concerning the oxidation state of the cobalt. The chemistry related to the synthesis of these complexes is further investigated,  and synthetic methods for the specific oxidation states of cobalt are developed. New cobalt (II) and/or (III) complexes of the water-soluble porphyrins T(MPy)PhP, T(MPy)(NPh)P, cis-B(MPy)B(NPh)P, PyT(SPh)P, (APh)T(SPh)P and cis(NPh)PyB(SPh)P (structures of these free bases are shown above) are synthesized and characterized. The pKa values of the coordinated aquo ligands of four diaquocobalt(III) complexes of water-soluble porphyrins in aqueous solutions are measured using proton NMR spectroscopy. The aggregation of cobalt(II) complexes of water-soluble porphyrin in aqueous solutions is observed by Ill NMR spectra for the first time. The accumulation of the porphyrin free-bases in HT-29 cells has been found to be closely related to the chemical structures, especially to sign, number and distribution of charges. Generally, porphyrins with lower charge accumulate more than ones with higher charge; porphyrins with positive charge accumulate more than those with the same negative charge; and the trans-isomer accumulates more than the cis-isomer. The porphyrin free-bases are found to be essentially non-toxic toward Chinese hamster ovary (CHO) cells. The cobalt(III) complexes of cationic porphyrins exhibit some selective toxicity toward hypmdc CHO cells, a property for which the compounds are designed, although the toxicities in hypoxic conditions are too small for use in bioreductive drugs. The porphyrins and metalloporphyrins have not shown high radiosensitization toward CHO cells under hypoxic conditions, and have not shown photosensitization toward HT-29 cells at wavelengths of 630 ± 10 run, although some weak effects are found. More biological studies (e.g., tumor accumulation studies in vivo) are needed before final evaluations of these drugs are made. Potential uses of the synthesized porphyrins in other aspects of cancer treatments and diagnosis should also be considered in future work.  iv  Table of contents Abstract ^ Table of contents ^  v  List of figures ^  xii  List of tables ^  xvii  Lists of abbreviations ^  xix  List of general abbreviations ^  xix  List of abbreviations of porphyrins ^  xxii  Acknowledgements ^  xxxii  Chapter 1 Introduction--The design of porphyrin compounds as potential anti-cancer agents ^  1  1.1 Porphyrin chemistry ^  1  1.2 Nomenclature and abbreviations of porphyrins used in this thesis ^ 2 1.3 Porphyrins and cancer ^ 1.3.1 Tumor localization of porphyrins ^  5 5  1.3.2 Porphyrins used in PDT (photodynamic therapy) ^ 6 1.3.3 Porphyrins used in tumor detection ^  7  1.3.4 Porphyrins used in chemotherapy ^  7  1.3.5 Porphyrins as radiosensitizers ^  8  1.4 Radio sensitizers ^ 1.4.1 Mechanisms ^  10 11  V  1.4.2 Properties of radiosensitizers ^  12  1.4.3 Nitroirnidazole radiosensitizers ^  13  1.5 Bioreductive agents ^  15  1.6 The design of porphyrins as anti-cancer agents ^  16  1.6.1 Electron affinity ^  16  1.6.2 Selectivity toward tumors ^  17  1.6.3 Cell uptake ^  19  1.6.4 DNA-binding ^  19  1.6.5 Solubility and lipophilicity ^  19  1.6.6 Multiple functions ^  20  1.7 Porphyrin syntheses ^  20  1.8 In vitro studies on synthetic porphyrins ^  25  1.9 The objectives of this thesis work ^  27  References-Chapter 1 ^  28  Chapter 2 The synthesis of the designed porphyrins ^ 35 2.1 Introduction ^  35  2.2 Experimental ^  37  2.2.1 Materials and method ^  37  2.2.2 The porphyrins with a general formula of PhnPy(4../)P ^ 38 2.2.3 Nitrations ^  42  2.2.4 Reductions of the nitro-porphyrins ^  47  2.2.5 Sulfonations ^  55  2.2.6 Methylations ^  62  2.3 Results and discussion ^  67  vi  2.3.1 Synthesis ^  68  2.3.1.1 Hydration of porphyrins ^  68  2.3.1.2 Synthesis of the porphyrins with general formula PhnPY(4-0P ^  70  2.3.1.3 Synthesis of nitro-porphyrins ^  71  2.3.1.4 Synthesis of mninophenylporphyrins ^ 73 2.3.1.5 Synthesis of sulfonated porphyrins ^ 73 2.3.1.6 Synthesis of methylpyridiniumporphyrins ^ 78 2.3.2 Proton-NMR spectra ^  81  2.3.2.1 Identification of substituents from 1H NMR spectra ^ 81 2.3.2.2 Assignments of signals to the protons of the mesosubstituent ^  83  2.3.2.3 Identification of isomers ^  90  2.3.2.4 Assignments of the signals for the pyrrole protons ^ 96 2.3.3 UV-visible spectra ^  105  2.3.4 Infrared spectra ^  108  2.3.5 Mass spectra ^  108  References-Chapter 2 ^  Chapter 3 Water- soluble metalloporphyrins ^ 3.1 Introduction ^  113  116 116  3.1.1 Cobalt complexes of water-soluble porphyrins ^ 116 3.1.2 Copper and zinc complexes of water-soluble porphyrins ^ 118 3.2 Experimental ^  118  3.2.1 Materials and methods ^  118  3.2.2 {Co11[Tet(MPy)P])C14.2H20 ^  120 vii  3.2.3 {Colll[Tet(MPy)P](OH2)}(C104)5.xH20 ^ 121 3.2.4 {Colret(MPy)P1(OH)}C14.2H20 ^  121  3.2.5 {CollIget(MPy)11(OH2)}C15-2H20 ^  121  3.2.6 {Co11[T(MPy)Phil}C13-1/2H20 ^  122  3.2.7 {Co111[T(MPy)Ph11(OH2)}04-2H20 ^  122  3.2.8 {CoRT(MPy)(NPh)PKOH2)}04.H20 ^ 122 3.2.9 {CollIcis-B(MPy)B(NPh)19(H20))C13-2H20 ^ 123 3.2.10 Na4{Co11[Tet(SPh)P]).nH20 (n=4 or 9) ^ 123 3.2.11 Na3{Colret(SPh)PKOH2)}.nH20 (n=3 or 14) ^ 124 3.2.12 Na2{Co111[PyT(SPh)P]} ^  124  3.2.13 Na2{Co111[(APh)T(SPh)11(OH2)} ^  125  3.2.14 Na{ Com[c-(NPh)PyB(SPh)P](OH2)} -2H20 ^ 125 3.2.15 Cu porphyrins complexes ^  125  3.2.16 Zinc porphyrin complexes ^  126  3.3 Results and Discussion ^  127  3.3.1 1H NMR spectra of the metalloporphyrins ^ 127 3.3.1.1 1H NMR spectra of Co complexes of cationic-porphyrins ^  127  3.3.1.2 111 NMR spectra of Co complexes of anionic-porphyrins ^  134  3.3.1.3 1H NMR of Cu-porphyrins ^  137  3.3.1.4 1H NMR of Zn-porphyrins ^  139  3.3.1.5 1H MAR studies of the aggregation of Con complexes^ 140 3.3.2 Syntheses ^  144  3.3.2.1 Synthesis of Co-cationic porphyrins ^ 144 3.3.2.2 Synthesis of Co-anionic-porphyrins ^ 145 viii  3.3.2.3 Reaction conditions for the synthesis of cobalt porphyrins ^  146  3.3.2.4 Synthesis of Cu-porphyrins and Zn-porphyrins ^ 151 3.3.3 Mass spectra ^  151  3.3.3.1 Mass spectra of cobalt complexes of anionic porphyrins ^ 151 3.3.3.2 Mass spectra of cobalt cationic porphyrins^ 154 3.3.4 Magnetism and coordination of the cobalt-porphyrin complexes ^ 155 3.3.5 UV-visible spectra ^  158  3.3.6 Measurement of the pKa values of cobalt(III) diaquo complexes ^ 163 3.3.7 Hydration and elemental analysis  ^  References-Chapter 3 ^  Chapter 4 Aggregation of porphyrins ^  170 172  174  4.1 Introduction ^  174  4.2 Experimental ^  175  4.3 Results and discussion ^  176  4.3.1. UV-visible spectra in aqueous solutions ^ 176 4.3.1.1 Tet(SPh)P ^  177  4.3.1.2 PyT(SPh)P and (APh)T(SPh)P ^  181  4.3.1.3 trans-BPyB(SPh)P ^  184  4.3.1.4 cis-BPyB(SPh)P, cis-B(APh)B(SPh)P and trans-B(APh)B(SPh)P ^  187  4.3.1.5 Tet(MPy)P ^  191  4.3.1.6 T(MPy)PhP ^  195  4.3.1.7 T(MPy)(NPh)P and cis-B(MPy)DPhP ^ 196 4.3.2. Affects of methanol on aggregation of porphyrins. ^ 198 ix  4.3.3 1H NMR studies on aggregation of porphyrins ^ 203 4.3.3.1 1H NMR studies on the aggregation of T(MPy)PhP ^ 203 4.3.3.2 1H NMR studies on the aggregation of PyT(SPh)P ^ 209 4.3.4 Aggregation models ^  214  4.3.4.1 Monomer dimerization or oligomerization ^ 214 4.3.4.2 Structural models ^  216  4.3.5 The equilibrium constants for dimerization ^ 219 4.3.6 Summary ^ References-Chapter 4 ^  Chapter 5 In vitro studies of selected synthetic porphyrins and metalloporphyrins ^  225 226  228  5.1 Introduction ^  228  5.2 Materials and methods ^  229  5.2.1 Cell growth, maintenance and treatment ^ 229 5.2.2 Drugs and drug solutions ^  232  5.2.3 Toxicity in oxic and hypoxic conditions^ 232 5.2.4 Radiosensitization in hypoxic conditions  ^ 234  5.2.5 Cell accumulation ^  237  5.2.6 Photosensitization ^  238  5.3 Results and discussion ^  239  5.3.1 Toxicity of porphyrins and metalloporphyrins ^ 239 5.3.2 Radiosensitization under hypoxic conditions ^ 242 5.3.3 Accumulation of porphyrin free-bases in HT-29 cells ^ 246 5.3.4 Photosensitization of porphyrin free-bases ^ 247  x  References-Chapter 5 ^  249  Chapter 6 Conclusions and suggestions for future work ^ 250 6.1 Synthesis of porphyrins ^  250  6.2 Metallation of porphyrins ^  251  6.3 In vitro studies ^  251  6.4 Suggestions for Future Work ^  252  6.4.1 Synthesis of porphyrins ^  252  6.4.2 Other metallations ^  253  6.4.3 Biological studies ^  254  References - Chapter 6 ^  Appendix A Solutions used in cell biology ^  255  256  A.1 a-medium ^  256  A-2 RPMI medium ^  256  A-3 PBS (phosphate buffer saline) solution ^  257  A-4 Methylene-blue solution ^  257  A-5 Trypsin solution (0.1 %) ^  257  Appendix B Partition coefficients of porphyrins ^  258  Appendix C 1H NMR titration curves for Co(111) diaquo porphyrin complexes ^  259  xi  List of figures Figure 1.1. The porphyrin core. ^  2  Figure 1.2. Porphyrins containing anti-cancer agents. ^  8  Figure 1.3. Structures of some water-soluble porphyrins. ^ 9 Figure 1.4. A proposed sensitization mechanism ^  11  Figure 1.5. Structures of some radiosensitizers. ^  14  Figure 1.6. Structures of tetrahydroxophenylpotphyrins. ^ 18 Figure 1.7. Structures of sulfonated derivatives of TetPhP^ 18 Figure 1.8. Structure of some synthetic porphyrins. ^  22  Figure 1.9. Modifications of TetPhP ^  24  Figure 1.10. Some possible modifications of TPhPyP. ^  26  Figure 2.1. Synthetic schemes for designed porphyrins ^  36  Figure 2.2. 1H NMR spectra of B(NPh)B(SPh)P, (APh)(NPh)B(SPh)P and B(APh)B(SPh)P in DMSO-d6 ^  77  Figure 2.3. 1H NMR spectrum of cis-(NPh)PyB(SPh)P in DMSO-d6. ^ 79 Figure 2.4. 1H NMR spectrum of (NPh)TPIT in CDC13. ^ 85 Figure 2.5. 1H NMR spectrum of trans-BPyB(SPh)P in DMSO-d6 ^ 86 Figure 2.6. 111 NMR spectrum of (NPh)T(MPy)P in CDC13. ^ 88 Figure 2.7. 1H NMR spectrum of cis and trans-DP111PyP in CDC13. ^ 91 Figure 2.8. The patterns for the 1H NMR spectra of the pyrrole protons ^ 92 Figure 2.9. 1H NMR spectra of cis- and trans-B(NPh)BPyP in CDC13. ^ 94 Figure 2.10. 1H NMR spectra of cis and trans-B(NPh)PhPyP in CDC13. ^ 95 Figure 2.11. Resonance structures of a nitrophenylpotphyrin ^ 97  xii  Figure 2.12. Resonance structures of an atninophenylporphyrin. ^ .98 Figure 2.13A. A comparison of the schematic spectra of TPhPyP, cis-DPhBPyP and PhTPyP. ^  99  Figure 2.13B. Illustration of the chemical shifts of the pyrrole protons of TPhPyP and some nitroporphyrins. ^  100  Figure 2.14. Assignment of the pyrrole protons in the 1H NMR spectrum of cis-(NPh)DPhPyP (CDC13). ^  103  Figure 2.15. 1H NMR spectrum of cis-(APh)PhBPyP in CDC13 ^ 104 Figure 2.16. Resonance forms of an aminophenylporphyrin and a nitrophenylporphyrin ^  107  Figure 3.1. Cell for anaerobic UV-visible spectroscopy ^  120  Figure 3.2. 1H NMR spectra of {Coll[Tet(MPy)P]}C14 in D20 ^ 128 Figure 3.3. 1H NMR spectra of Co(II) cationic porphyrins in DMSO-d6 under air ^ 131 Figure 3.4. 1H NMR spectra of Colli[Tet(MPy)P] species in DMSO-d6 ^ 132 Figure 3.5. 1H NMR spectra of Co[Tet(SPh)P] species in DMSO-d6 ^ 135 Figure 3.6. 1H NMR spectra of cobalt complexes of tris-sulfonatoporphyrins in DMSO-d6. ^  136  Figure 3.7. 1H NMR spectra of copper(II) complexes of cationic porphyrins in DMSO-d6. ^  138  Figure 3.8. 1H NMR spectra of Co11[Tet(MPy)P]C14 in D20. ^ 141 Figure 3.9. 1H NMR spectra of Na4{Con[Tet(SPh)P]} in D20 ^ 143 Figure 3.10. Mass spectrum of Na3{Colli[Tet(SPh)}P(OH2)}. ^ 152 Figure 3.11. The suggested molecular fragments for the major peaks in the mass spectrum of Na3{Co111[Tet(SPh)P](OH2)). ^ 153 Figure 3.12. Paramagnetic Co(DEE) complexes ^  156  Figure 3.13. UV-visible spectra of Con[Tet(MPy)P]. ^  160  Figure 3.14. UV-visible spectra of Coll[T(SPh)P] in H20. ^ 161  Figure 3.15. 1H NMR spectra of Colll[T(MPy)13] in D20 at different pD values. ^  165  Figure 3.16. 1H NMR spectra of Co111[Tet(SPh)P] in D20 at different pD values. ^  166  Figure 3.17. The pKa measurement for Co111[Tet(MPy)1]0320)2. ^ 168 Figure 3.18. The pKa measurement for Coul[Tet(SPh)P]0320)2 ^ 169  Figure 4.1. The normalized spectra of Tet(SPh)P at various concentrations in distilled water. ^  178  Figure 4.2. Beer's law diagrams for Tet(SPh)P in distilled water. ^ 179 Figure 4.3. The normalized spectra of Tet(SPh)P at various concentrations in a phosphate buffer. ^  180  Figure 4.4. Deviation from Beer's law for Tet(SPh)P in a phosphate buffer solution^  180  Figure 4.5. The normalized spectra of (APh)T(SPh)P at various concentrations in a phosphate buffer^  182  Figure 4.6. Beer's law deviation for PyT(SPh)P and (APh)T(SPh)P. ^ 183 Figure 4.7. The normalized spectra of trans-BPyB(SPh)P at various concentrations in distilled water ^  185  Figure 4.8. The normalized spectra of trans-BPyB(SPh)P at various concentrations in a phosphate buffer ^  186  Figure 4.9. The normalized spectra of cis-BPyB(SPh)P at various concentrations in a phosphate buffer^  188  Figure 4.10. The normalized spectra of cis-B(APh)B(SPh)P at various concentrations in a phosphate buffer^  189  Figure 4.11. The normalized spectra of trans-B(APh)B(SPh)P at various concentrations in a phosphate buffer^  190  Figure 4.12A. The normalized spectra of Tet(MPy)P chloride at various concentrations in distilled water ^  192  Figure 4.12B. Plot of A/b vs. concentration for Tet(MPy)13 chloride in distilled water^  193 xiv  Figure 4.13. The normalized spectra of Tet(MPy)P in a buffer solution at various concentrations ^  194  Figure 4.14. The normalized spectra of T(MPy)PhP at different concentrations in a phosphate buffer. ^  195  Figure 4.15. A plot of A/b vs. concentration for T(MPy)PhP in a phosphate buffer. ^  196  Figure 4.16. The normalized spectra of cis-B(MPy)DPhP at various concentrations in a buffer solution. ^  197  Figure 4.17. Absorbance spectra of (APh)T(SPh)P in aqueous buffer/methanol mixtures. ^  198  Figure 4.18. Absorbance spectra of trans-BPyB(SPh)P in aqueous buffer/methanol mixtures. ^  200  Figure 4.19A. Absorbance spectra of trans-B(APh)B(SPh)P in aqueous buffer/methanol mixtures. ^  201  Figure 4.19B. Absorbance spectra of cis-B(APh)B(SPh)P in aqueous buffer/methanol mixture ^  202  Figure 4.20. 1H NMR spectra of T(MPy)PhP in D20 and DMSO-d6 ^ 204 Figure 4.21. Correlation between the 1H chemical shifts and the concentration of T(MPy)PhP ^  206  Figure 4.22. Aggregation models for T(MPy)PhP in water. ^ 208 Figure 4.23. 1H NMR spectra of PyT(SPh)P. ^  210  Figure 4.24 Correlation between the 1H chemical shifts and the concentration of T(SPh)PyP. ^  211  Figure 4.25. Suggested aggregation model for PyT(SPh)P ^ 212 Figure 4.26. Aggregation models for Tet(MPy)P  ^ 217  Figure 4.27. Aggregation models for trans-B(MPy)DPhP ^ 220 Figure 4.28A Best fit curves for the dimerization of Tet(SPh)P and PyT(SPh)P in a buffer solution ^  223  Figure 4.28B Best fit curves for the dimerization of (APh)T(SPh)P in a buffer solution and Tet(MPy)P in distilled water ^  224  XV  Figure 5.1 The vessel for toxicity assay. ^  233  Figure 5.2. Set up for the radiosensitization assay ^  235  Figure 5.3. A representative example of survival curves (0 2 effect, ER — 3). ^ 236 Figure 5.4. Toxicities of compound 12 (100 gM) under hypoxic and oxic conditions. ^  241  Figure 5.5. Survival curves for radiosensitization by compound 18; a weak radiosensitizer. ^  244  Figure 5.6. Survival curves for effect of compound 3 with radiation; a weak protector. ^  245  Figure 5.7. Results of drug accumulation in HT-29 cells. ^ 246 Figure 5.8. Photosensitization using 630 nm radiation by compounds 1 (0.03 mM) and 9 (0.05 mM). ^ 248  xvi  List of tables Table 2.1. Elemental analyses of the PhnPY(4-n)P PorPhYrins ^ 40 Table 2.2. 1H-NMR data for the PhnPyo_nr,P porphyrins ^ 41 Table 2.3. UV-visible data for the PhnPyo_nyP porphyrins ^ 41 Table 2.4. Elemental analyses for the nitroporphyrins ^  48  Table 2.5. 1H NMR data for the nitroporphyrins ^  49  Table 2.6. UV-visible data for the nitroporphyrins ^  50  Table 2.7. Elemental analyses for the aminoporphyrins ^  52  Table 2.8. 1H NMR data for the aminoporphyrins ^  53  Table 2.9. UV-visible data for the aminoporphyrins ^  54  Table 2.10. Elemental analyses for the sulfonated porphyrins ^ 59 Table 2.11. 1H NMR data for the sulfonatoporphyrins ^  60  Table 2.12. UV-visible data for the sulfonatoporphyrins ^  61  Table 2.13. Elemental analyses for the methylpyridiniumporphyrins ^ 64 Table 2.14. 1H NMR data for the methylpyridiniumporphyrins ^ 65 Table 2.15. UV-visible data for the methylpyridiniumporphyrins ^ 66 Table 2.16. Reports on elemental analysis of Tet(SPh)P ^  69  Table 2.17. Calculated and observed chemical shifts of the nitrophenyl and aminophenyl protons ^  84  Table 2.18. The assignments of the pyrrole protons ^  101  Table 2.19. The chemical shifts of the H7 and H8 pyrrole protons ^ 102 Table 2.20. Soret bands of nitro and amino-porphyrins ^  107  Table 2.21. Data from IR spectra ^  109  Table 2.22. Data from CI mass spectra ^  111  xvii  Table 2.23. Cationic FAB mass spectra of some cationic porphyrins ^ 111 Table 2.24. Anionic FAB mass spectra of some anionic porphyrins ^ 112  Table 3.1. Elemental analyses and yields of copper-porphyrin complexes ^ 126 Table 3.2. Elemental analysis of zinc-porphyrin complexes ^ 127 Table 3.3. 1H NMR data of the Co cationic porphyrin complexes in DMSO-d6 ^ 134 Table 3.4. 1H NMR data for Zn porphyrin complexes in DMSO-d6 ^ 140 Table 3.5. Mass spectra of anionic Co-porphyrins ^  154  Table 3.6. Mass spectral data for cobalt cationic porphyrins ^ 154 Table 3.7. Magnetic moments of Co-Porphyrins ^  155  Table 3.8. Data of UV-visible spectra of Co-porphyrins ^  159  Table 3.9. Data of UV-visible spectroscopy ^  163  Table 3.10. The pKa values for cobalt porphyrins ^  167  Table 4.1. 1H Chemical shifts (ppm) of T(MPy)PhP at concentrations of 0.02 and 0.002 M^  205  Table 4.2. Chemical shifts in spectra of PyT(SPh)P ^  209  Table 4.3. Equilibrium K values for dimerization of porphyrins Table 5.1. In vitro tests and compounds tested ^  ^ 222 230  Table 5.2. Fluorescence maxima for excitation and emission ^ 238 Table 5.3. Radiation enhancement ratio for selected porphyrins and metalloporphyrins ^  243  xviii  Lists of abbreviations The abbreviations are given in two parts. The commonly used abbreviations in the chemical and biological literature are listed in the first part, while the abbreviations for the porphyrins used in this thesis are listed in the second part. List of general abbreviations  II-1 NMR^Proton nuclear magnetic resonance 2D^2-Dimensional ( NMR )  A^Angstrom ( 10-8 cm ) A^Absorbance Ac0^Acetate ( CH3C00- ) AIDS^The acquired immunodeficiency syndrome APh^4-aminophenyl Bis ( in chemical names ) Light-path length ( VU-visible spectroscopy ) br^broad signal ( NMR ) Concentration oc^Degrees centigrade CHO^Chinese hamster ovary ( a cell line ) cm^Centimeter ( 10-2 meter ) Di ( in chemical names ) Doublets ( NMR ) DMF^N,N-dimethylformamide DMSO -d6 Deuterated dimethylsulfoxide ( a NMR solvent ) DNA^Deoxyribonucleic acid DSS^Sodium-2,2-dimethy1-2-silapentane-5-sulphonate ( NMR reference )  xix  eqn^Equation ER^Enhancement ratio ESR^Electron spin resonance FAB^Fast-atom bombardment ( mass spectroscopy ) g^Gram(s) GY^Gray = 100 rads h^Hour(s) IR^Infrared kiH^Coupling constant ( NMR ) L^Liter(s) M^Molarity ( moles per liter solution ) m^Multiplet ( NMR ) Me^Methyl mg^milligram ( 10-3 gram ) MHz^Megahertz ( 106 Hertz ) min^Minute(s) mL^Milliliter(s) mol^Mole(s) MPy^4-Methylpyridinium nm^Nanometer ( 10-9 meter ) NMR^Nuclear magnetic resonance spectroscopy NPh^4-Nitrophey1 OER^Oxygen enhancement ratio PBS^Phosphate buffer saline PE^Plating efficiency Ph^Phenyl ppm^Parts per million ( NMR ) XX  Py^Pyridyl qt^Quartet (NMR) R^Alkyl or aryl group RPM^Revolutions per minute s^Singlet ( NMR ); strong signal ( IR) SER^Sensitizer enhancement ratio SPh^4-Sulfonatophenyl T^Tris ( in chemical names ) t^Triplet ( NMR ) Tet^Tetrakis ( in chemical names ) TMS^Tetramethylsilane ( NMR reference ) UV^Ultraviolet V^Volume ^  Volt  6^Chemical shift e  Extinction coefficient  X^Wavelength p.^Ionic strength PIL^Microliter 1-LM^Micromolar  Ix/  List of Abbreviations of porphyrins  13 15 17  porphyrin core  Abbreviations Full names^  Structures  cis-(APh)DPhPyP^544-aminopheny1)-10,15-4ipheny1-20-(4-pyridy1)porphyrin  cis-(APh)PhBPyP^5-(4-arninopheny1)-10-pheny1-15,20-his(4-poidy1)porphyrin  (APh)TPIIP^5-(4-aminopheny1)-10,15,20-trialeny1poiphyrin  (APh)T(SPh)P^5-(4-aminopheny1)-10,15,204ris(4-fiu1fonatopheny1)porphyrin  cis-B(APh)DPhP^5,10-12is(4-aminopheny1)-15,20-dipheny1porphyrin  trans-B(APh)DPhP^5,15-bis(4-aminopheny1)-10,20-ilipheny1porphyrin  cis-B(APh)B(SPh)P^5,10-bis(4-aminoplieny1)-15,20-bis(4-su1fonatopheny1)poiphyrin  trans-B(APh)B(SPh)P^5,15-bis(4-aminopleny1)-10,20-bis(4-sulfonatoaheny1)porphyrin  s03-  trans-B(APh)PhPyP^5,15-bis(4-ilminoaleny1)-10-0eny1-20-(4-gyridy1)polphyrin  cis-B(MPy)B(NPh)P^5,10-bis(4-methy1pyridinhun)-15,20-bis(4-nitrogheny1)porphyrin  N°2 trans-B(MPy)B(NPh)P  5,15-bis(4-methylpyridinhun)-10,20-bis(4-nitrophenyl)polphyrin NO2  +MeIQ  NH N_ N HN  0 NM  xxiv  cis-B(MPy)DP1113^5, 10-bi s(4-methylpyridinium)-15,20-diphenylpcnphyrin  + MiN  trans-B(MPy)DPhP^5,15-bis(4-methy1pyridiniutn)-10,20-dipheny1porphyrin  + mee  cis B(NPh)DPhP^5,10-bis(4-nitropheny1)-15,20-diaienylporphyrin -  trans B(NPh)DPhP^5,15-bis(4-nitropheny1)-10,20-ilipheny1porphyrin -  cis B(NPh)BPyP^5,10-bis(4-nitropheny1)-15,20-his(4-p_yridy1)gorphyrin -  trans-B(NPh)BPyP^5,15-Igs(4-nitropheny1)-10,20-his(4-pyridyl)porphyrin  cis-B(NPh)PhPyP^5,10-12is(4-nitropheny1)-15-pheny1-2044-pyridy1Vorphyrin  trans-B(NPh)PhPyP^5,15-bis(4-nitroaleny1)-10-phenyl-20-(4-pyridypporphyrin  cis-BPyB(SPh)P^5,10-12is(4-pytidy1)-15,20-his(4-su1fonatopheny1)otphytin  S  trans-BPyB(SPh)P^5,15-his(4-pyridy1)-10,20-his(4-fiu1fonatoplieny1)polphyrin  cis-DPhBPyP^5,104ipheny1-15,20-his(4-pyridy1)porphyrin  trans-DPhBPyP^5,15-dipleny1-10,20-his(4-ayridyl)porphyrin  cis-(NPh)DPPyP^5-(4-nitropheny1)-10,15-dip1ieny1-20-(4-pyridy1)porphytin  cis-(NPh)PhBPyP^5-(4-nitropheny1)-10-plenyl-15,20-his(4-pyridypporphyrin  cis-(NPh)PyB(SPh)P^5-(4-nitropteny1)-10-(4-atidy1)-15,20-his(4-su1fonatopheny1)porphyrin  (NPIOTPhP  5-(4-nitropheny1)-10,15,20-1risphenylomphyrin  (NPh)TPyP  5-(4-nitropheny1)-10,15,20-tris(4-pyridyl)poiphyrin  OEP  2,3,7,8,12,13,17,18-Qctagthy1porphyrin \  \ >--- N HN Q,..._\ ./  OMP  2,3,7,8,12,13,17,18-Qctaffiethy1porphyrin  PhTPyP^5-pheny1-10,15,20-irsi(4-pyridyl)porphyrin  PyT(SPh)P^5-(4-pyridy1)-10,15,20-tris(4-fiu1fonatopheny1)pozphyrin  T(APh)PhP^5,10,15,/ris(4-aminopheny1)-20-plenylporphyrin  Tet(MPY)P  ^  5,10,15,204etra1ds(4-methy1pyridinitun)porphyrin This porphyrin is abbreviated as TMPyP in the literature ME N  0  Tet(NPh)P^5,10,15,20-tetralcis-(4-nitrophenyl)porphyrin  TetPhP^5,10,15,20-letraphenylporphyrin This porphyrin is abbreviated as 7PP in the literature.  TetPyP^5,10,15,20-Letrakis(4-pyridy1)porphyrin This porphyrin is abbreviated as 7'PyP in the literature  Tet(SPh)P^5,10,15,20-tetrakis-(4-sulfonatophenyl)porphyrin This porphyrin is abbreviated as TPPS4 in the literature  T(MPy)(NPh)P^5,10,15-iris(4-methyloridiniurn)-20-(4-nitrophenyl)polphyrin  T(MPy)PhP  5,10,15-vis(4-methy1pyridinium)-201teny1porphyrin  T(NPh)PhP  5,10,15-vis(4-nitroaleny1)-20-pheny1porphyrin  cN  TPhPyP  5,10,15-iripheny1-20-(412.yridy1)porphyrin  Acknowledgments My gratitude and thanks go to both of my supervisors, Drs. Brian James and Kirsten Skov, for their guidance, encouragement and patience during the course of this thesis work; and for their considerations for the difficulties I have experienced as a foreign student of chemistry, biology, English and Canadian culture.  I am also grateful to my dear wife, Haibo, for her support, patience and sharing the experiences of the completion of this thesis. The help from the past and present members of both the "James Gang" and the "Heavy Metals", especially David, Andy, Philip, Paul, James, Jeff, Ken, Golnar, Terrance, Susan and Hans, is gratefully appreciated. Special thanks go to Don Yapp and Chris Alexander for their help both in the ways of learning sciences and learning the words Chris uses the most. Going for coffee with all these people has been an enjoyment. The help from support staff in chemistry department (especially Peter Border, Marietta Austria and Lianne Diarg) is gratefully appreciated. Help from a summer student, Heidy Lam, is also appreciated. Special thanks to other friends (Ziyi Kan, David Huang, Jinglong Du, J.C. Yu, Jane Zhu, QY Ma and their families) who have made my life in the years of working for this thesis enjoyable (at least sometimes). The opportunity to come to, live and study in this beautiful country, which has been made possible by my acceptance as a graduate student and the offer of finical support from the University of British Columbia, is highly appreciated.  Chapter 1 Introduction  Chapter 1 Introduction—The design of porphyrin compounds as potential anti-cancer agents 1.1 Porphyrin chemistry  Porphyrin compounds (the free bases and metalloporphyrins) play essential roles in several biological systems, including human beings. Metalloporphyrins are involved in the processes of oxygen transportation (hemoglobin); oxygen storage (myoglobin); oxygen activation (cytochrome P-450); and electron transport (the cytochromes).1 The importance of these processes in biological systems, plus the challenges of porphyrin metabolism disorders in human beings, such as porphyria, have drawn interest from many disciplines. Biochemists, biologists, biophysicists, chemists, geneticists, microbiologists, pharmacologists and toxicologists have all contributed to the understanding of porphyrin compounds. Porphyrin chemistry has been developing for more than a century since the isolation of hematoporphyrin in 1867.2 The early studies were mainly on natural products, particularly isolation and characterization of the porphyrins and metalloporphyrins contained in biomolecules, especially iron protoporphyrin IX from hemoglobin and myoglobin. The total synthesis of protoporphyrin in 1929,3 which proved the cyclic tetrapyrrole structure, and the syntheses of synthetic porphyrins, such as OMP (2,3 ,7,8,12,13 ,17,18-oct amethylporphyrin),4 TPP (5,10,15 ,20-tetraphenylporphyrin),5 and OEP (2,3,7,8,12,13,17,18-octaethylporphyrin),6 are the corner stones of modern porphyrin chemistry. Porphyrins have been employed by chemists to mimic biological reactions both in the interests of understanding these reactions in biological systems and of utilizing these reactions for chemical industry. Porphyrin compounds have been studied in clinical chemistry related to porphyrias, and as anti-cancer drugs for photodynamic  I  Chapter 1 Introduction  therapy. The chemistry of porphyrins has been well reviewed in the seven-volume series entitled "The Porphyrins", edited by Dolphin.7 1.2 Nomenclature and abbreviations of porphyrins used in this thesis  The nomenclature of porphyrins is described by Bonnett.8 Figure 1.1 shows the porphyrin core and the numbers of the carbon atoms which indicate the substitution positions.  13 15 17  2^20^18  Figure 1.1. The porphyrin core.  Synthetic porphyrins, such as TPP, OMP and OEP, are named according to this system. Besides the systematic numbering of the carbon atoms in a porphyrin core, the four carbon atoms between the pyrrole structures, which are carbons 5,10;15, and 20 are also said to be in the meso-positions. Abbreviations and the names of the porphyrins involved in this thesis are listed in the List of Abbreviations. These abbreviations are derived according to the systematic names, similarly to the way that TPP, OMP and OEP are derived, but in order to differentiate phenyl group and porphyrin, Ph is used for phenyl, and P is used for porphyrin; to differentiate tri or tris and tetra or tetralds, T is used for tri or tris, and Tet is used for tetra or tetrakis. The other abbreviations used are B for bis, D for di. 2  Chapter 1 Introduction  Abbreviations used for the substituents, and the numbering in the substituents are listed in Table 1.1. Table 1.1. Abbreviations of porphyrin substituents abbreviation  name  APh  4-aminoaieny1  MPy NPh  4-methylpyridinium 4-nitrophenyl  structure _  2- 3 \  —1 , —1\  6- 5  , ,  4 - NH2  2- 3  CH3  62- 3  ,  4 - NO2  6- 5  Py SPh  4-pyridyl 4-sulfonatophenyl  _  2- 3  ,  6- 5  _  —1 ,  6 -5  , ,  —  SO3-  Thus, 5,10,15,20-tetraphenylporphyrin (abbreviated as TPP in the literature) is abbreviated as TetPhP; 5-(4-nitropheny1)-10,15,20-trip_heny1porphyrin is abbreviated as (NPh)TPhP; 5,10,15,20-tetrakis-(4-methylpyridinium)porphyrin chloride is abbreviated as Tet(MPy)PC14 or as Tet(MPy)P when the emphasis is on the porphyrin (as with the other cationic porphyrins). In this thesis, Tet(SPh)P and PhT(SPh)P are used to represent the two porphyrins, 5,10,15,20-tetralcis(4-sulfonatoaienypporphyrin, and 5-pheny1-10,15,20tris(4-sulfonatophenyl)porphyrin, respectively, which are usually present as sodium salts; the cations are omitted when the emphasis is on the porphyrin (as with the other sulfonated porphyrins). In the literature,9,1°,11 TPPS, TPPS-4, TPPS4 or TSPP have been used for the tetra-sulfonated derivative, and TPPS-3 for the tri-sulfonated derivative. In this thesis, the abbreviations listed for the porphyrins represent the porphyrin free-bases, but the abbreviations are also used as the porphyrin dianion for  3  Chapter I Introduction  metalloporphyrins. This is commonly done in the literature'," and is illustrated by the following figure.  TetPhP  ^  M(TetPhP)  Trivial names are used for natural porphyrins. Structures of two natural porphyrins, protoporphyrin IX which is found in both hemoglobin and myoglobin, and hematoporphyrin, of which the derivatives are used as photo sensitizers for photodynamic therapy for cancer (Section 1.3), are shown below.  H2C=CH^CH3^ CH2 CH3  ^  I  CH^CH3  HO-CHCH 3^CH3 ^ NH  OH I CHCH3  \^/ HN C H3 ^\  CH3  CH2CH2C 02^C H2 C H2 C 02  protoporphyrin IX  ^  \  /  /  CH3  C H2 C H2 C 02^C H2 C H2 C 02  hematoporphyrin  4  Chapter 1 Introduction  1.3 Porphyrin and cancer 1.3.1 Tumor localization of porphyrins About half a century ago, Auler and Banzer12 examined the in vivo distribution of porphyrins in tumor-bearing animals and demonstrated accumulation of the porphyrins in tumors by fluorescence. Lipson et al. synthesized hematoporphyrin derivatives (HPD) in 1961 and initiated the use of this drug in photodynamic therapy (PDT).13 The selective accumulation of this porphyrin at the tumor became widely accepted and has been studied by many different experimental techniques." These experiments have shown that in animal models selectivity toward some tumors, compared to surrounding muscle tissue, does exist, although the uptake of this porphyrin product in tumor is lower than the uptake in some tissues including the kidney and the liver.14a,15 The high uptakes by kidney and liver is considered not crucial for the therapy because light received by these tissues is negligible, and the drug is basically non-toxic in the dark.16 The mechanism of the selective retention of this porphyrin product in different tissues, especially in tumors, is still not clear. 16 Synthetic porphyrin compounds have also been studied from the aspect of selective tumor accumulation. For example, it has been shown that Tet(SPh)P has an approximately 50-fold superiority over HPD as a tumor localizer." Other porphyrins, including 5, 10,15,20-tetralds(3-hydroxyphenyl)porphyrin and 5, 10,15,20-tetralds(4hydroxyphenyl)porphyrin (structures are shown later in Section 1.6.2) have been shown to have a good selectivity for tumor relative to muscle and skin." Levels of radiocopperlabeled 5,10,14,20-tetralds(4-carboxyphenyl)porphyrinatocopper(11) were reported to be 2 to 3 times higher in tumor than in skin, blood, fat and muscle.19  5  Chapter I Introduction  1.3.2 Porphyrins used in PDT (photodynamic therapy) PDT, a therapeutic method to treat tumors, requires a photosensitizing drug and a source of drug activation using light." The main toxic species is believed to be 102 produced by the light in the presence of the drug.21 Photofrin II, a purified form of hematoporphyrin derivatives (HPD), is currently the most widely used photosensitizer in PDT.22 The drug is a mixture of a few porphyrin compounds, the structures of which are still not clear.22 HPD has been studied as a photosensitizer for more than 15 years. PDT, which initially used this drug, seems very promising, but low absorption by this drug of red light, which is favored clinically vs. light with short wavelengths (for better penetration into the tumor), and the complex composition of the drug make the application limited.22 Many new drugs, based on benzoporphyrin derivatives (BPD), sulfonated derivatives of phthalocyanines and chlorin compounds, are under development.22 The attractive properties of these compounds are localization at the tumor, low toxicities, fluorescence, and photodynamic activities.22 Although work in developing new photosensitizers has been directed mainly at improvement of the absorption of light at longer wave lengths, other factors, especially high selectivities and efficacy, are also important and should also be considered.23 In animal models, a synthetic porphyrin Tet(SPh)P has been reported to have preferential uptake by some tumors;17 another synthetic porphyrin has been reported to be 25-30 times more efficient than HPD." Thus, a porphyrin designed with desired functional groups, charge and symmetry, could exhibit better photosensitizing abilities and accumulation at tumor sites than I-IPD, and these are the rationales for studying the chemistry of porphyrins as photosensitizers. In addition, chlorin compounds, which strongly absorb red light, can be easily made from porphyrins.23 6  Chapter 1 Introduction  1.3.3 Porphyrins used in tumor detection  Although HPD shows some promise for the detection of tumors based on the greater uptake in tumors and fluorescent properties, the phototoxicity of this drug is a problem in diagnosis. A non-toxic, less phototoxic, and more fluorescent drug would be preferable.24 The development of such new drugs, probably porphyrins or related compounds, is likely to depend on synthesis of new porphyrins. Motivated by the accumulation of porphyrin compounds at tumors, researchers have used manganese(III) complexes of porphyrins as contrast agents for magnetic resonance imaging (MRI), by employing the paramagnetic properties, the low phototoxicity (transition-metalloporphyrins are usually not photoactive), and tumor localization of these compounds. The most used compound in these studies is Mn[Tet(SPh)P].25 With this agent, the detection of tumors by MRI can be effectively enhanced.25 The Mn[PhT(SPh)P] complex is reported to be a better contrast agent than Mn[Tet(SPh)P].26 1.3.4 Porphyrins used in chemotherapy  Porphyrins are also of interest as tumor localizing agents for boron neutron capture therapy (BNCT) and chemotherapy. BNCT is a potential therapeutic technique for cancer treatment using a 1°B-containing drug which can accumulate in a tumor and the 1°B can be "activated" by thermal neutron radiation. The substantial advantage of BNCT over PDT is that neutrons have far greater penetrability than photons and thus can be used to treat deep-seated malignant cells.27 Boronated porphyrins referred to as SBK-II and BOPP (both contain a carborane linked to natural porphyrins) appear to be promising drugs.28  7  Chapter! Introducdon  Porphyrins containing the well-known chemotherapeutic drugs, 5-fluorouracil Figure 1.2a),29 dichloroethylenediamineplatinum(II) (Figure 1.2b)29 and bleomycin (Figure 1 .2c shows one example of a related compound)3° have been synthesized for use as anticancer agents.  b  a^  Na03S  Na0 S  SO Na  ^  SO Na  C  , CH3 / N4' OAc  /  (CH2)40^\\  —N+ h \  /—\\N+ —CH3  —/  \^H3C CH3  Figure 1.2. Porphyrins containing anti-cancer agents.  1.3.5 Porphyrins as radiosensitizers Compared to use in PDT, porphyrins have not been as well studied as radiosensitizers. Although the tumor selectivity of porphyrins has drawn the attention of radiobiologists, early studies on natural porphyrin free-bases suggested their use as radiosensitizers was not promising.31332  8  Chapter 1 Introduction  Based on the findings in various laboratories on the importance of the presence of a nitro group in a radiosensitizer (Section 1.4), the present thesis work focusing on synthesis of nitro-substituted porphyrins was initiated. During this thesis work, some encouraging results of synthetic porphyrins as radiosensitizers were published by O'Hara and coworkers.1° In this report, synthetic instead of natural porphyrins and metalloporphyrins were tested. The compounds tested were either cationic or anionic porphyrin free-bases [Tet(MPy)P, Tet(tMAP)P, Tet(SPh)P, Tet(CPh)P, Tet(SPhPh)P; Figure 1.31, and some of their complexes with metal ions [CoFeM, Cull, Pd", RhX, SnIV].  Tet(MPOP  Tet(tMAPh)P Tet(SPh)P  Tet(CPh)P  SOf^Tel(SPhPOP  tMAPh = 4-trimethylammoniumRhenyl CPh = 4-carboxylatoRhenyl SPhPh = 4-(4-sulfonatoRhenyl)Rhenyl  Figure 1.3. Structures of some water-soluble porphyrins.  9  Chapter 1 Introduction  Assessment as radiosensitizers was done in Chinese hamster fibroblast (V79N) cells. All the free-base porphyrins tested had essentially no sensitization in this system. The greatest effects on radiation-induced cell kill were achieved with the metalloporphyrins, labeled [Coliffet(MPy)1]C15 and Na3[Colli1'et(SPh)P]. It should be noted that porphyrins are also of interest in AIDS research.33  1.4 Radiosensitizers Radiotherapy is an important form of treatment for cancer. The radioresistance of some tumor cells is the main limitation of this therapy. The very efficient cell proliferation associated with malignant tumor growth reduces oxygen concentration, especially in the areas away from vascular capillaries, and this is believed to be one cause of the radioresistance.34 One approach for overcoming radioresistance of hypoxic (02-deficient) cells is the use of chemical radiosensitizers. A radiosensitizer is a drug which can enhance radiation kill of the tumor, especially for hypoxic cells. There are findings that sensitizers operate via several mechanisms besides the oxygen-mimicking mechanism which is derived from early thoughts about oxygen sensitization (see below). Thiol suppression, DNA-repair suppression and effects on DNA conformation are all important in the cell-killing processes related to ionizing radiation, and have been exploited in the design of radiosensitizers.35 Glutathione (GSH) in cells protects them from radiation. Drugs bound to glutathione or inhibiting its synthesis can enhance the killing effect of radiation.36,37 DNA molecules damaged by radiation can be repaired in the cells. Drugs such as platinum complexes, which can bind to DNA and may inhibit such repair processes, can also sensitize cells, particularly at low doses of radiation.38 This thesis work focuses upon oxygen-mimicking radiosensitization, which is described below (Sections 1.4.1-1.4.4). 10  chapter 1 Introduction  1.4.1 Mechanisms  When irradiation is applied to a biological molecule such as DNA, a series of reactions can occur, as shown in Figure 1.4.39,413 The radiation produces a charge separation, and the electron migrates to an "electron-affinic" site on the molecule and forms a dipolar molecule. The degree of damage due to radiation is related to the competition between ionization and charge recombination of the dipole molecule. The damage is fixed by the process where a proton dissociates from the cation to give a free radical. If a sensitizer S, which is an "electron-affinic" compound (dioxygen or its mimicking compound) is present, it can react with the dipolar molecule to give a cationic radical, and this increases the damage. This "free radical mechanism" proposed by Adam's group39'4° is supported by the results of ESR studies on the free radicals generated by fast mixing pulse radiolysis. 41,42 There are other possible mechanisms, such as the indirect mechanism, in which water is first excited to produce the damaging free radical species (such as .0H), and the presence of sensitizer then either makes more free radicals available to react with the target molecules or enhances the damage produced by the radicals.43 The sensitizer (e.g. 02) is also possibly involved in reaction with the biological free radicals produced by radiation (either through direct or indirect mechanism), and this can further fix the damage.44 charge recombination  irradiation  ionization  sensitization  S+  e— (og)  H (aq) free radical  Figure 1.4. A proposed sensitization mechanism (direct, S = radiosensitizer). 11  Chapter 1 Introduction  1.4.2 Properties of radiosensitizers  One of the criteria for effectiveness of radio sensitizers is the SER value (sensitizer enhancement ratio), which is defined as the ratio of radiation doses required to produce a given effect in the absence and presence of the drug: SER —  dose without drug for equal effect. dose with drug ,  The SER for dioxygen is called the OER (oxygen enhancement ratio). A high SER for a drug at a clinically achievable dose is desirable, but it does not have to be as high as the OER value (typically about 3 for mammalian cell lines at 1% survival).45 "Electron affinity", referring to the ability of a compound to mimic the sensitizing action of oxygen by accepting electrons, is an important property of a radiosensitizer as showed by the proposed sensitization mechanism in Section 1.4.1.46 Of the radiosensitizers developed, the majority are nitro-aromatic compounds which do have such electron-accepting properties.46 The ability to accumulate in a tumor, although not essential for a radiosensitizer, is a property which should be considered in the development of a new drug.35 It will obviously help to increase tumor kill and reduce side-effects such as toxicity and sensitization of normal tissues. The partition property of a drug, related to lipophilicity and solubility in water, can affect the distribution of the drug in the body, tumor selectivity, the ability to penetrate into the center of the tumor, and the ability to enter the cells. These properties in turn can affect the sensitivity to radiation and toxicity. 47,48 Water-soluble drugs are easier to manipulate in treatments and laboratory assessment. The ability of a drug to "target" DNA (or other important molecules in the cells) may play a role in radiosensitization, which depends on the concentration of the drug in the area adjacent to the target molecules. If a drug can be targeted to the required 12  Chapter 1 Introduction  molecules, then a lower drug dose can be used. Thus side-effects can be reduced. Metal complexes have been used as the carriers to target radiosensitizers to DNA.49,5° Besides the use of metal complexes to target DNA, metal-containing drugs which have crosslinking abilities and possibly inhibit the DNA repair, may also be of clinical benefit.38 Although there are insufficient data to show how molecular symmetry of a drug is related to the drug's ability to associate with a specific biomolecule (which is always asymmetric) and to the drug's selectivities to types of tumors, symmetry must play a role in the selectivity of a drug toward biological targets. Other properties such as low toxicity, stability under clinical conditions, and ease of synthesis, are also obviously important. 1.4.3 Nitroimidazole radiosensitizers One of the first drugs used in clinical trials was misonidazole (Figure 1.5), which was found to be an effective radiosensitizer,51 but results have shown the use of this drug is limited by its neurotoxicity.35 This may be related to the lipophilic properties of the materia1.52,53 Compounds potentially superior to misonidazole have since been designed, synthesized and tested." Some examples are shown in Figure 1.5. Pimonidazole (Ro-03-8799) developed in 1980 is slightly more efficient and less toxic than misonidazole.54 A promising aspect of this drug is that it shows a significant degree of selectivity toward human tumor tissues due to accumulation in low pH environments.55,56 However, clinical trials have been discontinued due to its side effects. Etanidazole (SR-2508) with a peptide-containing side group to change lipophilicity, and decrease neurotoxicity, is showing some promising effects in on-going clinical trials.57 Related to the development of radiosensitizers, drugs aiming to kill hypoxic cells in tumors are also under study. Recently, many such bioreductive drugs (more toxic in 13  ^  Chapter 1 Introduction  hypoxic conditions than in oxic conditions) have been developed. These drugs are reduced into more toxic forms via metabolic processes in hypoxic cells." Some dual functional radiosensitizers have also been developed. These combine radiation sensitizing and alkylating properties in the same structure. One example of this category is RSU 1069, which combines a 2-nitroimidazole with an aziridine group; the latter reacts with DNA, phosphate or glutathione to form ring-opened products in the side-chain.59,60 In vitro and in vivo studies show that this drug is more toxic to hypoxic cells than to oxic cells (a good bioreductive agent), and more efficient than misonidazole as a radiosensitizer.6° Unfortunately, this drug is too toxic for clinical use, but a second generation "prodrug" (RSU-6145, an analog with the aziridine ring open) is promising.61  ^A^?H  ^\^OH NCH2CHCH2OCH3  NCH2CHCH2N\^  NO2  ^  NO2  Pimonidazole(Ro-03-8799)  Misonidazole  I^1^  OH  0  I NC H 2C N HC H 2C H 20H  ^\  N  NO2  NO2  Etanidazole(SR-2508)  ^  OH  N 02  RSU-1069^  RSU4145  Figure 1.5. Structures of some radiosensitizers. All the drugs mentioned thus far in this section are nitroimidazole compounds, and  most radiosensitizers fall into the category of nitro-aromatics. This class of compounds, as mentioned above, is also being evaluated as hypoxic cytotoxins (bioreductive agents).62 Besides nitro-aromatics, metal-containing complexes as radiosensitizers are of special interest to this thesis work. Cis-DDP [cis-diamminedichloroplatinum(11)] has been 14  Chapter 1 Introduction  known since 1969 to have anti-tumor activity,63 and it was shown(e.g.64) that platinum complexes bind to DNA. The DNA binding abilities of platinum complexes suggested a rationale for the use of such species as radiosensitizers, and indeed Richmond and Powers reported in 1978 that cis DDP sensitized the spores of Bacillus megaterium to radiation.65 -  Since this time, many platinum complexes have been studied as radiosensitizers with different systems.38,48 The mechanism of the sensitization is still not clear: it could occur by inhibition of DNA repair through the binding of the complexes to DNA,66 or by the reaction of the complexes with radiation protectors present in biological systems.67 Another example of a metal-containing sensitizer is cis,cis,cis-RuC12(DMS0)2(4nitroimidazole)2, which was studied with the rationale of using the DNA binding abilities of such a metal complex to lead a radiosensitizer to a DNA site, and thereby increase the efficiency.5° Indeed this compound has a higher SER and lower toxicity in a CHO (Chinese hamster ovary) cell line than the sensitizer ligand 4-nitroimidazole.5° 1.5 Bioreductive agents As described in the last section, some nitro-imidazoles have been found to be selectively toxic to hypoxic cells. Other nitro-aromatics have also been found to have similar properties. An example of this class of compounds is nitracrine (figure below),62 a nitro derivative of acridine. This compound possesses high association constants for DNA, and has potential to accept electrons (due to the nitro group), thus, providing properties as bioreductive reagent. It has been found to be selectively toxic to hypoxic cells68 and sensitizing hypoxic cells to radiation.69 /- \  (H3C)2N^NH NO2  N  structure of nitracrine 15  Chapter 1 Introduction  1.6 The design of porphyrins as anti-cancer agents  Most anti-cancer drugs are toxic to both the tumor and the surrounding tissues, and sometimes even more toxic to the normal tissues:70,7i These problems may result in side-effects or even induce secondary malignancies.72 Solutions may lie in the use of drugs which preferentially accumulate in tumors (or hypoxic cells in tumors), and then sensitize tumor cells to radiation efficiently (as radiosensitizers) or are toxic to these cells (hypo)dc cytotoxins). There is a strong motivation to develop such drugs using porphyrin compounds because these compounds are known to have low toxicity, 10,72 to accumulate in tumor,9,24,17 and to sensitize cells to ionizing radiationlo (Section 1.3). Besides these properties, porphyrin compounds bind to DNA,73,74 and can be modified into nitroaromatic compounds which are found to be radiosensitizers and bioreductive agents (Sections 1.4 and 1.5). Porphyrins offer considerable flexibility in their organic and inorganic chemistry so that they can be modified in order to build-in some desirable functionalities. The idea for this thesis project was that specially designed porphyrin compounds could have useful anti-cancer properties. To design porphyrin compounds as radiosensitizers, several factors have to be considered, and these are discussed below. Although the design of porphyrin radiosensitizers is the major topic of this chapter, most of these considerations would also be relevant to the design of bioreductive agents and other anticancer agents. 1.6.1 Electron affinity  Researchers who are developing radiosensitizers and bioreductive agents based on nitro-aromatics, especially nitro-heterocyclic compounds, consider that such compounds have high electron affinities. 46 In this respect then, nitro groups could be introduced into the porphyrin structures (which are heterocyclic aromatics) in order to contribute a  16  Chapter 1 Introduction  reducible function which should increase the radiosensitizing activity of the materials. Although some nitro-porphyrins are known,75,76 they are not soluble in water. Thus an obvious aim was to synthesize water-soluble porphyrins containing nitro groups. Introducing metal ions into porphyrin systems is another way to introduce a reducible function in order to increase electron affinity.10 In this case, transition-metal ions with higher oxidation states should be considered, e.g. Co(III), Fe(III) and Mn(III), but other metal ions could also be considered; e.g., Zn[Tet(MPy)P] has been reported to have a relatively high SER.10 1.6.2 Selectivity toward tumors  Changes in porphyrin structures can affect the tumor-accumulating property of the drugs. This was first demonstrated by Winkelman17 with the comparison of the tumoraccumulation abilities of HPD and Tet(SPh)P. It was found that the ratio of HPD concentration in tumor and in muscle was less than 2, and that ratio of Tet(SPh)P was about 7. Studies on tetrahydroxophenylporphyrins (Figure 1.6)1' also demonstrated the effect of changes in porphyrin structures on tumor-accumulating property. The para- and meta-derivatives were found to have better selectivity than HPD (for accumulation at tumor vs. skin, which is very important for PDT therapy). The meta-derivatives were also found to be much more potent than HPD.18 Another example of the effects of porphyrin structure on drug distribution was reported in 1987 by Kessel and coworkers9, who determined, with in vitro studies, that sulfonated derivatives of TetPhP [TetP(SPh)P, cisDPhB(SPh)P, trans-DPhB(SPh)P, PhT(SPh)P, Tet(SPh)P] (the structures are shown in Figure 1.7), had different sites of photosensitization. The most lipophilic drug of the five derivatives, TPh(SPh)P, catalyzed lethal photodatnage mainly at intracellular loci, while the major phototoxic effect of the other drugs occurred at cell membranes. Studies of tumor localization of these drugs in vivo showed that they all had tumor preference, but 17  Chapter 1 Introduction  PhT(SPh)P and cis-DPhB(SPh)P were partitioned into neoplastic cells while the others were partitioned into tumor stroma. The efficiencies of photosensitization were also different for various porphyrins. Similar phenomena were found to be the case for sulfonated derivatives of phthalocyanines.77 The existing data are insufficient to demonstrate which structural changes would result in better selectivity, but the data have indicated generally that selectivity changes on modification of the structures. A balance of hydrophilic and lipophilic character, the symmetry of the structure, and the properties of the substituents, are all considered to play a role in selectivity. OH^para-derivative NH^N  meta-derivative OH R=  ortho-derivative HO  Figure 1.6. Structures of tetrahydroxophenylporphyrins.  R10  NH^N R5 HN  R20  R5 = RI 0 = Ri5 = Ph^R20 = SPh^ TPh(SPh)P R5 = R10 = Ph^R15 = R20 = SPh^cis-DPhB(SPh)P R5 = R15 = Ph^R10 = R20 = SPh^trans-DPhB(SPh)P R5 = Ph^Rio = Ri5 = R20 = SPh^PhT(SPh)P R5 = R10 = R15 = R20 = SPh^Tet(SPh)P  Figure 1.7. Structures of sulfonated derivatives of TetPhP. 18  Chapter 1 Introduction  1.6.3 Cell uptake  It is generally believed that radiosensitizers have to enter cells in order to be effective.74 Thus the ability for a drug to be absorbed by cells needs consideration. Accumulation of a drug in cells may relate to lipophilicity of the drug, which may be varied by introduction of substituents with charges or higher polarity (vs. non-polar substutuents). 1.6.4 DNA-binding  DNA molecules are the main targets of radiation damage,78 and thus the efficiency of radiosensitizers could be increased by improving their DNA-binding abilities. Poiphyrins are known to be DNA binders, and the binding models and the relationships of structures with the binding models have been reviewed.73,79 In the design of porphyrins with improved DNA-binding abilities, molecular symmetry and presence of groups with capacity to form a hydrogen-bond, such as -NH2, -NO2, -OH and pyridyl, should be considered. 1.6.5 Solubility and lipophilicity  The porphyrin core under neutral conditions is essentially a lipophilic structure. To make water-soluble porphyrins, ionic substituents have to be introduced, and the ones that can be easily introduced are methylpyridinium, trimethylphenylaminium, phenylsulfonate, and phenylcarbonate. When increasing water-solubility of a porphyrin structure, its lipophilicity has also to be considered, because this plays a key role in cell uptake.9 Thus the water-solubility and lipophicility should be "balanced". From this point of view, the lipophicilities of the well-known water-soluble porphyrins, such as Tet(MPy)P, Tet(SPh)P (see Figure 1.3 for the structures) are probably too low, given the presence of four ionic  19  Chapter 1 Introduction  groups. Fewer than four ionic groups in a structure of a porphyrin is suggested to be one of the criteria for design of porphyrins as radiosensitizers. 1.6.6 Multiple functions To synthesize porphyrins for use in radiosensitization, all of the factors discussed above and some others (such as stability, toxicity, economy, etc.) have to be considered. One potential approach was to introduce several different functional groups into one structure, in which the functions might be "balanced" (e.g water solubility vs. lipophilicity), although it was not clear which structure would be most beneficial. This suggested an approach in which a large number of porphyrins was to be synthesized in order to find an effective drug. Thus the methodology employed to synthesize the porphyrins had to be simple yet powerful enough to make a large variety of products. 1.7 Porphyrin syntheses  The synthesis of porphyrin free-bases has been well reviewed in various publications.80,81 The most convenient synthesis pathway is the monopyrrole-condensation method, which often gives symmetric products. The synthesis of TetPhP5 is an example: CHO +4  3/202  + 7H20  Many other symmetric porphyrins with four substituents of the same kind are also made with this method,82 including the so called "picket fence" porphyrins which were designed to mimic the bulky protein structure surrounding the heme active center (Figure 1 .8a).83  20  Chapter 1 Introducdon  To make a porphyrin with two different aryl substituents at the four mesopositions with this method requires the use of two different aldehydes. Such a synthesis can lead to 6 different porphyrin products. This so called mixed aldehyde approach was first developed by Little et al." Since then (1975), many interesting unsymmetric porphyrins have been made.85,86 These include: the "looping over" porphyrins with a sidearm providing a fifth intramolecular coordinating ligand (Figure 1.8b), in order to mimic deoxy-hemoglobin and -myoglobin in which iron has a fifth coordination site occupied by an imidazole of a histidine amino acid residue of the protein chain," a Zn complex of 5, 1 0, 1 5-tris(4-carboxylatopheny1)-20-(44 1 -bromobutylpyridinium))porphyrin (Figure 1.8c) which has been used in a study of redox systems for photochemical utilization of solar energy;88 a bifunctional metalloporphyrin (Figure 1.8d) which has been made from 5,10,15-tris(4-toly1)-20-(4-pyridyl)porphyrin, with the pyridyl coordinated to a Ru complex moiety in order to give a second redox-active center.89 Sari et al., using column chromatography, separated the 6 porphyrins shown in Figure 1.8e which were formed by condensation from a mixture of pyrrole, benzaldehyde and 4-pyridylcarboxaldehyde; the pyridyl nitrogen atoms were then methylated to make the porphyrins water-soluble in order to do DNA-binding studies." The details of this work were published during the course of this thesis work." The condensation of three different aldehydes with a pyrrole, in principle, could give a mixture of 21 porphyrins. The difficulty in the separation of such a mixture makes the method of little use for the synthesis of porphyrins with more than two different meso substituents. In fact, no porphyrins with three different meso substituents had been made prior to this thesis work.  21  Chapter 1 Introduction  a H3cy-13 c, C H3  CH3 CH3 H3C\^(-1.4 H3c ci H3^C O H3C `;,_, CH3 NH^ \ I^CH CO c-- 3 \VICO NH^Ico^/ /NH N  C02  CH3  C4 H8 Br  Riu (N H 3 )5 3 4"  1  R= R10 = R15 = R70 = Ph R5 =Rin=1115 =Ph R5 = R10 = Ph R5 =R15 =Ph R5 = Ph  R70 = Py R15 =R7.0 =Py R10 = R70 = Py R10 =R15 =R20=Py R5=R10=R15=R20=Py  TetPhP TPhPyP cis-DPILBPyP trans DPhBPyP PhTPyP TetPyP -  Figure 1.8. Structure of some synthetic porphyrins.  22  Chapter I Introduction  Other methods of porphyrin synthesis, such as the coupling of dipyrroles or cyclization of a tetrapyrrole, are for making porphyrins with more complicated structures," but the difficulties in preparing the intermediates make the methods generally less useful for the concerned purpose. To make a large variety of porphyrins especially with different substituents, two methods are used in this thesis work: (a) monopyffole-condensation and (b) direct modification on an isolated porphyrin. The latter method introduces a functional group into a synthesized porphyrin structure. Sulfonation of TetPhP is an example of a modification, which converts a non water-insoluble porphyrin (TetPhP) into a watersoluble one [Tet(SPh)P].92 Modification can be done in a controlled manner, and different porphyrins can be synthesized at different stages of modification. For example, the sulfonation of TetPhP can be stopped at one, two, three or four sulfonations; with proper purification procedures, all 5 possible sulfonation products [TPh(SPh)P, cis-DPhB(SPh)P,  trans-DPhB(SPh)P, PhT(SPh)P and Tet(SPh)P; see Figure 1.7 for structures] can be made, and the 5 porphyrins indeed have different biological activities.9 Nitration is another example of such modification. Although TetPhP has been known for many years, its nitration was reported only recently.76 According to this report, nitration of TetPhP yielded (NPh)TPhP as the main product. Reduction of the nitro group into amine, as a second modification, can be done after nitration, in order to synthesize a porphyrin with a peripheral amine.76,93 A third modification, such as sulfonation, can then be undertaken to make the amine-containing, water-soluble porphyrin (Figure 1.9).76 Although there are some examples of modifications on synthetic porphyrins as mentioned above, modification as a method to produce a large variety of porphyrins had not been well developed before this project. Many more porphyrins can been synthesized using the methodology of combining monopyrrole-condensation with subsequent  23  Chapter 1 Introduction  modification, as described herein. A small fraction of possibilities was chosen as the focus of this thesis. Modifications of porphyrins from many possible starting materials, such as the 6 compounds made from the condensation of pyrrole, benzaldehyde and 4pyridylcarboxaldehyde, plus metallation can make a large variety of porphyrins with different "functional groups", and a major part of this project addresses the syntheses and characterizations of these porphyrins and metalloporphyrins. Figure 1.10 shows some of the possible modification schemes for one of the the six porphyrins addressed above, 5,10,15-tripheny1-20-(4-pyridyl)porphyrin (TPhPyP). Nitrations can been performed  NH^N N^HN  / /  0 -,,--^ TetPhP  0  NH^N  H2 SO4  \^HN \^/  NH2  0 y^(APh)TPhP NH2  Figure 1.9. Modifications of TetPhP. 24  Chapter 1 Introduction  also on two or all three of the phenyl groups of this porphyrin; these nitrations and the following reduction, sulfonation and methylation are not shown. A complete scheme of the modifications of the six porphyrins performed in this thesis work is presented in Figure 2.1, in Chapter 2. Metallation of these porphyrins is another area included in this project. The importance of introducing metals into radiosensitizers was previously discussed in Sections 1.3 and 1.4. The chemistry of some metalloporphyrins is discussed in Chapter 3.  1.8 In vitro studies on synthetic porphyrins  The term "in vitro" refers to "in cells" and the term "in vivo" refers "in animals" in this thesis, as conventionally used in areas related to this thesis work. The testing of a new drug occurs mainly in three stages, in vitro studies, in vivo studies, and clinical trials. In vitro studies are usually sensitive, rapid and economical, and they can be used to predict some properties of drugs. Prior to in vivo studies and clinical trials, which give better predictions for success but are more difficult to do and more expensive, it is useful to know how toxic a compound is toward a particular cell line, and how effectively the compound sensitizes these cells to radiation. The proliferative capacity of a cell is examined using survival curves are generally used to screen new compounds as bioreductive agents, radiosensitizers or photosensitizers. The principles and the methodology of using these assays to evaluate the toxicity (in oxic or hypoxic conditions), radiosensitization and photosensitization ability of a compound are introduced in Chapter 5.  25  Chapter 1 Introduction  NMe3+  cis-(MAP)(MPy)DPhP HNO3  t  ,N .......,, ,.....,7,-.... „,.. .  N  \_- NH  '.1■11-1^N /--- N^HN---\  \  \  [H ]  H2  N °2  1 cis-(NPh)DPhPyP  --■  cis-(APh)DPhPyP  ikH204  N.  -03S  NH^N  NH^N---/ /^  \^HN r--- NI \ //  NO2  0^cis-(NPh)PyB(SP)P SO3-  \ 7---- N^HN C  H  0^cis-(AP)PyB(SPh)P Y SO3-  Figure 1.10. Some possible modifications of TPhPyP.  26  Chapter 1 Introduction  1.9 The objectives of this thesis work  A limitation of radiation therapy is the lack of selectivity toward tumors, especially toward the hypoxic cells in a tumor. PDT has good selectivity toward tumors but has a limitation from poor tissue penetration of the light. The purpose of this project was to exploit the possibility of synthetic porphyrin drugs which accumulate in tumors and sensitize cells to radiation. Using this kind of drug, some limitations of radiation therapy and PDT can potentially be overcome. The synthetic chemistry and the porphyrins synthesized also may be beneficial to the eventual discovery of new drugs as photosensitizers, hypoxic cytotoxins or tumor imaging agents. This thesis work focuses mainly on the synthesis and characterization of some designed porphyrins and metalloporphyrins, although some preliminary in vitro studies of these compounds as radiosensitizers or photosensitizers are also presented. The work is described within the following aspects: 1. Syntheses and characterization of porphyrins with functional groups; modifications of the 6 porphyrins with general formula PhnPy(4..n)P (Chapter 2). 2. Chemistry of some metalloporphyrins with the selected metal ions (cobalt and copper) (Chapter 3). 3. Behavior of the synthesized porphyrins in aqueous solutions (Chapter 4).  4. In vitro studies: radiosensitization, photosensitization, and toxicity of the synthesized porphyrins and metalloporphyrins (Chapter 5). 5. 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Chem., 23, 2370 (1984). 90^M.A. Sari, J.P. Battioni, D. Mansuy and J.B. Le Pecq, Biochem. Biophys. Res. Commun., 141, 643 (1986).  91^M.A. Sari, J.P. Battioni, D. Mansuy and J.B. Le Pecq, Biochemistry, 29, 4205 (1990). 92^J. Winkelman, Cancer Res., 22, 589 (1962); T. S. Srivastava and M. Tsutsui, J. Org. Chem., 38, 2103 (1973). 93^E. Tsuchida, J. Macromol. Sci.-Chem., A13(4), 545 (1979).  34  Chapter 2 Synthesis of porphyrins  Chapter 2 The synthesis of the designed porphyrins 2.1 Introduction  The synthesis and characterization of porphyrin free-bases are the main subject of this chapter. Porphyrins are synthesized with the monopyrrole-condensation method and subsequent modifications as discussed in Section 1.7. Condensation of pyrrole, benzaldehyde and 4-pyridinecarboxaldehyde gives a mixture of six porphyrins with general formula of PhnPyo_roP. The mixture is separated by column chromatography. Subsequent modifications as required by the design of the structures (Chapter 1) of the resultant porphyrins are: nitration of phenyls; reduction of the nitro groups to form amines; sulfonation of phenyls; and methylation of pyridyls. The synthetic schemes of the designed poiphyrins are shown in Figure 2.1. The full names and the corresponding structures for the abbreviations given in Figure 2.1 can be found in the List of Abbreviations. In this chapter, only the porphyrin free-bases are involved, so the abbreviations of porphyrins are used to represent the porphyrin free-bases. Purification of non-water-soluble porphyrins was mainly performed with column chromatography. For water-soluble porphyrins, dialysis was employed as the major purification process. Details can be found in the experimental section. Proton NMR spectroscopy was the main method used for characterization of porphyrins, especially for distinguishing isomers. Elemental analysis and UV-visible spectroscopy were also used to characterize all the porphyrins. Further characterization was carried out using mass and infrared spectroscopies for some of the porphyrins, and the data are presented in Sections 2.3.4 and 2.3.5. Aggregation has been found to be a general property of all water-soluble porphyrins, and is discussed in Chapter 4.  35  Chapter 2 Syntheds of porphyrins  ^• (NPh)TPhP^(APh)TPhP^PPOT(SPOP  TetPhP  ^ c-B(NPh)DPhP- -111- c-B(APh)DPhP^c-BPPWSPOP t-B(NPh)DPhP-^t-F3(APh)DPhP ^1-09910SPh,  ^ T(NPh)PhP^T(APh)PhP I""  TPhPyP  Tel(SPOP  c-(NPh)DPhPyP^c-(APh)DPhPyP c-B(NPh)PhPyP I^  c (/P03,9(5P47  t-B(NPh)PhPyP^t-B(APh)PhPyP T(NPh)PyP  PyISPOP c-DPhBPyP  c-(NPh)Ph3PyP^c-(APh)PhRPyP c-B(NPh)BPyP  c-B(44)8(VPOP  ^  c-85,8(SPOP ^ c-8400,0P4I) t-DPh8PyP^  1-8(4110,90POP  t-B(NPh)RPyP  1-8Py81SPO9 ^ /-80/9)DPhP PhTPyP  (NPh)WyP  1404,02.h.,1°  Iriii/9/)PhP TetPyP ^  •  7009y)P  Figure 2.1. Synthetic schemes for designed porphyrins  1^water-soluble porphyrin) (c = cis, t = trans, new_p_om1=,  36  Chapter 2 Synthesis of porphyrins  2.2 Experimental  2.2.1 Materials and method Pyrrole, benzaldehyde, 4-pyridinecarboxadehyde, methyl p-toluene sulfonate were purchased from Aldrich Chemical Company. Other chemicals (including, nitric acid, sulfuric acid, etc.) were purchased as analytical grade, and used without further purification. Anion (CO exchange resin (Amberlite IRA-402) was purchased from BDH. Molecular porous membrane tubing (molecular weight cut off = 1,000) was purchased from Spectra/Por. Alumina activity I (neutral) was purchased from Fisher Chemicals; alumina activity III and IV were obtained by mixing activity I with 10 % and 15 % water, respectively, 24 h before using. Silica gel was purchased from BDH. TLC plates (0.2 cm) of both alumina (neutral, activity I, alumina TLC plates stored in air were saturated with moisture and had lower activity, approximately activity HI) and silica gel were purchased from BDH. NMR spectra were recorded on a Varian XL-300 or a Bruker WH-400 instrument operating in the Fourier transform mode, using TMS (in CDC13 and DMSO-d6) or DDS (in D20) as reference. UV-visible spectra were recorded on a Perkin Elmer 552A spectrometer, with quartz cells (0.100 or 1.00 cm). Mass spectroscopy (El ; NH3 chemical ionization and FAB techniques) were performed by the mass spectroscopy laboratory, department of chemistry, University of British Columbia. Infrared spectra (Nujol mulls) were recorded on a Nicolet 5DX FT1R spectrometer. Microanalyses were performed by Mr. P. Borda of the department of chemistry, University of British Columbia. Dialysis was carried out in a 1000 tnL beaker. A piece (-10 cm long) of molecular porous membrane tubing (3 cm wide) was rinsed with distilled water, then one end of it  37  Chapter 2 Synthesis of porphyrins  was closed with a piece of string. The porphyrin sample was dissolved in a minimum amount of distilled water (2 - 10 mL), and the resultant solution was transferred into the tubing; the other end of the tubing was then closed with string. This dialysis "bag" was then put into the beaker containing distilled water (-800 mL) and the contents were stirred with a magnetic stir bar. The water outside the dialysis bag was changed to distilled water 2 h later, then dialysis was continued for another 2 h. After dialysis, the bag was cut open, the solution in the bag was transferred into a flask and evaporated to dryness on a Rotovap. Acetone was then added to the residue, which was then scraped off the flask wall; the suspension was filtered and the solid was collected and 6r-dried. 2.2.2 The porphyrins with a general formula of PhnPyo-oP These poiphyrins, comprising TetPhP, T'PhPyP, cis-DPhBPyP, trans-DPhBPyP, PhTPyP and TetPyP (see Figure 2.1), have four meso substituents which are phenyl and/or pyridyl. The porphyrins were synthesized from condensation of pyrrole, benzaldehyde and 4-pyridinecarboxaldehyde (2:1:1) as described generally by Little et aLl Benzaldehyde (3.0 mL), 4-pyridinecarboxaldehyde (4.0 mL) and pyrrole (4.4 mL) were added to hot propionic acid (250 mL) in a 500 mL round-bottom flask, and the mixture was refluxed for 1 h. A precipitate (0.55 g) was filtered off from the cooled down reaction mixture. The filtrate was concentrated on a Rotovap to about 50 mL and then acetone (500 mL) was added with stirring; the mixture was filtered to yield another 1.78 g precipitate, which was combined with the precipitate collected earlier. A portion of this crude product (0.5 g), which was a mixture of the 6 porphyrins, was dissolved in CHC13 (180 mL) and loaded on a column (5 x 30 cm) of silica gel. The solution of TetPhP and TPITyP was eluted off with CHC13 (— 1.5 L) successively; the mixture of the other four porphyrins was eluted off from this column using a mixed solvent (CHCI3 and methanol 9:1). This solution of the porphyrin mixture was evaporated to dryness, and then the residue was dissolved with a  38  Chapter 2 Synthesis of porphyrins  minimum volume of CHC13 (— 150 mL), loaded on a column (3 x 70 cm) of alumina activity III, and eluted with CHC13 (— 2 L). Four purple bands appeared on the column. Trans-DPIIBPyP was first eluted from the column, then the solvent in the column was forced out under a pressure of nitrogen, and the bands were removed using a spatula ("digging out" the bands saved time and eluant solvent). The portions of alumina containing porphyrin were washed on fret filters using a mixed solvent to dissolve the porphyrins (10% Me0H in CHC13 , ca. 0.2 L for each portion) . The bands in order from top to bottom of the column contained TetPyP, PhTPyP and cis-DPhBPyP. All of the solutions of porphyrins were washed with 5% Na2CO3 solution and then twice with water; the organic phases were evaporated to dryness on a Rotovap. Acetone was added to the residues, and these were filtered and air-dried. TetPyP was also synthesized by the condensation of pyrrole and 4-pyridinecarboxaldehyde (1:1) in propionic acid as in the procedure described above. The main portion of the propionic acid having been evaporated off first, then the porphyrin product was precipitated by adding acetone; this precipitate was then filtered, washed with acetone and air-dried. Elemental analyses (for samples stored and weighed in air) are shown in Table 2.1. The yields, also shown in Table 2.1, were calculated based on the pyrrole reagent. Data from NMR and UV-visible spectra are shown in Tables 2.2 and 2.3. The hydration of some of the porphyrins is discussed in Section 2.3.1.1; the assignments of the NMR signals are discussed in Section 2.3.2. Spectral data from IR and mass spectroscopies are presented in Sections 2.3.4 and 2.3.5, respectively.  39  Chapter 2 Synthesis of porphyrins  Table 2.1. Elemental analyses of the PhnPy(4-n)P porphyrins  TetPhP  expected found  C (%) 85.99 85.86  H (%) 4.89 4.82  N (%) 9.12 8.95  TPhPyP  expected found  83.90 83.81  4.72 4.69  11.38 11.34  9.0  trans-DPhBPyP.H20  expected found  79.48 79.66  4.76 4.64  13.23 13.12  1.6  cis-DPhBPyP^a expected found  81.84 78.40  4.55 4.70  13.64 12.74  4.3  PhTPyP^a expected found  79.73 77.13  4.40 4.38  15.87 15.09  2.0  TetPyP^a expected found  77.67 76.55  4.23 4.88  18.12 16.45  0.7  TetPyP.1/2H20^b expected found  76.56 76.56  4.56 4.34  17.67 17.86  25.0  Yield (%) 7.5  a: Samples were contaminated by alumina, which was found to be slightly soluble in the mixed solvent (10% Me0H in CHC13). Thus, 250 mL of CHC13 was passed through a column of alumina; then 250 mL of mixed solvent was passed through this column, and the elute was evaporated to dryness in a flask on a Rotovap. A white residue was observed on the wall of the flask. b: Sample was synthesized from condensation of pyrrole and 4-pyridinecarboxaldehyde.  40  Chapter 2 Synthesis of porphyrins  Table 2.2. 11I-NMR data for the PhnPy(4-n)P porphyrins (a)  3,5-Py  pyrrole  3,4,5-Ph  2,6-Ph  TetPhP  8.85 s(8)  7.78 m(12)  8.23 m(8)  TPhPyP  8.90 d; 8.87 s  7.78 m(9)  8.23 m(6)  7.80m(6)  2,6-Py  N-pyrrole  -  -2.78 s(2)  9.04 q(2)  8.18 q(2)  -2.80 s(2)  8.22m(4)  9.05q(4)  8.18q(4)  -2.84s(2)  7.80m(6)  8.22m(4)  9.05q(4)  8.18q(4)  -2.84s(2)  7.81 m(3)  8.22 m(2)  9.06 m(6)  8.18 m(6)  -2.88 s(2)  -  9.08 q (8)  8.19q(8)  -2.92s(2)  -  8.81 d (2,4,2) trans  -  8.91^d;^8.81d  DPhBPyP  (4,4)  cis-  8.92 d; 8.88 s  DPhBPyP  8.85 s; 8.81 d  (2,2,2,2) PhTPyP  8.93 d; 8.86 s 8.83 d (2,4,2)  TetPyP  -  8.88s(8)  a: In CDC13 at room temperature; chemical shift in ppm signal pattern (number of protons). Table 2.3. IN-visible data for the PhnPY(4-0" porphyrins(1)  Soret  visible^1  visible 2  visible 3  visible 4  TetPhP  416.7 (427)  513.6 (17.7)  548.7 (7.6)  590.7 (6.8)  649.7 (6.2)  TPhPyP  416.5 (474)  512.9 (22.6)  548.3 (10.7) 588.5 (9.2)  645.5 (8.5)  trans-DPhBPyP  415.5 (350)  511.5 (16.2)  546.0 (6.6)  587.1 (5.6)  642.5 (3.3)  cis-DPhBPyP  11 416.0 (354)  512.5 (15.6)  546.5 (5.1)  588.5 (4.7) . 641.8 (2.0)  PhTPyP  11 415.7 (301)  511.6 (14.9)  545.1 (5.4)  588.2 (5.7)  641.8 (2.5)  TetPyP  415.0 (348)  510.4 (18.3)  542.8 (6.2)  588.0 (7.3)  642.0 (2.97)  a: In CHC13 at room temperature at 1 x 10-5M; wavelength at maximum absorbance (Xmax) in nm, (extinction coefficient [E x10-3, M-1 cm-11). b: Samples contaminated with alumina (footnote a Table 2.1). Thus, the E values are minimum ones.  41  Chapter 2 Synthesis of porphyrins  2.2.3 Nitrations  The nitration reactions were performed with concentrated nitric acid in Schlenk tubes. Generally, a porphyrin (0.2 - 0.5 g) was dissolved in, or mixed with, acetic acid (30 - 50 mL) in a Schlenk tube, which was placed in a cold water-bath. Sulfuric acid (10 rnL), which was used as dehydration reagent in some cases, was added dropwise with stirring. Nitric acid (10 - 20 mL) was then added dropwise with stirring. Then the mouth of the Schlenk tube was stoppered; the stopcock on the side-arm was left open to avoid build-up of pressure, and the water-bath was removed. The color of the reaction mixture changed from purple to green when either sulfuric acid or nitric acid was added. The ratio of the reactant chemicals used depends on the porphyrin and the intended degree of nitration. The reaction time depends on the porphyrin and the degree of nitration. The nitration was monitored by TLC (silica gel or alumina). Samples for TLC taken from the reaction mixture were first neutralized with a solution of Na2CO3 and extracted with CHC13; then the CHC13 solutions were introduced onto TLC plates, which were developed using CHC13. The retention time of a porphyrin increased with the number of nitro groups. When the intended nitration was completed, cold water (about the same amount as the reaction mixture) was added slowly to quench the reaction (heat was produced when water was added). The hot mixture was cool by an ice-bath and neutralized firstly with concentrated NaOH, then with dilute Na2CO3 to pH — 8 (the color of the reaction mixtures changed from green to purple or brown at pH = 4-5). 42  Chapter 2 Synthesis of porphyrins  The product was extracted with CHC13 (100 - 200 mL), the CHC13 portion was washed with water three times (— 100 mL each time), and the CHC13 layers was evaporated to dryness on a Rotovap. The residues was washed with acetone (— 50 mL), filtered and dried. Nitration usually gave a mixture of products, and column chromatography was used to separate them. The compositions of the reaction mixtures, reaction times, details about column chromatography and other experimental conditions for each porphyrin are described below. (NPh)TPhP [5-(4-nitropheny1)-10,15,20-triphenylporphyrin]. TetPhP (0.5 g) was dissolved in acetic acid (50 mL), and concentrated nitric acid (20 mL) was added with stirring. Reaction was monitored with TLC (silica gel, 0.2 cm). When no TetPhP could be detected on a TLC plate (reaction time was 1 h), the reaction was stopped and the crude product was collected as described above. This product was passed through a column of alumina (activity IR 3 x 10 cm) with CHC13 as eluant to separate out some of the nonporphyrin materials, which remained on the top of the column. The eluted solution was evaporated to dryness on a Rotovap, and 0.51 g crude product was collected; this was loaded onto a column of alumina (activity I, 3 x 30 cm), eluted with CHC13, when three brown bands were observed. After a trace of TetPhP was eluted, solutions of (NPh)TPhP and B(NPh)DPIT were collected successively. The solutions were evaporated on a Rotovap to dryness and the residues were washed with acetone and air-dried. (NPh)TP1113 (0.40 g, 74% yield) and some B(NPh)DPhP (-0.03g as a by-product) were isolated. B(NPh)DPhP [mixture of 5,10-bis(4-nitropheny1)-15,20-diphenylporphyrin and 5,15-bis(4-nitropheny1)-10,20-diphenylporphyrin]. All the conditions were the same as above, except that the reaction was stopped when no (NPh)TPhP could be detected by TLC on silica gel (-5 h reaction time). After a trace of (NPh)TPhP was eluted, 43  Chapter 2 Synthesis of porphyrins  B(NPh)DPhP was eluted from a column of alumina (activity I, 3 x 30 cm). The bis-nitro product (0.40 g, 70% yield) was collected. Nitration of two of the phenyl groups in TetPhP gave a mixture of cis and trans products, as judged by 1H NMR (see Section 2.3.2). Attempts to separate them on an alumina column (activity I, 2 x 40 cm) failed. T(NPh)PhP [5,10,15-tri(4-nitropheny1)-20-phenylporphyrin]. All the conditions were the same as described above for the synthesis of (NPh)TPhP, but the reaction mixture was stirred until no B(NPh)DPhP was present according to TLC on silica gel (about two days were needed). The T(NPh)PhP product was collected in about 30% yield via the major eluted band from an alumina column (activity I, 3 x 30 cm). Traces of B(NPh)DPhP and Tet(NPh)P were also separated and isolated in small quantities (-2% yield each). An attempt to prepare Tet(NPh)P [5, 1 0, 1 5,20-tetra(4-nitrophenyl)porphyrin] failed because the required, more severe reaction conditions (reactions were monitored for 7 days under the conditions used for the synthesis of (NPh)TPhP) damaged the porphyrin structure. Purification of this porphyrin, isolated as by-product from the synthesis of T(NPh)PhP, failed because of the low solubility of this compound in common solvents. The by-product was only identified by 1H NMR data (singlet at 8.88 ppm, pyrrole protons; doublets at 8.63 and 8.38 ppm for nitrophenyl protons; and singlet at -2.84 ppm for N-pyrrole protons) for a dilute solution of this compound in CDC13. Cis-(NPh)DPhPyP [5-(4-nitropheny1)- 1 0, 1 5-dipheny1-20-(4-pyridyl)porphyrin]. All the conditions and procedures were the same as the ones used for the synthesis of (NPh)TPhP, except that TPhPyP (0.50 g) was used as the precursor porphyrin; and the reaction was stopped when no TPhPyP could be detected on a TLC plate (alumina, activity I, 0.2 cm, 5 h reaction time). After a trace of TPhPyP, cis-(NP)DPhPyP was eluted by CHC13 from a column of alumina (activity DI, 3 x 30 cm); the solution of cis44  Chapter 2 Synthesis of porphyrins  (NPh)DPhPyP was collected, washed with water, and the CHC13 layer was separated and evaporated to dryness. The residue was then washed with acetone and air-dried (60% yield). Cis and trans-B(NPh)PhPyP [5,10-bis(4-nitropheny1)-15-pheny1-20-(4pyridypporphyrin] and trans-B (NP)PhPyP [5,15-bis(4-nitropheny1)-10-pheny1-20-(4pyridyl)porphyrin]. All the conditions and precursor were the same as described above for the synthesis of cis-(NPh)DPhPyP, but the reaction was stopped when no cis(NPh)DPhPyP could be detected by alumina TLC (20 h reaction time). After a trace of (NPh)DPhPyP, trans-B(NPh)PhPyP, cis-B(NPh)PhPyP and a small amount of T(NPh)PyP were eluted from a column of alumina (activity lg 3 x 30 cm). The cis and trans isomers were observed as the two major bands on the column, but were collected together because poor separation (ca. 60% yield). A smaller amount (20 mg) of this mixture was separated using a longer column (alumina active a 2 x 40 cm), the trans isomer being eluted before the cis one; about equal amounts of the two isomers were obtained. T(NPh)PyP [5,10,15-tri(4-nitropheny1)-20-(4-pyridy1)porphyrin]. The reaction conditions and the precursor were again the same as described above for the synthesis of cis-(NPh)DPhPyP, but the reaction was stopped when a significant amount of T(NPh)PyP could be detected by alumina TLC (40 h reaction time). After traces of. cis and transB(NPh)PhPyP were eluted by CHC13, T(NPh)PyP was eluted out with 10% Me0H in CHC13 from a column of alumina (activity 1Ik 3 x 30 cm)(--30% yield). Trans B(NPh)BPyP [5,15-bis(4-nitropheny1)-10,20-bis(4-pyridyl)porphyrin}. -  Trans-DPhBPyP (0.20 g) was mixed with acetic acid (30 mL, the solubility of this porphyrin in acetic acid being low), concentrated sulfuric acid (10 mL) and then nitric acid (10 mL) was added dropwise, and the mixture was stirred. The reaction was stopped when no trans-DPhBPyP and no mono-nitration product could be detected by alumina 45  Chapter 2 Synthesis of porphyrins  TLC (24 h reaction time). The solubilities of the crude product (70% yield) in CHC13 or CH2C12 were low. The crude product looked fairly pure by TLC and 111 NMR data. A portion of this crude product (20 mg) was purified on a column of alumina activity IV (2 x 40 cm) using CHC13 as eluant (-200 mL). Cis-(NPh)PhBPyP [5-(4-nitropheny1)-10-pheny1-15,20-bis(4-pyridyl) porphyrin]. All the conditions were the same as described above for the synthesis of transB(NPh)BPyP, except that cis-DPhBPyP was used as the precursor and no sulfuric acid was used (i.e. 0.20 g porphyrin, 30 mL acetic acid and 10 mL nitric acid). The reaction was stopped when no cis-DPhBPyP could be detected by alumina TLC (20 h reaction time). The crude product, isolated from the reaction mixture, was separated with a column of alumina (activity N, 2 x 60 cm). After a trace of cis-DPhBPyP was eluted, cis(NPh)PhBPyP, and a trace of cis-B(NPh)BPyP were collected; the yield of cis(NPh)PhBPyP was 55%. Cis-B(NPh)BPyP [5, 10-bis(4-nitropheny1)-15,20-bis(4-pyridyl)porphyrin] . The conditions were exactly the same as the ones given for the synthesis of transB(NPh)BPyP, but cis-DPhBPyP was used as precursor. The reaction was stopped when no cis-(NPh)PhBPyP could be detected by alumina TLC (24 h reaction time). The crude product was separated with a column of alumina (activity IV, 2 x 40 cm). After a trace of cis-(NPh)PhBPyP was eluted, cis-B(NPh)BPyP was collected (-70% yield). (NPh)TPyP [5-(4-nitropheny1)-10, 15,20-tri(4-pyridyl)porphyrin] . All the conditions were the same as given above for the synthesis of trans-B(NPh)BPyP, but PhTPyP was used as precursor. The reaction was stopped when no PTPyP could be detected by alumina TLC (2 days reaction time). The pure product (-60% yield) was separated with a column of alumina (activity IV, 2 x 40 cm) from a green impurity.  46  Chapter 2 Synthesis of porphyrins  Elemental analyses (for samples stored and weighed out in air) of the nitrated porphyrins are shown in Table 2.4. The 1H NMR and UV-visible data are given in Tables 2.5 and 2.6, respectively. Spectral data from IR and mass spectroscopies are presented in Sections 2.3.4 and 2.3.5, respectively. The hydration of some of the porphyrins is discussed in Section 2.3.1.1; the assignments of the NMR signals are discussed in Section 2.3.2. 2.2.4 Reductions of the nitro-porphyrins  Reductions were performed according to the method reported in the literature.2,3,4 Generally, the porphyrin (0.1 g) was dissolved in CHC13 (20 mL) in a 250 inL roundbottom flask with magnetic stirring; acetic acid (30 mL) and then a solution of SnC12 (0.3 g in 30 mL concentrated HCI) were added. The mixture was stirred and refluxed overnight, and then was neutralized with concentrated NaOH and dilute Na2CO3 solutions to pH = 8-9. Heat was produced in the process of neutralization, so an ice-bath was used. When the mixture had cooled down to room temperature, FI20 (100 mL) and CHCI3 (100 inL) were added. The organic layer was separated, filtered, washed with water (100 mL) three times, and the organic layer was evaporated to dryness on a Rotovap. The crude product was purified on a column of alumina (activity IV, 2 x 30 cm), using —250 mL CHCI3 or a solvent mixture of Me0H and CHC13 as eluant. The yield was above 90% for all the porphyrins. To synthesize cis and trans-B(APh)DPIIP [5,10-bis(4-aminopheny1)-15,20diphenylporphyrin and 5,15-bis(4-aminopheny1)-10,20-diphenylporphyrin], a mixture of cis and trcms-B(NPh)DPhP was used as the precursor, because this mixture had not been  separated (see Section 2.2.3). A separation of the two reduced isomers on a column of  47  Chapter 2 Synthesis of porphyries  Table 2.4. Elemental analyses for the nitroporphyrins  (NPh)TPIT B(NPh)DPhP (mixture) T(NPh)PhP.1/2 H20 cis-(NPh)DPhPy13-1/2 H20 cis-B(NPh)PhPyP.H20 trans-B(NPh)PhPyP-1-120  C^%  H^%  N^%  expected  80.12  4.40  10.62  found  80.03  4.46  10.63  expected  75.00  3.98  11.93  found  74.96  3.95  11.79  expected  69.66  3.69  12.93  found  69.51  3.85  12.82  expected  77.20  4.33  12.56  found  77.46  4.22  12.58  expected  71.37  4.01  13.55  found  71.51  3.90  13.61  expected  71.37  4.01  13.55  found  71.74  3.80  13.61  68.25  3.44  15.61  found  63.95  4.35  11.45  expected  71.38  3.70  15.85  found  71.19  4.00  15.52  expected  75.66  4.18  14.40  found  75.22  4.21  14.63  expected  69.61  3.87  15.48  found  69.49  3.72  15.18  expected  73.33  4.05  16.68  found  73.20  4.09  16.78  T(NPh)PyP^a expected trans-B(NPh)BPyP cis-(NPh)PhBPyP 1/2.H20 cis-B(NPh)BPyP•H20 (NPh)TPyP 1/2•20  a: Sample was contaminated by alumina (see footnote a under Table 2.1).  48  Chapter 2 Synthesis of porphyrins  Table 2.5. 1H NMR data for the nitroporphyrins (a)  2,6-Ph  3,5-Py  3,5-NPh  3,4,5-Ph  2,6-Py  2,6-NPh  8.90 d; 8.87 s; 8.74 d  8.22 m (6)  -  8.64 d (2)  (2,4,2)  7.78 m (9)  pyrrole  (NPh)TPhP  B(NPh)DPhP h 8.90 d; 8.86 s; 8.77 s; 8.73 t 8.21 m (4)  -2.80 s (2)  8.41 d (2) -  8.64 m (4)  (5/2, 3/2, 3/2, 5/2)  7.80 m (6)  8.91 d; 8.79 s; 8.75 d  8.19 m (2)  (2, 4, 2)  7.99 m (3)  cis-  8.89 d; 8.86 s; 8.82 d  8.20 m (4)  9.04 q (2)  8.62 d (2)  (NPh)DPhPyP  8.79 d; 8.76 d; 8.73 d  7.77 m (6)  8.15 q (2)  8.37 d (2)  T(NPh)PhP  N-pyrrole  -2.83 s (2)  8.38 m (4) -  8.63 d (6)  -2.83 s (2)  8.38 d (6) -2.88 s (2)  (2, 2, 1, 1, 1, 1)  cis-  8.91 d; 8.85 d; 8.81 d  8.20 m (2)  9.04 q (2)  8.63 m (4)  B(NPh)PhPyP  8.79 d; 8.78 s; 8.75 d  7.78 m (3)  8.14 q (2)  8.37 m (4)  8.92 d; 8.84 d; 8.79 d; 8.75 8.19 m (2)  9.04 d (2)  8.63 m (4)  -2.87 s (2)  (2, 1, 1, 1, 2, 1) trans  -  B(NPh)PhPyP  d (2, 2, 2, 2)  7.78 m (3)  8.14 d (2)  8.37 m (4)  T(NPh)PyP  8.86 d; 8.80 m (2,6)  -  9.04 d (2)  8.64 d (6)  8.15 d (2)  8.38 d (6)  9.05 m  8.65 m  8.14 m  8.38 m  trans  -^  c 8.86 d;^8.80 d  -  B(NPh)BPyP cis-  8.90 d; 8.83 d; 8.83 s  8.19 q (2)  9.04 q (4)  8.63 m (2)  (NPh)PhBPyP  8.80 d; 8.78 d; 8.74 d  7.77 m (3)  8.14 q (4)  8.38 m (2)  -  9.05 d (4)  8.65 d (4)  8.14 d (4)  8.38 d (4)  9.02 d (4)  8.61 d (2)  8.10 d (4)  8.34 d (2)  -2.85 s (2) -2.87 s (2) -2.90 s -2.84 s (2)  (2, 1, 2, 1, 1, 1)  cis-  8.86 m; 8.80 m (4,4)  B(NPh)BPyP (NPh)TPyP  8.82 m; 8.77 d (6,2)  -  -2.90 s (2) -2.99 s (2)  a: In CDC13 at room temperature; chemical shift in ppm signal pattern (number of protons). b: A mixture of the cis and trans-isomers. c: Low concentration of the compound in the NMR sample.  49  Chapter 2 Synthesis of porphyrins  Table 2.6. UV-visible data for the nitroporphyrins (a)  Soret  visible 1  visible 2  visible 3  visible 4  418.0 (418)  515.3 (17.8)  550.5 (8.7)  589.3 (6.4)  646.6 (5.3)  12- 419.5 (314)  514.5 (19.3)  550.3 (9.3)  588.0 (6.3)  645.0 (3.7)  T(NPh)PhP  421.3 (262)  515.5 (17.5)  550.4 (8.7)  590.0 (6.2)  646.5 (3.8)  cis-(NPh)DPhPyP  417.6 (345)  513.9 (19.0)  549.8 (8.0)  587.6 (6.5)  645.5 (3.5)  cis-B(NPh)PhPyP  418.6 (301)  513.0 (19.7)  547.5 (8.0)  587.7 (7.1)  644.5 (3.4)  trans-B(NPh)PhPyP  419.0 (295)  513.5 (18.9)  548.5 (8.5)  588.9 (7.1)  644.1 (3.7)  (NPh)TPhP B(NPh)DPIT  T(NPh)PyP  c - 419.4 (306) 514.0 (19.7) 549.0 (9.3) 590.1 (7.7) 644.9 (4.1)  trans-B(NPh)BPyP  418.8 (258)  514.6 (17.2)  548.0 (7.3)  589.3 (6.8)  642.4 (2.9)  cis-(NPh)PhBPyP  416.4 (316)  512.7 (18.0)  548.0 (7.2)  589.0 (6.6)  644.0 (4.3)  cis-B(NPh)BPyP  418.9 (360)  513.3 (22.2)  548.5 (8.1)  591.6 (7.7)  645.5 (3.0)  (NPh)TPyP  416.8 (290)  512.2 (17.2)  545.7 (6.2)  588.5 (6.4)  642.5 (2.6)  a: In CHC13 at room temperature at 1 x 10-5M; wavelength at maximum absorbance(Xmax) in tun, (extinction coefficient [E x10-3, M-1 cm-1]). b: A mixture of the cis and trans-isomers. C: Sample contaminated by alumina (see footnote a under Table 2.1). Thus, the E values are minimum ones.  50  Chapter 2 Synthesb of porphyrins  alumina (CHC13 as eluant) was achieved. The syntheses of the isomers of cis and transB(APh)PhPyP also were started with a mixture of cis and trans-B(NPh)PhPyP as the precursor, and the isomers were again separated after reduction to the amino derivatives using a column of alumina (CHC13 as eluant). The separation of the reduced isomers was easier than the separation of the precursor bis(nitrophenyl) isomers. The trans isomer was eluted from the column before the cis one in both cases. Samples of cis-(APh)Phl3PyP was purified on a column of alumina using a mixed solvent as eluant (10% Me0H in CHC13), because these porphyrins having more polar substituents (two pyridyls and one or two aminophenyls) could not be eluted off using CHC13. All the other amino porphyrins were purified on an alumina column using CHC13 as eluent. The (APh)TPyP compound was synthesized from (NPh)TPyP, but only the 'II NMR spectrum was recorded. A pure sample of this compound was not obtained because its low solubilities in common solvents made column chromatography impractical. Thus, elemental analysis and UV-visible spectroscopy were not attempted. The elemental analyses (for samples stored and weighed out in air), Ill NMR and UV-visible data are shown in Tables 2.7, 2.8 and 2.9, respectively. The hydration of some of the porphyrins is discussed in Section 2.3.1.1; the assignments of the NMR signals are discussed in Section 2.3.2. The abbreviations are derived as described in Section 1.2 from the full names which can be found in the List of abbreviations.  51  Chapter 2 Synthesis of porphyrins  Table 2.7. Elemental analyses for the aminoporphyrins  (APh)TPhP  cis-B(APh)DPhP.H20  trans-B(APh)DPhP•H20  T(APh)Ph1P.1/2 H20  cis-(APh)DPhPyP.H20  trans-B(APh)PhPyP  cis-(APh)PhBPyP^a  C^%  H^%  N^%  expected  83.94  4.92  11.13  found  83.81  4.83  10.98  expected  80.86  5.05  12.86  found  80.79  5.13  12.87  expected  80.86  5.05  12.86  found  81.06  5.13  12.60  expected  79.04  5.09  14.67  found  79.34  5.23  14.58  expected  79.63  4.94  12.96  found  79.63  4.91  12.59  expected  77.83  4.98  14.78  found  77.89  5.03  14.46  expected  79.87  4.60  15.53  found  75.78  4.49  14.56  a: Sample contaminated by alumina (see footnote a under Table 2.1).  52  Chapter 2 Synthesis of porphyrins  Table 2.8. 111 NMR data for the aminoporphyrins (a)  pyrrole (APh)TPhP  2,6-Ph 3,4,5-Ph  8.93 d(2); 8.82 m(6) 8.25 m(6)  3,5-Py 2,6-Py  2,6-APh 3,5-APh  -  7.98 d(2) 4.00 s(2) -2.78 s(2)  7.74m(9) 8.91 m(4); 8.80 m(4) 8.20q(4)  cistrans  -  8.91d(4); 8.81d(4) 8.20q(4)  T(APh)PhP  -  8.89s(6); 8.77d(2)  8.19q(2)  7.98d(4) 4.00 s(4) -2.74s(2) 7.04 d(4)  -  7.98d(4) 4.00 s(4) -2.73s(2) 7.04 d(4)  7.75 m(6)  B(APh)DPhP  N-pyrrole  7.05d(2)  7.74 m(6)  B(APh)DPhP  -NH2  -  7.73m(3)  7.97d(6) 4.01 s(6) -2.73s(2) 7.04d(6)  8.99 d(1); 8.95 d(1)  8.21m(4)  9.02d(2)  7.98d(4) 4.05s(4) -2.77s(2)  (APh)DPhPyP 8.88 d(1); 8.84 s(2)  7.77 m(6)  8.16 d(2)  7.05 d(4)  8.19d(4)  9.01 d(2)  7.97d(4) 4.05s(4) -2.80s(2)  7.75 m(6)  8.15 d(2)  7.06 d(4)  8.99 d(1); 8.95 d(1)  8.19q(2)  9.04q(4)  7.98d(2) 4.05s(2) -2.82s(2)  (APh)PhBPyP 8.88 d(1); 8.84 d(1)  7.77m(3)  8.15q(4)  7.06d(2)  -  9.03 m  7.97 d  8.14m  7.08d  cis-  8.83 d(1); 8.78 d(2) trans  -  8.98-8.67m(8)  B(APh)PhPyP cis-  8.81 s(2); 8.77 d(2) (APh)TPyP12 9.00 d; 8.81 s 8.79d  4.05 s  -2.85 s  a: In CDC13 at room temperature; chemical shift in ppm signal pattern (number of protons). b: A dilute solution sample resulting from low solubility.  53  Chapter 2 Synthesis of porphyrins  Table 2.9. UV-visible data for the aminoporphyrins4)  Soret  visible 1  visible 2  visible 3  visible 4  (APh)TPhP  419.5 (395) 515.0 (18.7)  551.7 (10.6) 590.0 (6.8)  645.5 (5.8)  cis-B(APh)DPhP  421.5 (330) 517.5 (15.4)  555.0 (10.9) 592.5 (6.4)  650.0 (6.2)  trans-B(APh)DPhP  421.0 (336) 517.0 (15.8)  555.0 (11.0) 591.9 (6.1)  649.7 (5.9)  T(APh)PhP  423.8 (311) 518.2 (13.0)  558.3 (10.6) 593.5 (5.2)  651.5 (6.1)  cis-(APh)DPhPyP  418.8 (403) 515.3 (20.7)  551.0 (13.4) 589.4 (9.6)  648.4 (9.7)  trans-B(APh)PhPyP  422.3 (314) 518.0 (18.0)  555.0 (12.8) 592.0 (8.0)  650.0 (9.6)  419.8 (308) 515.4 (16.4)  552.7 (8.4)  648.3 (3.8)  cis-(APh)PhBPyP  b  590.0 (5.6)  a: In CHC13 at room temperature at 1 x 10-5M; wavelength at maximum absorbarice(kmax) in run, (extinction coefficient [C x10-3, M-1 cm4]). b:  Sample contaminated by alumina (see footnote a under Table 2.1). Thus, the E values are the  minimum ones.  54  Chapter 2 Synthesis of porphyrins  2.2.5 Sulfonations Sulfonation reactions were performed using a modified literature procedure.s The synthesis procedure for each of the porphyrins used in this work was about the same, and is described generally here as shown by the following scheme:  Porphyrin + H2SO4 green paste 100°c(4-12h) (”i H 0 2 green solution (2)  NaOH  Na 2 CO 3 purple solution evaporated "t cooled (3) purple solution + purple precipitate (4) filtered purple solution^purple precipitate washed with Me0H \ombined (5) Me0H added \rrple solution^yellow precipitate purple solution + precipitate (6) ifiltered  (inorganic salts)  purple solution^purple precipitate b\combined^washed with Me0H evaporated (7) to dryness^ purple solution^yellow precipitate (inorganic salts)  purple residue  (porphyrin + inorganic salts)  (8) washed with Me0H purple solution evaporated (9) to dryness  yellow precipitate (inorganic salts)  crude product  55  Chapter 2 Synthesis of porphyrins  The porphyrin (-0.50 g) was mixed well with concentrated sulfuric acid (10 mL) in a 50 mL round-bottom flask. (1) The flask was stoppered and heated in a oil-bath at 100110°C for 4-12 h (see below). After the reaction the mixture cooled down to room temperature, ice cold water (50 mL) was added slowly. (2) The mixture was cooled by an ice-bath and carefully neutralized with concentrated NaOH and dilute Na2CO3 to pH = 89. (3) After being concentrated by evaporation to about 20 inL, the solution was cooled in an ice-bath, when a precipitate of inorganic salts (mainly Na2SO4) formed. (4) The precipitate was filtered off and washed with methanol (50 inL). (5) To the filtrate (aqueous solution and methanol solution combined), 100 mL methanol was added, when more inorganic salt precipitated. (6) This mixture again was filtered (the precipitate was discarded) and then (7) the filtrate was evaporated to dryness on a Rotovap. (8) The purple residue (which was a mixture of porphyrin product and inorganic salt) was washed with methanol until only a light yellow solid remained. (If the purple color of the residue could not be washed out by methanol, then the residue was dissolved into a minimum amount of hot water, reprecipitated out with methanol, and then filtered.) (9) The purple methanol solution was evaporated to dryness on a Rotovap, and the purple residue was collected and dried under vacuum at 100°C overnight to give the crude product. For Na4Tet(SPh)P, the crude product was redissolved in methanol (-20 mL), and the solution was filtered to remove some insoluble impurity. To the filtrate, acetone (-200 inL) was added with stirring; a brown precipitate formed which was filtered off and airdried. This methanol-acetone reprecipitation procedure was repeated three more times. The final product was dried at 100 °C under vacuum. This product was a brown color and very hygroscopic. Another form of the product was made by evaporation of aqueous solutions of the brown material to dryness. Bright purple flakes resulted and there were collected; this material was essentially not hygroscopic.  56  Chapter 2 Synthesis of porphyrins  For the other sulfonated porphyrins, the crude products were either purified by dialysis or by repetitive methanol-acetone reprecipitation (3 times). Products purified by dialysis were collected by evaporation of the aqueous solutions and were essentially not hygroscopic. The actual purification method used for each porphyrin is indicated in Table 2.10. The overall yields of the reactions following purification of the products were 7080%. Reaction of the nitro precursor (NPh)TPhP with hot sulfuric acid for 30 min gave the amino product Na3(APh)T(SPh)P instead of the expected nitro product Na3(NPh)T(SPh)P; the yield of this porphyrin product was about —40%, which was relatively low compared to the yield of sulfonation of (APh)TPIT (80%). The amine product was identified by its 1H NMR spectrum (identification of the porphyrins by 1H NMR is discussed in Section 2.3.2), as well by elemental analysis (Table 2.10). A similar nitro-reduction occurred for sulfonation of the mixture of cis and transB(NPh)DPhP. Reaction of the mixture with sulfuric acid at 100 °C for 0.5 h gave a 13% yield of crude product, which was separated into three portions on a column of silica gel (3 x 30 cm) using a mixed solvent of methanol and CH2C12 (1:1). The three sections were identified by 1H NMR as B(NPh)B(SPh)P (the major product), (APh)(NPh)B(SPh)P and B(APh)B(SPh)P (the 1H NMR data are not listed in Table 2.11; the spectra of the three compounds are shown in Figure 2.2, p.'77, instead). The separation of the cis and transisomers of these three products using silica gel columns failed. These products were only isolated in sufficient amounts for samples for NMR spectra, because of the low yield and the difficulty of their separation. However, sulfonation of cis-(NPh)DPhPyP, another nitro porphyrin, for a 6 h reaction time, gave the sulfonated nitro porphyrin Na2[cis-(NPh)PyB(SPh)13] as the main product, although product distribution varied with reaction time (Section 3.3.1). The 57  Chapter 2 Synthesis of porphyrtos  crude product from this sulfonation reaction was dissolved in methanol (-30 mL), loaded on a column of silica gel (3 x 30 cm), and the column was eluted by a mixture of CH2C12 and CH3OH (1:3). The major band was identified by 1H NMR as cis-(NPh)PyB(SPh)P (Table 2.11; for identification of porphyrin products using NMR data, see Section 2.3.2). The sulfonated amino porphyrins (APh)T(SPh)P, cis and trans-B(APh)B(SPh)P were synthesized via the porphyrin precursors (APh)TPhP, cis and trans-B(APh)DPhP in higher yield (70-80%), than via sulfonation of (NPh)TPhP, cis and trans-B(NPh)DPhP, which gave these sulfonated amino porphyrins in lower yield as noted above. In Table 2.10, only the purified products with their elemental analyses are listed; the reaction time for sulfonation of cis, and trans-DPhBPyP was 12 h; for cis(NPh)PyDPhP, 6 h; and for all the others, 4 h. All the sulfonated porphyrins were isolated in the form of a sodium salt. Abbreviations were derived as described in Section 1.2 from the full names, which can be found in the List of Abbreviations. The naming of the counterion (Na+) is omitted in this thesis when the emphasis is on the porphyrin structure. The hydration of the porphyrins is discussed in Section 2.3.1; the assignments of the NMR signals are discussed in Section 2.3.2 and the 1H NMR data are given in Table 2.11. The UV-visible spectra of the sulfonated porphyrins in aqueous solution are complicated because of aggregation of the porphyrins, which is more fully discussed in Chapter 4. Table 2.12 presents the UV-visible spectral data at one concentration (1.0 x 10-5 M) in distilled water. Mass spectral data are presented in Section 2.3.5.  58  Chapter 2 Synthesis of porphyrins  Table 2.10. Elemental analyses for the sulfonated porphyrins  Na3[(APh)T(SPh)P].2H20^A expected found Na2[cis-B(APh)B(SPh)P].2H20^A expected found Na2[trans-B(APh)B(SPh)P).3H20^A expected found Na4[Tet(SPh)P].10H20^h expected found Na2[cis-(NPh)PyB(SPh)P].4H20^h expected found Na3[PyT(SPh)P].2H20^A expected found Na1[PyT(SPh)P].11H20^h expected found Na3[PyT(SPh)P].2H20^h and C. expected found Na2[cis-BPyB(SPh)P] - 1 OH20^h expected found Na2 [trans-BPyB(SPh)11.7H20^h expected found  C% H% 3.30 54.31 54.51 3.06 59.73 4.10 59.87 3.85 58.93 4.02 59.13 4.31 43.93 3.83 43.65 3.70 55.31 3.64 55.60 3.95 53.91 3.13 53.84 3.36 46.87 4.36 46.63 4.21 53.91 3.13 53.91 2.92 50.40 4.60 50.04 4.45 53.28 4.23 53.45 4.25  N% 7.20 7.40 9.41 9.50 9.38 9.43 4.66 4.39 9.00 8.76 7.31 7.20 6.36 6.23 7.31 7.22 8.40 8.41 8.88 8.66  S%  10.03 10.10 6.40 6.70 6.77 6.65  a: Sample collected from Me0H-CH3COCH3 precipitations. b: Sample collected from evaporation of water solutions. c: Sample dried at 100 °C under vacuum overnight and analyzed after being stored and weighed under nitrogen.  59  Chapter 2 Synthesis of porphyrins  Table 2.11. 1H NMR data for the sulfonatoporphyrins (a)  pyrrole Nal RAPh)T(SPh)P]  8.99 d(2); 8.85 s(6)  2,6-SPh  3,5-Py  2,6-APh  3,5-SPh  2,6-Py  3,5-APh  8.18 d(6)  -  8.92 s(2), 8.92 d(2)  B(APh)B(SPh)131  8.79 s(2), 8.78 d(2) i8.05 d(4)  Na,[trans-  8.94 d(4); 8.79 d(4) 8.17 d(4)  B(APh)B(SPh)P] Na4[Tet(SPh)P]  8.17 d(4)  -  8.18 d(8)  5.61 s(2) -2.80 s(2)  7.87 d(4)  5.55 s(4) -2.80 s(2)  7.00 d(4) -  7.86 d(4)  5.57 s(4) -2.84 s(2)  7.00 d(4)  8.03 d(4) 8.84 s(8)  N-pyrrole  7.04 d(2)  8.06 d(6) No [cis-  7.90 d(2)  -NFI7  -  -  -  -2.95 s(2)  8.04 d(8) No [cis  8.87 sb(8)  -  (NPh)PyB(SPh)Pl Nal [PyT(SPh)13]  8.86 m(8)  8.16 d(4)  9.02 d(2)  8.66 d(2) h  8.03 d(4)  8.25 d(2)  8.49 d(2)12  8.19 m(6) 9.03 d(2) 8.07 d(6)  Na,[cis-BPyB(SPh)P] 8.89 m (8)  -  -3.01 s(2)  -  -  -2.97 s(2)  -  -  -2.98 s(2)  -  -  -2.98 s(2)  8.27 m(2)  8.18 m(4) 9.04 d(4) 8.05 m(4) 8.28 d(4)  No [trans  -  8.89 m (8)  BPyB(SPh)P]  8.18 d(4)  9.04 d(4)  8.05 d(4)  8.29 d(4)  a: In DMSO-d6 at room temperature; chemical shift in ppm signal pattern (numbelof protons).  b: Signals for nitrophenyl.  60  Chapter 2 Synthesis of porphyrins  Table 2.12. UV-visible data for the sulfonatoporphyrins(a)  Soret  visible 1  visible 2  visible 3  visible 4  (APh)T(SPh)P  412.6 (314)  516 (12.7)  555 (8.4)  577 (7.1)  635 (5.7)  cis-B(APh)B(SPh)P  415  (98.8)  trans-B(APh)B(SPh)P  413  (61.7)  Tet(SPh)P  411.0 (464)  513 (15.5)  549 (7.0)  577 (6.5)  630 (3.9)  (NPh)PyB(SPh)P  416.0 (177)  515 (10.7)  552 (6.5)  578 (4.6)  640 (2.1)  PyT(SPh)P  410.6 (417)  513 (15.3)  555 (6.8)  577 (6.4)  632 (3.2)  cis-BPyB(SPh)P  409.3 (282)  514 (11.7)  550 (5.2)  579 (4.4)  636 (2.0)  trans-BPyB(SPh)P  409.3 (263)  514 (11.7)  550 (4.6)  579 (3.8)  636 (1.5)  491 (65.9)  -  -  665 (13.9)  -  -  700 (23.0)  a: In H20 at room temperature at 1 x 10-5M; wavelength at maximum absorbance(2) in nm, (extinction coefficient [C x10-3, M-1 cm-11).  61  Chapter 2 Synthesis of porphyrins  2.2.6 Methylations  The methylation reaction of TetPyP was carried out according to the literature procedure.6 Briefly, TetPyP (0.50 g) was mixed with 50 inL DMF, in a 100 mL roundbottom flask with a magnetic stir bar, and the mixture was heated to almost boiling (when the porphyrin dissolved); methyl p-toluene sulfonate (— 2 mL) was added with stirring and the solution was then refluxed for 4 h. The methylated pyridiniumporphyrin tetra(tosylate) formed as a precipitate when this reaction solution was cooled in a refrigerator at 4°C; this precipitate was filtered, washed with acetone and air-dried. 95% yield. The porphyrins PhTPyP, (NhP)TPyP, cis and trans-DP1BPyP, and cis and transB(NPh)BPyP were methylated under conditions similar to those described above (0.20 g porphyrin, 20 mL DMF, 1 mL methyl p-toluene sulfonate, 4 h). However, the tosylate salt of the methylated porphyrin did not form a precipitate when reaction mixture was cooled. The reaction mixture was thus reduced in volume by evaporation on a Rotovap to —10 mL; acetone (-100 mL) was then added to the mixture with stirring, and the resulting mixture was filtered; the precipitated purple, tosylate salt of the methylated porphyrin was washed with acetone (— 20 mL) and dried under vacuum at room temperature. The similarly formed methylated products from cis and trans-DPhBPyP, and cis and transB(NPh)BPyP were washed with cold water (— 20 mL) on filtration funnels; and then dried under vacuum at room temperature. All the tosylate salts were converted to the corresponding chloride salts by passing an aqueous solution of the porphyrin through an anion exchange column (Cl). The aqueous solutions of the tosylate salts of Tet(MPy)P, T(MPy)PhP and (NPh)TPhP were obtained by stirring the products in hot water (-20 tnL for 0.2 g porphyrin, 60-80 °C). The tosylate salts of cis and trans-B(MPy)DPIT, and cis and transB(MPy)B(NPh)P were dissolved in water by stirring these products with hot water (40 62  Chapter 2 Synthesis of porphyrins  mL for 0.2 g porphyrin, 50-60 °C) with the presence of the Ci ion exchange resin (-2 mL for 0.2 g porphyrin). The purple aqueous solutions from the anion exchange material were evaporated to dryness. Acetone (— 10 mL) were added to the purple residues; these were scraped off the wall of the flasks, filtered off and dried under vacuum at 100°C. The overall yield of methylation followed by anion conversion was around 90%. The chloride salts of cis and trans-B(MPy)DPhP, and cis-B(MPy)B(NPh)P were further purified by dialysis because the presence of impurities caused low C, H and N contents in the elemental analyses. Analysis of trans-[B(MPy)B(NPh)11C12 gave low C, H and N content, even after an attempt to purify this porphyrin by dialysis; this failed mainly because of the low solubility of this porphyrin in water. The elemental analyses (for samples stored and weighed out in air) and data from NMR spectra are shown in Tables 2.13 and 2.14, respectively. Discussions about the hydration of the compounds, and the assignments of the 1H NMR signals, are presented in Sections 2.3.1.1 and 2.3.2, respectively. The UV-visible spectra of these porphyrins again are complicated because of aggregation of porphyrins in aqueous solutions, and this is discussed in Chapter 4. The data of UV-visible spectra for these porphyrins at the concentration of 1.0 x 10-5 M are presented in Table 2.15. Data from mass spectroscopy are presented in Section 2.3.5. The abbreviations are derived as described in Section 1.2 from the full names which can be found in the List of abbreviations. All the porphyrins are isolated in the form of a chloride salt.  63  Chapter 2 Synthesis of porphyrins  Table 2.13. Elemental analyses for the methylpyridiniumporphyrins  [cis-B(MPy)DPhI]C12•5.5H20 ^a  C%  H%  N%  expected  64.73  5.52  10.30  found  64.91  5.32  10.14  60.07  4.55  12.74  59.86  4.80  12.50  66.17  5.38  10.52  66.21  5.22  10.55  60.07  4.55  12.74  found  53.50  5.22  10.18  expected  62.82  5.23  11.86  found  63.15  5.23  11.66  expected  59.63  4.86  12.65  found  59.72  5.00  12.47  expected  59.21  5.16  12.56  found  59.52  5.36  12.55  [cis-B(MPy)B(NPh)P]C12•4H20 ^a expected  found [trans-B(MPy)DPhP]C12.4.5H20^a expected found [trans-B(MPy)B(NPh)P]C12-4H20 ^a expected  [T(MPy)PhIlC13•4H20  [T(MPy)(NPh)P]C13.4H20  [Tet(MPy)P]C14•4H20  a: Sample purified by dialysis.  64  Chapter 2 Synthesis of porphyrins  Table 2.14. 1H NMR data for the methylpyridiniumporphyrins (!) pyrrole  [cis  9.12 s(2)  -  3,5-NPh  3,5-MPy  2,6-Ph  2,6-NPh  2,6-MPy  3,4,5,-Ph  -  9.46 d (4)  8.21 d (4) 4.73 s(6) -2.95 s (2)  9.00 d (4)  7.89 m(6)  8.71 d (4)  9.49 d (4)  -  8.51 d (4)  9.00 d (4)  -  9.48 d (4)  8.23 d (4) 4.72 s(6) -2.99 s (2)  9.03 d (4)  7.90 m(6)  8.72 d (4)  9.46 d (4)  -  8.50 d (4)  9.02(4) g  B(MPy)DPhP]C12^129.00 m(4)  -CH3  N-pyrrole  8.91 s(2)  [cis  9.0 m (8)  -  B(MPy)B(NPh)P]C12^12  [trans  -  9.0 m (8)  B(MPy)DPIT]C12^12. [trans  -  9.0 m (8) g  B(MPy)B(NPh)11C1? ^12 [T(MPy)PhI9C13  9.00 q (4)  -  9.20 s (4) [T(MPy)(NPh)1103 [Tet(MPy)I1C14  9.1 m(8) 9.2 s(8)  4.72 s(6) -3.02 s (2)  4.70 s(6) -3.02 s (2)  9.50 d (2)  8.23 d (2) 4.71 s(9) -3.04 s (2)  9.03 m(2)  7.90 m(3)  8.74 d (2)  9.51 d (6)  8.52 d (2)  9.00 d (6)  -  9.55 d (8) 9.00 d (8)  -  -  4.74 s(9)  -  3.06 s (2)  4.77 s(12) -3.10 s (2) .  a: In DMSO-d6 at room temperature; chemical shift in ppm signal pattern (number of protons). b: Sample purified by dialysis. c: Signals of the pyrrole and 2,6-methylpyridinium protons overlap.  65  Chapter 2 Synthesis of porphyrins  Table 2.15. UV-visible data for the methylpyridiniumporphyrins(a)  Soret  visible 1  visible 2 visible 3 visible 4  [cis-B(MPy)DPhl1C12  416.5 (211)  517 (12.1)  555 (6.6)  578 (6.5)  636 (2.2)  [cis-B(MPy)B(NPh)11C12  417.0 (241)  518 (13.4)  555 (7.6)  578 (7.2)  635 (2.4)  [trans-B(MPy)DP1111C12  415.5 (178)  516 (8.0)  555 (6.3)  578 (4.7)  637 (2.7)  [trans-B(MPy)B(NPh)11C12 417.0 (159) 11 516 (10.9) 12 552 (6.7)  582 (5.2)h 637 (2.3)  [T(MPy)Phl1C13  419.0 (292)  516 (15.2)  553 (7.5)  580 (7.2)  637 (2.2)  [T(MPy)(NPh)11C13  419.0 (276)  516 (15.3)  552 (6.5)  580 (6.7)  636 (1.7)  ffet(MPy)11C14  420.0 (250)  516 (14.3)  550 (6.7)  580 (6.2)  636 (1.4)  a: In H20 at room temperature at 1 x 10-5M; wave length at maximum absorbance(Xmax) in nm, (extinction coefficient [E x10-3, M-1 cm-11).  b: Minimum E values, because the compound is impure (see Table 2.13).  66  Chapter 2 Synthesis of porphytins  2.3 Results and discussion  The major goal of the work described in this chapter was to synthesis watersoluble porphyrins. A method of monopyrrole-aldehyde condensation followed by modification was developed and used in this work. Use of this method allows for the possible synthesis of many more porphyrins, especially water-soluble ones. Some of these possibilities are discussed in Chapter 6 outlining future work. A small amount of impurity was found to be present in a few of the porphyrins (cis-DPh8PyP, PhTPyP, T(NP)PyP, and cis-(APh)PIBPyP) reported here. The impurity was found to be alumina from column chromatography used for the porphyrin separations. Further purification on these potphyrins was generally not carried out because these porphyrins were used as precursors for the synthesis of the water-soluble porphyrins. 1H NMR spectroscopy was used as the major method for characterization of the porphyrins. UV-visible, infrared and mass spectroscopies were also recorded. However the IR and MS techniques were only applied to a limited number of compounds because of the limitation in time. The 1H NMR and UV-visible data have been presented in Section 2.2 together with the synthesis procedures. The assignments and other features of the 1H NMR signals are discussed in Section 2.3.2. The UV-visible spectra are discussed in Section 2.3.3, while the infrared and mass spectral data are presented and discussed in Sections 2.3.4 and 2.3.5, respectively. The retention times of the TLC of the porphyrins are not reported because irreproducible data result from variation in the activity of alumina TLC plates stored in air. Known porphyrins were always run with unknown samples for comparisons within the alumina TLC data.  67  Chapter 2 Synthesis of porphyrins  2.3.1 Synthesis 2.3.1.1 Hydration of porphyrins  Associated water molecules (hydration) were generally found in samples of the porphyrin free-bases. For the non-ionic porphyrins, samples were only dried under vacuum at room temperature, and elemental analyses were carried out by handling the compounds in air. Different degrees of hydration were found in these samples as judged by elemental analysis and the intensity of the water signals in '1.1 NMR spectra. The hydration is probably related to the possibility of hydrogen-bonding between associated water molecule(s) and the pyrrole-N, pyrrole-NH, -NH2, -NO2, or pyridyl moieties in the porphyrin structures. The elemental analyses of (NPh)TPhP, B(NPh)DPhP and (APh)rPhP have been reported as unhydrated,2 as found in the present work. The porphyrins TPhPyP7 and (NPh)TPyr have been reported in the literature as hydrated with one-half and one water molecule, respectively, while the present work supports zero and one-half water molecule, respectively. Comparison of the elemental analysis data for the other potphyrins to the literature can not be made because elemental analyses of the relevant compounds are not reported in the literature, although (APh)TPyP, T(APh)PhP, T(NPh)PhP and the porphyrins of general formula Ph4-Pv(4-n), were synthesized before.2,8, Hydration of ionic porphyrins is a general phenomenon described in the literature, as shown in Table 2.16, taking Tet(SPh)P as an example. It can be seen that even though different purification methods were used by different authors, hydration molecules were always present except when a "strong" drying process was used. Attempts to remove the hydrated water completely were not made because the handling of the hygroscopic, dry samples in biological experiments was thought to be inconvenient.  68  Chapter 2 Synthesis of porphyrins  Table 2.16. Reports on elemental analysis of Tet(SPh)P(0)  C44H26N4012S4Na4.12H20  C%  H% N% S%  42.66  4.04  4.50  10.29  (4.54)  (10.55) drying at 100 °C 5  4.17  9.66  10.80  (44.22)  (4.52)  (9.38)  (10.72) (6 times)1°  51.62  2.54  5.48  12.52  dialysis, and drying at 150°C in  (5.17)  (12.29)  vacuo 11  4.17  9.66  10.75  Celite column (pyridine-water-  (44.12)  (4.09)  (9.74)  43.93  3.83  4.66  (43.65)  (3.70)  (4.39)  C44H26N4012S4(NH4)4.9H20 44.23  C44H26N4012S4Na4  C44H26N4012S4(NH4)4.9H20 44.23  C44H26N4012S4Na4.10H20  Purificationreference. Et0H-1170 recrystallization and  Me0H-acetone^reprecipitation  CHC13 eluent) 12 10.67  dialysis, and drying at 100°C in vacuothis work  a: Calculated (found).  Hydration was also apparent from the 41 NMR spectra of the compounds. The water content in a sample of Na3PyT(SPh)P was measured by 111 NMR spectroscopy in DMSO-d6 as described below. Two NMR tubes, one with and one without the sample (— 2 mg) were sealed under N2 with septa, and then placed into a large Schlenk tube (3 x 20 cm, see Section 2.2.3) with a wide mouth, through which N2 flowed. DMSO-d6 was then injected into the NMR tubes from a syringe under N2, and the NMR spectra of the two samples were recorded. The integration values of the following peaks were measured: In the spectrum with the sample: water peak (W); solvent peak (S); peaks of the pyrrole, pyridyl and sulfonatophenyl (111, 24 protons); and peak of the N-pyrrole protons (H", 2 protons). In the spectrum of neat solvent:  69  Chapter 2 Synthesis of porphyrins  water peak (w); and the solvent peak (s). Then the value of W = W – — w • S represents the relative amount of water in the porphyrin sample. The number of water molecules associated with a porphyrin molecule was calculated as: (24 /2) x (VV71-1'); or (W'/H") The average of the two calculations was 11.5, which was close to the number of 11 estimated by elemental analysis (see Table 2.10). The addition of DMSO-d6 into the NMR tubes under N2 was necessary to obtain a reproducible result because this solvent absorbed moisture strongly. Errors in this measurement could arise also from the low accuracy of the integrations of the fairly widely spaced peaks; and from the possible differences in the sensitivities of the various protons because of possible differences in relaxation times. For Na3[PyT(SPh)13], two associated water molecules were present in a sample precipitated from Me0H by adding acetone, and in a sample formed by evaporation of aqueous solution with subsequent drying at 100°C under vacuum (Table 2.10). This may indicate that these two water molecules associate to the porphyrin molecule differently from the other nine water molecules present in a sample collected from evaporation of an aqueous solution (11 associated water molecules were estimated in this. sample, Table 2.10). The two water molecules may be associated with the porphyrin molecule by strong hydrogen-bonding via the pyrrole-N, the pyrrole-NH and/or the pyridyl-N. Pv(4)1' 2.3.1.2 Synthesis of the porphyrins with general formula Phn,, The synthesis of these porphyrins have been reported,9 following the procedure described by Little et al.' In the present work, different from the literature procedure, a significant amount of porphyrin product, besides that precipitated directly from the  70  Chapter 2 Synthesis of porphyrins  reaction mixture, was collected by precipitation from the concentrated condensationreaction mixture using acetone, and therefore the yield was higher. For the separation of the 6 porphyrins, alumina (activity III) and silica gel chromatographies with CHC13 as eluent were used instead of the reported method, which used silica gel column chromatography with a mixed solvent of CHC13, acetone and methanol as eluent. All the 6 porphyrins could be separated on a single column of the alumina activity M. However, use of silica sel to first separate TetPhP and TPhPyP from the other 4 porphyrins, followed by use of the alumina column to separate the other 4 porphyrin, was more efficient. There is an advantage of using neat solvent because the solvent can be readily recovered by distillation and drying. Mass, 111 NMR (in CDC13) and UV-visible (in CH2C12) spectroscopy data of these poiphyrins have been reported recently.9 The data obtained from MS (Section 2.3.5) and 111 NMR spectroscopy (Table 2.2, p.41) are in agreement with the reported data. There were some deviations of the UV-visible data from the reported data; e.g., the extinction coefficients for TetPyP, PhTPyP, and cis and trans-DPhBPyP were generally 10-20% lower in the present work, while for TPhPyP the values were —8% higher than the reported values.9 The impurity in samples of PhTPyP and cis-DPhBPyP might result in the low e values, although some difference may also result from the difference of the solvent used (CHC13 was used in this work, while CH2C12 was used in the literature work). The UV-visible (in CHC13) and Ill NMR (CDC13) spectra of TPhPyP have also been reported by another group;7 and data obtained in the present work generally agree with those in this report, though there is some disagreement about the assignments of the NMR signals (see Section 2.3.2). 2.3.1.3 Synthesis of nitro-porphyrins  71  Chapter 2 Synthesis of porphyrins  The nitration reagents used for the nitrations of the porphyrins varied according to the structures of the porphyrins. Generally, the more pyridyl and nitrophenyl groups present, the stronger the reagent needed. For example, the nitration of TPhPyP is more difficult than that of TPhP. The protonated pyridyl in the reaction medium probably reduces the activity of the porphyrin for the electronphilic substitution. The first nitration of TPhPyP gives almost solely the cis-isomer (as judged by 1 NMR, Section 2.3.2), which means that the phenyl trans to the pyridyl is more inert. In the course of this work, the reaction times for nitration were found to depend significantly on temperature, which was not controlled (room temperature), and so the reported reaction time varied with room temperature. It was more reliable to monitor the reaction by TLC than to control the reaction time. Strong nitration reagents have been used to nitrate porphyrins in some literature reports. NO2 in acetone and nitronium tetrafluoroborate were used for the nitration of OEP, 13514 and fuming nitric acid was used for the nitration of TetPhP.2 All these reagents are strong oxidants and can probably destroy the porphyrin ring structure, thus giving low nitration yields. Under the reaction conditions used here, NO2 was produced gradually in the reaction mixture, by using nitric acid mixed with a dehydration reagent such as acetic acid or a mixture of acetic acid and sulfuric acid; and thus relatively mild reaction conditions were provided leading to 60-85% yield from porphyrin precursors. The (NPh)TPIT poiphyrin has been synthesized by direct condensation of benzaldehyde, 4-nitrobenzaldehyde and pyrrole, but the yield is very low (2.7%);3 (NPh)TPyP has also been synthesized by direct condensation of the appropriate reagents with 7% yield.8 These low yields probably result from the high activity of 4nitrobenzaldehyde.15 The (NPh)TPhP compound (55% yield from TetPhP), a mixture of cis- and trans-B(NPh)DPhP (28% yield) and T(NP)PhP (no yield reported) have also been 72  Chapter 2 Synthesis of porphyrins  synthesized by nitration of TetPhP using fuming nitric acid.2 The methods used in this thesis gave higher yields of these porphyrins and seven other new nitrophenylporphyrins (see Figure 2.1, p.36; and Section 2.2.3). The 1H and 13C NMR, UV-visible, 1R and mass spectra of (NPh)TPIIP have been reported before.2,3 The spectral data obtained in this thesis work agree with those reported except that the extinction coefficient for the Soret band obtained here (in CHC13) was about 25% lower than the reported value (in CH2C12).2 The 'H NMR, UV-visible and mass spectra of the mixture of cis and trans-B(NPh)DPhP have also been reported by the same group,2 and the thesis data agree well. The synthesis, and 1H NMR and UV-visible spectra of (NPh)TPyP have been reported recently by another group;8 the extinction coefficients obtained in the present work were all about 100% higher than those reported, and there are some disagreements about the assignments of the 1H NMR signals (see Section 2.3.2). 2.3.1.4 Synthesis of aminophenylporphyrins The (APh)TPIT porphyrin has been synthesized and characterized by elemental analysis, 1H NMR and mass spectroscopy.2.3 The data for this porphyrin obtained here agree with those the literature. The other 6 aminophenylporphyrins (see Figure 2.1, p.36) are new compounds. 2.3.1.5 Synthesis of sulfonated porphyrins Several reports about the sulfonation of TetPhP to synthesize Tet(SPh)P can be found in the literature,5, 10,11 and the sulfonation procedures reported here are essentially the same as the literature one except that shorter reaction times are employed here. Preliminary studies on the reaction time for different types of porphyrins were carried out in this thesis work. Samples of the reaction mixture at different reaction times were taken; 73  Chapter 2 Synthesis of porphyrins  the porphyrin samples then were isolated (in the same way as described in the synthesis procedure), and Ill NMR spectra of these porphyrin samples were recorded. The degree of sulfonation was calculated by the integration ratio of the sulfonatophenyl peak to the phenyl (unsulfonated) peak. It is observed that the more pyridyl groups present, the longer reaction time is needed. For instance, sulfonation of (NPh)TPhP is complete in 0.5 h; replacing one phenyl by a pyridyl (i.e. for cis-(NPh)DPhPyP) requires 6 h reaction time; sulfonation time for cis or trans-DPIMPyP (12 h) is longer than the sulfonation time required for TetPhP (4 h). In the literature,5,10,11 there are major differences reported for purification of the sulfonated product. Following the initial studies in 1962 by Winkelman16 on the localization of sulfonated TetPhP in tumors, Fleischer et al.1° developed a synthetic method using six precipitations of (NH4)4Tet(SPh)P from methanol by adding acetone. Later on, Srivastava and Tsutsuis reported a method using lime to neutralize the excess sulfiiric acid with formation of a precipitate of calcium sulfate from the sulfonationreaction mixture, leaving the Na4Tet(SPh)P in the solution; the residue from the evaporation of this solution was then recrystallized from ethanol-water. Busby et a!. " pointed out problems with these processes, and reported a method using dialysis to purify Na4Tet(SPh)P (see Table 2.16, p.69, for the analyses of these products). Since this report, Celite column chromatography has been used to purify (NH4)4Tet(SPh)P,12 and Sephadex G-10 column chromatography used to purify (NH4)3[(APh)T(SPh)13].2 As part of this thesis work, Srivastava and Tsutui's method using lime to neutralize the H2SO4 was followed, but low contents of C, H, and N were found in the purified sample by elemental analysis; this probably means that inorganic salt was present as impurities, possibly resulting from the solubility of lime and CaSO4 in water. Alternatively, Ba(OH)2 solution was also used to precipitate sulfate anions, but a low yield of porphyrin  74  Chapter 2 Syntheab of porphyrins  resulted because of the coprecipitation of the barium salt of the sulfonated porphyrin. Sephadex G-10 column chromatography also gave a low yield because of absorption of the porphyrin by the stationary phase. Dialysis proved to be a good method for purification of all the sulfonated porphyrins described in this thesis except for Tet(SPh)P, when an observable amount passed through the membrane. The different behaviors of the sulfonated porphyrins in the dialysis process are probably related to the different aggregation properties of the sulfonated porphyrins, which are discussed in Chapter 4. Precipitation from methanol by adding acetone (as described in Section 2.2.5) was used to purify Na4Tet(SPh)P in this work. The 1H NMR spectra of the tetrasodium salt of Tet(SPh)P in D20 and in DMSOd6 have been reported before.5,11,17 The spectral data obtained here (Table 2.11, p.60) agree with the literature data. The (APh)T(SPh)P porphyrin has also been reported before.2 The 1H NMR spectral data (in DMSO-d6) of the sodium salt of the porphyrin diacid and of the ammonium salt of the porphyrin free-base, and the UV-visible spectrum of the ammonium salt in 0.1 M ammonium carbonate were reported in this paper.2 The 1H NMR spectral data in DMSO-d6 reported here (Table 2.11, p.60) were obtained from the sodium salt of the porphyrin free-base, and the UV-visible spectrum was obtained from the sodium salt in distilled water at 1.0 x 10-5 M. The data are not comparable because of the differences in the experimental conditions. The other 6 sulfonated porphyrins (see Figure 2.1, p.36,) are new compounds. Sulfonation of the phenyls on the porphyrins always left a small amount of unsulfonated phenyl group(s), which was detected by proton-NMR spectra, even if the  75  Chapter 2 Synthesis of porphyrins  reaction time was prolonged by two or three times. This observation tends to support the reversibility of the sulfonation reactions, which is common in organic chemistry." Sulfonation of (NPh)TPIIP gives an interesting reaction, in which the nitro group is reduced to an amine group, probably by an intermediate(s) formed by destruction of the porphyrin structure. Shorter reaction times increase the yield of the amino species (40% yield for 0.5 h reaction time and 13% for 4 h reaction time), but do not give a sulfonated nitro porphyrin product. The 11-1 NMR spectrum of the product in DMSO-d6 is essentially the same as the spectrum of the product from sulfonation of (APh)TP1113 which gives the characteristic signals for aminophenyl at 7.90 ppm (doublet), 7.04 ppm (doublet) and 5.61 ppm (singlet) (see Table 2.11 for assignments). From sulfonation of B(NPh)DPhP (a mixture of cis and trans-isomers), three products were isolated for N1VIR samples. The products are B(NPh)B(SPh)P, (APh)(NPh)B(SPh)P and B(APh)B(SPh)P, as judged by 1H NMR spectra, and the bis(nitro)porphyrin is the major product. These products are presumably mixtures of the  cis and trans-isomers because the starting material is a mixture of the cis and transisomers. The two isomers could not be identified by the spectra as described in Section 2.3.2.3 because of the overlapping of the signals of the pyrrole protons. The separation procedure is not capable of separating the cis and trans isomers. The spectra of these products are shown in Figure 2.2, in which the structures of cis-isomers are used to represent the products. The signals of the N-pyrrole protons, singlets at -2.91, -2.84 and -2.78 ppm, respectively, for the bis(nitro), mono(amino)-mono(nitro) and the bis(amino) species, are not shown; the signals at 5.61 ppm of the -NH2 protons in the amino species are also not shown. The porphyrins are not listed in Figure 2.1 (p.36) because purified samples were not isolated. Further studies on these products were abandoned because of the low yield of the synthesis reaction and the difficulty in the separation procedure.  76  Cluipter 2 Synthesis of porphyrins  • 9.0  8.0  • 7.0  Figure 2.2. 1H NMR spectra of B(NPh)B(SPh)P, (APh)(NPh)B(SPh)P and B(APh)B(SPh)P in DMSO-d6. Assignment (which are essentially identical for each species): 8.20 8.55 chem. shift(ppm) 8.8-9.1 8.70 dorm pattern d m d assignment. pyrrole 3,5-NPh 2,6-NPh 2,6-SPh  8.08 dorm 3,5-SPh  7.90 d 2,6-APh  7.04 d 3,5-APh  77  Chapter 2 Synthesis of porphyrins  Sulfonation of cis-(NPh)DPITyP in a 6 h reaction time gives the unreduced porphyrin cis-(NPh)PyB(SPh)P as the major product with a minor contamination of cis(APh)PyB(SPh)P; the 1H NMR spectrum of the sulfonation product is shown in Figure 2.3. Compared to (NPh)TPhP, the presence of the protonated pyridyl on cis(NPh)DPhPyP in acidic solution probably stabilizes this porphyrin structure from nucleophilic attack and therefore limits the formation of the reducing intermediate probably formed from degradation of the porphyrin structure. A reaction time of 6 h was optimal for synthesis because more amino porphyrin was formed with a longer reaction time, and incomplete sulfonation was evident with a shorter reaction time. In the spectrum of cis-(NPh)PyB(SPh)P (Figure 2.3), the signal of the pyrrole protons appears only as a broad singlet. This does not give enough information about the symmetry of the product (cis-isomer vs. trans-isomer; details about this identification are discussed in Section 2.3.2.3). However, the product is considered to be the cis-isomer because it is made from cis-(NPh)DPhPyP which is well defined by the pyrrole proton signals in the 1H NMR spectrum (see Figure 2.14, Section 2.3.2). 2.3.1.6 Synthesis of methylpyridiniumporphyrins  Methylation of the pyridyl moieties on a porphyrin can be easily performed with methyl 4-toluenesulfonate in DMF with a high yield, as described in the literature.6 A reaction time of 4 h was adequate for this reaction instead of overnight as reported, and prolonged reaction times caused complications in isolation of the product. The tosylate salts of the dicationic porphyrins are not soluble in cold water or acetone, but are soluble in the mixture of these two solvents. This probably results from the fact that organic solvents break up the aggregation of these porphyrins (Chapter 4) and therefore increase their solubility in water. A more detailed discussion about the effect of organic solvents on  78  Chapter 2 Synthesis of porphyrins '  S 03  0N  B.  9.2^9.0^9.8^8.6^8. 4^8.2^8.0^7 8 PPM  I  I^I^1^I^  I^II  a  Figure 2.3. 1H NMR spectrum of cis-(NPh)PyB(SPh)P in DMSO-d6. A. Before purification. There is some cis-(APh)PyB(SPh)P, identified by the peaks at 7.85 ppm (2,6-APh), 7.77 ppm (3,5-APh), and 5.80 ppm (NH2). B. Purified. Unsulfonated phenyl, identified by signals at 7.80 and 8.38 ppm. chem. shift(ppm) pattern assignment  9.02 d 3,5-Py  8.87 s(broad) pyrrole  8.66 d 3,5-NPh  8.49 d 2,6-NPh  8.25 d 2,6-Py  8.16 d 2,6-SPh  8.03 d 3,5-SPh  79  Chapter 2 Synthesis of porphyrins  porphyrin aggregation can be found in Chapter 4. In the process of synthesis, some watersoluble impurities in these dicationic products can be washed off using cold water, but acetone, which is used to wash away the reaction solvent and organic impurities, has to be completely removed first before the washing with H20 in order to avoid losing the product. The perchlorate salt of the diacid of Tet(MPy)P has been synthesized previously, and characterized by elemental analysis and UV-visible spectra in aqueous solution by Hambright and Fleischer in 1970.19 The tosylate salt of this porphyrin was synthesized by Pasternack et al. in 19726 and characterized by elemental analysis and UV-visible spectra in aqueous solutions. The chloride salt has been noted later in the fiterature,2° but characterization of this salt has been reported only recently9 as discussed below. The chloride salts of Tet(MPy)P, T(MPy)PhP, cis- and trans-B(MPy)DPhP, have been synthesized before.9 However, elemental analyses were not reported, while the 111 NMR spectra were reported in D20, CD3OD or CD30D-CD2C12 without specific reference to the solvent used for each compound, and the UV-visible spectra in aqueous solutions were reported without mention of concentration. The Ili NMR spectral data (Table 2.14, p.65) reported here were recorded in DMSO-d6. The UV-visible spectra in distilled water were found to be concentration dependent (Chapter 4) and are recorded at 1.0 x 10-5 M in this work (Table 2.15, p.66). These differences in the experimental conditions make the spectral data not easily comparable with the literature data. Dialysis was also employed in purification of the cationic methylpyridiniumporphyrins, but a major loss of porphyrin product was observed from the dialysis of T(MPy)PC14; this probably results from different aggregation properties of this compound in comparison to the other cationic porphyrins (Chapter 4).  80  Chapter 2 Synthesis of porphyrins  2.3.2 Proton-NMR spectra  The chemical shifts and patterns of the 1H NMR signals, the signal-intensity ratio of the substituents, plus the information from the syntheses were the main data used in this thesis to assign a structure to a synthesized porphyrin. The 1H NMR spectra of water-soluble porphyrins were measured in DMSO-d6 solvent. Measurements in D20 often gave complicated spectra which may result from aggregation of the porphyrins (Chapter 4). For non water-soluble porphyrins, CDC13 was used. The nitration and sulfonation always occur at the para positions of the mesophenyls. This was evident by the characteristic two doublets of the NMR spectrum corresponding to the meta and ortho protons of a 4-nitrophenyl or 4-sulfonatophenyl group. The formation of a 4-aminophenyl derivative was confirmed by the two doublets for the meta and ortho protons of aminophenyl and the appearance of the signal for the amine protons at 4.00-4.05 ppm in CDC13, which disappeared when D20 was added to the NMR samples. For the following discussion, a pyrrole proton refers to a proton bonded to a carbon atom of a pyrrole ring, while the proton associated with the pyrrole nitrogen is referred to as an N-pyrrole proton. 2.3.2.1 Identification of substituents from Ill NMR spectra All the substituents at the meso positions have characteristic chemical shifts  (ppm) which are listed in the following table with associated JHH coupling constants ( in Hz). The detailed assignments are considered in Section 2.3.2.2.  81  Chapter 2 Synthesis of porphyrins  solvent  , -NH2 APh  MPy  NPh  2,6-Ph  3,4,5- Py  SPh  .Ph CDC1/  4.0  DMSO-d6 5.6  8.0, 7.1 -  8.6, 8.4 8.2  (8.1)-4  (8.6)1  7.9, 7.0 9.5, 9.0 8.7, 8.5 8.1 (8.2)1  (5.7)!  (8.5)1  (7.5)!  Npyrrole -2.8— -3.1  7.8  9.1, 8.2  -  7.9  9.0, 8.3  8.2, 8.1 -2.8— -3.0  (6.3)a  (8.0)1  a^Coupling constant between ortho- and meta-protons.  The coupling constant for the pyrrole protons is 4.7 Hz in CDC13 and 4.3 Hz in DMSO-d6. The spectrum of the protons within a 4-substituted phenyl ring (APh, NPh, SPh), a 4-pyridyl ring (Py) or a 4-methylpyridinium ring (MPy) should be an ANBB' type, which is usually too complex to be analyzed for the coupling constants.21 However, the spectra of APh, MPy, NPh and SPh appear as AB quartets, which means the coupling constants between the 2 and 5, 2 and 6, and 3 and 5 protons are all small, so the system can be treated approximately as an AB system (i.e.,only the coupling of the 2 and 3 or 5 and 6 protons are considered). The coupling constants listed in the above table are obtained in this manner. The same approximation cannot be applied to the 4-Py and Ph systems because more complex spectra are obtained (see Figure 2.7, p.91, for examples). The various coupling constants noted do not vary from one porphyrin type to another. The assignments of the coupled signals to each group are achieved simply by comparisons with related spectra. For instance, a comparison of the spectrum of TetPhP to that of TPhPyP gives information about which signals correspond to which group, because on going from the former to the latter, a new set of signals [8.2 (211) and 9.1 (2H)] appears for the pyridyl group; the phenyl signals at 7.8 and 8.2 ppm reduce in intensity from 8 and 12H to 6 and 9H, respectively, and the pyrrole signal at 8.8 ppm (8H) splits into two doublets and one singlet; the N-pyrrole signal at -2.8 ppm scarcely changes. The other signals of the substituents are assigned in the same fashion by comparing the  82  Chapter 2 Synthesis of porphyrins  relevant spectra together with information from the syntheses (see the synthesis scheme in Figure 2.1, p.36). 2.3.2.2 Assignments of signals to the protons of the meso substituent -  The assignment of each signal to corresponding protons of the substituents presents a reasonably straightforward problem. The signals for the phenyl protons in the spectrum of TetPhP are easily assigned (as 7.78 ppm for the 3,4,5-Ph protons and 8.23 ppm for the 2,6-Ph protons) by consideration of the distances from the protons to the porphyrin ring, and the deshielding effect on the phenyl protons. These assignments have appeared in the literature.22 When the 4-phenyl proton is replaced by a nitro or an amino group, the chemical shifts can be calculated according to the shielding parameters of the nitro or amino group within aromatic compounds (figures shown below);23 these have been used by Sun et al.24 to calculate the chemical shifts for the protons of the 3nitrophenyl group of 5-(3-nitropheny1)-10,15,20-triphenylporphyrin. Table 2.17 presents the results of calculated and experimental chemical shifts (the numbering system is presented in Section 1.2, p.2, ans drawn again below).  4  /  \  \ 5  6  /  I —Porphyrin  +0.95 +0.17  ON  -0.75 -0.24 +0.33  +0.95 +0.17  HN  -0.63 -0.75 -0.24  83  Chapter 2 Synthesis of porphyrins  Table 2.17. Calculated and observed chemical shifts of the nitrophenyl and aminophenyl protons  2,6-nitrophenyl  3,5-nitrophenyl  2,6-aminophenyl  3,5-aminophenyl  calculated  8.23A+0.17=8.40  7.78A-1-0.95=8.73  8.231-0.24=7.99  7.78A-0.75=7.03  observedk  8.41  8.64  7.98  7.05  11: The 8.23 and 7.78 (ppm) values are for the 2,6 and 3,4,5-protons of the unsubstituted phenyl group. h: The averages of observed chemical shifts are listed here (see Tables 2.6 and 2.9).  Thus, a doublet is readily assigned to each kind of proton type (2,6- or 3,5-) for either the nitrophenyl or aminophenyl group; the 'least satisfactory' calculated value (for the 3,5nitrophenyl protons) is 0.09 ppm greater than the observed value. Figure 2.4 shows the spectrum of (NPh)TPhP and the assignments of the signals. The 1H NMR spectra of the sulfonated porphyrins are recorded in DMSO-d6. The 1H NMR spectrum of TetPhP in DMSO-d6 is not available (because of low solubility of this compound), and neither are the shielding parameters for the sulfonato group within a phenyl ring. However, the 1H NMR spectrum of T(MPy)PhP in DMSO-d6 gives the chemical shifts of the phenyl moiety at 8.13 ppm (2,6-phenyl protons) and 7.89 ppm (3,4,5-phenyl protons). The spectra of the sulfonatophenyl within the sulfonated porphyrins show two signals at 8.20 and 8.03 ppm in DMSO-d6 (Table 2.11, p.60). When the 4-phenyl proton is replaced by sulfonate, an electron withdrawing group, the chemical shifts of both of the phenyl signals (2,6- and 3,5-protons) should increase,23 which suggests that the 2,6-sulfonatophenyl protons resonate at 8.20 ppm (a shielding parameter of +0.07 ppm), and that the 3,5-sulfonatophenyl protons resonate at 8.03 ppm (a shielding parameter of +0.24 ppm). The opposite assumption would make the shielding parameters  84  Chapter 2 Syntheab of porphythas  for sulfonato to be abnormal (-0.10 for 2,6-protons and +0.31 for 3,5-protons).23 An example of the assignments for a sulfonated porphyrin is shown in Figure 2.5.  H20  N-pyrrole ..A,. ,,,,, ..A.. .......  " • • ' " • " 4 ' " ' "^MS  • • • "  Figure 2.4. 1H NMR spectrum of (NPh)TPhP in CDCI3. Assignments: Chem. shift (ppm)^8.7-9.01^8.64^8.41^8.22^7.78 pattern^d-s-d assignment^pyrrole^3,5-NPh^2,6-NPh^2,6-Ph^3,5-Ph 1 Assigned individually later in Section 2.3.2.4.  85  4,2sq.  111!1■!4^S.2^1.1 • .;.• PPM  N-pyrrole —  sove'  Figure 2.5. 1H NMR spectrum of trans-BPyB(SPh)P in DMSO-d6. Assignments: Chem. shift (ppm) pattern assignment  9.04 d 3,5-Py  8.89 m pyrrole  8.25 d 2,6-Py  8.18 d 2,6-SPh  8.05 d 3,5-SPh  86  ^ Chapter 2 Synthesis of porphyrins  The shielding parameter for the N atom within a pyridine is not listed in the fiterature.23 The spectrum of pyridine in CDC13 was recorded, and the shielding parameters for the pyridine nucleus were calculated using 7.27 ppm as the chemical shift for benzene.23 Then the chemical shifts of the 4-pyridyl on a porphyrin structure can be calculated using the chemical shifts for the phenyl groups of TetPhP (in the same way as shown in Table 2.17); the values are shown below:  \  /r-Th  9.06^8.17  +1.28 -0.06  8.55^7.21  7.60  \j  / \^+0.33  N  / \  porphyrin  chemical shifts shielding parameters calculated chemical shifts These calculated data agree very well with the observed data, which show average values of 9.05 and 8.18 ppm for the 3,5 and 2,6-pyridyl protons, respectively (see Table 2.2, p.41). The signals of the pyridyl within water-soluble porphyrins, the spectra of which are recorded in DMSO-d6, can be assigned with these data although these signals appear at slightly different positions (9.04, 8.25 ppm, Figure 2.5), presumably because of the difference in the solvent. The chemical shifts of the pyridyl protons of PyT(SPh)P in DMSO-d6, which are 9.03 and 8.27 ppm, are used generally here as those for pyridyl protons on a porphyrin in this solvent. These chemical shifts increase to 9.50 and 9.02 ppm when methylation occurs; the methylation of the pyridyl groups is expected to make both signals shift to lower field. Therefore the signal at 9.50 ppm is assigned to the 3,5 -MPy protons and the signal at 9.02 ppm to the 2,6-MPy protons. An example of the assignments in a spectrum of a cationic methylpyridiniumporphyrins is shown in Figure 2.6.  87  Chapter 2 Synthesis of porphyrins  The assignments of the signals of the protons within the porphyrin substituents are listed in Tables 2.2 (p.41), 2.5 (p.49), 2.8 (p.53), 2.11 (p.60) and 2.14 (p.65).  Me  NO2  Figure 2.6. 1H NMR spectrum of (NPh)T(MPy)P in CDCI3. Assignments: Chem. shift pattern assignment  9.50 d 3,5-MPy  9.17-9.00 m pyrrole  9.00 d 2,6-MPy  8.74 d 3,5-NPh  8.52 d 2,6-NPh  4.75 s Clii  88  Chapter 2 Synthesis of porphyrins  The signals of the pyridyl protons within pyridylporphyrins have been assigned in a few literature reports. Williams et al.7 assigned signals at 9.01 and 8.22 ppm to the 2,6and 3,5-Py protons of TPhPyP, respectively, and Ding et al. assigned signals at 9.06 and 8.15 ppm to the 2,6- and 3,5-Py protons of (NPh)TPyP, respectively.8 Differently, Sari et al.9 assigned signals at 9.1-9.4 and 8.2 ppm to the 3,5 and 2,6-Py protons, respectively, for porphyrins with a general formula PhnPy(4_n)P. All these assignment have been given without reasoning. The assignments given in the present work agree with the report of Sari et al. and not with the other two earlier reports. Ding et a!.8 also assigned the signals of the nitrophenyl protons within (NPh)TPyP as 8.66 ppm for the 2,6-NPh protons and 8.38 ppm for the 3,5-NPh protons. The assignments given here for the nitrophenyl signals are opposite to these, i.e., 8.61 ppm for the 3,5-NPh protons and 8.34 ppm for the 2,6-NPh protons, and these agree with the assignments for a 3-nitrophenyl porphyrin given by Sun et a!.24 The signals of the aminophenyl protons within (APh)TPyP are also assigned by Ding et al.,8 and are consistent with those given here. The assignments within some methylpyridinium porphyrins have also appeared in the literature.8,9 The assignments given in this thesis work agree with these reports in that the 3,5-MPy protons resonate at lower fields than the 2,6-MPy protons, although the NMR spectra are taken in different solvents. The two doublets of the sulfonatophenyl protons in the spectrum of Tet(SPh)P in DMSO-d6 have been assigned by using partial deuteration at the 2,6-position,17 and the assignments given here agree with the reported ones. It is more challenging to assign the split pyrrole signals to individual protons, and this is discussed later in Section 2.3.2.4.  89  Chapter 2 Synthesis of porphyrins  2.3.2.3 Identification of isomers  Another interesting feature of the 111 NMR spectra of the pyrrole protons is the information provided for structure assignments, especially for distinguishing between cisand trans-isomers. The following figure shows some of the symmetry elements of the two isomers of the A2B2P type; the treatment ignores the N-pyrrole protons because there is fast tautomer inter-conversion at room temperature in organic solvents." i  12  12  \  13  \ ._..-- NH  13  ^// A C 2  B^A N^H N  17^3^\  „\  „ 'C22  cis-isomer  1  2  ^  trans-isomer  It is clear that for the cis-isomer, there are four types of pyrrole protons, while for the trans-isomer there are only two types of pyrrole protons. Two identical protons on the same pyrrole ring within a porphyrin give rise to a singlet, and two different protons on the same pyrrole ring give two coupled doublets. So, two singlets and two doublets are expected for the cis-isomer and two doublets are expected for the trans-isomer in the 1H NMR spectra of pyffole protons. Figure 2.7 shows the spectra of cis- and transDPhBPyP. The trans isomer gives two doublets, and the cis one gives two doublets and  two singlets. Thus, the two isomers can be identified easily.  90  Chapter 2 Syntheah of porphyrins  9 0^8 8  8 . 6^8 . 4  8 . 2^8 . 0  7.8^7. 6 PPM  d^d  ^JL^ 111{!1111‘^  9 0^88^8.6^84^82^80^7. 8 ppm 7 6  Figure 2.7. 1H NMR spectrum of cis- and trans-DPhBPyP in CDCI3. The main difference in these spectra is in the 8.7-9.0 ppm region of the pyrrole protons. There are two doublets and two singlets for the cis-isomer and two doublets for the trans-isomer. For the assignments of these peaks, see Table 2.18, p.101; for the assignments of the other peaks, see Table 2.2, p.41.  91  Chapter 2 Synthesis of porphyrins  An observation that the two protons on the pyrrole ring appear identical in the 111 NMR spectrum, as long as the two adjacent meso substituents are identical, has been found to be true for all of the porphyrins studied in this thesis work. An example of this observation is the spectrum of (NPh)TPhP (Figure 2.4); in this compound the meso positions 5 and 20 have different substituents, but H12 and H13 give a singlet because the substituents at meso positions 10 and 15 are identical. Using this observation, the following patterns for the 1H NMR spectra of the pyrrole protons of porphyrins ( in the 8.6 - 9.0 ppm region), with different types of meso-substituents, may be drawn (Figure 2.8, relative intensities are also shown):  H7  H13  R5  R15  H3  H17  s [8]  #1 R5=R1 0R1 5=R20 #2 R5; R10=R15=R20  II  ^  #3 R5=R10; R15=R20  II  d[2] s[4] d[2]  H  d[2] s[2] s[2] s[2] d[4] d[4]  #4 R5=R15; R10=R20 #5 R5=R10; R15; R20  Ill'''  ^H^II^H #6 R5=R15; R10; R20  d[1] d[1] d[1] s[2] d[1] d[1] d[1]* d[2] d[2] d[2] d[2]  * The relative position of the singlet can vary.  Figure 2.8. The patterns for the 1H NMR spectra of the pyrrole protons. 92  Chapter 2 Synthesis of porphyrins  The spectra of all the porphyrins synthesized in this thesis work are consistent with these patterns, although some of the signals overlap giving an appearance of fewer signals. For instance, T(APh)PhP should give signals in pattern #2 as a doublet (2 protons), a singlet (4 protons) and a doublet (2 protons), but the spectrum appears to consist of a doublet (2H) and a broad singlet (6 protons); the latter arises from the overlapping of the singlet (4 protons) and one of the doublets (2 protons) (see Table 2.8, p.53). The spectra of the water-soluble porphyrins usually give broad and less informative signals (see Tables 2.11, p.60 and 2.14, p.65; Figures 2.5 and 2.6), but the cis- and trans-isomers can be assigned according to the spectra of the precursors (see Figure 2.1), whose isomers can be identified by the peaks of the pyrrole protons. More examples of the spectra of the pyrrole protons of cis- and trans- isomers are given in Figures 2.9 and 2.10. Figure 2.9 shows the spectra of the pyrrole protons of cisand trans-B(NPh)BPyP. In the spectrum of the cis- isomer, the two singlets overlap with the two doublets (see the inset), but there is no doubt about the identifications of the cisand trans-isomers by comparing the two spectra. In Figure 2.10, a more complex example (cis- and trans-B(MPy)PhPyP) is presented. There are four doublets for the trans-isomer  as shown by pattern #6 in Figure 2.8, but for the cis-isomer, the #5 pattern can been seen only with careful inspection. Nevertheless, the identification of the two isomers can be readily achieved using these spectra. The pyrrole protons of the mixture of cis- and trans-B(NPh)DPhP give a spectrum of two doublets and two singlets, the pattern expected for the cis-isomer, but the intensity ratio of the signals is 5/2:3/2:3/2:5/2 (see Table 2.5, p.49) instead of the required 2:2:2:2 for the cis-isomer (see # 3 in Figure 2.8). The observed ratio results from the overlapping of the signals for the cis- and trans-isomers. From the ratio, the isomer composition can be calculated:  93  Chapter 2 Synthesis of porphyrins  N H2  HN  N H2  9.0^8.0^7.0^6.0^5.0  ^  4.0 ppm  Figure 2.9. 1H NMR spectra of cis- and trans-B(NPh)BPyP in CDCI3. The pyrrole protons resonate in the 8.7-9.0 ppm region. Although the two doublets and the two singlets in the spectrum of the cis-isomer overlap, the #3 pattern can still be seen. The trans-isomer has the #4 pattern as expected.  94  Chapter 2 Synthesis of porphyrins  0N  ‘.0•1 fr^:51.!1;.1.11111^. 5.1:,.11;1 1 • .1!5.,;;m^,),,I i1i.,,I^Sill  N02  1.0^1.8^1.11^1.4^1.2^1.0^7.1 PPM  Figure 2.10. 1H NMR spectra of cis and trans-B(NPh)PhPyP in CDCI3. The pyrrole protons resonate in the 8.7-9.0 ppm region. The cis-isomer should have 1 singlet (2H) and 6 doublets (1H each) for the pyrrole proton (pattern # 5), but two of the doublets overlap to give a more intense doublet (2H) at 8.90 ppm, and the singlet overlaps with two of the other doublets; this can be seen when the spectrum is expanded. The trans-isomer gives four doublets (pattern #6) as expected.  95  Chapter 2 Synthesis of porphyrins  If X is the molar portion of the cis-isomer, then 2X = 3/2, and X = 75%, and therefore the molar portion of the trans-isomer is 25%. X [cis- isomer (2:2:2:2)]  1  1  (1 -X)[trans- isomer (4:4)] mixture(5/2:3/2:3/2:5/2)  2.3.2.4 Assignments of the signals for the pyrrole protons  The following discussion is devoted to the assignment of the 1H NMR signals to individual pyrrole protons. The numbering scheme is as shown in Figure 2.8. The substituents are always arranged alphabetically with respect to the numbered meso positions; i.e., the lowest numbered 5 meso position always accommodates the -  aminophenyl if present. There are two doublets and one singlet for the pyrrole protons in the spectrum of (NPh)TP11.13 (Figure 2.4). From the discussion above, the singlet is assigned to H12, H13, H17 and H18. The two doublets belong to protons H2 a Hg; and H3 H7. With the  assumption that C2 and Cg are more electron deficient than C3 and C7, the doublet at low field (8.90 ppm) is assigned to H2 and Hg, and doublet at higher field (8.74 ppm) to H3 and H7. The assumption is supported by the electronic distribution on 2-nitronaphthalene, as demonstrated by electronphilic substitution reactions," considering the whole nitrophenyl moiety as an electron-withdrawing group and the similarity of the relevant parts of 2-substituted naphthalene and the porphyrin (Figure 2.11 a). The assumption is also supported by the resonance forms of this nitroporphryin (Figure 2.11 b), and can be expanded to suggest that the pyrrole proton adjacent to an electron-withdrawing meso 96  ^ ^  Chapter 2 Synthesis of porphyrins  substituent (including 4-Py, 4-MPy, 4-NPh, and 4-SPh) resonates at a higher field than the other proton on the same pyrrole ring. a N O2+  ON  /--  0N  major product  Nph.^ j,,NH^N::-_\ i^ L 7-ìN HN /  b  --.1,...,  ^•-...,......^---,...„  0-  H  \^ 7^NH^N____  N/ \ -----^\/----- N^HN //^\-^0^H3 H2  0-\ o-^ ^ \ NH Nz.::^  NH^N  N 4--/^-ave. \N / ---"\^ / ---\^N^HN /^•^-...^ ----N^HN 0 -^H3 —\ ...i ^0 -^H3  1^ H2  \  H2  Figure 2.11. Resonance structures of a nitrophenylporphyrin. In contrast to the (NPh)TPIT example, the resonance forms of (APh)TP1113 are shown in Figure 2.12. The assumption is now that the pyrrole proton adjacent to an electron-donating meso substituent (4-APh) resonates at a lower field than the other proton on the same pyrrole ring, i.e., H3 and H7 resonate at lower field (at 8.93 ppm) than H2 and H8 (at 8.82 ppm) in the case of (APh)TPhP (Table 2.8, p.53).  97  Chapter 2 Synthesis of porphyrins  Ha  _/,..,---,.r.-  --y. H7^1^H H^ H ^ H7^NH N___ \^NH N__ \ N --/— ^ / N^ ...-...- N +— /^/ — \ /^ N HN N HN H^H3 \ H H ^ H3 L_/ ^ _^--H1  H1  ^  H1  Figure 2.12. Resonance structures of an aminophenylporphyrin. The signals of patterns # 2 and # 4 in Figure 2.8 can be assigned using these two assumptions, and so can the two doublets of pattern # 3. For the assignments of the two singlets in pattern #3, the following observations are employed: As shown schematically in Figure 2.13A, in the spectrum of TPhPyP, the singlet which corresponds to the pyrrole protons between two phenyls appears closer to the doublet at lower field; however, in the spectrum of PhTPyP, the singlet corresponding to the pyrrole protons between two pyridyls appears closer to the doublet at higher field. A conclusion made from these observations is that in the spectrum of cis-DPIA3PyP, the singlet at lower field for the pyrrole protons corresponds to the protons between the two phenyls, while the singlet at higher field relates to the protons between the two pyridyls. Checking through the data of all the porphyrins synthesized in this thesis work, the pyrrole protons situated between two meso electron-withdrawing substituents resonate at lower fields than the protons situated between two relatively electron-donating substituents. Another example is shown in Figure 2.13B. Figure 2.13B a shows the chemical shifts of the singlets for the pyrrole protons of TPhPyP, (NPh)TPhP, cis-(NPh)DPhPyP, T(NPh)PhP and cis-B(NPh)PhPyP. From this illustration, it can be proposed that in the spectrum of cis-B(NPh)DPhP the singlet at 8.86 ppm corresponds to H17 and H18, and the singlet at 8.77 ppm corresponds to H7 and Hg, as shown in Figure 2.13B b.  98  Chapter 2 Synthesis of porphyrins  8.88  8.92  NH ft-/881 ON \ N HN \^L../8  .85  Figure 2.13A. A comparison of the schematic spectra of TPhPyP, cis-DPhBPyP and PhTPyP.  The signals of the pyrrole protons of the porphyrins with two different substituents (patterns # 2-4) can all be assigned according to the assumptions regarding the resonance structures demonstrated in Figures 2.11 and 2.12 and using comparisons similar to the one demonstrated in Figure 2.13B. The results are included in Table 2.18.  99  Chapter 2 Synthesis of porphyrins  a  0  0N  8.79 0N  NH N \ N HN \ //  NO2  Figure 2.13B. Illustration of the chemical shifts of the pyrrole protons of TPhPyP and some nitroporphyrins.  100  Chapter 2 Synthesis of porphyrins  Table 2.18. The assignments of the pyrrole protons® 1-17  H3  H7  Hit  111  H13  H17  Hi g  TPhPyP  8.81 d  8.90 d  8.87 s  8.87 s  8.87 s  8.87 s  8.90 d  8.81 d  trans-DPhl3PyP  8.81 d  8.91 d  8.91 d  8.81 d  8.81 d  8.91 d  8.91 d  8.81 d  cis-DPhBPyP  8.81 d  8.92 d  8.88 s  8.88 s  8.92 d  8.81 d  8.85 s  8.85 s  PhTPyP  8.83 d  8.93 d  8.86 s  8.86 s  8.86 s  8.86 s  8.93 d  8.83 d  (NPh)TPIIP  8.90 d  8.74 d  8.74 d  8.90 d  8.87 s  8.87 s  8.87 s  8.87 s  cis-B(NPh)DPhP  8.90 d  8.73 d  8.77 s  8.77 s  8.73 d  8.90 d  8.86 s  8.86 s  trans B(NPh)DPhP  8.90 d  8.73 d  8.73 d  8.90 d  8.90 d  8.73 d  8.73 d  8.90 d  T(NPh)PhP  8.75 d  8.91 d  8.79 s  8.79 s  8.79 s  8.79 s  8.91 d  8.75 d  cis-(NPh)DPliPyP  8.82 d  8.76 d  8.73 d  8.89 d  8.86 s  8.86 s  8.89 d  8.79 d  cis-B(NPh)PhPyP  8.85 d  8.79 d  8.78 s  8.78 s  8.75 d  8.91 d  8.91 d  8.81 d  trans B(NPh)PhPyP -  8.84 d  8.79 d  8.75 d  8.92 d  8.92 d  8.75 d  8.79 d  8.84 d  T(NPh)PyP  8.86 d  8.80 d  8.80 s  8.80 s  8.80 s  8.80 s  8.80 d  8.86 d  trans-B(NPh)BPyP  8.86 d  8.80 d  8.80 d  8.86 d  8.86 d  8.80 d  8.80 d  8.86 d  cis-(NPh)PliBPyP  8.83 d  8.78 d  8.74 d  8.90 d  8.90 d  8.80 d  8.83 s  8.83 s  cis-B(NPh)BPyP  8.86 d  8.80 d  8.80 s  8.80 s  8.80 d  8.86 d  8.86 s  8.86 s  (NPh)TPyP  8.82 d  8.77 d  8.77 d  8.82 d  8.82 s  8.82 s  8.82 s  8.82 s  (APh)TPhP  8.82 d  8.93 d  8.93 d  8.82 d  8.82 s  8.82 s  8.82 s  8.82 s  cis-B(APh)DPhP  8.80 d  8.91 d  8.91 s  8.91 s  8.91 d  8.80 d  8.80 s  8.80 s  trans-B(APh)DPhP  8.81 d  8.91 d  8.91 d  8.81 d  8.81 d  8.91 d  8.91 d  8.81 d  T(APh)PhP  8.89 d  8.77 d  8.89 s  8.89 s  8.89 s  8.89 s  8.77 d  8.89 d  cis-(APh)DPITyP  8.78 d  8.99 d  8.95 d  8.83 d  8.84 s  8.84 s  8.88 d  8.78 d  cis-(APh)PhBPyP  8.77 d  8.99 d  8.88 d  8.77 d  8.95 d  8.84 d  8.81 s  8.81 s  (APh)TPyP  8.79 d  9.00 d  9.00 d  8.79 d  8.81 s  8.81 s  8.81 S^8.81 s  (APh)T(SPh)P 11  8.85 d  8.99 d  8.99 d  8.85 d  8.85 s  8.85 s  8.85 s  8.85 s  cis-B(APh)B(SPh)P  8.78 d  8.92 d  8.92 s  8.92 s  8.92 d  8.78 d  8.79 s  8.79 s  trans B(APh)B(SPh)P  8.79 d  8.94 d  8.94 d  8.79 d  8.79 d  8.94 d  8.94 d  8.79 d  cis B(MPy)DPhP  9.00 d  9.00 d  8.91 s  8.91 s  9.00 d  9.00 d  9.12 s  9.12 s  -  -  -  a: Chemical shift in ppm, peak pattern; in CDC13 unless stated otherwise. b: In DMSO-d6.  101  Chapter 2 Synthesis of porphyrins  For the porphyrins having three different substituents, the pattern for the pyrrole signals is either one singlet and six doublets for the cis-isomer (pattern # 5 in Figure 2.8) or four doublets for the trans-isomer (pattern # 6 in Figure 2.8). The assignments for these signals is an even more challenging problem. The difference between the chemical shifts of the two protons on the same pyrrole, defined as A, is characteristic of the two adjacent meso substituents. Table 2.19 lists these A values for examples of various porphyrins. The actual chemical shifts of the coupled doublets in other porphyrins may be different, but the A value remains the same. Table 2.19. The chemical shifts of the 117 and H8 pyrrole protons®  porphyrins  substituents  A value of H7 and H8  trans-DPhBPyP  phenyl, pyridyl  0.10 ppm, (8.91, 8.81)  trans -B(NPh)DPhP  nitrophenyl, phenyl  0.17 ppm, (8.90, 8.73)  trans-B(APh)DPhP  aminophenyl, phenyl  0.10 ppm, (8.91, 8.81)  trans-B(NPh)BPyP  nitrophenyl, pyridyl  0.05 ppm, (8.84, 8.79)  (APh)TPyP  aminophenyl, pyridyl  0.21 ppm, (9.00, 8.79)  a: In CDC13.  Other useful information is that the coupled doublets have more intense peaks for the 'inside' signals, this resulting from second order coupling. Using this information and the data in Table 2.19, the coupled doublets can be selected and assigned to a particular pyrrole ring. The two doublets corresponding to a pyrrole ring can be assigned using the assumptions discussed above. For example, the assignments of the pyrrole protons of cis(NPh)DPhPyP and cis-(APh)P1BPyP are shown in Figures 2.14 and 2.15, respectively.  102  Chapter 2 Synthesis of porphyrins  I  I  1 H8 H12 H17^H13  H2  H18 H3  H7  Figure 2.14. Assignment of the pyrrole protons in the 11-1 NMR spectrum of cis-(NPh)DPhPyP (CDCI3). The doublet (2H) at 8.89 ppm is coupled to both of the doublets at 8.79 ppm (A= 0.10 ppm) and 8.73 ppm (A= 0.16 ppm), as indicated by two dimensional NMR. The other coupled doublet is also shown above. Assignments are achieved as described in the text. 103  Chapter 2 Synthesis of porphyrtna  I H2N  u ..i  11111111^  1.  i  8.9  9.0  11111111111 ^ 8.8  II II I^I  Ii  H7 H13 H17 H2 H18 H8  H3 H12  o A pyridyl protons V phenyl protons • aminophenyl protons 0 amine protons 0 solvent  1111-11111111111111111111111111111  8^6^4  Figure 2.15. 1H NMR spectrum of cis-(APh)PhBPyP in CDCI3.  104  Chapter 2 Synthesis of porphyrins  Results from two dimensional NMEt of cis-(NPh)DPhPyP and cis-(APh)PhBPyP show which doublets couple to each other, and support the given assignments. The assignments of the pyrrole signals for all the porphyrins are presented in Table 2.18. The pyrrole signals for some of the porphyrins overlap [see Tables 2.8 (p.53), 2.11 (p.60) and 2.14 (p.65)] and are assigned accordingly. The split signals for pyrrole protons have been observed by many authors,1k2.7,8,9 but only the pyrrole signals for 5-(2-nitro-5-hydroxylpheny1)-10,15,20-tris(4tolypporphyrin have been assigned; Little" suggested that H3 and H7 resonate at higher fields than H2 and Hg, which agrees with the assignments of the nitroporphyrins presented here.  2.3.3 UV-visible spectra The intense absorbance of hemoglobin at 400 nm was discovered in 1883 by Soret,26 and this band was later observed in porphyrins.25 The optical spectra of porphyrins and related compounds have been reviewed in the literature.25,27 For porphyrin free-bases, there is a strong band in the near UV (around 400 nm), referred to as the Soret band, and four visible bands (from 500 to 650 tun) labeled as I, H, III, and IV on going from longer to shorter wavelengths. Four types of spectra have been classified as etio, rhodo, oxorhodo and phyllo, according to the relative intensities of the four visible bands.25 The porphyrins synthesized in this thesis work generally give the typical etio spectra (intensities of the visible bands: IV>IH>II>I). However, there are a few exceptions. The porphyrins PhTPyP, TetPyP, and (NPh)TPyP have phyllo type spectra (IV>II>IH>I). This type of spectra is believed to result from the development of asymmetry in the it electron cloud;25 however, the fact that TPliPyP has an etio-type spectrum and PhTPyP a phyllo-type suggests that other factors are involved, because both  105  Chapter 2 Synthesis of porphyrins  these porphyrins have the same symmetry. Cis-(APh)DPhPyP and trans-B(APh)PhPyP are also unusual in that the intensity of band I is greater than that of band II. This type of spectra does not fall into any of the four standard types. A third exception is apparent with the water-soluble porphyrins cis- and trans-B(APh)B(SPh)P in aqueous solution, the spectra of which have basically lost some of the characteristics of porphyrin structures: the intensity of the Soret bands is dramatically reduced from typically 3-4 x 105 M-'cm' to <1 x 105 M-'cm-1, while the intensities and wavelengths of the visible bands change dramatically from those of a normal porphyrin spectrum (Table 2.12, p.61). No theory to explain these deviations of the spectra from an etio-type has been developed in this work, though the third exception probably results from aggregation of the porphyrins in aqueous solution, which is discussed in Chapter 4. A considerable amount of work in the literature have been devoted into developing models to explain the optical spectra of porphyrins and metalloporphyrins. Reviews on this aspect can be found in the literature,27,28 but discussion on the models is beyond the scope of this thesis. The Soret band and the four visible bands of a porphyrin generally shifts to longer wavelengths after nitration of the porphyrins and, when the nitro group is reduced to an amine, the bands shift to even longer wavelengths. The wavelengths of the Soret bands of TetPhP, TPhPyP and their nitro and amino derivatives are listed in Table 2.20. The red shifting of the bands on alkylation of pyrrole hydrogens on going from porphin (the porphyrin core) to octa-alkylporphyrin has been explained by the electron-donating effects of the substituents.27 However, the red shifts noted in Table 2.20 cannot be explained similarly because both electron-withdrawing and -donating substituents give the same effect. Another approach to explain these red shifts is to consider the effect of increased conjugation in the systems when a nitro or amine group is introduced into the  106  Chapter 2 Synthesis of porphyrins  structure.15,29 The resonance forms of the aminophenyl- and nitrophenylporphyrin are shown in Figure 2.16 a; there are planar forms, and these are more stable, therefore contribute more, than the similar resonance forms for TetPhP (Figure 2.16 b). This will therefore increase the conjugation in the system, and lower the excitation energies. It is known that the phenyl ring is essentially perpendicular to the porphyrin ring in the case of TetPhP in the solid state.3° Table 2.20 Soret bands of nitro and amino porphyrins -  Amax (tun) 416.7 418.0 419.5  TetPhP (NPh)TPhP B(NPh)DPIT  ?.max (nm) (APh)TP1113 cis-B(APh)DPhP  419.5 421.5 421.0 423.8  trans-B(APh)DPhP  T(NPh)PhP TPhPyP cis-(NPh)DPhPyP cis-B('NPh)PhPyP trans-B(NPh)PhPyP  T(NPh)PyP  421.3 416.5 417.6 418.6 419.0 419.4  , T(APh)PhP cis-(APh)DPhPyP cis-B(APh)PhPyP  418.8 421.5 422.0  trans-B(APh)PhPyP  a NH N rN HN^—^H  N H N^/ ^K Vf \^_ rN HN^—^H  %7)N  NH N =f--)7/—\\ N^N 02  NH N ^  0 \\/ _  N  N H N  /0-  z  11)1NT-I 1\7N  NHN //^ rN HN  H NN _*) ^ / \  Figure 2.16. Resonance forms of an aminophenylporphyrin and a nitrophenylporphyrin 107  Chapter 2 Synthesis of porphyritu  Similar red shifts have also been observed with substitution of the para-protons of the four phenyls by -OCH3, -CH3, -Cl and -NO2, and these shifts were rationalized in the terms of greater conjugation.15,29 2.3.4 Infrared spectra The infrared spectra of TetPhP and some of its symmetric p-phenyl substituted derivatives have been reported,29 and the infrared spectra of some other porphyrins and metalloporphyrins have been reviewed.31 Table 2.21 lists the data obtained from IR measurements in the current studies. The assignments of the common peaks of the porphyrin ring structure can be found in the literature.29,31 The spectrum of TetPhP recorded is in general agreement with the literature data.29 Peaks at about 1517 and 1348 cm-1 (VNO2)3 are found for both cis-(NPh)PhBPyP (medium, from one NO2 group) and cis-B(NPh)BPyP (very strong, from two NO2 groups) within the nitro porphyrins. Peaks at around 1618 cm-1 (8NH)3 were detected for the amino porphyrins. The VcN of the pyrrole and pyridyl rings32 at —1595 cm-1 becomes more intense for the porphyrins having more pyridyl substituents. 2.3.5 Mass spectra The mass spectra of porphyrins have been reviewed in the literature.33 The porphyrins synthesized in this thesis work are mainly identified by 1H NMa spectroscopy, but the mass spectra of some samples were recorded. The results from a chemical ionization (NI-13) technique are shown in Table 2.22, which lists the peaks with relative intensity higher than 20 %. The peaks are basically M+, (M+1)+ and (M+2)+. For the amino-porphyrin, (M+NH)+ and (M+NH2)+ are also relatively strong peaks; these may result from the affinity of the atninophenyl group for the fragments from the iodizing chemical (NH3).  108  Chapter 2 Synthesis of porphyrins  Table 2.21. Data from IR spectra  TetPhP  TPhPyP  trans-  cis-  DPhBPyP  DPhBPyP  PhTPyP  TetPyP  cis-  cis-  cis-  (NPh)PhBPyP  B(NPh)BPyP  (APh)PhBPyP  T(APh)PhP  1623w  1618s  1607w  1604s 1560w  1597w 1598s  1594vs  1594vs  1592vs  1593vs  1575w 1575w 1580w  1548m  1545w br  1546w br  1592s  1592vs  1593s  1563  1564vw 1510m  1517m  1516vs 1508w 1499w  1444w 1444w 1444w 1404m 1408s 1350m 1351m 1352m 1310w  1444w 1405m  1400w  1401w  1349w  1350w  1351w  1310m  1402w  1405m  1405m 1349w  1348m  1349vs  1307w  1309w  1293m br  1286w 1251w  1252w 1255w  1255vw  1222w  1224w 1226w  1225w  1213w  1217w 1221w  1216w  1189m 1190w 1188w  1188w  1178m 1179w  1178w  1155m 1155m 1155m  1157w  1351m  1281s br 1253w  1229w  1226w  1228w  1210w  1213w  1220w  1214vw  1188w  1190w  1188w  1187w  1156  1158w  1155w  1158m  1078w  1081w  1111w  1110w  1069m  1069m  1217w 1177m  1179s  1159w  1160m  1143w 1082w  1082w  1069w  1031m 1033w 1032vw 1032w  1020w  1125w  1001m 1002m 1001w  1003m  1003m  1004w  1002w  998w  1002w  980m  979m  981m  980m  981m  980m  980m  980m  981w  984w  966s  966s  968s  970s  970s  971s  969s  969s  968s  967s  109  Chapter 2 Synthesis of porphyrins  Table 2.21 continued trans TetPliP  875m  TPhPyP  878m  -  DPhBPyP  881m  cis  cis  -  DPhBPyP  PhTPyP  891w  893w  880m  881m  TetPyP  -  (NPh)PhBPyP  852m  812m  817w  822w  799vs  796vs  801vs  786m  787m  794m  759m  759s  748m  752w  753s  744w  745s 739s  705m  705m  713m  699vs  699vs  706s  881m  656m  656m  B(NPh)BPyP  cis(APh)PhBPyP  T(APh)PhP  892vw 882m  880w  879w  848w  846m  866m 872w  872m  853w  853vw  850w  844w  844w  843w  849m  849s  841w 810w  795vs br  751m  796s br  799s br  784s  786s  796s br  800s br  799s br  805s br  751vw  755s  786s 749w  749w 743w  739m  738s 703w  698m  698m  673m  673m  675m 668w  656m  -  892w 882s br  867m 850m  cis  658m  658s  659m  710m  704vw  673vw  673vw  703w  664w  657w  659vw 646w  640m  640m  639w  638w  639w  639w  640w  641w  641vw br  631w 619w  619w  622w  622vw  560w  563w  564m  565w  565w  565w  564w  565w  560w  560w  552w  554w  553vw  553w  553w  554w  554w  553w  552w  552w  516w  518w  521w  527w  530w  531w br  519w  519w  517w  517w  w = weak; s = strong; m = medium; br = broad, vs = very strong, and vw = very weak.  110  Chapter 2 Synthesis of porphyrins  Table 2.22. Data from CI mass spectra  Ni+ (!)  (vi+1)+co  (m+2)+4)  cis-DPhBPyP  616 (81)  617 (100)  618 (38)  PhTPyP  617(57)  618 (100)  619(38)  cis-(NPh)PhBPyP  661 (25)  662 (100)  663 (44)  B(NPh)BPyP  706(35)  707 (100)  708(39)  cis-(APh)PhBPyP 631(80) 632 (100) 633 (41) a: Intensity as a percentage, relative to the major peak as 100 %.  (m+NH)(A)  (m+NH0+(!)  646 (20)  647 (28)  The El mass spectrum of [T(MPy)11C14 was also recorded; 678 ([1■4-4C1], 100%), 679 (52%), 680 (52%) and 681 (27%) were the main peaks detected. The main peaks in an El spectrum of [PhT(MPy)P]C13 were at 618 ([1■4-3C1-3CH3], 100%) and 619 (43%). Table 2.23 lists the data from cationic FAB (3-nitrobenzylalcohol matrix) spectra of some cationic porphyrins. The major peak in these spectra is that of the porphyrin fragment without the counterions. Table 2.24 lists the data from anionic FAB (3-nitrobenzylalcohol matrix) spectra of some anionic porphyrins. The major peak is [M-Nal- or [M-2Na+Hr, ignoring the matrix peaks. Table 2.23. Cationic FAB mass spectra of some cationic porphyrins  663 M-4C1-CH3 704 T(MPy)PhPC13 M-30-CH3 692 T(N1Py)(NPh)PC13 M-3C1-CH3 721 cis 2C1 CH3 M B(N1Py)B(NPh)PC11 631 cis-B(MPy)DPhPC12 6461 M-2C1 M-2C1-CH3 A: The major peak (ignoring the matrix peaks). Peak not identified.  Tet(MPy)PC14  -  6781 M-4C1 719! M-3C1 7071 M-3CI 7361 M 2C1 -  -  648 M-4C1-2CH3 689 M-3C1-2CH3 677 M-3C1-2CH3 75211  633 M-4C1-3CH3 735k  69411  713 M-3C1  754 M-2C1  723k  -  66112  111  Chapter 2 Synthesis of porphyrhts  Table 2.24. Anionic FAB mass spectra of some anionic porphyrins  Na2[cis-B(APh)B(SPh)13] Na2[trans-B(APh)B(SPh)11 Na2[cis-BPyB(SPh)P] Na2[cis-(NPh)PyB(SPh)11  8251  803  M-Na  M-2Na+H  8253  803  M-Na  M-2Na+H  7973  775  M-Na  M-2Na+H  8191  841  M-2Na+H  M-Na  3: The major peak.  The cationic spectrum (3-nitrobenzyl alcohol matrix, thioglycerol) of cisBPyB(SPh)P was also recorded, but it was of little use for characterization.  112  Chapter 2 Synthesis of porphyrins  References  1  ^  (a) R.G. Little, J.A. Anton, P.A. Loach and J.A. Ibers, J. Heterocyclic Chem., 12, 343, (1975).  (b) RG. Little, J. Heterocyclic Chem., 18, 129, (1981). 2 3  ^  ^  4  ^  W.J. Kruper, Jr., T.A. Chamberlin and M. Kochanny, J. Org. Chem., 54, 2753 (1989).  E. Tsuchida, E. Hasegawa and T. Kanayama, Macromolecules, 11, 947 (1978). J.P. Col'man, RR Gagne, C.A. Reed, T.R. Halbert, G. Lang and W.T. Robinson, J. Am. Chem. Soc., 97, 1427 (1975).  J.P. Collman, RR. Gagne, T.R. Halbert, J. Marchon and C.A. Reed, J. Am. Chem. Soc., 95, 7868 (1973). 5^T.S Srivastava and M. Tsutsui, J. Org. Chem. 38, 2103 (1973). 6  ^  R.F. Pasternack, P.R. Huber, P. Boyd, G. Engasser, L. Francesconi, E. Gibbs, P. Fasella, G. C. Venturo and L. deC. Hinds, J. Am. Chem. Soc., 94, 4511 (1972), and references therein.  7^G.N. Williams, R.F.X. Williams, A. Lewis and P. Hambright, J Inorg. Chem. 41, 41 (1979). 8^L. Ding, C. Casas, G. Etemad-Moghadam and B. Meunier, New J. Chem., 14, 421 (1990). 9^M.A. Sari, J.P. Battioni, D. Mansuy and J.B. Le Pecq, Biochemistry, 29, 4205 (1990). 10^E.G. Fleischer, J.M. Palmer, and T.S. Srivastava and A. Chatterjee, J. Am. Chem. Soc., 93, 3162 (1971). 11^C.A. Busby, R.K. DiNello and D. Dolphin, Can, J. Chem. 53, 1554 (1975). 12^A. Harriman and G. Porter, J. Chem. Soc. Faraday Trans. 2, 75, 1532 (1979).  113  Chapter 2 Synthesis of porphyrins  13^L. Gong and D. Dolphin, Can. J. Chem., 63, 401 (1985). 14  ^  J. Cavaleiro, M. Neves, M. Hewline and A, Jackson, J. Chem. Soc. Perkin Trans. I, 1986, 575.  15^D.W. Thomas and A.E. Martell, J. Am. Chem. Soc., 78, 1335 (1956). 16^J. Winkelman, Cancer Res., 22, 589 (1962); 17^A. Corsini and 0. Harrman, Talanta, 33, 335 (1986). 18  ^  Q. Xiong, R. Xu and Z. Zhou, "Basic Organic Chemistry", High Education Press, Beijing, 1983, p.686.  19^P. Hambright and E. B. Fleischer, Inorg. Chem., 9, 1757 (1970). 20^J.A. O'Hara, E.B. Douple, M.J. Abrams, D.J. Picker, C.M. Giandomenico and J.F. Vollano, Int. J. Radiat. Oncol. Biol. Phys., 16, 1049 (1989).  E.J. Gibbs, I. Tinoco, Jr., M.F. Maestre, P.A. Ellinas and R.F. Pasternack, Biochem. Biophys. Res. Commun., 157, 350 (1988).  K. Kano, T. Nakajima, M. Takei and S. Hashimoto, Bull. Chem. Soc. Jpn., 60, 1281 (1987). 21^E.D. Becker, "High Resolution NMR", Academic Press, Orlando, 1980, p167. 22^C.B. Storm, T. Teklu and E.A. Sokolski, Ann N.Y. Acad. Sci., 63, 206 (1973). 23^M. Zanger, Org. Magn. Reson., 4, 1(1972). 24^Y. Sun, A.E. Martell and M. Tsutsui, J. Heterocyclic Chem., 23, 561 (1986).  114  Chapter 2 Synthesis of porphyrins  25^K.M. Smith, in "Porphyrins and Metalloporphyrins", H.E. Falk, ed., Elsevier Scientific Publishing Co., Amsterdam, 1975, p.3. 26  ^  Soret, Compt Rend., 97, 1267 (1883).  27^M. Gouterman, in "The Porphyrins", Vol III, D. Dolphin, ed., Academic Press, New York, 1978, p.1. 28^M. Gouterman and G.H. Wagniere, J. MoL Spectrosc., 11, 108 (1963). M. Goutennan, J. MoL Spectrosc., 6, 138 (1961). 29^D.W. Thomas and A.E. Martell, J. Am. Chem. Soc., 78, 1338 (1956). 30^E.G. Fleischer, C.K. Miller and L.E. Webb, J. Am. Chem. Soc., 86, 2342 (1964). 31^J.0. Alben, in "The Potphyrins", Vol III, D. Dolphin, ed., Academic Press, New York, 1978, p.323. 32^R.J.H. Chan, Y.O. Su and T. Kuwana, Inorg. Chem., 24, 3777 (1985). 33^H. Budzikiewicz, in "The Porphyrins", Vol III, D. Dolphin, ed., Academic Press, New York, 1978, p.395.  115  Chapter 3 Water-soluble metalloporphyrins  Chapter 3 Water-soluble metalloporphyrins 3.1 Introduction  A porphyrin can coordinate to almost any transition metal.' Due to the limitation of time and the interests of this project, only complexes of cobalt, copper and zinc complexes of the designed porphyrins were studied in this thesis work. 3.1.1 Cobalt complexes of water-soluble porphyrins Cobalt complexes of water-soluble porphyrins were reported to have the best effects among the metalloporphyrins of eight metals tested for radiosensitizers.2 Because of these findings, the cobalt complexes of the water-soluble porphyrins synthesized in this thesis work (Chapter 2) became of interest for potential use as radiosensitizers. The cobalt complexes of some water-soluble porphyrins, in particular of Tet(MPy)P and Tet(SPh)P have long been of interest in chemistry and biochemistry. The electrochemical properties,3,4 redox properties,5,6,7 interactions with dioxygen,8,9 association with DNA and amino acids,1°,Il and their potential as anti-cancer agents,2 have all been addressed in the literature. The increased lability of axial ligands of Com[Tet(MPy)P], relative to those of the usually inert Com complexes, was also studied12,13," The complex, formulated as Co111[Tet(MPy)P]C15.(H20)2, has been synthesized and characterized by Pastemack et al.12ab However, Neta5 found later that when a solution of this complex was evaporated to dryness, part of this material was reduced to Co11[Tet(MPy)13], presumably by water. It was also pointed out by Abwao-Konya et al." that the cobalt-porphyrin complex synthesized by the Pastemack-Cobb method did not assure a cobalt(I11) product. Abwao-Konya et al." used Hg(NO3)2 to oxidize Co" to Com, 116  Chapter 3 Water-soluble Inetalloporphytiaa  but there was no evidence to support a Co" formulation in their report. A subsequent synthesis of Co111[Tet(MPy)P](C104)5.2H20 was reported by Ashley et a1.13 in which Co(C104)2 and [Tet(MPy)P](CI04)4 were reacted in DMF, followed by precipitation with ethanol; however, the elemental analysis of this material quoted for carbon was 2.66% greater than the value calculated for Com[T(MPy)P](C104)5.2H20. The Co11[Tet(MPy)114+ species has been generated in solutions under an inert atmosphere.8,16 The complex was also isolated by Hambright and Fleisher from an aqueous solution as CollfTet(MPy)PNC104)4;17 however, these authors presented no physical data. Synthesis of Coll[Tet(MPy)P](PF6)4 has also been addressed in the literature.18 The compound Na4Co11[Tet(SPh)19.8H20 was first prepared in 197619 by refluxing cobalt(II) acetate and the sodium salt of Tet(SPh)P in water. The resultant material was purified by recrystallization from ethanol-water, and characterized by elemental analysis and ESR. The synthesis of this complex has also been reported by other authors.  20,21  Although Com[Tet(SPh)P] has been used in various studies,2,21,22 the synthesis of this complex has not been reported in detail. In an earlier report,23 this complex was claimed to be synthesized by reaction of CoSO4 and Na4[Tet(SPh)13] in DMF, followed by work-up of the DMF solution on an alumina column (eluted with dilute aqueous NaOH solution), and work-up of the aqueous solution on a Sephadex column; however, the elemental analysis quoted value for carbon was low by 3.23% and for hydrogen was high by 0.48% from calculated values for Na3Com[T(SPh)11(H20)2. Hambright and Langley14 reported the synthesis of Na3ColiT(SPh)11.12H20 by reaction of the porphyrin with CoO, however the formulation of a Con complex fit better for the reported elemental analysis data.  117  Chapter 3 Water-soluble metalloporphyrks  As can been seen from this brief overview, a detailed procedure for isolation of a specific oxidation state, cobalt water-soluble porphyrin complex has yet to be reported. In this chapter, the synthesis and characterization of the Coll and Com complexes of Tet(MPy)P and Tet(SPh)P, and some results on their solution chemistry, are presented. In addition, the synthesis and characterization of the novel cobalt (II and/or III) complexes of the other water-soluble porphyrins addressed in the previous chapter, including T(MPy)PhP, T(MPy)(NPh)P, cis-B(MPy)B(NPh)P, (APh)T(SPh)P, PyT(SPh)P, and cis(NPh)PyB(SPh)P, are also presented. 3.1.2 Copper and zinc complexes of water-soluble porphyrins Copper and zinc complexes of water-soluble porphyrins are also reported to have relative high radiosensitizing abilities toward V79 cells.2 The complexes Cul[Tet(IvIPy)P] and CullfTet(SPh)P] have been made before by reaction of Cu0,20 Cu(C104)2," or Cu(Ac0)219 with the porphyrins in water. The aggregation properties of these complexes have also been studied.24,25 In this thesis work, the copper complexes of 6 water-soluble porphyrins have been synthesized starting with CuC12. A Zn[Tet(MPy)P] species has been synthesized by methylation of Zn[TetPyP]," and a Zn[Tet(SPh)P]compound 20 has been synthesized by reaction of the porphyrin with ZnO in aqueous solution. The Zn(II) complexes of these two porphyrins and of three other new porphyrins are synthesized in this thesis work.  3.2 Experimental 3.2.1 Materials and methods Description is limited to material not covered in Section 2.2 in Chapter 2.  118  Chapter 3 Water-soluble metalloporphyrins  The chloride and tosylate salts of the cationic porphyrins and the sodium salts of the anionic porphyrins were synthesized as previously described in Chapter 2. All other chemicals were analytical grade and were used without further purification. The cation exchange resin (Na+ form) was prepared from H+ ion exchange resin (Amberlite IR-120, BDH) by eluting with a concentrated NaC1 solution until the eluent was neutral to pH test paper. The pH values for pKa measurements were measured with a pH meter (Radiometer Copenhagen PHM25) using a glass combination electrode (Cole-Parmer Chemical Co.). The general dialysis procedure described in Section 2.2 was followed, some modifications for specific syntheses being described later in this section. External references (DDS = 0.00 ppm in D20, T'MS = 0.00 ppm in DMSO-d6) were used for paramagnetic samples in 1H NMR measurements. The NMR samples under vacuum were made by vacuum transfer of degassed solvent, and the NMR tube was flame-sealed. NMR samples under N2 were made by adding degassed solvent to solid samples degassed under N2. Anaerobic UV-visible spectra were measured with a specially designed cell (Figure 3.1). Samples were weighed into the cell, and a known amount of distilled water was added to the side-arm flask. Water was degassed three times by a freeze-pump-thaw process, then mixed with the complex. A spectrum was then taken. To observe the reactions of the complex with 02, the gas at 1 atm was bubbled through the sample for 20 s using a needle, and then the system was sealed and a series of spectra were taken. Magnetic susceptibilities of solid samples were measured using a magnetic susceptibility balance (Johnson Matthey Fabricated Equipment). The diameter of the sample tube was 0.30 cm, and the length of sample was about 1.5 cm (-0.04 g). The actual sample length and weight in the tube were measured using a ruler and an analytical balance. 119  Chapter 3 Water-solahle metalloporphyrlas  All elemental analyses were done on samples handled under N2, unless stated otherwise.  Figure 3.1. Cell for anaerobic UV-visible spectroscopy..  3.2.2 {Con[Tet(MPy)PJ)C14.2H20  A solution of CoC12.6H20 (500 mg, 2.1 mmol, in 20 mL water) was added to a solution of the Tet(MPy)P tosylate salt (500 mg, 0.35 nunol, in 50 mL water), and the mixture was refluxed for 45 min. The reaction mixture was cooled and 10 rriL saturated NaC104 solution was added, resulting in the formation of a brown precipitate, presumably a perchlorate salt. The precipitate was filtered, washed twice with cold water (-5 mL each  120  Chapter 3 Water-soluble metalloporphyrins  and passed through a column (2.5 x 25 cm) of Cl" ion exchange resin, which was prewashed with distilled water. The purple eluent was evaporated to dryness on a Rotovap at 30-40°C. The residue was washed with acetone then dried at 100°C under vacuum (< 0.1 ton) overnight. Elemental analysis: Found: C, 57.63; H, 4.09; N, 12.47; Cl, 15.80%; calculated for C44H40C14CoN802: C, 57.84; H, 4.38; N, 12.47; Cl, 15.55%. Yield 90%.  3.2.3 {Com[Tet(MPy)P1(OH2))(C104)5•xH20 The brown precipitate obtained by adding NaC104 to the reaction solution as described in Section 3.2.2 was dissolved in warm (-50°C) 0.001 M perchloric acid solution (50 mL) and the solution stirred under air for 1 h; this solution was then lyophilized to dryness (-0.25 g product).  3.2.4 {Comffet(MPy)PROHDC14-2H20 Co11[T(MPy)11C14-2H20 (200 mg, 0.22 mmol) was dissolved in 20 mL of 0.001 M HC1 and stirred in air for 1 h; the solution was dialyzed in 1 L water for 1 h, and then the solution in the dialysis bag was rotary evaporated at 25-30 °C to dryness. The resultant brown residue was dried at 100°C under vacuum (< 0.1 ton) overnight. Elemental analysis: Found: C, 56.78; H, 4.13; N, 12.00; Cl, 15.37 %; calculated for C44H4104CoN803: C, 56.74; H, 4.41; N, 12.04; Cl, 15.27 %. Yield 75%. •  3.2.5 {Colllffet(MPy)PROH2)}C15.2H20 [CorT(MPy)P]C14.2H20 (200 mg, 0.22 mmol) was dissolved in 20 mL distilled water and dialyzed in 1 L water for 1 h. Then the solution in the dialysis bag was rotary evaporated at 25-30 °C to dryness. The residue was dissolved in 0.001 M HC1 solution (20 mL), the solution was stirred in air for 1 h, and then lyophilized to dryness under vacuum. The resultant purple material was dried at 100°C under vacuum (< 0.1 ton)  121  Chapter 3 Water-soluble metalloporphyrins  overnight. Elemental analysis: Found: C, 54.58; H, 4.44; N, 11.92; Cl, 18.06 %; calculated for C44H42C15CoN803: C, 54.72; H, 4.35; N, 11.61; Cl, 18.39 %. Yield 75%. 3.2.6 (Con[T(MPy)PhPDC13.1/2H20 T(MPy)PhP trichloride (200 mg, 0.24 mmol) and CoC12-6H20 (200 mg, 0.84 mmol) were dissolved in 20 mL water and the resultant solution was reuxed for 2 h. Saturated NaC104 solution was added to the reaction mixture and a brown precipitate formed; this was filtered and washed with cold water twice (-5 mL each). The brown powder was dissolved in hot water, and passed through a column (3.5 cm x 30 cm) of C1 exchange resin. The solution was evaporated to dryness with rotary evaporation at 2030°C, and the residue was dried at 100°C under vacuum. Elemental analysis: Found: C, 60.61; H,4.04 , N,11.30 %; calculated for C44H35C13CoN7O112: C, 60.70; H, 4.02; N, 11.27%. Yield: 92%. 31.7 {Com[T(MPy)PhP1(OH2))C14•2H20  Co11[T(MPy)PhP]C13.1/21120 (100 mg, 0.11 mol) was dissolved in 0.001 M HC1 solution (10 mL); the solution was stirred in air for 1 h, and then lyophilized to dryness. This residue was washed with acetone and dried under vacuum at 100°C. Elemental analysis: Found: C, 58.94; H, 3.99, N, 11.13%; calculated for C44H38C14CoN702: C, 58.87; H, 4.24; N, 10.93%. Yield: 82%. 3.2.8 {Co9T(MPy)(NPh)11(OH2))C14.1120  A cobalt(11) complex was first made using the same procedure as that used for the synthesis of Coll[T(MPy)PhFIC13.1/2 H20 (Section 3.2.6). Starting with this Co(l) product, the synthetic procedure for {Com[T(MPy)PhP](OH2)1C14.2H20 was then followed. The title compound was collected with a yield of 90%. Elemental analysis:  122  Chapter 3 Water-soluble metalloporphyrins  Found: C, 56.19; H, 3.76, N, 11.90% calculated for C44H37C14CoN803: C, 56.06; H, 3.93;N, 11.89%.  3.2.9 {Col11[cis - B(MPy)B(NPh)P1(1120))C13.2H20 A solution made by dissolving cis-[B(MPy)B(NPh)11C12 (200 mg, 0.22 mmol) and CoC12.6H20 (200 mg, 0.84 mmol) in 50 mL distilled water was refluxed for 5 h. Then to this reaction mixture, 30 niL saturated NaC104 was added; the resultant precipitate was collected by filtration, washed with distilled water (10 niL x 3) and air-dried. The brown powder and -20 inL prewashed Cl- exchange resin were suspended in 150 mL distilled water, and stirred at -50°C for 4 h. This mixture was filtered and the purple filtrate was loaded on a prewashed column (3 x 15 cm) of Cl- exchange resin. The purple eluant was concentrated to -10 inL by roto-evaporation, and the resultant solution was transferred into a dialysis bag and dialyzed for 2 h in 1 L distilled water. The solution in the dialysis bag was then evaporated to dryness. To this residue, 0.001 M HC1 solution (10 inL) was added, and 02 was bubbled through the resultant solution for 0.5 h. This solution was lyophilized to dryness, and the residue was dried under vacuum at 100°C. Elemental analysis: Found: C, 55.22; H, 3.76, N, 11.90; Cl, 10.89%; calculated for C44H36C12CoN807: C, 55.43; H, 3.77; N, 11.76; Cl, 11.18%. Yield -60%.  3.2.10 Na41Coll[Tet(SPh)PD•nH20 (n=4 or 9) A solution of Co(acetate)2-6H20 (500 mg, 1.8 mmol, in 10 nth water) was added to a solution of Na4[Tet(SPh)P].10H20 (500 mg, 0.42 mmol, in 50 mL water). The mixed solution was refluxed in air for 60 min, cooled and passed through a Na+ ion exchange column (2 x 20 cm). The eluant was evaporated to dryness, and the resultant purple residue was redissolved in 50 friL methanol; the methanol 'solution was filtered and the filtrate was concentrated to -20 inL. To the concentrated methanolic solution, acetone (150 niL) was slowly added with stirring to give a brown precipitate which was collected by vacuum filtration. This methanol-acetone reprecipitation procedure was repeated twice 123  Chapter 3 Water-aohtble metalloporphyrins  more and the resultant product was dried at 100°C under vacuum (<0.1 ton) overnight. Elemental analysis: Found: C, 45.88; H, 2.78, N, 4.87%; calculated for C44H32CoN4Na4016S4 (n=4): C, 46.01; H, 2.51; N, 5.11%. Yield 85%. This product was  exposed to air for a week, and then a second elemental analysis was carried out (sample was handled in air): Found: C, 42.55; H, 3.38, N, 4.51%; calculated for [C44/142C0N4Na4021S4 (n=9)]: C, 42.60; H, 3.42; N, 4.28%. 3.2.11 Na3{Com[Tet(SPII)P1(OH2)}.nH20 (n=3 or 14)  A solution of Na4[Tet(SPh)11-10H20 (500 mg, 0.42 nunol in 50 mL water) and a solution of Co(acetate)2.6H20 (500 mg, 1.8 mmol in 10 mL water) were mixed and refluxed in air for 60 min. The reaction mixture was passed through a Na+ ion exchange column (2 x 20 cm), and the eluant was evaporated to dryness. The ion exchange colunm was used to ensure that any Co2+ as the counterions for the porphyrin was removed, which is proved by mass spectral data (Section 3.3.3.1). The resultant residue was dissolved in 0.01 M HC1 solution (30 mL), and the solution was washed with chloroform three times (30 mL each time) to remove acetic acid formed; the aqueous layer was concentrated to —15 mL by roto-evaporation at 25-30°C, and the resultant solution was dialyzed in a dialysis bag surrounded by 1 L distilled water for 2 h. The solution recovered from the dialysis bag was evaporated to dryness on a Rotovap at 25-30°C to give a purple residue. Elemental analysis: Found: C, 39.82; H, 4.07, N, 4.22%; calculated for C44H54CoN4Na3027S4 (n = 14): C, 39.81; H, 3.91; N, 4.17%. Yield —50%. This sample was then dried at 100°C under vacuum (<0.1 ton) overnight; elemental analysis of the dried sample: Found: C, 46.81; H, 2.84, N, 4.96%; calculated for C44H32CoN4Na3016S4 (n = 3): C, 47.11; H, 2.93; N, 5.04%. 3.2.12 Na2{C09PyT(SPh)11)  This complex was synthesized by following the synthetic procedure used for Na3{ConfTet(SPh)PK0H2)).3H20 (Section 3.2.11). Elemental analysis: Found: C, 54.10; 124  Chapter 3 Water-soluble metalloporphyrins  H, 2.80, N, 6.54%; calculated for C43H24CoN5Na209S3: C, 54.04; H, 2.51; N, 6.33%. Yield —90%. 3.2.13 Na2{ComRAPh)T(SPh)PI(OH2))  This complex was synthesized by following the synthetic procedure used for Na3{Coifi[Tet(SPh)P](OH2)}.3H20 (Section 3.2.11). Elemental analysis: Found: C, 53.33; H, 2.80, N, 7.14%; calculated for C44H28CoN5Na2S3010: C, 53.50; H, 2.84; N, 7.09%. Yield —90%. 3.2.14 Na{ComIc (NPh)PyB(SPh)11(OH2)}.2H20 -  This complex was synthesized by following the synthetic procedure used for Na3{Com[Tet(SPh)PKOH2)}-3H20 (Section 3.2.11). Elemental analysis: Found: C, 54.69; H, 3.06, N, 8.40%; calculated for C43H30CoN6Na01 1 S2: C, 54.20; H, 3.15; N, 8.82%. Yield —90%. 3.2.15 Cu porphyrin complexes  Copper complexes of cationic water-soluble porphyrins were synthesized with a procedure similar to the synthetic procedure for {Com[Tet(MPy)PhP](OH))C14.2H20 (Section 3.2.4). Briefly, the porphyrin (200 mg, —0.2 nunol) and CuC12.6H20 (200 mg, 0.69 tnmol) were dissolved in 50 mL distilled water, and this solution was refluxed for 1 h. The reaction mixture was evaporated to dryness, and the residue was washed with acetone (3 x 10 mL; CuC12 is soluble in acetone), and then air-dried. The product was dissolved in distilled water (-10 mL); the resultant solution was transferred into a dialysis bag, and dialyzed in 1 L distilled water for 2 h. The solution in the dialysis bag was roto-evaporated to dryness, and the residue was washed with acetone (-20 mL), and dried under vacuum. Yields and elemental analyses (for samples handled in air) of the products are listed in Table 3.1.  125  Chapter 3 Water-soluble metalloporphyrins  The copper complexes of anionic porphyrins were synthesized by reaction of the porphyrins with CuC12. Briefly, the porphyrin (200 mg, -0.2 mmol) and CuC12•6H20 (200 mg, 0.69 nunol) were dissolved in distilled water (50 mL), and this solution was refluxed for 1 h. The reaction mixture was evaporated to dryness, and the residue was washed with acetone (3 x 10 mL), and air-dried. The product was dissolved in -10 mL distilled water; the resultant solution was passed through an Na+ exchange column (-3 x 20 cm), and the purple eluant was transferred into a dialysis bag, and dialyzed in 1 L distilled water for 2 h. The solution in the dialysis bag was transferred into a flask and evaporated to dryness on a Rotovap, and the residue was washed with acetone (-20 mL), and dried under vacuum. Yields and elemental analyses (for samples handled in air) of the products are listed in Table 3.1. 3.2.16 Zinc porphyrin complexes  Zinc complexes were synthesized using the procedures as described for the syntheses of the copper complexes. OH' or Cl- was found to be the axial ligand according to the elemental analyses as listed the Table 3.2, but the OH' was not detected by IR spectroscopy. Yields for the syntheses were 80-90%. Table 3.1. Elemental analyses and yields of copper-porphyrin complexes  { Cu[Tet(MPy)P]} C14. 10H20 { Cu [T(MPy)PhP] } C13- 10H20 { Cu [T(MPy)(NPh)P] }C13•6H20 Na4{Cu[Tet(SPh)P] } • 10H20 Na3 { Cu [(APh)T(SPh)P] } • 9H20 Na3{Cu[PyT(SPh)P]) •9H20  expected found expected found expected found expected found expected found expected found  C% 49.74 49.47 52.27 52.56 53.71 53.67 41.75 41.57 45.58 45.81 45.32 45.57  H% 5.28 5.00 5.35 5.24 4.93 4.56 3.63 3.40 3.80 3.85 3.69 3.68  N% 10.55 10.42 9.70 9.54 11.39 11.01 4.43 4.37 6.04 5.99 6.15 6.02  yield % 78 90 79 81 90 92  126  Chapter 3 Water-soluble metalloporphyrins  Table 3.2. elemental analysis of zinc-porphyrin complexes  Zn112 {Zn(C1)[Tet(MPy)P] } Cl4. 9H20 Znin{Zn(CD[T(MPy)PhP] } C13. 7H20 Na5{Zn(OH)[Tet(SPh)P] } .4H20 Na4{Zn(OH)[(APh)T(SPh)P] } .9H20 Na4{Zn(OH)[PyT(SPh)P] } .10H20  expected found expected found expected found expected found expected found  C% 45.29 45.59  H% 4.63 5.67  51.36 51.43 44.02 44.16 43.99 44.01 42.37 42.21  4.86 4.78 2.92 3.34 3.75 3.60 3.84 3.89  N% 9.61 9.63 9.53 9.10 4.67 4.38 5.52 5.83 5.73 5.51  3.3 Results and Discussion  3.3.1 111 NMR spectra of the metalloporphyrins  In this thesis work, both Co" and Coll complexes are found to be air-stable in DMSO-D6 and the species do not aggregate, as judged by the concentration independence of the 1H NMR spectra; 1H NMR spectroscopy in DMSO-d6 is very useful for characterizing these complexes, although 1H NMR data in the literature are rare. The 1H NMR spectra of some of these complexes in D20 are also presented. 3.3.1.1 1H NMR spectra of Co complexes of cationic-porphyrins  Figure 3.2 shows 1H NMR spectra of {Co11[Tet(MPy)P])C14 in D20 in air and under vacuum. Spectrum 3.2a was measured 24 h after the sample was made by dissolving 2 mg the complex in 0.5 mL D20 (c2', 4 x 10-3 M) in air; oxidation to a diamagnetic Co(III) species has clearly taken place. In this spectrum, the pyrrole protons give a singlet at 9.11 ppm, which overlaps with a doublet corresponding to the 3,5-MPy protons at 9.13 ppm (JHH = 6.6 Hz), a doublet for the 2,6-MPy protons appears at 8.80 ppm (JHH = 6.6  127  Chapter 3 Water-soluble metalloporphirins  Hz), and the signal of the methyl protons overlaps with the water peak at 4.63 ppm. This is considered to be a spectrum of {Co111[Tet(Mr.Py)P](H20)(OH))04 (although a peroxidebridged dimer cannot be ruled out completely, see Section 3.3.5), mainly because the spectrum of this porphyrin is pD-dependent (Section 3.3.6); the pD value of this solution is about 8 according to Figure 3.17, as determined by the chemical shift of the pyrrole protons (Section 3.3.6).  a  bilmilmilininlymp ill^ni i^1 ni 9.2^2.1^9.0^U.S^CO PF911.7  liiiilitilitilittitimillittitittitI 11111 14^  12^  I^I 14^  12^  10  11111111111111  6^  10  4 PPM  -v1111111111^1:^11,111 PPM  Figure 3.2. 1H NMR spectra of {Co1l[Tet(MPy)PDC14 in DO. a: in D20 in air; the spectrum changes slowly with time as the Co(fl) is oxidized; b: under vacuum in degassed D20. 128  Chapter 3 Water-soluble metalloporphyrins  Similarly, the 1H NMR spectra in D20 of {Com[T(MPy)PhP]}C14 and {CollT(MPy)(NPh)PEC14 are also pD-dependent. In every spectrum of these complexes, the signal for pyrrole protons splits to one signet and two doublets. Similarly to the cases for the porphyrin free-bases (Section 2.3.2, p.81), the singlet and the two doublets can be assigned to the 7, 8, 12, 13 and, to 2, 3, 17, 18 pyrrole protons, respectively. The other signals can also be assigned by comparing the spectra to those for the porphyrin freebases. The 1H NMR data and assignments for the two complexes at pD-7 are listed in the table below: 7,8,12,13 pyrrole {C,olll[T(MPy)Ph11}C14  9.12 s  {Com[T(MPy)(NPh)PI}C14  9.12 s  2,3,17,18 pyrrole 9.23 d ' 9.04 d 99..1064 bbrr  3,5-MPy 2,6-MPy 3,4,5-Ph or 3,5-NPh  2,6-Ph or 2,6-NPh  9.12 d .  8.81  7.76 m  8.16 d  9.11 d  8.79  8.55 br  8.36  The plots of the chemical shifts for the 7, 8, 12, 13 pyrrole protons (singlet) provide an opportunity to measure the pKa values of the diaquo complexes, which is discussed in Section 3.3.6. Spectrum of {Com[cis-B(MPy)B(NPh)PDC13 in D20 did not give sharp signals, therefore was not further studied. Figure 3.2b is a spectrum of 2 mg of {Coli[Tet(MPy)P]}C14 in 0.5 mL degassed D20 under vacuum; no oxidation is evident and the spectrum is that of a paramagnetic Co(H) species. The broad signal at 13.7 ppm corresponds to the pyrrole protons which are closest to the paramagnetic center; the signals of the 2,6-MPy and 3,5-MPy protons overlap with each other and result in the signal at 9.88 ppm; and the resonance of the methyl protons is at 5.15 ppm. The proton chemical shifts of this species are dependent on the concentration and the ionic strength, these findings being discussed later (Section 3.3.1.5). The Evan's method, which uses 1H NMR spectroscopy to measure the magnetic susceptibility of a compound in solution, was used in the literature by Pasternack et a!. 12b 129  Chapter 3 Water-soluble metalloporphyrina  to show that the cobalt was in its diamagnetic, +3 oxidation state in a complex of Tet(MPy)P. However, the experiment was performed in D20 and presumably in air, and thus this evidence does not show that the complex in solid state was a cobalt(III) species because, as shown by Figure 3.2, a cobalt(II) complex in D20 under air is oxidized to a cobalt(III) complex. Figure 3.3 shows the spectra of {Co11[Tet(MPy)P]}C14 and {Con[T(MPy)PhP]}C13 in DMSO-d6, recorded in air. In spectrum 3.3a for {Coll[Tet(MPy)P]}C14, the pyrrole protons give a broad signal at 13.9 ppm, and the 2,6-MPy protons a broad signal at 9.8 ppm; the 3,5-MPy protons which are farther away from the paramagnetic center give a relatively sharp signal at 10.1 ppm, while the protons of the methyl groups, which are farthest from the paramagnetic center, give an even sharper signal at 5.30 ppm. This spectrum shows that this cobalt(II) complex is air-stable in this solvent. In spectrum 3.3b, The pyrrole and IstPy protons of {C0llT(MPy)PhP}}C13 give peaks similar to those found in spectrum 3.3a for {CoR[T(MPy)PhP]}C14: a broad peak at 13.3 ppm corresponds to the pyrrole protons; another broad peak at 9.9 ppm and a relatively sharp peak at 10.1 ppm correspond to the 2,6-MPy and 3,5-MPy protons, respectively; and two extra signals corresponding to the phenyl group appear at 8.25 and 8.45 ppm. The resonance of the methyl protons appears at 5.24 ppm as a singlet. Again the Co(II) complex remains in this oxidation state under these conditions. In Figure 3.4, the spectra of {Com[Tet(MPy)P](H20)}(C104)5 and {Co111[Tet(MPy)P](H20)}C15 in DMSO-d6 are shown. The two sets of signals in each spectrum indicate that two species are present in the solution of each complex, and the difference between the two species is presumably due to the variation of axial ligands. The resonances of the 3,5-MPy protons appear at 9.35-9.55 ppm, and the resonances of the 2,6-MPy protons appear at 8.75-9.05 ppm. The resonances of the pyrrole protons appear 130  a  II f I I I I  f  I I I^I I I I^I I 1 1  11  I  I  111111-1111^1111iIIII  14^12^10^  Figure 3.3. 1H NMR spectra of Co(II) cationic porphyrins in MASO-d6 under air. a: 1Co11[Tet(MPy)P]}C14; b: {Co11[T(MPy)PhP]}C13. (The solvent signal is at 2.49 ppm, and the water signal is at 3.3 ppm)  6PPM  Chapter 3 Water-soluble metalloporphyrins  111111  9.4^9.2^9.0  8.B PPM  4.8^  4.6  Figure 3.4. 1H NMR spectra of Co1li[Tet(MPy)12] species in DM$0-d6. a: {Com[Tet(MPy)11(H20))(C104)5, b: (Com[Tet(Iv1Py)P](H20))C15.  at 9.48 ppm (species 1) and 9.27 ppm (species 2) in the case of the perchlorate, and at 9.27 ppm (species 3) and 9.09 ppm (species 4) in the case of the chloride. The possible axial ligands for species 1 and 2 giving spectrum a are water and DMSO, ignoring possible coordination of C104. The axial ligands for the species 2 and 3 must be the same because their pyn-ole protons give the same signal (9.27 ppm). The 2,6-MPy protons corresponding to these species give peaks at slightly different positions, presumably centered at 8.89 ppm for the perchlorate (spectrum a) and at 8.91 ppm for the chloride 132  Chapter 3 Water-soluble metalloporphyrins  (spectrum b), because these protons are closer to the different counterions. Similarly, there are small differences in the resonances of the 3,5-MPy and methyl protons of species 2 and 3 in spectra a and b. Chloride must be coordinated to cobalt in species 4 simply because this species does not exist in the solution of the perchlorate. The actual assignments for the various species in these solutions can not be made with the information available. Replacing a neutral axial ligand by a negatively-charged ligand (C1) is likely to shift the pyrrole protons to higher field. This is certainly observed in the 1H NMR - pH studies, discussed in Section 3.3.6: when H20 is replaced by OH' as a axial ligand, all the signals shift to higher field. By this observation and a study of the spectra shown in Figure 3.4, it is clear that is involved in the coordination shell of cobalt(III) in DMSO solutions of the chloride salt. Proton NMR spectra of the {Coffi[T(MPy)PhP](H20))C14 and {Co'11[T(MPy)(NPh)P](H20))C14 porphyrins in DMSO-d6 also show that there are two species present in the solution for each system. As discussed above, the actual species in solution are not well defined; the species corresponding to the signals at relatively low field is maned #1 (the axial ligand(s) is probably H20 and/or DMSO), and the species corresponding to the signals at relatively higher field is named species #2 (at least one of the axial ligands is Cl). The chemical shifts of these signals are listed in Table 3.3. The methyl signals appear at 4.7-4.8 ppm and are not listed. The 1H NMR spectrum of {Colll[cis-B(MPy)B(NPh)PKH20))C13 in DMSO-d6 only has one set of signals (Table 3.3), and is probably not involved in coordination, judging by a comparison of the chemical shift of the AB quartet in this spectrum to those of the other complexes listed in Table 3.3. In this cis- complex, the overall cationic charge (assuming no coordination of Cl-) is +3, as opposed to +4 in the other species listed in  133  Chapter 3 Water-soluble metalloporphyrins  Table 3.3; this will be a factor in deciding the relative tendencies to bind the negatively charged Cl- (vs. water or DMSO). Table 3.3. 1H NMR data of the Co cationic porphyrin complexes in DMSO-d6  chloride salts  pyrrole  3,5MPy  2,6MPy  2,6-Ph or 3,4,5-Ph^or 2,6-NPh 3,5-NPh  Colll[T(MPy)PhP] #1  9.23 s, 9.15(AB qt)  9.48 d  8.92 d  8.17 m  7.89 m  Col11[T(MPy)PhP1 #2  9.06 s, 8.96(AB qt)  9.40 d  8.82 d  8.11 m  7.85 m  CollirT(MPy)(NPh)P] #1  9.26 s, 9.19(AB qt)  9.50 d  9.43 d  8.72 d  8.43 d  Cox[T(MPy)(NPh)13] #2  9.08 s, 9.00(AB qt)  9.42 d  8.82 d  8.69 d  8.37 d  Com[cis-B(MPy)B(NPh)P]  9.22 s, 9.16(AB qt),9.09 s  9.43 d  8.90 d  8.71 d  8.43 d  The 1H NMR spectral data for {Coultret(MPy)PMC104)5 in DMSO-d6 have been reported before as 9.37 (s, 8H), 9.27 (d, 8H), 8.92 (d, 8H) and 4.80 (s, 12H), but without assignments.3 These data do not correspond to those obtained for either of the species discussed here (Figure 3.4). The authors of this report did not mention any drying procedure or elemental analysis; one possibility is that the reported sample contained more water, and that the observed spectrum is for the species with two axial water ligands. 3.3.1.2 1H NMR spectra of Co complexes of anionic-porphyrins Figure 3.5 shows the spectra of Na4{Con[Tet(SPh)P)).4H20 and Na3(Com[Tet(SPh)P](OH2)}.3H20 in DMSO-d6 under air. In the paramagnetic spectrum (a) for Coli[Tet(SPh)P], the very broad signal at 12.8 ppm is assigned to the pyrrole protons, because these are closest to the paramagnetic center; the sharper signal at 8.8 ppm is assigned to the remote 3,5-SPh protons, and the underlying broad signal at 9.2 ppm to the 2,6-SPh protons. In the diamagnetic spectrum (b), the sulfonatophenyl protons give an AB quartet (8.0 to 8.2 PIN11, Jim = 8.0 Hz), while the resonance of the pyrrole protons appears at 9.20 ppm as a singlet. The 1H NMR spectra of Na2(Com[PyT(SPh)P]) and Na2{Cou'l(AP)T(SPh)P(OH2)] in DMSO-d6, shown in Figure 3.6, are less defined than the 134  Chapter 3 Water-soluble metalloporphirrhos  corresponding spectrum of Conet(SPh)P. The broadening of the peaks may result from the coordination of the pyridyl or amine group of one porphyrin molecule as an axial ligand at the metal center of a second porphyrin molecule. The peaks due to water in both spectra are obviously broadened and shifted down field from the resonance of free water (5 = 3.36 ppm); this may result from a exchange process between coordinated and free water, or from the possible formation of hydrogen bonds between water and the pyridyl or the amine moieties of the porphyrin structures. The Ili NMR spectra of the complexes in  a  9.2 9.0 •.11 U.S •.4 9.2 PPli  ^J IIIIf I1I I^I I  14^12^10^  4^PPM  Figure 3.5. 1H NMR spectra of Corl'et(SPh)Pj species in DMSO-d6. a: Na4{Co11[Tet(SPh)11).4H20; b: Na3{Com[Tet(SPh)11(H20)).3H20 (Peaks at 2.49 and 3.36 ppm correspond to DMSO-d5 and water, respectively).  135  16^14^12^10^8^4^2^0^-2^-4 PPM Figure 3.6. 1H NMR spectra of cobalt complexes of tris-sulfonatoporphyrins in DMSO-d6. a: Na2{Com[PyT(SPh)P]); b: Na2{ColIVAP)T(SPh)Pli0H2)}•H2O.  I.  Chapter 3 Water-soluble metalloporphyrina  D20 show two even broader peaks centered at 9.2 and 8.3 ppm. The broadening of these peaks in D20 again could result from the coordination of the pyridyl or amine groups. The spectra of Collcis-(NPh)PyB(SPh)13] in DMSO-d6 and in D20 have the same characteristics as described for the tris-sulfonato species. Attempts to synthesize Na2{Coll[PyT(SPh)P]} and Na2{Con[(AP)T(SPh)11) via the same procedure used for the synthesis of Na4(Co11[Tet(SPh)P]}.4H20 resulted in isolation of mixtures of Co(II) and Co(III) species, as judged by Ili NMR data. This may result from the existence of coordinating groups (pyridyl or amine) at the Co center of the porphyrin structures, and such coordination may alter the redox properties of the systems; a 5-coordinate Co(II) porphyrin species is much more sensitive than 4-coordinated Co(II) toward dioxygen.26 A broad peak probably corresponding to the pyrrole protons of the Co(II) species was observed at 12.7 ppm in a spectra of a mixture of Co(II) and Co(III) complexes of PyT(SPh)P in DMSO-d6; a peak at the same chemical shift is also observed in the spectrum of a mixture of Co(II) and Co(III) complexes of (APh)T(SPh)P in DMSO-d6.  3.3.1.3 111 NMR of Cu-porphyrins The 11-1 NMR spectra of the Cu-porphyrins give broad signals for the porphyrin protons. The appearance of these broad signals and the disappearance of the N-pyrrole protons (at approximately -3.0 ppm, Section 2.2) are evidence for coordination of copper(II). The II-I NMR spectra for the complexes of the copper(H) complexes of cationic porphyrins in DMSO-d6 are shown in Figure 3.7. In spectrum a for Cu[Tet(MPy)P], two signals are observable at 9.2 and 4.5 ppm; the latter is assigned to the methyl groups, while the former is probably due to the 3,5-MPy protons. The signals for the pyrrole and 2,6-MPy protons, which are closer to the paramagnetic center, are probably too broad to 137  Chapter 3 Water-soluble metalloporpbyrins  111111111i1111111111111111117111111111111111111r1111111111IIIIII111111.11,1  12^10^8^  4^  0^-2 PPM  Figure 3.7. 1H NMR spectra of copper(II) complexes of cationic porphyrins in DMSO-d6. a: Cu[Tet(MPy)11; b: Cu[T(MPy)Plin c: Cu[T(MPy)(NPh)11 (Peaks at 2.5 and 3.4 correspond to DMSO-d5 and water, respectively; the peak at 1.2 ppm is from an unknown impurity.) 138  chapter 3 Water-soluble metalloporphyrins  be observed. In spectrum b for Cu[T(MPy)PhP], signals at 9.2, 7.7 and 4.5 ppm are assigned to the 3,5-MPy, 3,4,5-Ph and the methyl protons, respectively. Again the signals of pyrrole, 2,6- MPy and 2,6-Ph protons are considered too broad to be observed. In spectrum c for Cu[T(MPy)(NPh)P], the peak at 8.5 ppm is assigned to the 3,5-NPh protons, and the other signals are assigned similarly to those of spectra a and b, as 9.2 ppm to the 3,5-MPy protons and 4.5 ppm to the methyl protons. The assignments generally are supported by the relative intensities of the signals; for instance, the ratio of the protons for the 3,5-MPy, 3,4,5-Ph and CH3 groups is 2:1:3 for Cu[T(MPy)PhP], which is observed approximately in spectrum b. The spectra of the copper complexes of the anionic porphyrins in DMSO-d6 have a pattern for the aromatic protons similar to that of the cationic ones: one broad peak at 7.8 ppm for Cu[Tet(SPh)P], two broad peaks at 7.8 and 7.0 ppm for Cu[(AP)T(SPh)P], and two broad peaks at 7.8 and 7.3 ppm for Cu[PyT(SPh)P]. In the last two cases, the peak at 7.8 ppm is much more intense than the peaks at higher field (7.0 and 7.3 ppm, respectively), and is assigned to the 3,5-SPh protons (6H); the signal at 7.0 ppm in the spectrum of Cu[(AP)T(SPh)P] is assigned to the 3,5-APh protons (2H), and the signal at 7.3 ppm in the spectrum of Cu[PyT(SPh)P] to the 3,5-Py protons (2H). The only signal observed in the spectrum of Cu[Tet(SPh)P] (at 7.8 ppm) is assigned to the 3,5-SPh protons. Again, the signals for the pyrrole, 2,6-SPh, 2,6-APh and 2,6-Py protons are probably too broad to be observed in this spectrum. 3.3.1.4 1H NMR of Zn-porphyrins  The 1H NMR spectra of the Zn complexes have the same characteristics as those of the porphyrin free-bases, except that the signals for the N-pyrrole protons (approximately at -3.0 ppm) do not appear, and all the signals, especially the pyrrole signals, shift to the higher field. The signals are assigned similarly to the assignments of the free bases (Tables 2.11 and 2.14), and are listed in Table 3.4. 139  Chapter 3 Water-soluble metalloporphyrina  Table 3.4. 111 NMR data for Zn porphyrin complexes in DMSO-d6 Zn[Tet(MPy)P]  9.42 d 3,5-MPy 9.42 d Zn[T(MPy)PhP] 3,5-MPy Zn[Tet(SPh)P] 8.78 s pyrrole Zn[(APh)T(SPh)P] 8.88 d, 8.75 m pyrrole Zn[PyT(SPh)P] 8.78 m pyrrole  9.00 s pyrrole 9.03 s; 8.93 AB qt, pyrrole 8.13 d 2,6-SPh 8.12 d, 2,6-SPh 7.99 d, 3,5-SPh 8.13 d, 2,6-SPh 8.01 d, 3.5-SPh  8.87 d 2,6-MPy 8.93 d 2,6-MPy 8.00 d 3,5-SPh 7.83 d 2,6-APh 9.04 s(br) 3,5-Py  8.18 d 2,6-Ph  4.73 Cfli 7.86 m 4.71 3,4,5-Ph CH/  5.46 s 6.97 d 3,5-APh NH, 8.27 s(br) 2,6-Py  3.3.1.5 1H NMR studies on the aggregation of Con complexes  Aggregation of Co(II) porphyrins has not been directly reported upon before; however, an aqueous solution of CoiTet(SPh)P] did not give rise to an ESR signal at room temperature, while addition of DMF to the solution under air prior to freezing resulted in a spectrum due to the presence of the monomeric Co(11)-porphyrin and its dioxygen complex (C(III)-02).'9 These findings suggested that the Co(ll) porphyrin aggregates in aqueous solution and, when organic solvent is added, the aggregation breaks down, thus leading to the ESR spectrum of the monomer and (in the presence of 02) the superoxide complex. In this thesis work, the aggregation of {ConfTet(MPy)PDC14 and Na4{Coll[Tet(SPh)P] were studied in a preliminary manner by 11-1 NMR in D20 under anaerobic conditions. Figure 3.8 shows the 1H NMR spectra of {Co11[Tet(MPy)P])C14 in D20 under vacuum at concentrations of 2 x 10-3 M (a), 2 x 10-3 M at g = 0.2 M (NaC1) (b), and 1 x 10-2 M (c). The signal of the pyrrole protons (13-14 ppm) shifts to higher field, and the signal of the MPy protons (9.5-10 ppm) remains essentially at the same position (very small shifts to lower field can be observed), when either ionic strength or concentration increases. These findings possibly result from aggregation, though no model can be proposed from the data available. 140  Chapter 3 Water-soiable metalloporphyrins  Limh....e........00  11111111111FIIIIIIIiiiiimiiiiIIII  14^12^10^8  I^  5  '  11^11111^II  4 PPM  Figure 3.8. 1H NMR spectra of Co1l[Tet(MPy)I9C14 in D20. a: C = 2 x 10-3 M, b: C = 2 x 10-3 M at 1.i = 0.2 M (NaC1), c: C = 1 x 10-2 M.  141  Chapter 3 Water-soluble metalloporphyrins  Spectra of Con[Tet(SPh)P] in D20 under N2, and the conditions for each spectrum, are shown in Figure 3.9. The sharp signals at 9.23, 8.45 and 8.25 ppm (indicated by arrows in spectra a and b) perhaps result from Com[Tet(SPh)1] formed by oxidation of the Co(II) complex by residual oxygen in D20. In spectrum a, the very broad signal centered at —12.0 ppm can be assigned to the pyrrole protons, the broad signal centered at about 9.6 ppm (labeled V) can be assigned to the 2,6-SPh protons, and a relatively sharp signal at 9.15 ppm which can be assigned to the 3,5-SPh protons. There is some degree of overlap for the last two signals. When the concentration increases from 0.004 to 0.008 M (from spectrum a to b), the signal for the pyrrole protons shifts significantly to higher field (-12.0 to —11.3 ppm) and the signal for the 3,5-SPh protons shifts slightly to lower field. The shift of the signal for the 2,6-SPh protons is not observable because the signal overlaps with the signal of the 3,5-SPh protons. Spectrum c is obtained at the same concentration as spectrum b, but NaC1 is added to a concentration of 0.2 M. Again, the pyrrole signal shifts to even higher field, while that for the 2,6-SPh protons shifts further to lower field (now at 9.32 ppm), and the two signals overlap. There may be a broad peak in the region of 7-10 ppm in this spectrum because the base line cannot be phased to give a straight line, and this peak could be the signal for the 2,6-SPh protons. Spectrum d, obtained at higher concentration (0.03 M) without salt, is almost the same as spectrum c. When salt is present in a solution at this higher concentration, the spectrum changed dramatically as shown in spectrum e; the peak of the 3,5-SPh protons shifts back to higher field (from 9.35 ppm in spectrum d to 9.00 ppm in spectrum e), and a peak which might be assigned to the 2,6-SPh protons appears at 8.1 ppm. The signal for the pyrrole protons is probably buried under the observed peaks. Again, no model can be proposed based on these data; however, more than one process appears to be involved in the aggregation of this metalloporphyrin over the range of concentrations and ionic strengths used because of the complexity of the signal shifts for the 3,5-SPh protons.  142  C  e^PfalooMomay.mmommlima1001010111000061~010 Ma  111111111111111111111111111111iiiiiiiiiiiilliiiiirilliwiiiiiiIIIIIIIIIIITilifilifillimili  15^14^15^1k^11^10^  7 PPM^6  Figure 3.9. 1H NMR spectra of Na41Colffet(SPh)PD in D20. a: C = 4 x 10-3 Nt, b: C =8 x 10-3 Ni; c: C =8 x 10-3 M, p. = 0.2 M (IslaC1); d:C=3x10-2M;e:C=3x10-2M,Lt=0.2M(NaC1).  143  Chapter 3 Water-soluble metalloporphyrins  3.3.2 Syntheses 3.3.2.1 Synthesis of Co-cationic porphyrin complexes One original purpose of this project was to synthesize Com complexes of the watersoluble porphyrins in order to evaluate these compounds as potential anti-cancer agents. For the synthesis of Com[Tet(MPy)13], the method of Pastemack et al.12ab was followed. Briefly, the porphyrin and a cobalt(II) salt were refluxed in water, and then NaC104 was added to precipitate the metallated porphyrin as a perchlorate salt; this was washed with water. 12a, b Then the perchlorate salt was converted into a chloride salt by ion exchange. Information from UV-visible spectroscopy of the chloride salt in aqueous solution under air in the present work showed the presence of a Co(III) complex, which agreed with the literature. 12a' b The 111 NMR spectrum of this product in D20 in air also showed the compound was a Com complex (spectrum a, Figure 3.2, p.128). Further characterization by both 111 NMR in degassed D20 under vacuum (spectrum b, Figure 3.2) and 11-1 NMR spectrum in DMSO-d6 (spectrum a, Figure 3.3, p.131) showed the compound made was in fact paramagnetic, implying a complex of Co(Il); the perchlorate salt was shown by 1H NMR in DMSO-d6 to be a mixture of complexes of Co(ll) and Co(III) (see Figure 3.3a, p.131, and Figure 3.4, p.132, for the NMR characterization of the Co(l) and (III) complexes). The method of Abwao and Konya et al.,15 in which Hg2+ is replaced by Co2+ from a Hg2+ porphyrin complex and then supposedly the Hg2+ oxidizes the Co(TI) complex to {Co111[Tet(MPy)P]}C15, was also followed; the perchlorate product was found to be a mixture of Co(II) and Co(III) while the chloride salt was a Co(II) species. The acidity of the solutions, from which the products were formed by evaporation, was found to be important for these syntheses (Section 3.3.2.3). Thus, for the syntheses of Co(III) complexes, as described in Section 3.2, the solutions after purification procedures 144  Chapter 3 Water-soluble metafloporphyrins  were always acidified, and lyophilization was used in some cases to preserve the acidity of the solution (see Section 3.3.2.3 for a discussion about the reaction conditions). 3.3.2.2 Synthesis of Co-anionic porphyrin complexes In an effort to prepare Na3{ComiTet(SPh)Ph, a literature method reported for the synthesis of Na3(Fe111[Tet(SPh)P]}27 was followed but with minor changes. Co(acetate)2.6H20 instead of FeSO4 was used. The acetate group was found to coordinate to the Com-porphyrin, as shown by a 1I-1 NMR spectrum in DM50-d6 of the crude product which showed a peak at -1.57 ppm; this disappeared in a spectrum of the purified sample and reappeared when sodium acetate was added to this purified sample. Starting with CoSO4-6H20 in the same conditions resulted in a mixture of Com and Con complexes with the latter dominating. The existence of acetate in the reaction mixture may facilitate the oxidation of the Con complex to Com through coordination.26 Coordinated and free acetate were removed from the crude product by extraction with CHC13 under acidic conditions, and the inorganic salts left over were removed by dialysis. The low yield for Com[Tet(SPh)P], compared to the yields for cobalt complexes of the other anionic porphyrins (Section 3.2), mainly resulted from the loss of the complex in the dialysis process. Attempts to synthesize Na{Com[c-BPyB(SPh)11) failed in that a mixture of cobalt(II) and cobalt(III) complexes was always isolated, as judged by 1H NMR in DMSO-d6 (Co(l) species give rise to a broad peak at —13 ppm, see Section 3.3.1.2). Unlike the other cobalt anionic porphyrin complexes, a solution of this mixture at 1 x 10-5 M in distilled water could not be oxidized by air to a purely cobalt(III) species, as judged by the presence of a shoulder peak on the shorter wavelength side of the Soret band (typically, a Co(II) species gives a Soret band at a shorter wavelength than the corresponding Co(III) species, Section 3.3.5). 145  Chapter 3 Water-soluble metalloporphyrins  3.3.2.3 Reaction conditions for the synthesis of cobalt porphyrins In order to understand the process of the synthesis, especially regarding the oxidation state of cobalt, the effects of a number of reaction conditions were studied.  Reaction time The reaction of [Tet(MPy)P]C14.4H20 and CoC12.H20 (1:5 mole ratio) in refluxing aqueous solution under air was monitored by UV-visible spectroscopy. After 45 min reaction time, metallation was essentially completed, as the four visible maxima between 500 and 700 rim had been replaced by a singe absorbance at —540 rim; and the spectrum was typical of a Co(II) complex (see Table 3.8, p.158). The oxidation state of Co could be inferred by the position of the Soret bands in the UV-visible spectra, according to the literature.28 The Soret band shifted to longer wavelengths (from 429 to 431 rim) at prolonged reaction times, indicating enhanced formation of the Com complex; however, a pure Com complex, which has a Soret band at 434 run in neutral aqueous solution28 was never observed, even up to 24 h. Prolonged reaction times also resulted in a lower yield of product and more impurities. In fact, when the porphyrin and excess CoC12 were refluxed in water for 2 days, UV-visible spectroscopy indicated that almost all of the porphyrin had been destroyed. 1H NMR spectra of DMSO-d6 solutions of the precipitate , formed by adding sodium perchlorate to the mixture of [Tet(MPy)11C14 and CoC12 at different reaction times, were also recorded in order to study the effect of reaction time on the oxidation state of the cobalt. After reaction time of 45 min, the precipitate was a Coll species; at 2 11, a Co(III) species could be observed; and at 10 h or longer reaction times, a mixture containing about the same amount of Co" and Com species was observed.  146  Chapter 3 Water-soluble metalloporphyrins  Based on the observations above, possible reactions occurring in aqueous solution are tentatively proposed to be [H2P4+ is used to present the porphyrin free base cation Tet(MPy)11: (1) Co2+(aq) + H2P4+  Co1134+ + 2H+  (2) CoffP4+ + H+ + 1— 02 4  ConIP5+ + 1H20 2  3.1 3.2  (3) CoInP5+ + Co2+(aq) —  Col[134+ + Co3+(acp  3.3  (4) Co3+(aco + — 21 H20  1 CO2+(acp + —02 + H+  3.4  4  The metallation (eqn 3.1) first occurs, and then the Co(II) complex is oxidized by air (eqn 3.2, see Section 3.3.5). The oxidation equilibrium favors the Co(III) complex as observed by 1H NMR (spectrum b, Figure 3.2, p.128) in aqueous solution. However, a subsequent metal ion exchange reaction might result in the formation of some Co(10-porphyrin species, whatever reaction time is used. The Co3+ formed from eqn 3.3 is a strong oxidant, which can oxidize water to 02 (E°CO3+/CO2+ 1.842 v)29, and also can presumably destroy the porphyrin structure. Aggregation of the Co(II) species at the concentration and ionic strengths used for the synthesis might also play a role in the observed phenomena; this is discussed later in this section (eqns 3.2 and 3.5). Acidity For the synthesis of Cow[Tet(MPy)P], samples collected from evaporation of the solutions after Cl- ion exchange were always mainly the Co(II) complex, while evaporation of acidified solution gave a Co(III) complex, as judged by 1H NMR spectra in DMSO-d6. Use of the tosylate salt of the porphyrin or use of Co(acetate)2 resulted in more Co(III) in the product mixture of porphyrin complexes precipitated by NaC104 than when using chloride salts as the starting materials (for a reaction time of 10 h). This may be 147  Chapter 3 Water-soluble metalloporphyrina  explained in that, in the refltudng process, protons (produced from the replacement of the N-pyrrole protons by cobalt) may be lost in chloride media in the form of HC1, while the formation of acetic or tosylic acid may help to preserve the acidity. Possible coordination of anions may also have some effect on the Co-redox equilibrium, as discussed in the case of the Co complexes of anionic porphyrins (Section 3.3.2.2). Acidity is generally important in the synthesis of the Co(H') complexes of all the cationic and anionic porphyrins as described in the synthesis procedures (Section 3.2). For instance, when a sample of Na4Coll[Tet(SPh)13] was dissolved in water to a concentration of approximately 10-5 M, and the solution stirred in air until the UV-visible spectrum showed that Cou[Tet(SPh)P] (the Soret band at 411.8 nm, Figure 3.14) was oxidized to Com[Tet(SPh)13] (the Soret band at 423.9 nm), evaporation of the solution to dryness on a rotovap gave a residue which was a mixture of Co(II) and Co(III) (according to the NMR spectrum). However, when this solution was acidified and evaporated to dryness at lower temperature (25-30°C), a Co(III) product resulted. It is hard to rationalize why the oxidation of a Co(II) to Co(III)-porphyrin can be completed in aqueous solution at neutral conditions (Figure 3.2, p.128), and yet acidity is needed to ensure a Co(Ill) product in the synthesis procedure. It may be that aggregation of Co(II) complexes is important in the redox process. Aggregation of water-soluble porphyrins and metalloporphyrins in aqueous solutions is well known.30 Although reports on the aggregation of Con water-soluble porphyrin complexes are rare in the literature (Section 3.3.1.5), related Cu" and Znil water-soluble porphyrin complexes have been reported to aggregate.25,32,38 Some Ili NMR evidence for the aggregation of Co" complexes is presented in Section 3.3.1.5.  148  Chapter 3 Water-soluble metalloporphyrim  Co(111)-porphyrins have been reported not to aggregate in aqueous solutions, 12a,13,31 and this was confermed with an anionic Co(III)-porphyrin in the present work (Section 3.3.5). The reactions involving the Co(ll) and Co(I11) redox process and the evaporation process are possibly related to the equilibria shown below using the Co complexes of [Tet(MPy)P]4+ as an example: Co1134+ + H+ + -102 —... 4  1  Com135+ + — H20 3.2 2  n [Co11114+ [ConP4+ln 3.5 Eqn 3.2 is the redox reaction and eqn 3.5 represents the aggregation process for the Co(l) complexes. If a solid sample of {Co11[Tet(MPy)PDC14 is dissolved in water to form a dilute solution (pH —8) under aerobic conditions, the 02 can oxidize the Co(ll) complex to a Co(III) complex as shown in equation 3.2. When this solution is concentrated by evaporation, Co1134+, although presents in a small amount, will start to aggregate (eqn 3.5), and this shifts eqn 3.2 to the left. Consequently, at the point of dryness the complex is in its reduced, aggregated Coll state. If the pH is changed from 8 to 3 (0.001 M acid was used for the synthesis, Section 3.2), the ratio of Co(II) to Co(III) species governed by eqn 3.2 is reduced by 105. The increase in concentration of the total cobalt from a dilute to a saturated solution of the complex (e.g., at pH 3) is probably not sufficient to shift the equilibrium of eqn 3.3 to effectively form the Co(II) complex. This suggested mechanism is also supported by the following experiments: A concentrated solution of Co9ITet(SPh)11 (100 mg in 10 mL water, —0.01 M) was stirred under air for one hour; at this stage the Soret band in the UV-visible spectrum appeared at 423.9 nm (the maximum for the Co(III) complex, Section 3.3.5). In the following 2 h, a sample was taken out every 15 min and a spectrum was recorded immediately after dilution to approximately 10-5 M; and then another spectrum of the  149  Chapter 3 Water-soluble metalloporphyrins  same dilute sample was run again 5 min later. The latter spectrum was found to have an absorbance approximately 10% higher at 423.9 nm than the first spectrum for every sample, which was always essentially the same. This can perhaps be explained by assuming that in the oxidized concentrated solution (-0.01 M), only a small portion (-10%) of the complex was present as Con9jTet(SPh)11 (the majority being in the aggregated forms), and thus the maximum absorbance appeared at the wavelength of the Soret band of Co(III); however, when the solution was diluted, the aggregated forms of Co(II) complex were dissociated into monomer and were oxidized to Co(III) (eqn 3.2), and this resulted in the absorbance increase. Unfortunately, this portion of the Co(II), if it does exist, can not be definitely and directly detected by UV-visible spectroscopy because of the poor sensitivity of the method, nor by NMR spectroscopy because a Coo species, especially in the aggregated forms, gives only broad signals in the 1H NMR spectrum.  Temperature Neta5 has demonstrated that when an aqueous solution of Na3Com[Tet(SPh)P] is evaporated to dryness, and the residue then heated to 120 0C, part of the resulting product is reduced to Col[Tet(SPh)P]. This agrees with the finding in the present work that a mixture of Co(II) and (III) complexes resulted from evaporation of a neutral solution of Com[Tet(SPh)P], this is originating from air-oxidation of ColiTet(SPh)P1 (p.149). The observation of Neta has been stated by Hambright and Langley14 to result from the heating of the solid sample which causes partial reduction to the Co(II) state. In this thesis work, all the Co(III) complexes were dried at 100°C under vacuum overnight, and no reduction was found. Also, neutral solutions of {Com[Tet(MPy)P1) C15 were evaporated to dryness either by lyophilization or roto-evaporation, and the residues dried at 100°C or room temperature under vacuum; {Co9ITet(MPy)PDC14 was the only cobalt-containing product, as indicated by 1H NMR. These findings indicate that reduction 150  Chapter 3 Water-soluble metalloporphyrins  occurs during the process of concentration, and not via the heating process as suggested in the literature." The conversion found by Neta probably resulted from the evaporation process at the relatively high pH value (> 6) of the solution (see previous page). Lyophilization has the advantage of maintaining the higher oxidation state of cobalt probably because it prevents an increase in concentration and retains the acidity of the solution, both of these factors having effects on the oxidation state of the isolated product as discussed above. 3.3.2.4 Synthesis of Cu porphyrins and Zn porphyrins -  -  Synthesis of the copper and zinc complexes was first tried using a reaction involving Cu(OH)2 (or CuO) and ZnO, respectively. The advantage of using these metal sources with low solubilities was that the excess starting metallic material could be filtered off at the end of the reaction. However, reaction of PyT(SPh)P with Cu(OH)2, CuO or ZnO gave a very low yield of the required complex, because of the observed association of the product with the metal precursor (Cu(OH)2, CuO or Zn0), possibly via coordination of the pyridyl group. Samples synthesized with Cu(OH)2, CuO or ZnO had to be dialyzed to obtain analytically pure products. It was also difficult to filter the muddy looking suspensions of Cu(OH)2, CuO or ZnO. The syntheses starting with CuC12 and ZnC12 were more convenient. Dialysis of the resulting copper and zinc porphyrin complexes resulted in high yield, probably because of the aggregation of these complexes at the dialysis concentrations used (0.01 -0.005  M).32  3.3.3 Mass spectra 3.3.3.1 Mass spectra of cobalt complexes of the anionic porphyrins Figure 3.10 shows the anionic FAB mass spectrum (thioglycerol matrix) of Na3{Com[Tet(SPh)P](OH2)} .3H20 and Figure 3.11 shows the suggested molecular 151  100 — -  50  1034  974  956  926  90  902  I  0— 900  950  1000  71,  1097  111  1050^  1100  Figure 3.10. Anionic FAB mass spectrum of Na3{Co11i[Tet(SPh)]P(OH2)}•3H20.  152  Chapter 3 Water-soluble metalloporphyrins  Na 03S  No 03S  Figure 3.11. The suggested molecular fragments for three of the major peaks in the mass spectrum of Na3(Co111[Tet(SPh)P](OH2)}.3H20.  153  Chapter 3 Water-soluble metalloporphyrins  Table 3.5. Mass spectra of some anionic Co-porphyrins Na2{Cox[(APh)T(SPh)1311 Na2{Com[PyT(SPh)11} Na{ Coli(cis-NPh)PyB(SPh)11 )  947  9251  NaH{CoRAPh)T(SPh)13])  H2{CoRAPh)T(SPh)11}  933  911!  NaH{Co[PyT(SPh)Pj}  H2{Co[PyT(SPh)11)  8981  876  Na{CoRNPh)PyB(SPh)P11  H{Co[(NPh)PyB(SPh)P])  1: The major peak in the spectrum besides peaks due to the matrix.  3.3.3.2 Mass spectra of cobalt cationic porphyrins  The mass spectra of the cobalt cationic porphyrins were recorded using the cationic FAB technique (3-nitrobenzylalcohol matrix). The major peaks above mass number 500 are listed in Table 3.6. Table 3.6. Mass spectral data for some cationic cobalt porphyrins {Conl[Tet(MPy)P(OH)}C14 {C09IT(MPy)Ph(OH2)}C14 (Com[T(MF'y)(NPh)P(OH2)1C14 {ConTB(MPy)B(NPh)P(OH2)}C13  734  720!  704  M-4C1-0H-1  M-4C1-0H-CH1  M-4C1-0H-2CH3-1  7191  704  689  M-4C1-14, 0  M-4C1-1-170-CH/  M-4C1-H70-2CH1  7641  748  734  M-4C1-H70  M-4C1-1170-CH/  M-4C1-H70-2CH1  792  7781  732  M-3 C1-1470-1  M-3 C1-HO-CH  M-3 C1-11,70-CH1 -NO7  II: The major peaks.  A peak at 690 in the spectrum of {Colll[Tet(MPy)P](OH))C14 is also relatively intense, and may be assigned as M-4C1-0H-3CH3. There is a peak at 735 in the spectrum of {CoztT(MPy)PhP)(OH2))C14, but the molecular fragment for this peak is not obvious; a possible suggestion is (M-4CI+0), the oxygen atom being a fragment of a coordinated water after loss of two protons. 154  Chapter 3 Water-sotable metalloporphyrias  3.3.4 Magnetism and coordination of the cobalt-porphyrin complexes The magnetic susceptibilities of solid samples of some of the cobalt porphyrins were measured using a magnetic susceptibility balance at room temperature (21.0°C). xg = 01-(R-R0)/109.m where^xg = weight magnetic susceptibility (c.g.s. units) C = balance calibration constant (calibrated against N1C12-6H20; xg = 1.82 x 10-5 c.g.s.units) 1= sample length (cm) R = reading for tube plus sample Re = reading for empty tube M = sample mass in g. The molar susceptibilities (xm) obtained by multiplying xg by the molecular weight of the compound are listed in Table 3.7. Table 3.7. Magnetic moments of some Co-Porphyrins  Na4 {Coll[Tet(SPh)P]} .4H20 Nal {Com[Tet(SPh)P](OH7)) •1411,0 Na7{Com[(APh)T(SPh)P(OH,)]).1170 Nal {Coni[PyT(SPh)P]) (Cog[Tet(MPy)P]}C14•21-1?0 {Com[Tet(MPy)P](OH0)C15-2H70  Xm x 103  Xm' x 103  1-1 (3-M.)  7.39 10.6 3.94 1.39 3.34 0.45  7.87 11.1 4.42 1.87 3.82 0.93  4.32 5.04 3.24 2.11 3.01 1.48 .  unpaired electrons 3 4 2 1 2 1(?)  Diamagnetic susceptibilities of porphyrin free-bases have been reported.32 The reported molar diamagnetic susceptibility of TetPhP (Xm = 4.81 x 10-4 c.g.s. units) is used to correct the Xm values. The corrected Xm values (Xm') are also listed in Table 3.4, as well as magnetic moment I/ which can be calculated as: IA = 2.84 (Xmi•T)1/2 B.M.(T is the temperature in K). The values of the magnetic moments listed in Table 3.7 are all significantly higher than the theoretical values calculated from the spin-only expression 155  Chapter 3 Water-soluble metalloporphyrins  = [n(n + 2)}1/2, for the listed numbers of unpaired electrons, except for the value for {CoififTet(MPy)PKOH2))C15 which is low. These deviations may indicate some d-ir interactions between the metal and the porphyrin system. Further investigations on the magnetochemical aspects of these complexes are essential. The magnetic moments of Co-water-soluble porphyrins have not been reported in the literature. However, some five-coordinated Co(III) complexes (Figure 3.12) have been found to be paramagnetic with 2 unpaired electrons.33  X  X = Cl, Br, I. Figure 3.12. Paramagnetic Co(III) complexes. The ligand in the complex shown in Figure 3.12 is similar to a porphyrin in providing a planar, macrocycfic N4 ligand set; further, the four nitrogen have two negative charges which are delocalized within the aromatic systems. When an extra ligand binds to these five-coordinate complexes, the resulting six-coordinate species becomes diamagnetic.33 Examples of five-coordinate Co(III) porphyrin complexes are known.34 The magnetic data presented here strongly suggest that cobalt in these Co(1.1.1) complexes of water-soluble porphyrins is five-coordinate in the solid state, while it is shown later that in aqueous solutions two water molecules are present as axial ligands (Section 3.3.6). It is possible that in the process of lyophilization or drying, one of the coordinated water molecules is lost, i.e., the fifth ligand in these five-coordinate Co(III) porphyrin complexes (Table 3.7) is water. When such cobalt complexes are dissolved in water or DMSO, the paramagnetism is lost (as judged by 1H NMR data, Section 3.3. 1) following coordination of the solvent in the sixth site. A six-coordinate formulation for the Co(III) porphyrin  156  Chapter 3 Water-solabk metidloporphyrine  complexes in the solid state (e.g., {Com[Tet(SPh)19(OH2)2}) would be inconsistent with the observed paramagnetism. There is a general agreement that, in the absence of strongly coordinating ligands, Co(ll) porphyrins are isolated in solid state as four-coordinate species;35 in solution, the complexes are nearly always five-coordinate.26 The paramagnetism of the Co(III) complexes presented in Figure 3.12 has been rationalized, in simple ligand field terms, as a system with a strong in-plane ligand-field of the Schiff base and a weak axial ligand of the halogen, which results in the dxy and dz2 orbitals being of similar energy and hence a (dxz)2(dyz)2(dxy)1(dz2)1 configuration for the meta136 (see the energy splitting diagram37 shown below, intermediate position). In the porphyrin systems, the axial ligand (H20) is stronger than a halogen, and the d orbitals may split in a manner closer to that of a square pyramid system. In the case of Com[Tet(SPh)P](OH2), 4 unpaired electrons are detected, consistent with a high spin species shown on the right-hand side of the energy splitting diagram. For the Co11[Tet(SPh)13] complex a high spin square planar Co(1)N4 system (shown on the lefthand side of the diagram) would fit the experimental data. —A— dx2..y2  4  dx 2 -y 2  —I— dxy^  ^  dx2_y2  .-' 7-T 4 dz2  ii,,,:,.-  dxy^ .--la .-.^......_. .dz2^d72^.^... d^d^dyz -----"-'" inewav= xz, yz^xz,d  square planar COI)  _L_  --ir dx z dyz ^  .---  dxy  d orbital splitting for ^ square planar^ square pyramid Co(M) --> stronger axial ligand  The same schemes do not fit the data for the Co(ITI and II) complexes of Tet(MPy)P. One possible explanation for this is that there may be some metal-metal 157  Chapter 3 Water-soluble metalloporphyrins  interactions which couple the unpaired electrons to some degree. For example, Coli[Tet(VIPy)P] could be a dimer with one Co-Co bond, which would leave two unpaired electrons for each molecule. Such interactions, if they do exist, are less defined for Coilr[Tet(MPy)P]; there is no obvious explanation for the measured, low paramagnetism. For CoifiRAPh)T(SPh)P(OH2)] and Cox[PyT(SPh)13], there are the possibilities of the pyridyl or amine substituent coordinating, which would change the energy splitting diagrams compared to that for Com[Tet(SPh)P]; the possibilities for a Co-Co interaction promoted by aggregation of the porphyrin structures are higher than in the case of Com[Tet(SPh)P] because there is less static electronic repulsion when one of the charged groups is replaced by a neutral group. The rationale used for the 2e magnetism of the complexes shown in Figure 3.12 could also be applied to Com[(APh)T(SPh)P(OH2)], with the aminophenyl possibly acting as the weak axial ligand. More detailed investigation is required to develop even a qualitative theory in order to explain why CoffiRAPh)T(SPh)P] apparently has 2 unpaired electrons while Com[PyT(SPh)P] apparently has 1 unpaired electron, although a difference in geometry between the coordination of the pyridyl and the coordination of the aminophenyl can be imagined (Section 3.3.7). 3.3.5 UV-visible spectra The UV-visible spectral data for the Co-porphyrin complexes dissolved in water at 1.0 x 10-5 M (at room temperature) are presented in Table 3.8. There are some differences in the data for Colir[Tet(MPy)P] and Com[Tet(SPh)P] compared with those in the literature. For example, Pasternack and Cobbi2a reported X = 437 nm with E = 1.68 x 105 cm-1AV, for Coni[Tet(MPy)P] at pH = 8 (0.001 M borate buffer), while Ashley et al. 13 reported X^= 433 run with E = 1.11 x 105 cnr1 M-1 at pH =7.65 or 6.56, g =1.00 M (NaC104). These deviations may result from the different experimental conditions, such as the pH value, ionic strength and concentration.  158  Chapter 3 Water-soluble ntetalloporphyrins  Table 3.8. UV-visible spectral data for Co-porphyrins (a)  ___Xmax [Ex10-3] 428.7^[119] 434.4 [190] 432.0 [162] 431.9 [158] 431.9 [117] 411.8 [198] 423.9 [219] 430.8 [145] 429.0 [150] 431.0 [125]  Xmax [Ex10-3] 540.0^[12.7] 548.0 [16.2] 545.0 [14.8] 545.0 [14.3] 545.0 [10.5] 529.5 [13.4] 539.5 [14.4] 539.5 [13.1] 545.0 [11.4] 547.0 [11.6] A: wavelength (Xmax) in tun, [extinction coefficient (E) in cm-1M-1].  {Co11[Tet(MPY)Pi}C14•2F170 {Com[Tet(MPy)P](H0)}04-21-170 {Com[T(MPy)PhP}(1-170))C14.2H,0 { Coll T(MPy)(NPh)P] (1-170)) C14-H70 { Com[B(MPy)B (NPh)P] (11, 0) } Cla .21170 Na4 ( Con[Tet(SPh)P]1•9H20 Nal {Colll[Tet(SPh)P](H70)} • 141170 Na, {Com[(APh)T(SPh)P](H70) } Nal {Com[PyT(SPh)P] } Na { C om[cis-(NPh)Py(SPh)P](1-1? 0)) • 2H, 0  The Soret band of CoIf Tet(SPh)P] is obviously sharper and more intense than the corresponding Soret band for the other cobalt anionic porphyrins, and the latter (the last three entries of Table 3.8) appear at longer wavelengths. The shifting of this X and the broadness of the bands might result from some coordination of the pyridyl or atninophenyl moiety at the cobalt center. The coordination of stronger ligands than water at axial sites of such metalloporphyrins generally results in the shifting of the Soret X to longer wavelengths.23 The Com[PyT(SPh)P] species obeyed Beer's law in distilled water from 1 x 10-6 to 1 x 10-4 M. This agrees with an earlier finding that Co(111) complexes of water-soluble porphyrins do not aggregate in aqueous solutions. 12a,13,  31  The ColliTet(MPy)P] and Cog[Tet(SPh)P] species are stable in the solid state toward air for at least days, but are air-sensitive in aqueous solution; this is demonstrated by the II-I NMR spectra in Figure 3.2 (p.128), and by the UV-visible spectra shown in Figures 3.13 and 3.14. The data in Figure 3.13 show the process of oxidation of Coll[Tet(MPy)P] by 02, with isosbestic points appearing at 428, 475 and 540 nm. 159  Chapter 3 Water-soluble metalloporphyrins  350  ^  450  ^  550  ^  65011M  Figure 3.13. UV-visible spectra of Con[Tet(MPy)11 at 8.7 x 10-5M, under various conditions at room temperature. a: under argon; b: 10 min later after 02 Was bubbled through the solution at 1 atm for 20 s; c: another 10 min later; d: another 20 min later; e: another 40 min later. Further spectral changes were observed after the solution was exposed to air (see text).  160  Chapter 3 Water-soluble metalloporphyrins  350  ^  450  ^  550  ^  650n  Figure 3.14. UV-visible spectra of Coll[Tet(SPh)P] in H20 at 6.9 x 10-5M, at various conditions at room temperature. Spectra were taken: under argon; 1 min later after 02 was bubbled through the solution for 20 s; another 10 min later; another 15 min later; and another 45 min later in sequence as the arrows show. No more spectral change was observed either at later times during the following 2 h, or at a later time after the solution was exposed to air.  161  Chapter 3 Water-soluble metalloporphyrins  A similar set of spectra was reported for the electrochemical reduction of a solution of Coaget(MPy)I1,28 under anaerobic conditions, where different isosbestic points were seen at 425 and 450 nm in the Soret region. The differences are probably due to the involvement of a superoxo intermediate which has been observed previously by ESR when Con[Tet(MPy)P] is oxidized by dioxygen;8 a Co(III) peroxide dimer and a Co(III) monomer final product were also incorporated in to an overall mechanism,8 which could be presented as: [ConP(OH2)]4+ + 02 ---- [(02-)ComP(OH2)]4+ ^  3.6  [(02-)Con?(OH2)}4± + [CouP(OH2)]4+^[(H20)PCo11'--022--ComF'(OH2)]8+^3.7 [(H20)PCom-022--Coll[P(OH2)]8+ + 2H20^2[C0mP(OH2)(OH)]4+ + H202 3 . 8 In the conditions used for synthesis (0.001 M HC1), the last reaction becomes: [(H20)PCo111--.022--ComP(OH2)J8+ + 2H+ + 2H20 ^2[CoRP(OH2)2]4+ + H202 3.9 Spectrum e in Figure 3.13 is likely to correspond to the peroxy dimer according to the findings of Evan and Wood.8 The solution corresponding to spectrum e (e = 1.34 x 105 cm-'M-1) was then exposed to air, and the spectrum of the solution changed slowly in the next 10 h, in that the Soret band shifted to slightly longer wavelength and increased in intensity by –35%, finally reaching the spectrum of the Co(III) monomer.28 A solution of Con[Tet(MPy)11 was acidified with HC1 solution to pH = 3 and exposed to air, when a Co(III) monomer spectrum was achieved in 1 h, and there was no more subsequent change; this reaction is probably the combination of those of eqns 3.6, 3.7 and 3.9. The data suggest that the Co(Il') complex synthesized is highly likely to be a monomer and not a peroxy dimer. Figure 3.14 shows the oxidation of ColTet(SPh)P] by 02 in aqueous solution. There is an isosbestic point in the Soret region at 417 nm. Very similar 'reverse' spectra were reported when Coni[Tet(SPh)P] was reduced by methanol in a photochemical reaction under vacuum, but more isosbestic points were observed in this methanol system  162  Chapter 3 Water-soluble metalloporphyrins  (at 360, 419, 454, 534 and 615 nm).21 The similarity of the final spectrum in Figure 3.14 to the initial spectrum in the methanol system suggests that the final product in the automddation process is same as the starting compound in the methanol system, which is considered21 to be a Co(III) monomer. The four negatively charged sulfonatophenyl groups will make the metal center more electron rich compared with that in Coll[Tet(MPy)P] which has four electron-withdrawing methylpyridinium groups; the oxidation reaction should be more favorable for the sulfonatophenylporphyrin system. The copper metallation of the porphyrins can be readily monitored by UV-visible spectroscopy. The replacement of the four visible bands (500-650 nm) of the free porphyrins by one major band (530-550 nm) with a weak shoulder band (575-585 nm) in this region is very characteristic for the metallation. Because of the known aggregation properties of copper complexes of water-soluble porphyrins in aqueous solutions,32,38 the UV-visible spectra are almost certainly dependent on concentration and ionic strength, but the details of these dependences are beyond the scope of this thesis work. Table 3.9 shows the UV-visible data in distilled water at 1.0 x 10-5 M at 20°C. Table 3.9. UV-visible spectral data for Cu-porphyrins (a) Xmax [Exl 0-3] (CUITet(MPY)P1)C14: 101170  {Cu[T(MPy)Ph13] } C11 -101170 {Cu[T(MPy)(NPh)PDC11.6H10 Na4{Cu[Tet(SPh)13]) -101170 Nal { Cu [(APh)T(SPh)P] ) .91170 Nal {Cu[PyT(SPh)P] } .91170  422 421 421 410 409 411  [200] [95] [158] [466] [376] [314]  Xmax [Exl 0-3]  Xmax [EX 1 0-3]  545 546 546 538 538 540  585 583 583 575 575 578  [16.1] [8.3] [12.9] [19.2] [17.5] [16.7]  [3.8] [2.4] [2.8] [3.1] [2.6] [4.0]  a: wavelength (Xmax) in tun, [extinction coefficient (E) in cm-IM-1]. 3.3.6 Measurement of the pKa values of cobalt(III) diaquo porphyrin complexes The 11-1 NMR spectra of the Com porphyrin complexes in D20 show a pH dependence which is attributed to the equilibria presented in equations 3.10 and 3.11: C o lliP (1120)2n  ^  ComP(H20)(OH)11-1 + H+^Kai^3.10 163  Chapter 3 Water-soluble utetsdloporphyrins  ConiP(H20)(OH)n-1^ComP(OH)2n-2 Fi+^"2^3.11 P = Tet(MPy)P, n = 5+; P = T(MPy)PhP/P , T(MPy)(NPh)P, n = 4+ P = T(SPh)P, n = 3The^N1VIR spectra in D20 at various pD values for Com[Tet(MPy)P] and Com[Tet(SPh)P] are shown in Figures 3.15 and 3.16, respectively. In these spectra, all the signals shift to higher field when the pD increases, and this is attributed to conversion of coordinated D20 to OD-; presumably enhanced electron donation to the metal center results in increased shielding of the porphyrin protons, especially the pyrrole protons which are closest to the metal. At higher pD, the resolution of the signals for the MPy protons in the spectrum of Cos[Tet(MPy)11 becomes somewhat poorer, presumably due to OD- associating with the MPy groups. On the other hand, in the case of ColiTet(SPh)13], at lower pD possible association of protons with the sulfonate groups decreases the resolution of the sulfonatophenyl signals. The 1H NMR spectra of Con[T(MPy)PhP] and Coffi[T(MPy)(NPh)P] at different pD values have the same characteristics as that of Com[Tet(MPy)13]. Spectra of the other Co"-porphyrins in D20 solution do not give sharp signals (Section 3.1), and therefore were not studied at different pD values. The dependence of the chemical shift of the pyrrole protons on the pD values means that the pKa values for eqns 3.10 and 3.11 can be measured by NMR. A solution of the Cog-porphyrin (1.0 x 10-3 M) in D20 was prepared; then to half of the solution was added solid NaOH to make CNaoH = 0.1 M, and to the other half was added 6.0 M HC1 to make Clio = 0.1 M. Aliquots were drawn into NMR tubes during the titration of the acidic solution with the basic solution, and the pD values were monitored by a 'pH' meter. NMR samples prepared in this way had essentially identical concentrations of the porphyrin and ionic strengths (C 1.0 x 10-3 M, 1.1 = 0.1 M) with known pD values. 1H NMR spectra were recorded at 23°C. The pKa values were 164  Chapter 3 Water-soluble metaDoPorPhYrhu  al I^  4e  a;^cy,^ I^  ,,f'  /  pD = 2.05  e0 , ,  /  pD = 6.00 S11611%millail° .4141"P444".....b...„,„,,,,,,.....001a• qr. qr.  •••• a;  1  ild  ei  1  pD = 12.00  milinquillimulipuquillimpupliquumpupulinupuquumnimil 9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8.2 PONO  Figure 3.15. 1H NMR spectra of 10-3 M Colli[T(MPy)Pj in D20 at 23°C at different pD values. Assignments are discussed in Section 3.3.1.  165  Chapter 3 Water-soluble metalloporphyrias  pD = 3.00  to°^0%.°° 1P13 $,sgx‘9 tysitt` ,9s1c°^k  p. pD = 7.02  1601011WMI^\1111"114~14114111141#A1  dkimminerwpoithoo,  pD = 10.10  a  11111^[111111111111^111111  id.°^9.5^9.0^8.5^8.0 PPM  Figure 3.16. 1H NMR spectra of 10'3 M Com[Tet(SPh)PJ in D20 at 23°C at different pD values. Assignments are discussed in Section 3.3.1. 166  Chapter 3 Water-soluble metalloporphyrins  obtained from a graph of the chemical shifts of the pyrrole protons versus the pD values. The graphs for Com[Tet(MPy)11(D20)2and ConifTet(SPh)PJ(D20)2 are shown in Figures 3.17 and 3.18, respectively. The corresponding graphs for the other porphyrins studied, Com[T(MPy)PhP] and Co111[T(MPy)(NPh)11, are presented in Appendix C. A conventional method" was used to determine the equivalent points, and the pKa values obtained together with available literature values are shown in Table 3.10. Table 3.10. pKa values for some cobalt(M) diaquo porphyrin complexes  pKal  pKa2  conditions (ref.)  Conget(MPy)11(H70),  6.0 5.9 ±0.1 5.46 ± 0.09  10.0 9.9±0.1 10.7 ± 0.8  ComfTet(MPY)PND20)2  5.5 ± 0.1  10.7 ± 0.1  Con11[T(Wy)PhP](D20)2  6.2 ± 0.1  10.7 ± 0.1  Co111[T(MPy)(NPh)P](D20)2  5.9 ± 0.1  10.8 ± 0.1  C=2 x 10-5 M (12a)^a C=4.34 x 10-6 M (3)^a 1.t =1.0 M [NaC104] 25°C (13) C=1.0 x 10-3M 23°C (present work) C=1.0 x 10-3M 23°C (present work) C=1.0 x 10-3M 23°C (present work)  CollTet(SPh)P](-120)2  5.72± 0.01  ConitTet(SPWP20)2  7.02  9.76  7.6 ± 0.1  11.8 ± 0.1  11 =1.0 M (NaC104) 25°C (23) . IA =1.0 M (NaC104) 25°C (40) C=1.0 x 10-3M 23°C (present work)  a: temperature is not reported. The pKa values obtained in this work for Com[Tet(SPh)P](D20)2 are higher than those obtained for Com[Tet(MPy)P](D20)2, and this can be understood because the latter has four electron-withdrawing cationic groups on the porphyrin ring resulting in a  167  Chapter 3 Water-sohthie metalloporphyrim .  9.35  9.3 -  9.25 -  9.2 -  MMINIMMI  9  8.95 -  8.9  3^5^7^9^11^13  pD  Figure 3.17. pKa measurements for Co0i[Tet(MPy)F1(D20)2 from II-I NMR data at 23°C.  168  Chapter 3 Water-salable metalloporphyrias  9.45  9.4  9.35 —  ONIM,  9.15  9.1 —  I^  9.05  2^4^6^8  I 10^12  14  pD  Figure 3.18. pKa measurements for Co1itTet(SPh)Pp20)2from 1H NMR data at 23°C.  169  Chapter 3 Water-soluble metalloporphyrins  relatively electron deficient cobalt center, which makes the coordinated water more acidic. In the case of Com[Tet(SPh)ITD20)2, the negatively charged sulfonatophenyl substituents result in a relatively electron rich cobalt center which makes the coordinated water less acidic. Similarly, when a MPy group is replaced by the less electron-withdrawing phenyl group, i.e., in going from Conget(MPy)11(1320)2 to Com[T(MPy)PhI](D20)2, the pKal value increases; and when this phenyl group is replaced by the more electron-withdrawing nitrophenyl group (Com[T(MPy)P1111(D20)2--->Com[T(MPy)(NPh)P](D20)2), the pKai value decreases. The pKa2 values of the three cationic cobalt porphyrins studied were essentially the same. All of the literature pKa values listed in Table 3.10 were measured by UV-visible spectrophotometric titrations in H20. There is some disagreement between some of these published results with those obtained in this work, particularly for the Tet(SPh) system. The reasons for the difference are not clear. The isotopic difference between D20 and H20 should be negligible in these measurements. 3.3.7 Hydration and elemental analysis The hydration of water-soluble metalloporphyrins has been observed by several authors in the literature. 14,15,19,20 Hydration is also a general observation for water-soluble porphyrin free-bases (see Section 2.3.1). The degree of hydration is determined by elemental analysis, and is observed also by Ill NMR measurements in DMSO-d6. Of interest, no hydration is observed in the sample of Na2{Co111[PyT(SPh)P]), although hydration (including possibe coordination ) is observed in all the other metalloporphyrins synthesized in this work. This is perhaps related to the coordination of the pyridyl group, which could eliminate the existence of coordinated water. There is also the possibility that, in the solid state, this complex forms oligomers (e.g., a tetramer as shown below). The Com[(APh)T(SPh)P] species would not be able to form such a tetramer because of the 170  Chapter 3 Water-soluble metalloporphyrins  different coordination direction of the arninophenyl group and, in the solid state, the axial ligand for this metalloporphyrin is most probably a water molecule, as shown by the correct elemental analysis for Na2{ComRAPh)T(SPh)PK0H2)).  r  171  Chapter 3 Water-soluble metalloporphyrina  References-Chapter 3  1^J•W• Buchler, in "The Porphyrins and Metalloporphyrins", K.M. Smith, ed., Elesvier Scientific Publish Co., 1976, p.157. 2^J.A. O'Hara, E.B. Douple, M.J. Abrams, D.J. Picker, C.M. Giandomenico and J.F. Vonano, Int. J. Radiat. OncoL Biol. Phys., 16, 1049 (1989). 3^R.J.H. Chan, Y.O. Su and T. Kuwana, Inorg. Chem., 24, 3777 (1985). 4^C. Aroullo-McAdazns and K.M. Kadish, Inorg. Chem., 29, 2749 (1990). D. Sazou, C. Araullo-McAdams, B.C. Han, M.M. Franzen and K.M. Kadish, J. Am. Chem.  Soc., 112, 7879 (1990).  R.M. Kellett and T.G. Spiro, Inorg. Chem., 24, 2373 (1985). C. Ni and F.C. Anson, Inorg. Chem., 24, 4754 (1985). 5^P. Neta, J. Phys. Chem., 85, 3678 (1985). 6^R. Langley, P. Hambright and R.F.X. Williams, Inorg. Chem., 24, 3716 (1985). 7^S. Mosseri, P. Neta, A. Harriman and P. Hambright, J. Inorg. Biochem., 39, 93 (1990). 8^D.F. Evans and D. Wood, J. Chem. Soc. Dalton Trans., 1987, 3099. 9^F.C. Anson, C. Ni and J. Saveant, J. Am. Chem. Soc., 107, 3442 (1985). 10^R.F. Pastemack, E.J. Gibbs and J.J. Villafranca, Biochemistry, 22, 2406 (1983). 11^E. Mikros, F. Gaudemer and A. Gaudemer, Inorg.Chem., 30 1806 (1991). 12^(a) R.F. Pastemack and M.A. Cobb, J. Inorg. NucL Chem., 35, 4327, (1973). (b) R.F. Pastemack, E.G. Spiro and M. Teach, J. Inorg. NucL Chem., 36, 599 (1974). (c) R.F. Pastemack, M.A. Cobb and N. Sutin, Inorg. Chem., 14, 866 (1975). 13^K.R. Ashley, M. Berggren and M. Cheng, J. Am. Chem. Soc., 97, 1422 (1975). 14^P. Hambright and It Langley, J. Inorg. Biochem., 32, 197 (1988). 15^J. Abwao-Konya, A. Cappelli, L. Jacobs, M. Krishnamurthy and M. Smith, Transition Met. Chem., 9, 270 (1984). 16^S. Baral and P. Neta, J.Phys Chem., 87, 1502 (1983). 17^P. Hambright and E.B. Fleischer, Inorg. Chem., 9, 1757 (1970). 18^F.C. Anson, C.-L. Ni, and J.M. Saveant, J. Am. Chem. Soc., 107, 3442 (1985). 19^I.A. de Bolfo, T.D. Smith, J.F. Boas and J.R. Pilbrow, J. Chem. Soc. Dalton Trans., 1976, 1495. 20^0. Herrmann, S.H. Mehdi and A. Corsini, Can. J. Chem., 1978, 1084. 21^K. Hatano, K. Usui and Y. Ishida, Bull. Chem. Soc. Jpn., 54, 413 (1981). 22^T.J. Fullerton, P.A. Watson and L.J. Wright, Appita, 43, 23 (1990). 23^K.R. Ashley and S. Au-Young, Inorg.Chem., 15, 1937 (1976). 24^G. Dougherty and R.F. Pastemack, Inorg. Chim. Acta, 195, 95 (1992). 172  Chapter 3 Water-soluble metalloporphyrias  25^K.M. Kaciish, G.B. Maiya, C. Araullo and R. Guilard, Inorg.Chem., 28, 2725 (1989). A. Cosini and 0. Herrmann, Talanta, 33, 335 (1986). 26^B.R James, in "The Porphyrins", D. Dolphin, ed., Academic Press, New York, 1978, Vol.5, p.205. 27^E.B. Fleischer, J.M. Palmer, T.S. Srivastava and A. Chatterjee, J. Am. Chem. Soc., 93, 3162 (1971). 28^D.F. Rohrbach, E. Deutsh, W.R Heineman and R.F. Pastemack, Inorg, Chem., 16, 2650 (1977). 29^"CRC Handbook of Chemistry and Physics", 49th ed., The Chemical Rubber Co., Cleveland, 1968, p.D86. ^ 30 W.I. White, in "The Porphyrins", Vol. V. D. Dolphin, ed., Academic Press, New York, 1978, p.303. C.A. Hunter and J.K.M. Sanders, J. Am. Chem. Soc., 112, 5525 (1990). 31^M. Krishnamurthy, J.R Sutter and P. Hambright, J. Chem. Soc. Commun., 1975, 13. 32^T.P.G. Sutter, P. Hambright, A.N. Thorpe and N. Quoc, Inorg. Chim. Acta, 195, 131 (1992). 33^N.A Bailey, E.D. McKenzie and J.M. Worthington, J. Chem .Soc. Dalton, 1977, 763. E.D. McKenzie and J.M. Worthington, Inorg. Chim. Acta, 16, 9 (1976). E.D. McKenzie, RD. Moore and J.M. Worthington, Inorg. Chim. Acta, 14, 37 (1975). 34^RD. Chapman and E.B. Fleischer, J. Am. Chem. Soc., 104, 1582 (1982). T. Sakurai, K Yamamoto, H. Naito and N Nakamoto, Bull. Chem. Soc. Jpn., 49, 3042 (1976). 35^J.W. Buehler in "The Porphyrins and Metalloporphyrins", K.M. Smith, ed., Elesvier Scientific Publish Co., 1976, p.157. 36^B.M. Higson and E.D. McKenzie, J. Chem. Soc. Dalton, 1972, 269. 37^"Advanced Inorganic Chemistry", 4th Ed., F.A. Cotton and G. Wilkinson, John Wiley & Sons, New York, 1980, p.642. 38^K.M. Kadish, B.G. Maiya and C. Araullo-McAdams, J. Phys Chem., 95, 427 (1991). 39^"Chemistry Laboratory Manual", University of British Columbia, 1992/1993, p.73. 40^K.R. Ashley and J.G. L,eipoldt, /norg. Chem., 20, 2326 (1981).  173  Chapter 4 Aggregation of porphyrins  Chapter 4 Aggregation of porphyrins 4.1 Introduction  The importance of porphyrin chemistry is briefly reviewed in Chapter 1. In the studies of porphyrin chemistry, especially in aqueous solutions, aggregation is often encountered.' An understanding of the extent, the kinetics, and the models of the aggregation of porphyrins and metalloporphyrins is crucial to interpretation of kinetics and thermodynamic data obtained in studies on these compounds concerning their substitution and oxidation-reduction reactions,2 DNA-porphyrin associations,3 and in vivo behaviors.4 Because of the practical and intrinsic importance of aggregation in the chemistry and characterization of porphyrin compounds, this chapter is devoted to the aggregation of the water-soluble, porphyrin free-bases synthesized in this thesis work (Chapter 2). Resulting from the hydrophobic properties of the porphyrin ring, aggregation is a general property of porphyrin compounds in aqueous solutions. Three types of interactions have been proposed in connection with the aggregation of porphyrin compounds:5 (1) n-rc interactions for porphyrin free-bases, (2) metal-n interactions for metalloporphyrins, and (3) metal-side-chain interactions (coordination) for metalloporphyrins. The last two types of interactions are often found in non-aqueous solutions of metalloporphyrins,2,6 and are not included in the focus of this chapter. The aggregation of porphyrin free-bases has been of interest for several decades." The early studies have been mainly on natural porphyrins, and a basic model of a "face to face" interaction is proposed. With the synthesis of water-soluble porphyrins, which started in the early seventies, the focus of studies on aggregation of porphyrins shifted to this new class of compounds.' Many methods have been employed in these studies  174  Chapter 4 Aggregation of porphyrins  including: UV-visible spectrometry,3,8 fluorescence spectrometry,9,0 temperature-jump kinetic studies," and NMR spectroscopy.12,13 However, limited by the availability of the number of synthetic, water-soluble porphyrins, most of the studies have been on just a few symmetric porphyrins. The synthesis of a large variety of water-soluble porphyrins in the present work provided an opportunity for further studies on the relationship between the aggregation and structures of such porphyrins. In this thesis work, UV-visible and NMR. spectroscopy have been used in the studies on porphyrin aggregation. 4.2 Experimental  All the porphyrins used in this chapter were synthesized as reported in Chapter 2. Only the sodium salts of anionic porphyrins and the chloride salts of cationic porphyrins were studied. A buffer solution was made by dissolving NaH2PO4 (0.69 g, 0.0050 mol) and Na2HPO4 (0.71 g, 0.0050 mol) in distilled water (1.00 L). The pH value of this buffer solution, measured by a pH meter (Radiometer Copenhagen PHM25) with a glass combination electrode (Cole-Parmer Chemical Co.), was 7.03 ± 0.01, and the ionic strength was 0.013 M. Stock solutions of the porphyrins (1.0 x 10-4 M) were made by dissolving a known amount of the solid porphyrin (— 2 mg) in the required amounts of the buffer solution (-15-25 mL). The solutions at other concentrations were made by dilution of the stock solutions with buffer or buffer and methanol using volumetric analysis apparatus. Solutions of the porphyrins in pure water were made in the same way but using distilled water instead of the buffer solution. Absorbance spectra were measured on a Perkin Elmer 552A spectrometer using quartz cells (0.100, 0.500, 1.00, 2.00 cm) at 20.0°C. The ordinate scale of the absorbance  175  Chapter 4 Aggregation of porphyrins  plots, controlled electronically in the spectrometer, was set proportional to the product of concentration (C) and light-path length (b) for each of the solutions of a porphyrin of absorbance A; thus the vertical axis was proportional to A/bC (equal to e, the molar absorptivity or extinction coefficient), and if Beer's law was obeyed, the spectra of these solutions would be identical. For instance, a solution at concentration C was measured using a cell of path length b at a scale S; another solution at concentration of 0.500 C was measured using either a 2 b path length cell at scale S or a b path length cell at 0.5 S, depending on the availability of the cells; another solution at 0.0300 C was then measured using a 10 b cell path length at scale 0.3 S. This method was checked by recording spectra of Tet(SPh)P in DMSO at various concentrations (10-6 -10-4 M); identical spectra resulted. Spectra recorded in this way are noted in this work as the "normalized spectra". 11.1 NMR spectra in D20 were recorded on a 300 MHz Varian spectrometer, while 2-D 1H NMR data were recorded on a 400 MHz spectrometer; spectra were measured at room temperature (23-25°C) and with external reference (DDS = 0 ppm). 4.3 Results and discussion  4.3.1. UV-visible spectra in aqueous solutions The classic method for indicating aggregation of porphyrins is a Beer's law study, in which aggregation is the only phenomenon that gives rise to deviations from Beer's law.1 Dimerization has been proposed as a structural model for most of the aggregations. 8,11,14,15 The most important spectral evidence for dimerization is the observation of an isosbestic point in the normalized spectra in dilution studies where the product of concentration and light-path length is kept constant.16 Dimerization of monomers is the simplest model to rationalize such evidence, but higher aggregation (for example tetramerization from initially present dimers) can also explain the data. Little 176  Chapter 4 Aggregation of porphyrins  evidence supporting the monomer-dimer model could be found in the literature for synthetic porphyrins. In this work, the appearance of the spectra should be of the same type as those recorded in the literature method where the product of concentration and light-path length was kept constant at one absorbance scale,16 but the experiments could be controlled more conveniently using the variation of absorbance scale and the variation of cell path length. 4.3.1.1 Tet(SPh)P The spectra of this porphyrin in distilled water at different concentrations are shown in Figure 4.1. The molar absorptivity of the Soret band at 410.0 nm remains essentially the same (e = 5.0 x 105 M-1 cm-1) when the concentration increases from 0.50 x 10-6 to 4.0 x 10-6 M (see inset in Figure 4.2), and increases when the concentration increases from 6.0 x 10-6 to 4.0 x 10-5 M; the molar absorptivity at 392 nm increases throughout with increase of concentration. The Beer's law plots for the spectra in distilled water are shown in Figure 4.2 (410 nm and 392 nm data; some spectra are not shown in Figure 4.1 for the sake of clarity). In Figure 4.2A (X = 410 nm), the inset is a plot of the data from 2.5 x 10-7 to 4.0 x 10-6 M, when the law is obeyed with e = 5.0 x 105 M-1 cm-1; the rest of the data (at higher concentrations) deviate positively from this straight line. [e increases with increasing concentration: the molar absorptivities calculated for the spectra at 1.0 x 10-5 M (e = 5.3 x 105), 2.0 x 10-5 M (e = 5.5 x 105) and 4.0 x 10-5 M (e = 5.6 x 105) are 6-12% higher than the e value at lower concentrations]. Fleisher et a/.14 had interpreted the data for this porphyrin as obeying Beer's law (at 10-4 - 10-9 M) at 411 nm with e = 5.33 x 105 M-1 cm-1, while Corsini and Herrmann12 reported that this porphyrin obeys Beer's law (at 2.1 x 106  M, phosphate buffer) at 412 nm with e = 5.10 x 105 M4 cm-1; these values essentially 177  Chapter 4 Aggregation of porphyrbu  agree with the average of the e values obtained here. The deviation from Beer's law is more obvious in Figure 4.2B (X = 392 nm). 6.0  Figure 4.1. The normalized spectra of Tet(SPh)P at various concentrations in distilled water. The concentrations are 5.0 x 10-7, 1.0 x 10-6, 2.0 x 10-6, 4.0 x 10'6 6.0 x 10-6, 8.0 x 10-6, 1.0 x 10-5, 2.0 x 10-5 M in sequence as the arrows show.  Figure 4.3 shows the normalized spectra of Tet(SPh)P in buffer solutions at different concentrations. As the concentration increases, A.max remains the same (411.3 nm), although the molar absorptivity decreases. There is an isosbestic point at 404.8 nm, which indicates that only two species are in equilibrium in these conditions. The Beer's law deviation is shown in Figure 4.4. 178  Chapter 4 Aggregation of porphyrina  A  ^  data at 410 nm  24 16  -  12  20  s 44  01 0.4  16  01  12  P  8  4  e  r., 1  AO°  02  03  0  ..di PFP-A  rr 4  AO°  4  .  ^^ ^^ ^ 0.5 1 1.5^2^2.5 3 3.5 4  Concentration x 105 (M) data at 392 nm 5 4 3 2 1 0 0  ^  0.5  ^^ ^^ ^ 1 1.5^2^2.5 3.5 4 3  Concentration x 105 (M) Figure 4.2. Beer's law diagrams for Tet(SPh)P in distilled water.  179  Chapter 4 Aggregation of porphyrins  Figure 4.3. The normalized spectra of Tet(SPh)P at various concentrations in a phosphate buffer. The concentrations are 0.10 x 10-5, 0.50 x 10-5, 1.0 x 10-5, 2.0 x 10-5, 3.0 x 10-5, 4.0 x 10-5, 5.0 x 10-5, 6.0 x 10-5 M in sequence as the arrows show.  25 20 15 10 5  i  ^  2^3^4  ^ ^ 5 6  Concentration a 105 (M)  Figure 4.4. Deviation from Beer's law for Tet(SPh)P in a phosphate buffer solution. 180  Chapter 4 Aggregation of porphyrhis  These findings are contrary to the early report, as noted above, that Tet(SPh)P obeys Beer's law at concentrations between 1 x 10-4 to 1 x 10-9M, at pH = 7.0 [p p = 0.10 M (NaC104)] or pH = 13 (0.1 M Na0H).  14  P4  0 and  However, the data agree with later  reports in that Tet(SPh)P aggregates at 20°C at ionic strength p. = 0.05 M (NaNO3) as observed by temperature-jump kinetic studies,1' and in buffer solutions at p, = 0.1 M [KNO3, phosphate, or Na(0Ac)] as observed by UV-visible spectra.i2 These reports used a dimerization model to rationalize the data, and the relevant equilibrium constants (K values) for the monomer/dimer equilibrium are summarized in Section 4.3.5). The aggregation observed here in distilled water and in the buffer solution most probably is a dimerization process as rationalized in the literature.8,12 Alternatively, there might be two stages of the aggregation process; e.g. the aggregation in the buffer solution could be an oligmerization process. However, a monomer-dimer model (discussed Section 4.3.4) is supported by studies of the behavior of this porphyrin in mixtures of methanol and buffer solutions (Section 4.3.2). The equilibrium constant for this presumed dimerization in the buffer solution is presented in Section 4.3.5. 4.3.1.2 PyT(SPh)P and (APh)T(SPh)P The patterns of the normalized spectra at different concentrations. for the buffer solutions of PyT(SPh)P and (APh)T(SPh)P are similar. The former has an isosbestic point at 402.8 nm (Xmax = 410.7 nm at C = 1.00 x 10-6 M), and the latter has two isosbestic points at 404.8 and 428 nm (Amax = 412.4 nm at C = 1.00 x 10-6 M). The Xmax values shift to shorter wavelengths for both porphyrins with increasing concentration. Again, the isosbestic points indicate two species in equilibrium in both systems. The normalized spectra of (APh)T(SPh)P are shown in Figure 4.5 as an example. Negative deviations from Beer' law are obviously observed in the spectral data for both porphyrins. The Beer's law plots (A/b vs. C) for these porphyrins in the buffer solution are shown in Figure 4.6 181  Chapter 4 Aggreption of porphyrins  (not all the spectral data collected for the Beer's law plot are shown in Figure 4.5 for reasons of clarity in this figure). Negative deviations from Beer's law can be also observed for both porphyrins from the Beer's law plots, although the plot in Figure 4.6B might well be taken to be a straight line; the corresponding spectral data of Figure 4.5 more clearly show the deviation.  3.0  3  3{30^410  40  4K)-500nm  Figure 4.5. The normalized spectra of (APh)T(SPh)P at various concentrations in a phosphate buffer. The concentrations are 0.10 x le, 0.30 x 10'5, 0.50 x le, 1.0 x 10'5, 2.0 x 10'5, 3.0 x 10-5, 4.0 x 10'5, 6.0 x 10"5, 8.0 x 10"5, 10.0 x 10'5 M in sequence as the arrows show. 182  Chapter 4 Aggregation of porphyrins  A^  PyT(SPh)P (410.7 nm)  25  20  15  10  5  0 0  ^^ ^ ^ ^ ^ ^ ^ ^ 9 7 8 6 4 5 2 3 1 Concentration x 105 (M)  (APh)T(SPh)P (412.4 run) 20  15  5  0 0  ^ ^ ^ ^ ^^ 7 8 9 10 1 2^3^4^5^6 Concentration x 105 (M)  Figure 4.6. Beer's law deviation for PyT(SPh)P and (APh)T(SPh)P. 183  Chapter 4 Aggregation of porphyrins  The aggregation of PyT(SPh)P porphyrin in distilled water has also been examined. The maximum absorbance (410 nm) essentially obeys Beer's law (e = 4.3 x 105 M-1 cm-', at concentrations from 0.05 x 10-5 to 1.0 x 10-5 M), but the molar absorptivities in the 350-390 nm region increase with increasing concentration. No isosbestic point was observed. Again, the observed aggregation in the buffer for these two porphyrins might be a monomer-dimer processes, and the equilibrium constants for presumed dimerization are presented in Section 4.3.5.  4.3.1.3 trans-BPyB(SPh)P The normalized spectra in distilled water for this porphyrin (Figure 4.7) are much more complicated than those for Tet(SPh)P. There are two bands in the Soret region, one at 400-410 nm and the other at 442 nm. It also can been seen that there are probably two peaks overlapping to give the band at 400-410 nm. At low concentration (1.0 x 10-6 M), there appears to be a single peak at 409 nm in this region; when the concentration increases by 5 times to 5.0 x 10-6 M, a shoulder band at approximately 400 nm appears with this peak, while the molar absorptivity in this region decreases and that of the band at 442 nm increases; on increasing the concentration above 5.0.x 10-5 M, the molar absorptivities of the broad bands at 400 - 410 nm increase as the the shoulder peak becomes more intense, and that of the band at 442 nm now decreases. This behavior with varying concentration was quite reproducible. All these observations indicate that this porphyrin shows complicated aggregation processes under these conditions, although an isosbestic point is observed at 423 nm In buffer solutions, the aggregation behavior is completely different. Only one band is observed in the region of 400-410 nm in the normalized spectra(Figure 4.8). At low 184  Chapter 4 Aggregation of porphyrins  concentrations (<1 x 10-5 M) the major absorbance is at 410.0 mn, which is close to the Xmax  values of the tetralds- and tris-sulfonatoporphyrins, while at higher concentrations  the absorbance at 443.0 nm becomes the major one. There is a clean isosbestic point at 421.3 nm.  Figure 4.7. The normalized spectra of trans-BPyB(SPh)P at various concentrations in distilled water. The concentrations are a: 1.0 x 10'6, b: 5.0 x 10-6, c: 1.0 x 10-5, d: 5.0 x 10-5, e: 1.0 x 104 M.  185  Chapter 4 Aggregation of porphyrins  Figure 4.8. The normalized spectra of trans-BPyB(SPh)P at various concentrations in a phosphate buffer. The concentrations are 0.10 x 10-5, 0.30 x 10'5, 0.50 x 10'5, 1.0 x 10"5, 2.0 x 3.0 x 10'5, 4.0 x 10-5, 5.0 x 10-5, 6.0 x 10'5 M in sequence as the arrows show.  As discussed later in this Chapter (Section 4.3.4), it is likely that the aggregation process observed here in the buffer solution is a monomer-dimer equilibrium. The process observed in distilled water probably then involves the formation of dimer and timer; i.e., the aggregation process is less well defined than it is in buffer. 186  Chapter 4 Aggregation of porphyrins  4.3.1.4 cis-BPyB(SPh)P, cis-B(APh)B(SPh)P and trans-B(APh)B(SPh)P  The normalized spectra at various concentrations in a buffer solution of these three porphyrins show that they do not conform to a simple aggregation model in the range of concentrations studied. Figure 4.9 shows the normalized spectra of cis-BPyB(SPh)P; there would be two isosbestic points at 398 and 419 nm if the spectrum at 1.0 x 10-6 M were ignored. This probably means that above this concentration, one aggregation equilibrium dominates and, below this concentration, another equilibrium becomes significant. There appears to be two bands overlapping in the Soret region, the A, values being approximately 409 and 402 rim. At lower concentration (0.10-3.0 x 10-5 M), the 409 rim band dominates, while at higher concentration (4.0-8.0 x 10-5) the 402 rim band dominates. Figure 4.10 shows the normalized spectra of cis-B(APh)B(SPh)P. The data are more complicated than for cis-BPyB(SPh)P. From 1.0 x 10-6 to 1.0 x 10-5 M, the Xmax shifts from 410 to 418 rim, but when the concentration increases from 1.0 x 10-5, to 8.0 x 10-5 M, Xmax shifts back to shorter wavelengths from 418 to 412 rim. There would be two isosbestic points at 397 and 440 nm if the spectra of 1.0 x 10-6 M (a) and 2.0 x 10-6 M (b) were ignored. Again more than one aggregation equilibrium is involved for this porphyrin. The case of trans-B(APh)B(SPh)P is again complicated. There are at least two absorbance maxima in the Soret region as shown in Figure 4.11. The extinction coefficient at —415 rim decreases with increasing concentration from 0.50 x 10-5 to 1.0 x 10-5 M and then increases with increasing concentration from 1.0 x 10-5 to 4.0 x 10-5 M, before decreasing again from 4.0 x 10-5 to 5.0 x 10-5 M; the Amax for this peak also changes "back and forth". The molar absorptivity at 455 rim remains approximately unchanged at these concentrations. This figure again shows that the aggregation is a multiple-step 187  Chapter 4 Aggregation of porphyrins  process (see also Section 4.3.2). It should be noted that the molar absorptivities of this porphyrin at either of the absorbance maxima are only about 10% of that for the 410 nm maxium of Tet(SPh)P.  Figure 4.9. The normalized spectra of cis-BPyB(SPh)P at various concentrations in a phosphate buffer. The concentrations are 0.10 x le, 0.50 x 10-5, 1.0 x 10-5, 2.0 x 10-5, 3.0 x 10-5, 4.0 x 10'5, 5.0 x 10'5, 6.0 x 105, 7.0 x 10'5, 8.0 x 10-5 M in sequence as the arrows show.  188  0  350^380^410^440^ 500nm  Figure 4.10. The normalized spectra of cis-B(APh)B(SPh)P at various concentrations in a phosphate buffer. The concentrations are: a: 1.0 x 10-6 M; b: 5.0 x 10'6 M; the others: 1.0 x 10-5, 2.0 x 10-5, 3.0 x 10, 4.0 x 10, 5.0 x 6.0 x 10'5,7.0 x 10-5, 8.0 x 10 M in sequence as shown by the arrows.  189  Chapter 4 Aggregation of porphyrhn  Q50  0  350^390  430^470  510  Figure 4.11. The normalized spectra of trans-B(APh)B(SPh)P at various concentrations in a phosphate buffer. The concentrations for the spectra from the top to the bottom at X — 415 nm are: 4.0 x 10-5, 5.0 x le, 0.5 x 10'5, 2.0 x 10-5, and 1.0 x 10-5 M (text, p.187).  190  Chapter 4 Aggregation of porphyrins  4.3.1.5 Tet(MPy)P The tosylate salt of this porphyrin was reported by Pastemack et al. in 19728 to obey Beer's law and was considered to remain as a monomer in aqueous solution at concentrations of 1 x 10-6 to 6 x 10-5 M at 1 M ionic strength. Later on, it became generally accepted that this porphyrin did not aggregate in this concentration range.' In 1983, Kano et al. reported that aggregation of the tosylate salt of this porphyrin can be detected at low concentrations (1 x 10-8 - 2 x 10-7 M) by fluorescence spectroscopy.9 The existence of aggregated forms at 1 x 10-6 M was also shown in this report by using fluorescence spectra as a function of temperature and methanol content. Beer's law behavior was also reported in this paper for both the chloride and tosylate salts at concentrations between 10-5 - 10  "3  M. However, these findings were strongly disputed  subsequently by Pasternack et al. in 1985.17 Also in 1985, a report on the aggregation of the chloride salt of this porphyrin was published by Brookfield et a!. 18, in which deviation from Beer's law was observed at lower concentrations where A/b vs. C gave a curve; at concentrations of 5 x 10-6 to 7 x 10-5 M, A/b vs. C gave a straight line, but this straight line did not pass through the origin which is another criterion for Beer's law deviation. Again, aggregation was also observed using fluorescence spectroscopy.  18  In 1987, more  aggregation data were published by Kano et al.1° to support their earlier findings9 that the porphyrin tosylate aggregates at low concentrations. The dispute about the aggregation still continued in a paper published again by Pastemack's group in 1988.3 Recently, 1H NMR data have provided evidence for some interaction between the porphyrin and the tosylate counterion.19 1H NMR spectroscopy has been also employed in studies on the aggregation of the porphyrin chloride salt in D20, 10,17,20 and there is general agreement among these authors that aggregation is only observed by 1H NMR at concentrations above —0.01 M, although the data reported by different authors were quite different. The copper(II) complex of this porphyrin has also been used as a probe in order to comment 191  Chapter 4 Aggregation of porphyrins  on the aggregation of the free base, and the complex was found to aggregate at 0.5 M concentration and 0.2 M ionic strength by ESR.21 As shown in Figure 4.12A, marked changes are found in the spectra of the porphyrin chloride in distilled water. The Amax shifts to shorter wavelengths and the molar absorptivity increases with increasing concentration. These results simply indicate that the chloride salt of this porphyrin does not obey Beer's law in distilled water over the whole range of concentrations from 2.5 x 104 to 1.0 x 10-4 M, and the deviation from Beer's law is positive. 3.0  ,-... E c..) .., cb ■••••■ C.)  siZ  •••■ 1.  41t  0  350  400  ^i^  450  Figure 4.12A. The normalized spectra of Tet(MPy)P chloride at various concentrations in distilled water. The concentrations are: 2.5 x 10'7, 5.0 x le, 1.0 x 10'6, 2.0 x 10'6, 4.0 x 10-6, 6.0 x 106, 8.0 x 10'6, 2.5 x 10, 5.0 x 10'5, 1.0 x 104 M in sequence as the arrow shows.  192  Chapter 4 Aggregation of porphyrins  The plot of A/b vs. C at 422.0 nm is shown in Figure 4.12B, and the inset of Figure 4.12 B is an enlargement of the lower concentration region. 28 12 13  24  12 0.9  20  0.6 0.3  16  o 0  01  02  0.3  OA  0.5  OA  07  0.8  12  8  4  o 0^1^2^3^4^5  7^8^9^10  Concentration x 105 (M)  Figure 4.12B. Plot of A/b vs. concentration for Tet(MPy)P chloride in distilled water.  The results shown in Figure 4.12B agree with the report by Brookfield et al. on the Beer's law plot in similar conditions," but do not agree with the reports that this porphyrin obeys Beer's law by Pasternack et al. (tosylate salt, p = 1 M, C> 10-6 M)8 and by Kano et al. (chloride and tosylate salts C> 10-5 M).9 In conclusion, this porphyrin, at least as the chloride salt, aggregates in distilled water over the whole range of 193  Chapter 4 Aggregation of porphyrhis  concentrations examined; the presumed monomer-dimer equilibrium constant is presented in Section 4.3.5. Aggregation is also observed in a buffer solution (IA = 0.013 M). The buffer solution spectra of the Soret band region are shown in Figure 4.13. As in the distilled water system, the extinction coefficient of the Soret band increases with increasing concentration from 1.3 x 10-6 to 8.0 x 10-5 M. The Xmax first shifts to slightly longer wavelength, and then shifts to shorter wavelengths when the concentration increases from 2.5 x 10-6 to 8.0 x 10-5 M. This behavior appears more complicated than in distilled water.  Figure 4.13. The normalized spectra of Tet(MPy)P in a buffer solution at various concentrations. The concentrations are: 1.3 x 10'6, 2.5 x 10-6, 5.0 x 10-'5, 1.0 x 10-5, 2.0 x 10-5, 4.0 x le, 6.0 x 10-5, 8.0 x 10-5 M in sequence as the arrow shows. 194  ampter 4 Aggregadon of porphyrins  4.3.1.6 T(MPy)PhP  Figure 4.14 shows the normalized spectra of the chloride salt of this porphyrin at various concentrations in the buffer solution. There is a positive deviation from Beer's law for the Soret band of this porphyrin, from C = 1.0 x 10-6 to 4.0 x 10-5 M, while no more spectral changes are observed at C = 4.0, 6.0, 8.0 and 10 x 10-5 M. These data indicate that the aggregated form in the observed equilibrium dominates exclusively above concentrations of 4.0 x 10-5 M, and this aggregated form might just be the dimer.  350  ^  400  ^  450n  Figure 4.14. The normalized spectra of T(MPy)PhP at different concentrations in a phosphate buffer. The concentrations are: 0.10 x 10'5, 0.50 x 10-5, 1.0 x 10'5, 2.0 x 10'5, 4.0 x 10'5 M in sequence as shown by the arrow; the spectra at 6.0 x 10-5, 8.0 x 10-5 M overlap with the spectrum at 4.0 x 10'5 M.  A plot of A/b vs. concentration is shown in Figure 4.15, in which a straight line might be drawn approximately, although deviation from Beer's law is observed at the lowest concentrations (0.10 to 4.0 x 10-5 M) as evident in Figure 4.14. This indicates well that the plot of A/b vs. concentration sometimes is not as sensitive as the normalized 195  Chapter 4 Aggregation of porphyrins  spectra for detecting aggregation phenomena. Deviation from Beer's law is also observed more clearly in the normalized spectra (Figure 4.5) than in the plot of Ab vs. C (Figure 4.6B) in the case of (APh)T(SPh)P in the buffer solution. 24 20 16 -› 12 8 4 0 0^1^2^3^4^5^6  ^^ ^ ^ 9 10 8 7  Concentration x 105 (M)  Figure 4.15. A plot of A/b vs. concentration for T(MPy)PhP in a phosphate buffer. 4.3.1.7 T(MPy)(NPh)P and cis-B(MPy)DPhP These two porphyrins were also studied in the same range of concentrations in a buffer solution. The C values at Xmax for both increase with increase of concentration from 1.0 x 10-6 to 4.0 x 10-5 M, and then decrease with increase of concentration from 4.0 x 105 to 10.0 X 10-5 M. The itinwc at 1.0 x 10-5 M is at 419 nm for the mononitro compound  and at 420 nm for the cis- compound. The Xmax shifts "back and forth" at the lower concentrations (Figure 4.16B) but is constant at 420 nm at the higher concentrations (Figure 4.16A). Once again, the aggregation in this concentration range must be a multistage process. The normalized spectra of cis-B(MPy)DPhP are shown in Figure 4.16 in the two concentration regions, as an example of these two porphyrins. 196  Chapter 4 Aggregation of porphyrins  Figure 4.16. The normalized spectra of cis-B(MPy)DPhP at various concentrations in a buffer solution. The concentrations in sequences as indicated by the arrows are: 4.0 x 10-5, 6.0 x 10-5,8.0 x 10-5 10.0 x 10-5 M in A; and 0.10 x 10'5, 0.50 x 10-5, 1.0 x 10'5, 2.0 x 10'5, 4.0 x 10-5 M in B. 197  Chapter 4 Aggregation of porphyrins  4.3.2. Affects of methanol on aggregation of porphyrins.  As noted by others, some organic solvents can break down aggregation of porphyrins, and methanol, glycerol and acetone have been found to have a dramatic effect.9,21,22 The effects of adding methanol to the buffer solution of some porphyrins were studied in this thesis work. In Figure 4.17, the absorbance spectra of 1.0 x 10-5 M (APh)T(SPh)P in mixtures of the buffer solution and methanol are shown. 4.0  4  I  4  I 0  350  45  4 ^!  SOOnm  Figure 4.17. Absorbance spectra of (APh)T(SPh)P in aqueous buffer/methanol mixtures. The content of methanol: 0, 10, 20, 30, 50% (v/v) in sequence as indicated by the arrows.  198  Chapter 4 Aggregation of porphyrins  The Soret band increases significantly in intensity (by 44%) and shifts slightly to longer wavelengths (410.9 to 412.7 nm) when the methanol content increases from 0 to 50%. Isosbestic points are observed at 395, 401, 431 and 455 nm in Figure 4.17 (on expansion), and these isosbestic points may imply that the data result from a shifting equilibrium of two species. The spectra of 1.0 x 10-5 M Tet(SPh)P in the mixtures of buffer solution and methanol (0, 10, 20, 30, 40%, v/v) have characteristics similar to those of the (APh)T(SPh)P system. Isosbestic points are observed at 409 and 424 nm; the Soret band increases in intensity by 8% and A.„.x shifts to longer wavelengths (411.3 to 412.0 nm) when the content of methanol increases from 0 to 30%. The spectrum in 40% methanol is the same as in 30% methanol. Similar characteristic spectral changes also occur for PyT(SPh)P (at 1.0 x 10-5 M) in the mixtures of the buffer solution and methanol (0, 10, 20, 30%, v/v). Isosbestic points are observed at 405.5 and 425.5 nm; the Soret band increases in intensity by 32% and Xmax shifts to longer wavelength (410.1 to 411.3 nm) when the content of methanol increases from 0 to 30%. More marked spectral changes occur in the case of 1.0 x .10-5 M transBPyB(SPh)P when the content of methanol changes (Figure 4.18). In pure buffer solution, there are two peaks (at 410 and 443 nm) at about the same intensity (Figure 4.8, p.186). In a 10% methanol solution, the band at 410 nm becomes 2.2 times more intense than in pure buffer, while the intensity of the band at 455 nm decreases by 27%; when 30% methanol is present, the intensity of the band at 410 nm is 7 times that in the buffer, and the band at 455 nm disappears completely. There is an isosbestic point at 424 nm.  199  Chapter 4 Aggregation of porphyrnn  2.5  o.^  350  390  430  4^ 470  500nm  Figure 4.18. Absorbance spectra of trans-BPyB(SPh)P in aqueous buffer/methanol mixtures. The content of methanol. 0 10, 20, 30% (VAT) in sequence as indicated by the arrows. 200  Chapter 4 Aggregation of porphyrina  Spectral changes for trans-B(APh)B(SPh)P (at 1.0 x 10-5 M) under the same conditions are shown in Figure 4.19A. In the spectrum at 0% methanol, the major absorbance is at 455 iun, and there is a minor broad peak at about 415 mn (Figure 4.11, p.190). When methanol is present, the intensity of this minor peak (at 415 nm) increases dramatically and, in a mixture of 1:1 methanol and buffer solution, the intensity of this band is about 12 times higher than in neat buffer solution, while the other band (at 455 nm) disappears completely. There is no isosbestic point.  40  A  2.0-  ,  0  350  390  430  470  510  550nm  Figure 4.19A. Absorbance spectra of trans-B(APh)B(SPh)P in aqueous buffer/methanol mixtures. The content of methanol: 0, 10, 20, 30, 50% (VA') in sequence as indicated by the arrows.  No isosbestic points are observed in the spectra for cis-BPyB(SPh)P and cisB(APh)B(SPh)P in the mixtures of buffer and methanol (0-50%, v/v). The spectra of cis201  Chapter 4 Aggregation of porphytins  B(APh)B(SPh)P in the mixtures are shown in Figure 4.19B as an example. In the spectrum at 1.0 x 10-5 M at 0% methanol, the Soret band is unsynunetric which may indicate that there are two overlapping bands. When 10% methanol is present, the Xmax shifts to shorter wavelength (from 419 to 407 rim) to give a more symmetric band with a 25% increase in intensity. When the content of methanol increases from 10 to 50%, the X max shifts back to longer wavelengths (from 407 to 417 rim), and the intensity of the Soret band increases by additional 71%.  350  Figure 4.19B. Absorbance spectra of cis-B(APh)B(SPh)P in aqueous buffer/methanol mixtures. The content of methanol: 0, 10, 20, 30, 50% (V/V) in sequence as indicated by the arrows.  In the spectrum of cis-BPyB(SPh)P (1.0 x 10-5 M) at 0% methanol, the band at about 409 nm is slightly more intense than the band at about 402 rim (Section 4.3.1.4 and 202  Chapter 4 Aggregation of porphyrins  Figure 4.9, p.188). When the methanol content is increased, the band at 409 nm increases in intensity and shifts to longer wavelengths. In the spectrum in 50% methanol, the intensity of this band has increased by —100% and shifted to 411 tun, while the band at 402 nm cannot be observed.  4.3.3 1H NMR studies on aggregation of porphyrins 4.3.3.1 1H NMR studies on the aggregation of T(MPy)PhP The Ill NMR spectra of T(MPy)PhP in D20 at various concentrations are shown in Figure 4.20 (a-d), and the 1H NMR spectrum of this porphyrin in DMSO-d6, which was found to be concentration independent (0.002-0.02 M), is also shown in this figure (z). As listed in Table 2.14 (p.65) and discussed in Section 2.3.2, the signals in spectrum z are assigned as follows: the doublet at 9.50 ppm to the 5,10,15-(3,5-MPy) protons; the doublet at 9.03 ppm to the 5,10,15-(2,6-MPy) protons; the singlet at 9.2 ppm and the AB quartet at 9.2 ppm (part of the quartet is overlaps with the signal of the 3,5-(MPy) protons) to the pyrrole protons; the doublet at 8.23 ppm to the 20-(2,6-Ph) protons; and the multplet at 7.9 ppm to the 20-(3,4,5-Ph) protons. The methyl protons appear as a singlet at 4.71 ppm and the N-pyrrole protons appear as singlet at -3.04 ppm, and these peaks are not shown in this figure. In a spectrum in D20 (spectrum d for instance), each of the signals of the 5,10,15-(3,5-MPy), 5,10,15-(2,6-MPy) and 20-(3,4,5-Ph) protons is split into two signals; the pyrrole protons give rise to a broad signal at 8.9 ppm, and the signal of the methyl protons overlaps with the solvent peak. The assignments of the signals in spectrum a and d are listed in Table 4.1, and labeled in Figure 4.20. Results from two dimension NMR spectrum of this porphyrin at — 0.01 M in D20 show that all the signals assigned to the same substituent couple to each other, thus supporting the assignments.  203  Chapter 4 Aggregation of porphyries  CH I 1^'' OH^N-4" H3 L H6  ^H2 /y 0 \ X 0 H^H3 --NH^N---------^ 5 i,^H 0 Ni- CH3 N HN / 6 H 5 \^26-^ H^H2  0  + H5^H3  111111/111^111111411111111111111111  9.5^9.0^8.5^8.0^7.5^7.0 PPM^6.5  Figure 4.20. 1FI NMR spectra of T(MPy)PhP in D20 and DMSO-d6. a: C = 2 x 10-2, b: C = 8 x 10-3, c: C = 4 x 10-s, d: C = 2 x 10-3M in D20; z: in DMSO-d6. 204  Chapter 4 Aggregation of porphyrina  When the concentration increases, all the signals shift to higher fields, but to different degrees. Plots of chemical shifts against concentration are shown in Figure 4.21. The values of tics (changes of the chemical shifts of the signals) from spectrum a to spectrum d are listed in Table 4.1. Table 4.1. 1H Chemical shifts (ppm) of T(MPy)PhP at concentrations of 0.02 and 0.002 M  A 0 0 10-(3,5-MPy) 10-(2,6-MPy) 5,15(3,5-MPy)  9.14 9.07 a pattern d 0.07 Ars d  8.72 8.35 d 0.37  9.07 8.73 d 0.34  a  *  0  +  5,15(2,6-MPy)  2044-Ph)  20-(2,6-Ph) 2043,5-Ph)  8.57 7.71 s(broad) 0.86  7.76 7.53 t 0.23  7.83 6.78 d 1.05  7.64 7.15 t 0.49  The broad signal of the pyrrole protons, which can be observed in spectra c and d, perhaps becomes broader and less visible when the concentration increases. The broadness of the 1H NMR signal of the pyrrole protons, in the case of Tet(SPh)P in D20, has been attributed to slow tautomeric exchange of the central deuterium between the pyrrole nitrogen atoms.12 However, the observed concentration dependence of the pyrrole signal in Figure 4.20 may be some evidence that this broadness is, at least partially, related to aggregation of this porphyrin. From Figures 4.20 and 4.21, it can be seen that the splitting of the signals of the 10-(3,5-MPy) and 5,15-(3,5-MPy) protons becomes larger when the concentration increases; the extension of the two lines in Figure 4.21 corresponding to these two signals may reach the same chemical shift (-9.15 ppm) at a concentration of zero (when aggregation should be eliminated); this agrees with the fact that these two signals appear identical when aggregation is eliminated, as observed in the spectrum in DMSO-d6. The same observation can be made for the signals of the 5,10,15-(2,6-MPy) protons which may give a common resonance at -8.80 ppm at C = 0, and the 20-(3,4,5-Ph) protons which may give a common resonance at -7.80 ppm at C = 0. The chemical shifts at 205  Chapter 4 Aggregation of porphyrins  concentration of 0 obtained by extending the lines in Figure 4.21 are not the same as the chemical shifts observed in the spectrum in DMSO-d6; this difference presumably results from differences in the interactions in the porphyrin monomer with the solvents. 9.5  6.5  o  ^  0.004^0.008^0.012^0.016  ^  0.02^0.024  concentration (M) —A— 1042,6—0-- 5,1543,5- —X— 5,15-(2,61043,5MPy) MPy) MPy) MPy) —X— 20-(4-Ph) —0— 20-(2,6Ph) --I— 20-(3,5-Ph)  Figure 4.21. Correlation between the 1H chemical shifts and the concentration of T(MPy)PhP. Aggregation can be observed by comparing spectrum d (0.002 M in D20) and spectrum z (in DMSO-d6) in Figure 4.20. This observation agrees with the deviation from Beer's law at much lower concentrations (10-6 - 8 x 10-4 M in buffer solution) observed by UV-visible spectra (Section 4.3.1.5). A structural model for this porphyrin can be suggested based on the changes of the chemical shifts of the signals from spectrum d (at 0.002 M) to spectrum a (at 0.02 M). The aggregation is pictured as starting from formation of a dimer in which one porphyrin sits on top of the other with a 180° rotation, and with one of the porphyrin planes  206  Chapter 4 Aggregation of porphyrins  displaced along the axis connecting carbons 10 and 20 (Figure 4.22A). Higher aggregation occurs in a similar fashion as shown in Figure 4.22B, in which a trimer is given as an example. Because aggregation (possible dimerization) is observed at 10-6 to 10-4 M, at least in a buffer solution (Section 4.3.1.5), the aggregation observed here is probably oligomerization. This model can explain well the data of Figure 4.20 obtained from NMR studies. In this model, the pyridinium rings are able to rotate freely, as indicated by the NMR spectra where only one signal is observed for the 5,15-(2,6-MPy) protons (this should be a doublet, but is observed as a broad singlet) and only one doublet is observed for 5,15(3,5-MPy) protons. The phenyl groups have to rotate to be parallel to the porphyrin rings in order to have the strongest  7C-7C  interaction (the distance for a strong  7E-7C  interaction is  —4 A1,23). The 20-(2,6-Ph) protons, in comparison to the other protons of the same phenyl ring, are closest to the centers of the adjacent porphyrin rings, and this makes their signal shift the most as the concentration increases, (Table 4.1, Figure 4.21); the 20-(3,5-Ph) protons have the second shortest distance from the centers of the adjacent porphyrin rings, and their signal shifts the second most. The signals of the rest of protons also shift accordingly to their distances from the adjacent porphyrin rings; for example, the signal of 10-(3,5-MPy) protons is little shifted at the various concentrations, as the protons are quite far from either the phenyl or the porphyrin rings on adjacent porphyrin molecules. A structural model has been suggested by Kano et atm for the dimerization of Tet(MPy)P, in which one porphyrin sits on top of the other with a 45 degree rotation, and all the methylpyridinium groups are identical. This model is not applicable to T(MPy)PhP at this concentration because of the difference between the 10-MPy and 5,15-M-Py groups observed in the 1H NMR spectra. The aggregation model for Tet(MPy)P is further discussed in Section 4.3.4. 207  Chapter 4 Aggregation of porphyrina  A  B \ 41■11.11W^  /4  (^  \ ^  (^ /  Figure 4.22. Aggregation models for T(MPy)PhP in water. A: a dimer; B: a trimer. (It seems plausible that the chloride anion could be situated between the charged pyridinium centers.) 208  Chapter 4 Aggregation of porphyrins  4.3.3.2 1H NMR studies on the aggregation of PyT(SPh)P Figure 4.23 shows the spectra for PyT(SPh)P in 1320 at different concentrations (a4) and a spectrum of this porphyrin in DMSO-d6 (y), which is concentration independent. As discussed in Section 2.3.2 and listed in Table 2.11, the signals in spectrum (y) have been assigned and are listed again in Table 4.2 for comparison purposes. The signals in spectra b (at 0.002 M) and f (at 0.02 M) can be assigned as shown in Table 4.2 (these two concentrations are chosen because they are the same as those for spectra d and a in Figure 4.20 and this makes comparison of the changes in the two cases easier). Different from the assignments for a spectrum of this porphyrin in DMSO-d6 in which the 2,6-SPh protons resonate at lower field than the 3,5-SPh protons, the signals at the higher field (at 7.22 and 7.43 ppm in spectrum b) are assigned to the 2,6-SPh protons. This assignment has been proven by partial deuteration of the 2,6-SPh protons within Tet(SPh)P for spectra measured at similar concentrations.12 The pyrrole protons give a multiplet at 8.87 ppm in spectrum y; these protons give a very broad signal at 8.6 ppm in spectrum a, and this shifts to higher field and broadens as the concentration increases. The signal of the pyrrole protons becomes too broad to be seen in spectra e and f. In spectrum f, most of the signals are broad. Table 4.2. Chemical shifts in spectra of PyT(SPh)P.  5-(3,5-Py)  0  A  0  5-(2,6-Py)  10,20-(3,5-SPh)  0  a  10,20-(2,6-SP1i) 15-(3,5-  + 15-(2,6-  SPh)  SPh)  Y  9.03  8.27  8.04  8.28  8.04  8.28  b f pattern  8.72  7.50  7.85  7.22  7.96  7.43  7.92  6.55  7.33  6.55  7.75  7.2  d-+s(br)  s-)s(br)  d--)s(br)  s(br)-->s(br)  d  d->s(br)  ACS  0.80  0.95  0.52  0.67  0.21  0.23  209  Chapter 4 Aggregation of porphyrios  H5  o°x  S03-  1^r1^1^1^1^r^i^II^III^IIIIII^11,11111!IIIIII  9•0^8.5^8.0^7.5^7.0^6.5^6.0  PPM  Figure 4.23. 1H NMR spectra of PyT(SPh)P. a: C = 1.0 x 10-3, b: C = 2.0 x 10-3, c: C = 4.0 x 10-3,d: C = 8.0 x 10-3 e: C = 1.0 x 10-2, f: C = 2.0 x 10-2 M in D20; y: in DMSO-d6.  210  Chapter 4 Aggregation of porphyrins  The differences of the chemical shifts of the signals (Acs) in spectra b and f are also listed in Table 4.2. The chemical shifts of the signals are plotted vs. concentration in Figure 4.24. 9  6  o^0.004^0.008^0.012^0.016 concentration (M) P5-(3,5-Py)  ^  0.02^0.024  L 5-(2,6-Py)^010,20-(3,5-SPh)  010,20-(2,6-SPh) X15-(3,5-SPh)^+15-(2,6-SPh)  Figure 4.24 Correlation between the 1H chemical shifts and the concentration of T(SPh)PyP.  Similar to the spectral changes in Figure 4.20, all the signals shift to higher fields when the concentration increases, and a similar aggregation model (Figure 4.25) can be suggested for this porphyrin in these conditions.  211  Chapter 4 Aggregation of porphyrins  A  5 ^5 5 5  — /^ -^  \  12(3^ \  _/^  ^  _ " -- r-  -  /  /\----} Oil^t i^  i^12  -  (3- -\  0  Figure 4.25. Suggested aggregation model for PyT(SPh)P.  A: a dimer; B: a tetramer with a perspective view of the 5,15-substituents; C: a tetramer with a perspective view of the 10,20-substituents. (Nat ions could presumably be situated between the charged sulfonato centers) 212  Chapter 4 Aggregation of porphyrina  Somewhat different from the cases for T(MPy)PhP, the extensions of the lines (of Figure 4.24) for the 10,20-(3,5-SPh) protons and for the 15-(3,5-SPh) protons do not converge at zero concentration, and neither do the lines for the 2,6-SPh protons. This perhaps indicates that the aggregation of this porphyrin at the noted concentrations is beyond the monomer-dimer process, and the trends for the changes in the chemical shifts with concentration are not easily rationalized in terms of a monomer-dimer equilibrium. Another important observation is that in Table 4.1, the Acs values (from 0.002 M to 0.02 M) for the protons on the same substituent show more significant differences than the related values of A" (from 0.002 M to 0.02 M) in Table 4.2. For example, the value for the 10-(2,6-MPy) protons (0.37 ppm) and the 10-(3,5-MPy) protons (0.07 ppm) in Table 4.1 are significantly different compared to the corresponding values in Table 4.2 for the 15-(2,6-SPh) protons (0.23 ppm) and the 15-(3,5-SPh) protons (0.21 ppm). This may indicate that the side displacement in the model for this sulfonatoporphyrin is smaller than in the model for the pyridiniumporphyrin (Figure 4.22), and that the 15-(2,6-SPh) protons are also significantly shielded by the ring current of the pyridyl ring of the adjacent porphyrins. Thus, the distance between two adjacent SPh groups might be close enough to shift upfield all the SPh group signals, which also means that the 10,20-SPh groups might be unable to rotate freely. This suggestion is supported by the broadness of the signals for the 10,20-SPh protons (Figure 4.23), and is also plausible because the SPh rings would have more 1C-7C interaction than the MPy rings which are positively charged because of the delocalization of the charge from the nitrogen atoms. Figure 4.25A shows a dimer model; Figure 4.25B shows the plausible interaction of the pyridyls with the porphyrin rings and the ability of 15-SPh to rotate; and Figure 4.25C shows the potential interactions between the 10 and 20-SPh groups.  213  Chapter 4 Aggregation of porphyrins  4.3.4 Aggregation models  Several aggregation models have been suggested for metalloporphyrins in nonaqueous solution,2,6,24 and for natural porphyrins in aqueous solution,' but discussion on models for synthetic, water-soluble porphyrins is rare in the literature, though the aggregation of symmetric, synthetic porphyrin free-bases including Tet(SPh)P,11.12 Tet(mpy)p,2,10,9,1 8 Tet(CPh)P (5,10,15,20-tetracarboxyphenylporphyrin),8 and Tet(tAPh)P [ 5, 10,15,20-tetra(4-trimethylammoniumphenyl)porphyrin]  15  has been  intensively studied. 4.3.4.1 Monomer dimerization or oligomerization  The monomer-dimer equilibrium model was first proposed for the aggregation for a natural porphyrin because isosbestic points were observed in the normalized spectra in dilution experiments. However, as noted by White' in 1978 in a review about aggregation of porphyrins and metalloporphyrins: "it is conceivable that the isosbestic points may be fortuitous, or that the two species in solution might not be the monomer and the dimer, but in the absence of contrary evidence, the best explanation is a monomer-dimer equilibrium." Since then, there have been more reports on the aggregation of porphyrins, but no more direct evidence either for or against this monomer-dimer model has been reported, and thus this monomer-dimer equilibrium has been used as a general aggregation model for all porphyrin free-bases. In this work, there is evidence against the monomer-dimer process as a general model because of the complexity of the normalized spectra in the dilution experiments for some porphyrins including: cis-BPyB(SPh)P (Figure 4.9, p.188), cis-B(APh)B(SPh)P (Figure 4.10, p.189), trans-B(APh)B(SPh)P (Figure 4.11, p.190), T(MPy)(NPh)P and cis-  214  Chapter 4 Aggregation of porphyrina  B(MPy)DPhP (Section 4.3.1.7, p.196; Figure 4.16, p.197). The complexity of these spectra just cannot be explained using a monomer-dimer model. Evidence has also been found to support the monomer-dimer model for certain porphyrins: Tet(SPh)P, (APh)T(SPh)P, PyT(SPh)P and trans-BPyB(SPh)P. For these porphyrins, isosbestic points have been observed in the normalized spectra in both dilution experiments in a buffer solution and in the methanol-effect experiments. The existence of the isosbestic points in the spectra in the methanol experiments for Tet(SPh)P, for example, shows that in this system just two species are most probably in equilibrium. When 30% methanol is present, it can be assumed that just one species, the monomer, is present because organic solvents are known to break down aggregation quite efficiently.9 This assumption is also supported by the fact that there is no more spectral change when the methanol content increases from 30% to 40%. Thus, the two species in equilibrium at 0% methanol should be monomer and dimer. The spectrum at 0% methanol also corresponds to one in the figure of the normalized spectra in the buffer solution (Figure 4.3, p.190), in which isosbestic points are also present, and the two species in equilibrium at different concentrations should again be monomer and dimer. This discussion can be extended to the porphyrins (APh)T(SPh), PyT(SPh)P and trans-BPyB(SPh)P. For the porphyrins Tet(MPy)P and T(MPy)PhP, no solid evidence has been found to either support or oppose the monomer-dimer model in the buffer solution, although the processes observed by UV-visible are likely to be a monomer-dimer process; this is because the aggregation is 'limited' in both cases, i.e., the points in the Beer's law diagram at higher concentrations (>2 x 10-5 M) tend to give a straight line plot (Figures 4.12B and 4.15). It has been clearly shown in this work that model and degrees of aggregation are affected strongly by the nature of the substituents, the number of charged substituents and 215  Chapter 4 Aggregation of porphyrins  the position of the substituents. Thus, the aggregation model can be quite different from one porphyrin to another. 4.3.4.2 Structural models 4.3.4.2.1 A model for the tetra-ionic porphyrins A "face-to-face" dimer with an angle of 45° between corresponding porphyrin axes has been suggested by Kano et al.10 according to NMR and fluorescence spectral data. A figure of this model is presented in Figure 4.26. A model for higher aggregation at higher concentrations (> 0.01 M according to Kano et al.) is also suggested in this figure. A similar model (Figure 4.26) can be suggested for the aggregation of Tet(SPh)P, and NMR datau in the literature support such a model for this porphyrin: (1) the proton NMR signals of the 2,6-SPh protons and the 3,5-SPh protons of shift to higher field when the concentration increases, and the former shifts more than the latter; (2) there is only one doublet for the 2,6-SPh protons and one doublet for the 3,5-SPh protons, which implies that the planes of the sulfonatophenyl groups are either parallel to the porphyrin ring or are freely rotating, and in the "face-to-face" model the sulfonatophenyl groups should be able to rotate freely. This model is optimized for other factors, such as charge distribution and steric hindrance. 4.3.4.2.2 Models for the tri-ionic porphyrins For T(MPy)PhP and PyT(SPh)P, a "slide-over" model (Figures 4.22 and 4.25) has been suggested in this thesis work according to 1H NMR data (Section 4.3.3). Different from the the "face-to-face" model, this "slide-over" model takes advantage of the existence of a hydrophobic phenyl group, which can form a relatively strong interaction  216  Chapter 4 Aggregation of porphyrins  A  ,  +_  /:)-----\ Figure 4.26. Aggregation models for Tet(MPy)P. A: a model for a dimer; B: a model for a trimer.  217  Chapter 4 Aggregation of porphyrina  with an adjacent porphyrin ring(s), and this interaction could facilitate the rotation of this ring to make it parallel to the adjacent porphyrin ring(s). There are no obvious factors that prevent the dimer from further aggregation. Shifts of the 1H NMR signals of porphyrins with variation of concentration have been reported for porphyrins in organic solvents, and similar "slide-over" models have also been suggested.2,5 This "slide-over" model may be appropriate for the aggregation of other tri-ionic porphyrins, including T('MPy)(NPh)P and (APh)T(SPh)P (this work), and PhT(SPh)P.8 There is similarity among these porphyrins in that they both possess three charged mesosubstituents and one lipophylic meso-substituent.  4.3.4.2.3 Models for bis-ionic porphyrins  UV-visible studies on the aggregation of the bis-ionic porphyrins show that the compounds [cis-BPyB(SPh)P, cis- and trans-B(APh)B(SPh)P and cis-B(MPy)DPIill, except trans-BPyB(SPh)P, are involved in higher aggregation processes than the monomer-dimer equilibrium in the range of concentrations studied. Although in the buffer solution, the behavior of trans-BPyB(SPh)P may conform to a simple monomer-dimer aggregation, in distilled water this porphyrin may also participate in higher aggregation. It can be concluded generally that these porphyrins tend to aggregate more than the tetra- or tri-ionic porphyrins because they have more lipophilic substituents. Unfortunately, 1H NMR spectra in D20 at room temperature of these porphyrins are uninformative because only less defined broad peaks can be observed, which again probably result from oligomerization processes. Several plausible aggregation models can be suggested for these porphyrins, and in solution one porphyrin could exist as a mixture of some of these forms. Among these possibilities, a model for trans-B(MPy)DPhP, based on the "slide218  Chapter 4 Aggregation of porphyrho  over" model for T(MPy)PhP (Figure 4.22), is presented in Figure 4.27; as shown, the formation of a trimer, tetramer or higher aggregated forms can be achieved without steric hindrance. The UV-visible and NMR spectra suggest that a mixture of the forms may be present in an aqueous solution, and this makes further characterization of this porphyrin in aqueous solution very difficult. 4.3.5 The equilibrium constants for dimerization  If dimerization of monomers is the only process observed in the concentration ranges used for solutions of some of the porphyrins, including Tet(SPh)P (Figure 4.3), Py(SPh)P, (APh)T(SPh)P (Figure 4.5) and trans-BPyB(SPh)P (Figure 4.8) in a buffer solution, and Tet(MPy)P in distilled water (Figure 4.12A), the following equations can be written for analysis of the UV-visible spectral data: CT = 2 CD + Cm^  4.1  2M^D^K = CD / Cm2^4.2 4.3  A = emCm + eDCD^ Where CT = the total concentration of the porphyrin as monomer, CD = the concentration of the dimer present at equilibrium,  Cm = the concentration of monomerpresent at equilibrium, K = the equilibrium constant for the dimerization, em = molar absorptivity of the monomer, CD = molar absorptivity of the dimer,  A = the total absorbance at a total concentration of  CT,  when the path length b = 1.00 cm. An equation can be derived as:8 Am -A = [(2em - eD)(4KCT +1 -1/1+ 8KC )]/8K, where Am= Cm X CT. Rearrangement gives: 219  Chapter 4 Aggregation of porphyrina  a  MI■111.--MINMEON■1■1  C}■•■■•■■-■■•■  411■1•1111M--M■■•■•■■---(11)  a4■■■■■■F-III■  MONIN11■0111111■1■11  0---•■••■••■!---■ ■1•11M-.■111■11■INIMMEN■-■0  0■01■1■Ml■  MMENIMMINHINIIMINI■•■••1=11.0  0--11.1•■■-•  ■1■---•■•■11  Figure 4.27. Aggregation models for trans-B(MPy)DPhP. a: a model for a dimer; b: possibilities for a tetramer. 220  Chapter 4 Aggregation of porphyrhu  A -A (4C T -2& 8 m^)K + 1 = 1.11 +8KC T _ 6 M D  Putting (4C -8 T  A -A  2e m^ ^e ) B, this equation becomes B2K + 2B 8CT =0, -  —  M D  2B 8C and = K T B2 -  4.4  Thus, once £m and ED values are chosen, then the K value, and the A values at each concentration can be calculated using eqn. 4.4; and the calculated curve can be compared to the experimental curve for a best-fit. For Tet(SPh)P, the molar absorptivity in distilled water at low concentrations 4 x 10-6 M) at 410 nm (inset in Figure 4.2A) was used as  Em for the calculation of K in  the buffer solution and ED was varied a best-fit curve (data from Figure 4.3, p.180). The resultant curve with the experimental data and the Beer's law straight line for the assumed monomer are shown in Figure 4.28A. The K and  ED  values obtained in this way, together  with the ones obtained by other authors, are listed in Table 4.3. For PyT(SPh)P, the Cm value was obtained from the molar absorptivity in distilled water at 410 nm at concentrations from 5 x 10-7  to 1.0 X 10-5  M, where Beer's law was  found to be obeyed (Section 4.3.1.2, p.181). Such data presented in Figure 4.6A (p.183) are analyzed. For (APh)T(SPh)P (data of Figure 4.5, p.182) and trcms-BPyB(SPh)P (data of Figure 4.8, p.186), the em values were first estimated according to the molar absorptivity of these porphyrins in mixtures of the buffer solution and methanol, and then optimized for best-fit curves. For Tet(MPy)P in water, both  Em and eD were varied, and a  satisfactory best-fit curve resulted; however, the calculated value at 10 x 10-5 M was 3% lower than the experiement data, this might result from the error of this method or possibly from further aggregation beyond dimerization at this concentration. The best-fit curves are shown in Figures 4.28A and 2.28B. The K values, the Em and ED values are listed in Table 4.3. 221  Chapter 4 Aggregation of porphyrins  Table 4.3. Equilibrium K values for dimerization of porphyrins.  K (M-1)  Conditions  Tet(SPh)P  1.5 x 104  Tet(SPh)P Tet(SPh)P  9.6 x 104 2.2 x 104  Tet(SPh)P  1.5 x 104  Tet(SPh)P  1.6 x 104  PyT(SPh)P  8.9 x 104  (APh)T(SPh)P  1.0 x 105  PhT(SPh)P  4.28 x 104  pH= 7.04 0.010 M phosphate buffer pH = 7, 0.05 M NaNO3 pH = 6.4 0.100 M KNO1 pH = 7.0 0.028 M phosphate buffer + 0.050 M KNO3 pH = 9.0 0.100 Na(0Ac) buffer pH = 7.04 0.010 M phosphate buffer pH = 7.04 0.010 M phosphate buffer pH = 7.5, 0.1 M KNO3  trans BPyB(SPh)P Tet(MPy)P  4.9 x 107  -  7.3 x 105  pH = 7.04 0.010 M phosphate buffer distilled water  &ma, epa X (nm) 5.0, 1.8 411 4.91, 4.2 412 5.1, 3.9 412 4.7, 4.0 412 4.2, 1.8 411 3.0, 2.9 412 4.00, 2.46 413 2.6, 0.49 410 0.50, 5.0 422  Ref. present work temp.-jump" UV-visible12 UV-visible12 UV-visible12 present work present work UV-visible8 present work present work  a Em and ED in units of 105 M-1 cm-1. The K value for Tet(SPh)P obtained here is the same as that obtained under similar conditions (pH = 7.0, 11 = 0.1 M) using the same technique.12 However, this value is about 6 times lower than the value obtained by a temperature-jump technique." This difference may result from a difference in experimental conditions or most likely from the low accuracy of the temperature-jump teclmique.8 The em value for this porphyrin obtained here is basically the same as the reported ones,12 but the ED value is only about half of the reported value.12 Again this difference might result from the slightly different experimental conditions or from the errors in the treatments. From a comparison of the values obtained under the same conditions for different porphyrins in the present work, the K values are in the order of Tet(SPh)P < PyT(SPh)P  5. (APh)T(SPh)P << trans-BPyB(SPh)P. This order 222  Chapter 4 Aggregation of porphyrina  Tet(SPh)P at 411.3 24 20 — 16 —  fi. 12 — 8 4  _ 0  0 Experimental ^ Calculated —I^ Bee rs law for the monomer  ^^  1  2^3^4  ^ ^ 5 6^7  Concentration x 105 (M) PyT(SPh)P at 412.4 nm 25  20 —  15 —  1 10 — 0 Experimental ^ Calculated —I— Beer's law for the monomer I 0  1  ^  I^I^I^I 2^3^4  ^  I  ^^ I I  5^6  7^8^9  Concentration x 105 (M)  Figure 4.28A. Best-fit curves for the UV-visible spectral data of Tet(SPh)P and PyT(SPh)P in an aqueous buffer.  223  augger 4 Aggregation of porphyrins  (APh)T(SPh)P in a phosphate buffer (412.4 nm) 20  15  • • •  --t-Z 10  • • •  5  0^Experimental  •  -- -1-  ,  0  !a  2^3^4^5^6  —  Calculated Beer's law tor the monomer I^i  ^ ^ ^ ^ 7 9 8 10  Concentration x 105 (M) Tet(MPy)P in distilled water at 422.0 nm  0^1^2^3^4^5^6  ^  7^8^9^10  Concentration x 105 (M) Figure 4.28B. Best-fit curves for the UV-visible spectral data of (APh)T(SPh)P in an aqueous buffer and Tet(MPy)P in distilled water. 224  Chapter 4 Aggregadon of porphyrins  corresponds inversely with the number of charges, i.e., the less charged structure gives the higher degree of aggregation (dimerization) at equivalent conditions. The K values for (APh)T(SPh)P and PyT(SPh)P are close, both porphyrins being tri-anionic, and the values are also similar to that reported for another tri-anionic porphyrin, PhT(SPh)P.8 The K values obtained here are most probably the equilibrium constants for the monomer-dimer process, because evidence has been found in this work to support the monomer-dimer model under the conditions used (Section 4.3.4.1, p.214) for the porphyrins listed above, except Tet(MPy)P; a monomer-dimer equilibrium also seems likely, however, for Tet(MPy)P, as judged by the literature.9.10,18 4.3.6 Summary  Aggregation is a general phenomenon for porphyrin free-bases in aqueous solutions, and is found for all the water-soluble porphyrins studied in this thesis work. The degree of aggregation is probably related to many structural factors, including the ionic charge (a tetra-ionic porphyrin probably aggregates in a fashion different from that a triionic porphyrin--compare the "face-to-face" model in Figure 4.26 vs. the "slide-over" model in Figures 4.22 and 4.25); the distribution of charge (trans-and cis-BPyB(SPh)P appear to aggregate differently under the same conditions--see Figures 4.8 and 4.9); and the nature of the substituents (a difference is observed between T(MPy)(NPh)P and T(MPy)PhP--see Sections 4.3.1.6 and 4.3.1.7). Although some structural models related to the porphyrin structures have been suggested in this thesis work, the findings present a preliminary attempt to study a correlation between structure and aggregation models; more evidence is necessary to support the models suggested in this chapter. However, once aggregation proceeds beyond dimerization, species at various stages of oligomerization may exist simultaneously in solution making such studies difficult.  225  Chapter 4 Aggregation of porphyrins  References-Chapter 4  1^W.I. White, in "The Porphyrins", D. Dolphin, ed., Academic Press, New York, 1978, Vol. 5, p.303. 2^R.J. Abraham and K.M. Smith, ./. Am. Chem. Soc., 105, 5734 (1983). 3^E.J. Gibbs, I. Tinoco, Jr., M.F. Maestre, P.A. Ellinas and R.F. Pastemack, Biochem. Biophys. Res. Commun., 157, 350 (1988).  4^J.J. Katz, L.L. Shipman, T.M. Cotton and T.R. Janson, in "The Porphyrins" D. Dolphin, ed., Academic Press, New York, 1978, Vol 5, p.402. M.R. Waselewski, U.H. Smith, B.T. Cope and J.J Katz, J. Am. Chem. Soc., 99, 4173 (1977). S.G. Boxer and G.L. Gloss, J. Am, Chem. Soc., 98, 5406 (1976). 5^P. Leighton, J.A. Cowan, R.J. Abraham and J.K.M. Sanders, J. Org. Chem., 53, 733 (1988). 6^R.J. Abraham, F. Eivazi, H. Pearson and K. M. Smith, J. C. S. Chem. Commun., 1976, 698. R.J. Abraham, F. Eivazi, H. Pearson and K. M. Smith, J. C. S. Chem. Commun., 1976, 699. 7^C.A. Hunter and J.K.M. Sanders, J. Am. Chem. Soc., 112, 5525 (1990). 8^R.F. Pastemack, P.R. Huber, P. Boyd, G. Engasser, L. Francesconi, E. Gibbs, P. Fasella, G.C. Venturo and L. deC. Hinds, J. Am. Chem. Soc., 94, 4511 (1972). 9^K. Kano, T. Miyake, K. Uomoto, T. Sato, T. Ogawa and S. Hashimoto, Chem. Letters, 1983, 1867 10^K. Kano, T. Nakajima, M. Takei and S. Hashimoto, Bull. Chem. Soc. Jpn., 60, 1281 (1987). 11^M. Krishnamurthy, J.R. Sutter and P. Hambright, J. Chem. Soc. Chem. Commun., 1975, 13. 12^A. Corsini and 0. Herrmann, Talanta, 33, 335 (1986). 13^K.M. Kadish, G.B. Maiya, C. Araullo and R. Guilard, Inorg. Chem., 28, 2725 (1989). A. Salehi, A. Shirazi and T.C. Bruice, Inorg. Chim. Acta, 194, 119 (1992).  226  Chapter 4 Aggregation of porphyrina  14  ^  E.B. Fleisher, J.M. Palmer, T.S. Srivastava and A. Chatterjee, J. Am. Chem. Soc., 93, 3162 (1971).  15^M. Krishnamurthy and J.R. Sutter, Inorg. Chem., 24. 1943 (1985). 16^W.M Gallagher and W.B. Elliott, Ann. N. Y. Acad. Sci., 206, 463 (1973). R.F. Pastemack, L Francesconi, D. Raff and E. Spiro, Inorg. Chem. 12, 2606 (1973) W.I. White and R.A. Plane, Bioinorg. Chem., 4, 21 (1974). 17  ^  R.F. Pastemack, E.J. Gibbs, A. Gaudemer, A. Antebi, S. Bassner, L. De Poy, D.H, Turner, A. Williams, F. Laplace, M. H. Lansard, C. Merienne and M. Perree-Fauvet, J. Am. Chem. Soc., 107, 8179 (1985).  18^R.L. Brookfield, H. Ellul and A. Harriman, J. Photochem. 31, 97 (1985). 19^K.M. Kadish, B.G. Maiya and C. Araullo-McAdams, J. Phys. Chem., 95, 427 (1991). 20^N. Foster, J. Magn. Reson. 56, 140 (1984). 21^G. Doughty and R. F. Pastemack, Inorg. Chim. Acta, 195, 95 (1992). 22^K.E. Kellar and N. Foster, Inorg. Chem., 31, 1353 (1992). 23^L.R. Milgrom, S. Bone, D.W. Bruce and M.P. Macdonald, J. Mol. Electronics, 7, 95 (1991). 24^RV. Snyder and G.N. La Mar, J. Am. Chem. Soc., 99, 7178 (1977).  227  Chapter 5 In vitro activities  Chapter 5 In vitro studies of selected synthetic porphyrins and metalloporphyrins 5.1 Introduction Some in vitro studies on the radiosensitizer activities of synthetic porphyrins and metalloporphyrins have appeared in the literature' (Chapter 1). Preliminary investigations on the in vitro activities of some of the porphyrins and metalloporphyrins synthesized in this thesis work are presented in this chapter. Toxicity is an essential property of a drug. Drugs targeting the tumor hypoxic cells (which may be resistant to conventional drugs) may be evaluated by measuring the toxicity of the drugs in oxic vs. hypoxic conditions. The toxicities of most of the synthesized water-soluble compounds in both oxic and hypoxic conditions are presented in this chapter. As discussed in Chapter 1, one of the goals of this project was to design porphyrins as radiosensitizers. With the assay routinely used to assess radiosensitization abilities of drugs, low radiosensitization efficiencies were found in this thesis work in mammalian cells for porphyrins and metalloporphyrins, including those metalloporphyrins which have been reported earlier in the literature to have high efficiencies." The degree of lipophilicity of the porphyrins synthesized in this thesis work was varied by changing the number of ionic groups and probably by the symmetry (cis-isomer vs. the trans-isomer). The partition coefficients between 1-octanol and water of some porphyrins were determined and are presented in Appendix B. The effects of charges on the porphyrin free-bases on cell accumulation were also tested.  228  Chapter 5 In vitro activities  A few of the porphyrin free-bases were also tested for their photosensitization properties. Table 5.1 shows the assays and the compounds tested. The selections of the compounds for each assay are discussed in the later sections of this chapter. Partition coefficients of the porphyrin free-bases are presented in Appendix B. Due to time limitation, the biological investigations have only been carried out to a limited extent. Other relevant assays, which explore the DNA-binding properties and reduction potentials, were not carried out. Investigations were not extended to the compounds with low watersolubilities, such as trcms-B(MPy)DPhP, trans-B(MPy)B(NPh)P, and cis- and transB(APh)B(SPh)P. Some of the results were consistent with the original ideas of designing the chemical structures, some were not. Further investigations are essential in order to establish possible correlations between chemical structures and the biological activities. 5.2 Materials and methods 5.2.1 Cell growth, maintenance and treatment The cells used in the toxicity and radiosensitization assays were obtained from a CHO (Chinese hamster ovary) cell line. The cells were routinely grown in a spinner culture flask in a+/+ medium, and the cell solutions were maintained at 37°C in an "Incu-cover" incubator (Associated Biomedic Systems) under an atmosphere of 95% air and 5% CO2 (Canadian Liquid Air Co.). The cell cultures were diluted daily to a cell concentration of about 1 x 105 cells/mL to maintain an exponential growth (doubling time was approximately 13 h). Drug accumulation and photosensitization assays were performed using cells from an HT-29 (human-cancer tumor) cell line which was maintained as monolayers in  229  Chapter 5 In vitro activities  Table 5.1. In vitro tests and the tested compounds  code  compound^a  toxici ty  radiosen.^.^. sttization x x x  x x x x x x x x x x x x x x  15  Tet(MPy)P T(MPy)PhP T(MPy)(NPh)P cis-B(MPy)DPhP cis-B(MPy)B(NPh)P Tet(SPh)P PyT(SPh)P (APh)T(SPh)P cis-BPyB(SPh)P cis-(NPh)PyB(SPh)P trans-BPyB(SPh)P Co[Tet(MPy)P] Co[T(MPy)PhP] Co[T(MPy)(NPh)P] Co [cis-B(MPy)B(NPh)P]  16  Co[Tet(SPh)P]  x  x  17  Co[PyT(SPh)P]  x  x  18  Co[(APh)T(SPh)P]  x  x  19  Co[cis-(NPh)PyB(SPh)P]  20  Cu[Tet(MPy)P]  x  21  Cu[T(MPy)PhP]  x  22  Cu[T(MPy)(NPh)P]  x  23  Cu[Tet(SPh)P] Cu[PyT(SPh)P]  x x  1 2 3 4 5 6  7 8 9 10 11 12 13 14  24  x x x x x  cell uptake x x x x x x x x x x  photosensitization x x x  x x x x  x x x x  x x  Cu[(APh)T(SPh)P] x 25 a^The counterion for the cationic porphyrins and metalloporphyrins is C1-;  The counterion for the anionic porphyrins and metalloporphyrins is Nat. The oxidation state of the cobalt in complexes 12 - 19 is "+3" with water as the axial ligands in aqueous solutions. The oxidation state of copper in complexes 20 - 25 is "+2". Assays carried out. 230  Chapter 5 In vitro activities  RPMI+/+ medium at 37°C under an atmosphere of 95% air and 5% CO2 in an incubator (National Inc.). The cultures were trypsinized (0.1% trypsin solution) twice a week (doubling time was approximately 24 h), and typically 1 x 105 and 0.5 x 105 cells were plated in 25 cm2 tissue culture flasks (Falcon) for a cell culture and for a backup, respectively. The cell samples in dilution tubes taken from treatments (drug incubation, X-ray irradiation, or light irradiation) were vortexed, and then centrifuged (7 min); the cells were then resuspended in 8 mL of medium (a-/- for CHO cells or RPM1-I+ for HT-29 cells) through vortex mixing and counted for cell concentration. Aliquots of these cell solutions were plated into 5 cm Petri dishes prepared previously (filled with 5 mL a+/+ medium for CHO cells or RPM1+/+ for HT-29 cells, and kept in a tray incubator for 24 h). The number of cells plated into the Petri dishes was such as to ensure 100-200 colonies per dish. Two parallel dishes were plated with the same number of cells from the same sample. The dishes were incubated in a tray incubator for 7 days (CHO cells) or 13 days (HT-29 cells), and the colonies were then stained using a methylene blue solution and counted. At least two or three parallel experiments were performed. The plating efficiency (PE) and surviving fraction (SF) were calculated as: number of colonies PE — ^ , for all the treatments; number of cells plated PE (at time t) SF —^, for toxicity experiments; and PE (at zero time) PE (at dose D) SF —^, for radiosensitization or photosensitization experiments. PE (at zero dose) A centrifuge (Sorvall RC-3) was operated at 600 RPM at 4°C for separating cells from solutions (usually 7-9 min). The cell concentration (cells/mL) was determined using a "Coulter Cell Counter" (Coulter Electronic Inc.). Tray incubators (National Inc.) operated at 37°C with a 95% air and 5% CO2 gas flow. Nitrogen used to produce hypoxic 231  chapter 5 In vitro activities  conditions was oxygen-free grade (Linde Specialty Gas, Union Carbide). Plastic Petri dishes and tissue culture flasks (Falcon, Becton Dickinson and Co.) were used for all the experiments. The preparations of the solutions, including PBS, media (a+/-, a-/-, a+/+, RPMI-/+, and RPMI+/+), trypsin, and methylene blue, are described in Appendix A. A 10% (v/v) ScintiGest solution was made from ScintiGest (a tissue solubilizer, Fisher Scientific Co.) and doubly distilled water. 5.2.2 Drugs and drug solutions The tested compounds (see Table 5.1) were synthesized and purified as described in Chapters 2 and 3. Stock solutions of the tested compounds at 1.0 mIvl were made by dissolving the compounds in double distilled water (warming was occasionally required to make these solutions). Solutions used for testing of the compounds (conventionally called drug solutions in biological laboratories) were made from dilution of the stock solutions using the appropriate medium, and the solutions were sterilized by filtering using 10 mL disposable syringes and Nalgene 0.22 p.m filter units. 5.2.3 Toxicity in oxic and hypoxic conditions The toxicities of the compounds were measured by incubating CHO cells with the drug solutions for various time intervals under 1 atmosphere of either air or nitrogen, as reported in the literature.2 Approximately 2 x 106 cells were harvested from a spinner culture for each of several vessels (Figure 5.1) using a centrifuge. The cells were resuspended by vortexing in 1 tnL a+/- medium per vessel. Drug solutions of about 20 mL (at a concentration of 105 ii.M) for each vessel were made with a+/- medium, then filtered. An aliquot of 9 tnL of each drug solution was added into each of two vessels (for oxic and hypoxic conditions, respectively). Medium (a+/-, 9 mL) was added to each of the 232  Chapter 51n vitro activities  control vessels (for oxic and hypoxic conditions respectively). The vessels were maintained at 37.4°C in a Labline Instruments "Orbit Shaker Bath", which was in a warm room at 37°C. The hypoxic and oxic conditions were created by flowing humidified nitrogen and air, respectively, through the vessels for 1 h. Then the resuspended cell solution (1 rnL per vessel) was added, the concentration of the drugs becoming 100 JAM at this time by the dilution of the cell solution. A small amount of the solution was left in the pipette to avoid introducing air into hypoxic vessels. Samples of 1 mL were taken from each vessel at incubation times of 0, 1, 2, and 3 h. The samples then were treated as described in Section 5.2.1.  Gas Inlet  ^  Gas Outlet  Glass Tube for Sampling  Cell Suspension  Figure 5.1. The vessel for toxicity assay.  233  Chapter 5 In vitro activities  Toxicity experiments for HT-29 cells were performed with subconfluent monolayers of cells. Cells (0.5 x 106) were seeded in 5 cm Petri dishes two days before experiments, and the dishes were incubated in a tray incubator. On the day of experiment, the drug solutions (10 inL) at 100 gM were made from the drug stock solutions using RPM14+ medium. The cell subconfluent monolayers in the Petri dishes were rinsed with PBS; and then each of the drug solutions was added to two Petri dishes (5 mL drug solution to each Petri dish). The Petri dishes were then immediately covered with aluminum foil to avoid receiving light. One of the two Petri dishes with the same drug solution was incubated in a tray incubator for 1 h, and the other for 2 h. After incubation, under the minimum laboratory light, the drug solutions were poured off; the cells were rinsed with PBS, trypsinized and added into dilution tubes which contained 10 mL PBS solution; approximately 2 x 105 cells were taken from each dilution tube and added to another dilution tube containing 10 inL RPM14+ medium and; the cells in the medium solutions were treated as described in Section 5.2.1. 5.2.4 Radiosensitization in hypoxic conditions  A published procedure was followed for the radiosensitization assays.2,3 Irradiation was performed using an X-ray source (Philips, 250 kv, 0.5 mm Cu), using the set-up shown in Figure 5.2. Glass "duck vessels"3 with magnetic stir bars were used. Radiation doses of the set-up were measured using a Precision Electrometer (Victoreen, Model 500). Drug solutions of about 17 niL for each drug were made by dissolving the compounds (drugs) in a medium solution (a+/- or a-/-, depending on the design of the experiment), at a concentration of C'. These solutions were filtered for sterilization, and 14.5 inL of each solution were added to each of the duck vessels. For the control vessel, 14.5 inL medium solution (a+/- or a-/-) were added instead of the drug solution. The 234  Chapter 5 In vitro activities  vessels were then immersed into a water bath at 37.4°C with magnetic stir bars. Nitrogen gas was blown through the arms of the vessels for 1 h to produce hypoxic conditions. Approximately 4 x 106 cells, harvested from a spinner cell culture using a centrifuge and resuspended in 0.5 mL medium (a+/- or a-/-, depending on the design of the experiment), were added into each vessel (a small amount of solution was left in the pipette to avoid 14.5 introducing air into the vessel). The concentration of the drugs became C (i.e., 150 C') at this time resulting from the dilution of the cell solution. The cells were incubated in the water-bath (37.4°C) with the drug solutions (or medium only for the control) for 1 h, then chilled with ice-water for at least 5 min before irradiation. The vessel to be irradiated was immersed in 1 L ice-water in a plastic container which was placed on top of the head of the X-ray source, and the stir bar was activated with a motor from above. Samples of 0.5 1.5 mL (more cells were needed for higher doses) were taken out through the gas-outlet arm of the duck vessel at doses of 0, 5.0, 10.0, 15.0, 20.0, and 25.0 Gy into dilution tubes containing 9 mL a+/- medium; the dose rate was typically —5 Gy/min. These samples were then treated as described in Section 5.2.1. Gicitlet \\\  Stir motor Cell suspension  Gas inlet /  ' \\  Ice water  Magnetic stir bar  X—ray beam Figure 5.2. Set up for the radiosensitization assay.  235  Chapter 5 In vitro activities  The survival curves were obtained by plotting log SF vs. dose.4 The effect of oxygen in CHO cells is shown in Figure 5.3 as a example of radiosensitization (oxygen being a natural sensitizer, see Chapter 1). The sensitization enhancement ratio (SER) was calculated at 1% survival (SF = 0.01), which is commonly chosen for comparison.4 Dose without drug SER —^(at 1% survival) Dose with drug  1  CHO CELLS  - \^4c1) \^N.A.  _  Y+.  T  :  02  0^5^10 15 20 25 30 DOSE (Gray) Figure 5.3. A representative example of survival curves (02 effect, SER - 3). (adapted from ref. 4) 236  Chapter 5 In vitro activfties  5.2.5 Cell accumulation Cell accumulation was determined using a fluorometric assay.5 Cells (1 x 106 HT29) were seeded into 10 cm Petri dishes with 15 mL RPMI+/+ medium two days before the experiments, and the dishes were incubated in a tray incubator. The drug solutions (17 mL) at 100 plvl were made from each of the drug stock solutions using RPM14+ medium on the day of the experiment. The Petri dishes with subconfluent monolayers were rinsed with PBS solution three times to remove traces of serum in the medium for cell growth to avoid interactions between the drugs and serum; then 5 inL of a drug solution were added into each of three Petri dishes. The dishes were wrapped with aluminum foil immediately after the drug solutions were added, and then incubated in a tray incubator for 1 h. Thereafter, the drug solutions were poured off, and the Petri dishes were rinsed with PBS solution three times. The cells were then harvested using a rubber policeman instead of trypsinization in order to avoid any possible interactions between trypsin solution and the drugs, and resuspended into 10 mL of PBS solution by vortexing (for another wash) in dilution tubes. Cells in a Petri dish without the drug treatment were harvested into a dilution tube containing 10 mL of PBS solution as a control. Cells in two control Petri dishes (without the drug treatment) were also harvested in the same way and counted; and the average of the cells in the control dishes was used as the number of cells. per Petri dish. Cells in the PBS solution were centrifuged, and the PBS solution was poured off as carefully and as completely as possible. ScintiGest solution (10%, 5.00 mL) was added to each of the dilution tubes, which were then wrapped with aluminum foil to prevent possible photo-destruction of the porphyrin drugs. The solutions were kept at 37°C (in a warm room) for at least 24 h to digest the cells. Standard solutions at 0.10 - 5 tM, made by diluting 100 11M drug solution using 10% ScintiGest, were also wrapped with aluminum foil and kept at 37°C for the same time as the unknown samples. Some samples were found to be too concentrated for the measurements, and were diluted by 10-50 237  Chapter 5 In vitro activities  times, and the diluted samples were again wrapped with aluminum foil and kept at 37°C for at least 12 h to assure reproducible results. The concentrations of the drugs in the digested solutions were measured by fluorescence using a Farrent Optical System 3 Scanning Spectrometer at the maxima for excitation (scanned from 380-500 nm) and emission (scanned from 600 -700 nm); the wavelengths are listed in Table 5.2 for each of the compounds tested. Results were calibrated by standard solutions of each drug. At least two or three experiments were performed for each test. Table 5.2. Fluorescence maxima for excitation and emission (a)  Compound  Xexcitation (nm)  1  445  2  emission (nm)  Cornpound  Xexcitation (nm)  Xemission (nm)  635  8  417  647  435  637  9  413  641  3  445  645  10  416  645  6  415  642  11  413  642 •  7  413  642  a: See Table 5.1 for identification of the compounds; compounds 4 and 5 are not listed in this table because irreproducible data were collected (see Section 5.3.3).  5.2.6 Photosensitization  These experiments were carried out following an established procedure6 with minor changes. HT-29 cells (1 x 106) were seeded into tissue culture flasks two days before experiments. Drug solutions (20 mL) at 50 pdv1 were prepared by diluting the stock solutions (1.0 mM in distilled water) with RPM14+ medium on the day of experiments, 238  Chapter 5 In vitro activities  and filtering. The flasks with cells were rinsed with sterilized PBS solution three times to remove the serum, and then a drug solution (15 mL) was added to each of two flasks. The flasks were wrapped with aluminum foil immediately after addition of the drug solutions, and incubated in a tray incubator for 1 h. Then drug solutions were poured oft the flasks rinsed with 0.1% trypsin solution, and cells were trypsinized (6 min) and counted. 2 x 106 cells were taken and washed with 10 mL PBS solution, resuspended in 1 mL cold PBS solution, and chilled in ice. A tunable light source (Photo Technology International Inc., Model 500) was adjusted to the wavelength (±10 nm) required for the experiments. A Petri dish was placed on ice which was covered with a black cloth. To this Petri dish, 120 pl. of cell solution were delivered and irradiated to a certain dose; 100 ilL solution was recovered and added to a dilution tube containing 9.9 nth of RPMI-/+ medium. The doses used were typically 0, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0 J/cm2 (maximum exposure being --1 minute) and a new Petri dish was used for each sample. The samples in the dilution tubes were treated as described in Section 5.2.1. 5.3 Results and discussion  5.3.1 Toxicity of porphyrins and metalloporphyrins Toxicities of 6 of the porphyrin free-bases (1-3 and 6-8) and 12 metalloporphyrins (12-14, 16-18 and 20-25) were determined in both hypoxic and oxic conditions at 100 1AM after 1 to 3 h incubation (37°C) using CHO cells. The toxicities, under oxic conditions, of compounds 4, 5, 9, 10 and 11 toward HT-29 cells in the absence of light at incubation times of 1 and 2 h were also tested. Generally, under conventional experimental conditions in which laboratory light had not been avoided, porphyrin free-bases tested were found to be non-toxic to CHO 239  Chapter 5 In vitro activities  cells; however, non-reproducible results were observed for compounds 2 and 3. Photodynamic toxicity induced by light at certain wavelengths might explain the cell killing in the experiments where these two compound were found to be toxic, although none of the porphyrin free-bases was found to be photo-dynamically toxic toward HT-29 cells with red light (630 ± 10 tun, Section 5.3.4). The control data in photo-dynamic toxicity experiments also show that the porphyrin free-bases (including compounds 2 and 3) are not toxic to HT-29 cells under mdc conditions for 1 h incubation without light. The porphyrin free-bases (4, 5, 9, 10 and 11) were also found to be non-toxic to HT-29 cells without light under oxic conditions. The copper complexes (20-25) and cobalt complexes of the anionic porphyrins (16-19) were found to be non-toxic at 1001AM toward CHO cells under both hypoxic and co& conditions, even after 3 h incubation. However, the cobalt complexes of the cationic porphyrins (12-14) were found to be slightly toxic after incubation periods of 2 and 3 h under hypoxic conditions and non-toxic under mdc conditions; the toxicities at 100 1.11v1 were found to be similar (e.g., the plating efficiency was about 0.4 after 2 h incubation, and about 0.3 after 3 h incubation). A representative diagram showing the toxicity results for compound 12 is presented in Figure 5.4. Compounds 1, 6 and 16 have been reported as non-toxic to V79N cells, while compound 12 has been reported to be slightly toxic to these cells (50% survival after 1 h incubation) under oxic conditions.' The results found in this thesis work essentially agree with these reported data. That the cobalt(III) complexes of the cationic porphyrins are more toxic under hypoxic than under oxic conditions toward CHO cells may be related to the expected ease of reduction of the complexes compared to the other compounds tested, because of the  240  Chapter 5 In vitro activilles  relatively high oxidation state of the metal and the positive charges on the porphyrin ligands.  0  Control Air  —X— Control Nitrogen —0-- Comp.12 Air --+— Comp.12 Nitrogen  0.1  0  ^^ i  2^ 3  Time ( h )  Figure 5.4. Toxicities of compound 12 (100 pM) under hypoxic and oxic conditions.  Although some selective toxicities of cobalt complexes of cationic porphyrins toward hypoxic cells have been detected, these compounds are unlikely candidates as bioreductive drugs because the toxicities are relatively low at these concentrations. In spite of this, the results show that modification of the chemical structures has some biological effect. 241  Chapter 5 In vitro acdthies  The toxicities of compounds 15 and 19 were not tested because the materials were only synthesized and purified at a late stage of this thesis work. These compounds at 100 jz.M were not toxic in CHO cells after 1 h incubation under hypoxic conditions in radiosensitization experiments (Section 5.3.2). 5.3.2 Radiosensitization under hypoxic conditions  The cobalt complexes (12-19), one copper complex (20) and the nitro-porphyrin free-bases (3, 5, 10) were tested as potential radiosensitizers, because the introduction of reducible metal ions and/or nitro group(s) is thought to contrabute to radiosensitization (Chapter 1). A few other porphyrin free-bases (1-2, 6-8) were also tested for comparison. Different media were used because the effect of serum in a medium (a+/-) was realized (see discussion below) at a late stage of this thesis work. Generally, porphyrin free-bases are either weak radiation protectors or show no effect at all; and metalloporphyrins are weak sensitizers (see Table 5.2). All of the metalloporphyrins are slightly more effective in a medium without serum (a-/-) than in a medium with serum (a+/-). Serum has been reported to reduce accumulation of Photofrine (hematoporphyrin derivatives) in cells significantly,7 and thus may have similar effects on the cell accumulation of the porphyrins concerned in this work and therefore affect the radiosensitization abilities of these drugs. Porphyrin free-bases containing nitro-groups (3, 5, 10) were found to be slightly radio-protective, contrary to expectation (Chapter 1). For the cobalt porphyrin complexes (12-19), the most effective radiosensitizer (in a-/- medium) was compound 15 (SER =  1.22), which contains two nitro-groups and two methylpyridinium groups, and this might provide relatively high lipophilic and electron affinic properties (Chapter 1). Compound 20, a copper complex, showed a radiosensitization efficiency similar to that of the cobalt complexes (12 - 19).  242  Chapter 5 In vitro acdvitlea  Table 5.3. Radiation enhancement ratio for selected porphyrins and metalloporphyrins Porphyrin free-bases (100 mM) Code 1 2 3 5  SER (a+/-) 0.98  6 7 8  1.04 1.00 0.96  10  ,  Metalloporphyrins (100 InM)  SER (a-/-)!  Code 12  1.00 0.92 0.92  13 14 15 16 17 18 19 20  0.94  SER (a+/-) 1.15 1.0511 1.08  —  SER (a-/-) 1.19 1.11 1.09 1.22  1.08 1.08 1.07 1.08  1.08 1.13 1.13 1.14  a: Exposure to light was carefully avoided in these experiments. b: Concentration =25 JAM (limited by solubility).  Compounds 12, 16 and 20 have been reported to be effective radiosensitizers (SER = 2.4, 2.3, 1.8, respectively) in Chinese hamster fibroblast (V79N) cells.' This current thesis project had been actually encouraged by these literature results. However, the results obtained with the noted compounds are quite different from those reported.' The differences may result from differences in experimental conditions. In the literature report, a different cell line was used; cell monolayers instead of cell suspensions were irradiated; and Hanks' balanced salt solution (MSS) (Gibco) instead of medium solutions was used for the drug solutions. It is possible that comparable results would be obtained using the same experimental conditions as reported. Of note, the characterization of complexes 12, 16 and 20 in the literature report' was non-existent. Figures 5.5 and 5.6 demonstrate the two kinds of behaviors: compound 18 is a weak radiosensitizer, while compound 3 is a weak radioprotector.  243  Chapter 51n vitro activities  0.1  0.01  —0— Control —0— Compound 18 I  0.001  o  ^  5  ^  10^15  ^  20  ^  25  Dose (Gr)  Figure 5.5. Surviving curves for radiosensitization by compound 18, a weak radiosensitizer.  (hypoxic conditions in a-/- medium, SER = 1.13)  244  Chapter 5 In vitro activities  0.1  0.01  0.001  — C>— Control —0-- Compound 3  0.0001 ^ 0  5  ^  10^15^20^25 Dose (Cr)  Figure 5.6. Surviving curves for effect of compound 3 with radiation, a weak radioprotector.  (hypoxic conditions in a-/- medium, SER = 0.92)  245  Chapter 5 In vitro act/vides  5.3.3 Accumulation of porphyrin free-bases in HT-29 cells The accumulation of the porphyrin free-bases in HT-29 cells was measured, and the results are shown in Figure 5.7.  2.5  0 1^2^3^6^7^8^9^10^11  Compounds Figure 5.7. Results of drug accumulation in HT-29 cells. Compounds 1 3 are cationic porphyrins, while the rest are anionic porphyrins. It -  can be seen by comparing the accumulation values of compound 1 to those of compounds 2 and 3, that the accumulation values change dramatically when the total number of charges on the porphyrins changes from 4 to 3. The accumulation of compounds 4 and 5 (total charge is 2) in the cells was also studied; the values varied for repeat experiments and were in the range of 12-25 mmol/million-cells (i.e., 5 - 10 times the values for 2 or 3). 246  Chapter 5 In vitro activities  The variations in the uptake values for 4 and 5 probably result from the aggregation properties of these porphyrins (Chapter 4). Aggregation of porphyrins affects the size of the species in the solution and therefore may well affect the accumulation in cells, and possibly the spectral measurements for the concentration. The aggregation could depend on experimental conditions, particularly on the concentrations of the cells and the particles from cell digestion which could vary in the experiments. For the anionic porphyrins, the accumulation value also increases when the number of charges per porphyrin is reduced, as in going from compound 6 (-4) to 7 (-3) and 8 (3), and further to 9 11 (-2). The existence of other groups, such as the nitro-group(s) (3 -  and 10), the amine group (8), and the pyridyl group(s) (7 and 9 11) seems to have little -  effect on drug accumulation in cells. "Charge geometry" [cis (9) vs. trans (11)], shows a large effect, which may be related to the different modes of aggregation of the porphyrins (Chapter 4), or to a difference in the mode of the interaction of the porphyrins with cell membranes. For porphyrins having the same number of charges (1 vs. 6; 2 and 3 vs. 7 and 8), the cationic porphyrins have much higher accumulation values than the anionic porphyrins. 5.3.4 Photosensitization of porphyrin free-bases Synthetic porphyrins, including Tet(SPh)P and tetrakis(3-hydroxyphenyI)porphyrin, have been reported8,9 to have some photosensitizer properties superior to those of the clinically used drug Photofin II® (see Chapter 1). The quantum yield of singlet oxygenl° and the DNA damage" produced by light and some other synthetic porphyrins have also been studied with respect to PDT. An in vitro assay using human tumor cells6 was used to test the photosensitization of some porphyrin free-bases synthesized in this thesis work.  247  Chapter 5 In vitro activities  Among the porphyrins tested [1(0.03 and 0.05 mM), 2(0.03 mM), 3(0.03 and 0.05 mM), 7(0.05 mM), 9(0.05 mM), and 11(0.03 mM)] using red light (X = 630 ±10 nm), only 9 shows some photosensitization (Figure 5.8). Although 1 has been reported to have high efficiency for producing singlet oxygen in a chemical assay," this porphyrin shows no effect in the cellular assay under the conditions used. I  0  0  .2  0  I  tc 0.1 a .t t 0 rn *MI  0.01  o  ^  i^ ^ ^ 0.5^1 1.5 2  Dose (J/cm2)  Figure 5.8. Photosensitization using 630 nm radiation by compounds 1 (0.03 mM) 0; and 9 (0.05 mM) 0.  The results presented here are very preliminary and not particularly exciting; nevertheless, further investigations on these porphyrins including variations of the light wavelength, the concentration of drugs, and the cell lines should be carried out for complete evaluation of the compounds as photosensitizers. The other porphyrin free bases, besides the ones tested here, are also of interest as possible photosensitizers.  248  Chapter 5 In vtro Activities  References-Chapter 5  1  ^  J.A. O'Hara, E.B. Douple, M.J. Abrams, D.J. Picker, C.M. Giandomenico and J.F. Vollano,  Int. J. Radiat. OncoL Biol. Phys., 16, 1049 (1989). 2 3  ^  4 5 6  ^  ^  ^ ^  B.A. Moore, B. Palcic and L.D. Skarsgard, Radix. Res., 67, 459 (1976).  L. Parker, L.D. Skarsgard and P.T. Emmerson, Radiat. Res., 38, 493 (1969)  B. Palcic, J. Brosing and L.D. Skarsgard, Br. J. Cancer, 46, 980 (1982).  M. Korbelik and J. Hung, Photochem. PhotobioL, 53, 501 (1991).  M. Korbelik, G Krosl, H. Adomat and K.A.Skov, Photochem. PhotobioL, 55 Suppl., 54S (1992).  7 8  ^  ^  M. Korbelik, Photochem. Photobiol., 56, 391 (1992).  M.D. Berenbaum, S.L. Akande, R. Bonnett, H. Kaur, S. Ioannou, R.D. White and U.J. Winfield, Br. J. Cancer, 54, 717 (1986).  9  ^  J.W. Winkelman, in "Methods in Porphyrin Photosensitization", D. Kessel, ed., Plenum Press, New York, 1985, p.91.  10  ^  S.J. Milder, L. Ding, G. Etemad-Moghadam, B. Meunier and N. Paillous, J. Chem. Soc., Chem. Commun., 1990, 1131.  J.B. Verlhac and A. Gaudemer, Now.'. J. Chim., 8, 401 (1984). 11  ^  B.R. Munson and R.J. Fiel, Nucl. Acids Res., 20, 1315 (1992).  R.J. Fiel, N. Datta-Gupta, E.H. Mark and J.C. Howard, Cancer Res., 41 3543 (1981).  249  Chapter 6 Conelink= and future work  Chapter 6 Conclusions and suggestions for future work The purpose of this project was to design, synthesize and test new porphyrin compounds as potential radiosensitizers. Other possible applications of the new porphyrins to cancer treatment were also of interest. In conclusion: (1) modification of porphyrins as a methodology in porphyrin synthesis has been developed, (2) aggregation of porphyrin free-bases has been studied and the understanding of this aspect of porphyrins has been advanced, (3) some chemistry of metalloporphyrins, especially of cobalt porphyrin, has been studied in depth, (4) and some in vitro investigations have been initiated. 6.1 Synthesis of porphyrins  Although examples of modifications of synthetic porphyrins can be found in the literature,' it is the first time that a wide range of porphyrins has been synthesized using the method of pyrrole condensation and subsequent modification. In this thesis work, more than 40 porphyrins have been synthesized, and most of them have been fully characterized. Among these porphyrins, 22 are new compounds, and 15. are soluble in water. A novel class of porphyrins with three different meso substituents has been synthesized. The 1H NMR spectra of the pyrrole protons of the porphyrin with lower symmetry are particularly of interest, and some work has been devoted to the assignments of the signals in these spectra. Aggregation of the water-soluble porphyrins was subsequently encountered in the studies of both the porphyrin free-bases and metalloporphyrins, and studied. It can be generally concluded that all of these porphyrin free-bases aggregate to a certain degree in 250  Chapter 6 Coneintions and future work  aqueous solutions. Aggregation models are suggested, and a novel "slide-over" model is suggested for tris-ionic porphyrins; some equilibrium constants for the monomer.-..-.--dimer process were evaluated. 6.2 Metallation of porphyrins  The synthesis and characterization of two known cobalt pozphyrins, two known copper porphyrins, seven new cobalt potphyrins and four new copper porphyrins were carried out. Although the two known cobalt porphyrins [CoTet(MPy)P and CoTet(SPh)P] had been used prior to this study, some questions remained regarding their synthesis and characterization, because of the complexity of the systems resulting from air oxidation and aggregation. A reproducible synthesis for Co(fl) or Co(ll) porphyrin complexes has been developed here, and the mechanisms involved in this synthesis reaction have been explored to some degree. Some properties of the cobalt complexes in aqueous solutions, for example the pKa values of the diaquocobalt(1.11) porphyrins, have been studied. 6.3 In vitro studies  The porphyrin free-bases and the metalloporphyrins are non-toxic to CHO cells at concentrations of 100 IIM or 25 gM. The accumulation of the porphyrins in HT-29 cells is related to the porphyrin structure. Generally, the fewer charges on molecules, the greater the accumulation. For example, the accumulation of the T(MPy)PhP porphyrins with three plus charges is about 10 times higher than that of a porphyrin [Tet(M13y)P] with four similar charges. For porphyrins with the same number of charges, the positively charged porphyrins accumulate to a greater extent than the negatively charged porphyrins. For example, the uptake of T(MPy)PhP (2, +3 charge) is about 23 times higher than the uptake of PyT(SPh)P (7, -3 charge). 251  Chapter 6 Conch:dons and future work  Although some of the porphyrins showed moderate effects as radiosensitizers and/or photosensitizers, none of these encouraged further assessment in vivo. These findings are contrary to a literature report2 in which different experimental conditions were used. The introduction of cobalt(11) and nitro groups does improve the radiosensitizing abilities of the porphyrins, but this effect is smaller than anticipated. The results of the in  vitro activities of the porphyrin compounds are very preliminary, and further assessment required to permit any conclusions regarding the potential of these porphyrin compounds as anticancer drugs. 6.4 Suggestions for Future Work 6.4.1 Synthesis of porphyrins  Using the method developed in this work, many porphyrins of interest can be synthesized. For example, porphyrins with nitro-imidazole moieties may have higher radiosensitizing ability. An example of the structure of such a porphyrin is shown below, which may be synthesized by either a condensation of 4-pyridinecarboxaldehyde, 4(5)nitro-(2-imidazolecarboxaldehyde) and pyrrole with subsequent methylation (and  M = 2H or Co(III), etc.  252  Chapter 6 Conclutions and future work  metallation), or a condensation of 4-pyridinecarboxaldehyde, 2-imidazolecarboxaldehyde and pyrrole with subsequent nitration, methylation (and metallation).  6.4.2 Other metallations Analogues of cisplatin have been of interest as second or third generation drugs for many years.3 Taking advantage of the reported tumor-accumulation ability of the porphyrins, tumor selectivity of the drug might be realized by a synthesized porphyrin analogue of cisplatin. A structural example of the suggested compounds is drawn below (A). The peripheral coordination of the porphyrins to other metal complex moieties (for example Ru(NH3)5) is also of interest for formation of potential anti-cancer drugs; an example is shown below (B). The lipophilicities of these compounds can be varied by chaffing the number of charged groups, or changing the coordinating peripheral group, [e.g., the amine group in (APh)T(SPh)P, or the pyridyl in PyT(SPh)P]; further, more than one "active" center may be introduced using porphyrins with more peripheral coordinating groups (e.g., cis- or trans-BPyB(SPh)P). SO 3-  H2N  ^  SO 3"  \ CI PtH 2N  N  S03N  SO 3-  ^  M = 2H or Co(III), etc.^  SO 3"  M = 2H or Co(III), etc.  A  253  Chapter 6 Conc.Inflow and future work  Other metalloporphyrins (with metal ions in the center of the porphyrin structure) may also be of interest, including those of Pt(II and IV), Ru(II and III) and Zn(II). 6.4.3 Biological studies  Further biological studies should be carried on the newly synthesized, porphyrin compounds. Of immediate interest would be to study the tumor accumulation properties of the compounds. If these porphyrin compounds have very good tumor selectivities, then they have several potential applications in oncology: (a) as targeting agents, (b) as drugs in chemotherapy (for example, like the one suggested for the cisplatin analogue), (c) as drugs in boron neutron capture therapy (BNCT)4 or (d) as contrast agents in magnetic resonance imaging (MRI).5  254  Chapter 6 Conelndona and future work  References - Chapter 6  1  ^  For example: W.J. Kruper, Jr., T.A. Chamberlin and M. Kochanny, J. Org. Chem., 54, 2753 (1989).  L. Ding, C. Casas, G. Etemad-Moghadam and B. Meunier, New J. Chem., 14, 421 (1990).  J.A. O'Hara, E.B. Douple, M.J. Abrams, D.J. Picker, C.M. Giandomenico and J.F. Vollano, Int. J. Radiat Oncol. Biol. Phys., 16, 1049 (1989) For example: "Platinum and Other Metal Coordination Compounds in Cancer Chemotherapy", M. Nicolini, ed., Martinus Nijhoff, Boston, 1988. K.A. Skov and N.P. Farrell, Int. J. Radiat Biol., 57, 947 (1990). J.S. Hill, S.B. Kahl, A.H. Kaye, S.S. Stylli, M. Koo, M.F. Gonzales, N.J. Vardaxis and C.I. Johnson, Proc. Natl. Acad. Sc!. USA, 89, 1785 (1992) S.B. Kahl, M. Koo, B.H. Laster and R.G. Fairchild, Strahlenther. Onkol., 165, 134, (1989). D.A. Place, P.J. Faustino, K.K. Berghmans, P.C.M. van Zijl, A.S. Chesnick and J.S. Cohen, Magn. Reson. Imaging, 10, 919 (1992). It Emestus, L.J. Wilmas and M. Hoehem-Berlage, Clin. Exp. Metastasis, 10, 345 (1992).  255  Appendix  Appendix A Solutions in cell bioloov  A.1 a-medium  An alpha-modification of Eagle's minimum essential media (MEM, Gibco) was used in all procedures involving the maintenance or incubation of CHO cells. Three different forms of the media were used, oc-/-, cc+/- and a+/+ depending on the requirements of the procedure. One packet of a-medium powder and 10,000 units of Penstrep antibiotic (Gibco) were added to 10 L of double distilled water, and the solution was stirred for 2 h at room temperature, then sterilized by filtration through a 0.22 micron filter to make a-/- media. Fetal bovine serum (10%, Gibco, 1 L) and NaHCO3 (20 g) were added to the above solution before filtration; the pH of the resultant solution was adjusted to 7.30 with 4 M NaOH, and then filtered to produce a+/+ media. The a+/- media were made up by adding 10% (v/v) of bovine serum to the cc-/- media. All media were stored at 4°C. A-2 RPMI medium  One packet of RPMI powder, NaHCO3 (20 g) and 10,000 units of Penstrep antibiotic (Gibco) were added to 10 L of doubly distilled water and the solution was stirred for 2 h at room temperature, then sterilized by filtration through a 0.22 micron filter to make RPMI -/+ medium. RPMI +1+ medium was made by adding 10 % of FC II (Fetal Clone II, HyClone Laboratories Inc.) to the RPM14+ medium.  256  Appendix  A-3 PBS (phosphate buffer saline) solution  NaC1 (160 g), KC1 (4g), Na2HPO4 (23 g) and KH2PO4 (4 g) were dissolved in distilled water (20 L), and the solution was sterilized by filtration through a 0.22 gm filter (Nalgene). The solution was stored at room temperature and cooled to 4 °C when required. A-4 Methylene-blue solution  Methylene-blue (2 g) was dissolved in distilled water (1 L), and the solution was allowed to stand for 1 h prior to filtration. A-5 Trypsin solution (0.1 %)  Trypsin (1.0 g, Difco), KC1 (10 g), D-glucose (1 g) and sodium citrate dihydrate (5.0 g) were dissolved in double distilled water, and the resultant solution was sterilized by filtration. The solution was stored at - 10 °C.  257  Appendix  Appendix B Partition coefficients of porphyrins  The porphyrin free-bases were dissolved in octanol-saturated distilled water to make porphyrin solutions (10 mL each porphyrin) at 5 x 10-6 M. This concentration was chosen for the convenience of spectral measurements. The absorbances of these solutions at the Soret-band maxima were measured as Ae values. Each of the solutions (5.00 mL) was mixed with octanol (5.00 mL) and shaken for lh. The mixtures were centrifuged (600 RPM, 5 min) in order to separate the layers. The absorbances of the aqueous layer at the same wavelengths as the measurements of the Ae values were recorded as the Af values. A - Af Partition coefficient (P) values were then calculated as: P= ` The P values obtained Af are listed in the following table. .  porphyrin Tet(MPy)P T(MPy)PhP T(MPy)(NPh)P cis-B(MPy)DPhP cis-B(MPy)B(NPh)P trcms-B(MPy)DPhP trans-B(MPy)B(NPh)P Tet(SPh)P PyT(SPh)P (APh)T(SPh)P cis-BPyB(SPh)P cis-(NPh)PyB(SPh)P trans-BPyB(SPh)P cis-B(APh)B(SPh)P trans-B(APh)B(SPh)P  label used in Chapter 5 1 2 3 4  5  6  7 8 9  10 11  charge  A.max(nm)  P  +4 +3 +3 +2 +2 +2 +2 -4 -3 -3 -2 -2 -2 2 2  421 421 422 421 421 418 420 411 411 413 410 413 409 413 418  0.44 0.88 1.2 17 2.8 13 3.0 <0.01 <0.01 <0.01 0.23 0.82 0.16 0.71 0.29  -  -  It should be noted that these P values are accurate only if Beer's law is obeyed by these porphyrins in aqueous solutions up to 5 x 10-6 M; this has been found to be true for the tricationic, tetraldsanionic and trisanionic porphyrins (Chapter 4 of this thesis), and not true for the other porphyrins. In any case, these measurements provide a good indication of the trends of the change of the P value as the structure changes, although the error is estimated to be 10-25% for the porphyrins not obeying Beer's law.  258  Appendix C ill NMR titration curves for Co(III) diaouo porphvrin complexes  9.3  9.2 —  -  9.9  I^I^I^I^I 2^4^6^8^10^12^14 I  o  pD  (Col11[T(MPy)PIT](0D2)2}C14 ( pKai = 6.2 ± 0.1; pKa2 = 10.8 ± 01).  259  Appendix  9.3  0 ^  a  9.2  ANL  9  8.9 0  ^ ^ ^ 2 4  10  ^ ^ 12  14  pD  { Colli[T(MPy)(NPh)P(OD2)2] ) C14 ( pKa = 5.9 ± 0.1; pKa2 = 10.8 ± 01).  260  

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