Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1993_fall_phd_meng_grant.pdf [ 10.85MB ]
Metadata
JSON: 831-1.0061766.json
JSON-LD: 831-1.0061766-ld.json
RDF/XML (Pretty): 831-1.0061766-rdf.xml
RDF/JSON: 831-1.0061766-rdf.json
Turtle: 831-1.0061766-turtle.txt
N-Triples: 831-1.0061766-rdf-ntriples.txt
Original Record: 831-1.0061766-source.json
Full Text
831-1.0061766-fulltext.txt
Citation
831-1.0061766.ris

Full Text

PORPHYRIN CHEMISTRY PERTAINING TO THE DESIGN OFANTICANCER AGENTSbyGRANT GUANGZHEN MENGB . Sc., Peking University, Beijing, 1983M.Sc., Peking University, Beijing, 1986A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULT OF GRADUATE STUDIESDEPARTMENT OF CHEMISTRYWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIASEPTEMBER 1993©Grant Guangzhen Meng, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of ^CA &IP/ .,5Z-.^The University of British ColumbiaVancouver, CanadaDate OCt.^3DE-6 (2/88)AbstractA limitation of radiotherapy is the lack of selectivity (in term of cell killing) towardtumor cells, especially toward hypoxic tumor cells. Porphyrin drugs used in photodynatnictherapy (PDT) have good selectivity (in term of drug accumulation) toward tumors, butthe therapy has a limitation due to poor penetration of light through tissues. The initialobjective of this thesis work was to design, synthesize and test for potential porphyrinradiosensitizers with improved tumor selectivity. The rationale was that theradio sensitization abilities of the porphyrins might be improved by introduction of certainfunctional groups (including nitro groups and metal ions), while retaining or improving (bybalancing lipophilicity and hydrophilicity) selective properties of porphyrins. Such a drugwould first selectively accumulate in tumors, then sensitize the effects of X-rays, whichpenetrate tissues well, to kill cells, thus overcoming some of the limitations of bothradiotherapy and PDT. If such compounds can be synthesized, they may also be used inother aspects in cancer treatments and diagnosis, including PDT, chemotherapy, boronneutron capture therapy, and magnetic resonance imaging diagnosis. The potential of suchcompounds to be bioreductive drugs by targeting the hypoxic cells in tumors is also ofinterest in this project. This thesis work focuses mainly on the synthesis andcharacterization of some designed porphyrins and metalloporphyrins. Some preliminary invitro studies of these compounds are also presented.A method of combining pyrrole condensation with aldehydes and subsequentmodification is developed and used to synthesize a large variety of porphyrins. Among 40porphyrins synthesized, 22 are new and 15 are water-soluble. A new class of porphyrinswith three different meso substituents is synthesized (e.g., cis-(NPh)PyB(SPh)P, thestructure of which is shown below). Substituents which are presumed to improve theproperties of radiosensitization (NO2), DNA association (NH2, pyridyl), and water-solubility (methylpyridinium and sulfonatophenyl) are introduced into the porphyrin101520cis-(NPh)PyB(SPh)P = 5-(4-nitropheny1)-10-(4-pyridy1)-15,20-(4-sulfonatophenyl)porphyrinTet(SPh)P = 5,10,15,20-tetralcis(4-sulfonatophenypporphyrinPyT(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)porphyrintrans-BPyB(SPh)P = 5,15-bis(4-pyridy1)-10,15,20-bis(4-sulfonatophenyl)porphyrinTet(MPy)P = 5,10,15,20-tetralcis(4-methylpyridinium)porphyrinT(MPy)PhP = 5,10,15-tris(4-methylpyridinium)-20-(4-phenyl)porphyrinT(MPy)(NPh) = 5,10,15-tris(4-methylpyridinium)-20-(4-nitrophenyl)porphyrincis-B(MPy)B(NPh) = 5,10-tris(4-methylpyridinium)-15,20-bis(4-nitrophenyl)porphyrinstructures. The Ili NMR spectra of these porphyrins are presented with each of the signalsassigned. The aggregation of the porphyrin free-bases in aqueous solutions is reported. Allthe water-soluble porphyrin free-bases aggregate in aqueous solutions, but to differentdegrees. Different models for the aggregation are suggested, including a new "slide-over"model for the aggregation of tris-ionic porphyrins. Equilibrium constants (K) for anassumed monomer dimer process for 6 porphyrins have been calculated, and the Kvalues 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).Cobalt complexes of the water-soluble porphyrins Tet(MPy)P and Tet(SPh)P havebeen reported in the literature in the investigation of several chemical and biologicalsystems. However, questions remain in the literature regarding the synthesis andcharacterization of these compounds, especially concerning the oxidation state of thecobalt. The chemistry related to the synthesis of these complexes is further investigated,and synthetic methods for the specific oxidation states of cobalt are developed. Newcobalt (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 andcharacterized. The pKa values of the coordinated aquo ligands of four diaquocobalt(III)complexes of water-soluble porphyrins in aqueous solutions are measured using protonNMR spectroscopy. The aggregation of cobalt(II) complexes of water-soluble porphyrinin 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 beclosely related to the chemical structures, especially to sign, number and distribution ofcharges. Generally, porphyrins with lower charge accumulate more than ones with highercharge; porphyrins with positive charge accumulate more than those with the samenegative charge; and the trans-isomer accumulates more than the cis-isomer.The porphyrin free-bases are found to be essentially non-toxic toward Chinesehamster ovary (CHO) cells. The cobalt(III) complexes of cationic porphyrins exhibit someselective toxicity toward hypmdc CHO cells, a property for which the compounds aredesigned, although the toxicities in hypoxic conditions are too small for use inbioreductive drugs. The porphyrins and metalloporphyrins have not shown highradiosensitization toward CHO cells under hypoxic conditions, and have not shownphotosensitization toward HT-29 cells at wavelengths of 630 ± 10 run, although someweak 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 thesynthesized porphyrins in other aspects of cancer treatments and diagnosis should also beconsidered in future work.ivTable of contentsAbstract^Table of contents^ vList of figures xiiList of tables^ xviiLists of abbreviations^ xixList of general abbreviations^ xixList of abbreviations of porphyrins xxiiAcknowledgements^ xxxiiChapter 1 Introduction--The design of porphyrin compounds as potentialanti-cancer agents^ 11.1 Porphyrin chemistry 11.2 Nomenclature and abbreviations of porphyrins used in this thesis^ 21.3 Porphyrins and cancer^ 51.3.1 Tumor localization of porphyrins^ 51.3.2 Porphyrins used in PDT (photodynamic therapy)^ 61.3.3 Porphyrins used in tumor detection^ 71.3.4 Porphyrins used in chemotherapy 71.3.5 Porphyrins as radiosensitizers^ 81.4 Radio sensitizers^  101.4.1 Mechanisms 11V1.4.2 Properties of radiosensitizers^  121.4.3 Nitroirnidazole radiosensitizers  131.5 Bioreductive agents^  151.6 The design of porphyrins as anti-cancer agents^  161.6.1 Electron affinity^ 161.6.2 Selectivity toward tumors^ 171.6.3 Cell uptake^ 191.6.4 DNA-binding 191.6.5 Solubility and lipophilicity^  191.6.6 Multiple functions 201.7 Porphyrin syntheses^ 201.8 In vitro studies on synthetic porphyrins^ 251.9 The objectives of this thesis work 27References-Chapter 1^ 28Chapter 2 The synthesis of the designed porphyrins^352.1 Introduction^ 352.2 Experimental 372.2.1 Materials and method^ 372.2.2 The porphyrins with a general formula of PhnPy(4../)P^ 382.2.3 Nitrations^ 422.2.4 Reductions of the nitro-porphyrins^ 472.2.5 Sulfonations^ 552.2.6 Methylations 622.3 Results and discussion^ 67vi2.3.1 Synthesis^ 682.3.1.1 Hydration of porphyrins^ 682.3.1.2 Synthesis of the porphyrins with general formulaPhnPY(4-0P^ 702.3.1.3 Synthesis of nitro-porphyrins^ 712.3.1.4 Synthesis of mninophenylporphyrins 732.3.1.5 Synthesis of sulfonated porphyrins^ 732.3.1.6 Synthesis of methylpyridiniumporphyrins^ 782.3.2 Proton-NMR spectra^ 812.3.2.1 Identification of substituents from 1H NMR spectra^ 812.3.2.2 Assignments of signals to the protons of the meso-substituent^ 832.3.2.3 Identification of isomers 902.3.2.4 Assignments of the signals for the pyrrole protons^ 962.3.3 UV-visible spectra^ 1052.3.4 Infrared spectra 1082.3.5 Mass spectra^ 108References-Chapter 2 113Chapter 3 Water-soluble metalloporphyrins^ 1163.1 Introduction^ 1163.1.1 Cobalt complexes of water-soluble porphyrins^ 1163.1.2 Copper and zinc complexes of water-soluble porphyrins^ 1183.2 Experimental^ 1183.2.1 Materials and methods^ 1183.2.2 {Co11[Tet(MPy)P])C14.2H20 120vii3.2.3 {Colll[Tet(MPy)P](OH2)}(C104)5.xH20^ 1213.2.4 {Colret(MPy)P1(OH)}C14.2H20 1213.2.5 {CollIget(MPy)11(OH2)}C15-2H20^ 1213.2.6 {Co11[T(MPy)Phil}C13-1/2H20 1223.2.7 {Co111[T(MPy)Ph11(OH2)}04-2H20^ 1223.2.8 {CoRT(MPy)(NPh)PKOH2)}04.H20 1223.2.9 {CollIcis-B(MPy)B(NPh)19(H20))C13-2H20^ 1233.2.10 Na4{Co11[Tet(SPh)P]).nH20 (n=4 or 9) 1233.2.11 Na3{Colret(SPh)PKOH2)}.nH20 (n=3 or 14)^ 1243.2.12 Na2{Co111[PyT(SPh)P]} ^ 1243.2.13 Na2{Co111[(APh)T(SPh)11(OH2)}^ 1253.2.14 Na{ Com[c-(NPh)PyB(SPh)P](OH2)} -2H20^ 1253.2.15 Cu porphyrins complexes^ 1253.2.16 Zinc porphyrin complexes 1263.3 Results and Discussion^ 1273.3.1 1H NMR spectra of the metalloporphyrins^ 1273.3.1.1 1H NMR spectra of Co complexes ofcationic-porphyrins^ 1273.3.1.2 111 NMR spectra of Co complexes ofanionic-porphyrins^ 1343.3.1.3 1H NMR of Cu-porphyrins 1373.3.1.4 1H NMR of Zn-porphyrins^ 1393.3.1.5 1H MAR studies of the aggregation of Con complexes^ 1403.3.2 Syntheses^ 1443.3.2.1 Synthesis of Co-cationic porphyrins^ 1443.3.2.2 Synthesis of Co-anionic-porphyrins 145viii3.3.2.3 Reaction conditions for the synthesis ofcobalt porphyrins^ 1463.3.2.4 Synthesis of Cu-porphyrins and Zn-porphyrins^ 1513.3.3 Mass spectra^ 1513.3.3.1 Mass spectra of cobalt complexes of anionic porphyrins ^ 1513.3.3.2 Mass spectra of cobalt cationic porphyrins^ 1543.3.4 Magnetism and coordination of the cobalt-porphyrin complexes ^ 1553.3.5 UV-visible spectra^ 1583.3.6 Measurement of the pKa values of cobalt(III) diaquo complexes ^ 1633.3.7 Hydration and elemental analysis ^ 170References-Chapter 3^ 172Chapter 4 Aggregation of porphyrins^ 1744.1 Introduction^ 1744.2 Experimental 1754.3 Results and discussion^ 1764.3.1. UV-visible spectra in aqueous solutions^ 1764.3.1.1 Tet(SPh)P^ 1774.3.1.2 PyT(SPh)P and (APh)T(SPh)P^ 1814.3.1.3 trans-BPyB(SPh)P^ 1844.3.1.4 cis-BPyB(SPh)P, cis-B(APh)B(SPh)P andtrans-B(APh)B(SPh)P^ 1874.3.1.5 Tet(MPy)P^ 1914.3.1.6 T(MPy)PhP 1954.3.1.7 T(MPy)(NPh)P and cis-B(MPy)DPhP^ 1964.3.2. Affects of methanol on aggregation of porphyrins. ^ 198ix4.3.3 1H NMR studies on aggregation of porphyrins^ 2034.3.3.1 1H NMR studies on the aggregation of T(MPy)PhP^ 2034.3.3.2 1H NMR studies on the aggregation of PyT(SPh)P^ 2094.3.4 Aggregation models^ 2144.3.4.1 Monomer dimerization or oligomerization^ 2144.3.4.2 Structural models^ 2164.3.5 The equilibrium constants for dimerization^ 2194.3.6 Summary^ 225References-Chapter 4 226Chapter 5 In vitro studies of selected synthetic porphyrins andmetalloporphyrins^ 2285.1 Introduction^ 2285.2 Materials and methods^ 2295.2.1 Cell growth, maintenance and treatment^ 2295.2.2 Drugs and drug solutions^ 2325.2.3 Toxicity in oxic and hypoxic conditions^ 2325.2.4 Radiosensitization in hypoxic conditions  ^ 2345.2.5 Cell accumulation^ 2375.2.6 Photosensitization 2385.3 Results and discussion^ 2395.3.1 Toxicity of porphyrins and metalloporphyrins^ 2395.3.2 Radiosensitization under hypoxic conditions 2425.3.3 Accumulation of porphyrin free-bases in HT-29 cells^ 2465.3.4 Photosensitization of porphyrin free-bases^ 247xReferences-Chapter 5^ 249Chapter 6 Conclusions and suggestions for future work^2506.1 Synthesis of porphyrins^ 2506.2 Metallation of porphyrins 2516.3 In vitro studies^ 2516.4 Suggestions for Future Work^ 2526.4.1 Synthesis of porphyrins 2526.4.2 Other metallations^ 2536.4.3 Biological studies 254References - Chapter 6^ 255Appendix A Solutions used in cell biology^ 256A.1 a-medium^ 256A-2 RPMI medium 256A-3 PBS (phosphate buffer saline) solution^ 257A-4 Methylene-blue solution^ 257A-5 Trypsin solution (0.1 %) 257Appendix B Partition coefficients of porphyrins^ 258Appendix C 1H NMR titration curves for Co(111) diaquoporphyrin complexes^ 259xiList of figuresFigure 1.1. The porphyrin core.^  2Figure 1.2. Porphyrins containing anti-cancer agents. ^  8Figure 1.3. Structures of some water-soluble porphyrins. 9Figure 1.4. A proposed sensitization mechanism^  11Figure 1.5. Structures of some radiosensitizers.  14Figure 1.6. Structures of tetrahydroxophenylpotphyrins. ^  18Figure 1.7. Structures of sulfonated derivatives of TetPhP  18Figure 1.8. Structure of some synthetic porphyrins.^  22Figure 1.9. Modifications of TetPhP^  24Figure 1.10. Some possible modifications of TPhPyP.^  26Figure 2.1. Synthetic schemes for designed porphyrins^  36Figure 2.2. 1H NMR spectra of B(NPh)B(SPh)P, (APh)(NPh)B(SPh)P andB(APh)B(SPh)P in DMSO-d6^  77Figure 2.3. 1H NMR spectrum of cis-(NPh)PyB(SPh)P in DMSO-d6.^ 79Figure 2.4. 1H NMR spectrum of (NPh)TPIT in CDC13.^  85Figure 2.5. 1H NMR spectrum of trans-BPyB(SPh)P in DMSO-d6 ^ 86Figure 2.6. 111 NMR spectrum of (NPh)T(MPy)P in CDC13.  88Figure 2.7. 1H NMR spectrum of cis and trans-DP111PyP in CDC13.^ 91Figure 2.8. The patterns for the 1H NMR spectra of the pyrrole protons^ 92Figure 2.9. 1H NMR spectra of cis- and trans-B(NPh)BPyP in CDC13.^ 94Figure 2.10. 1H NMR spectra of cis and trans-B(NPh)PhPyP in CDC13.^ 95Figure 2.11. Resonance structures of a nitrophenylpotphyrin ^ 97xiiFigure 2.12. Resonance structures of an atninophenylporphyrin.^ .98Figure 2.13A. A comparison of the schematic spectra of TPhPyP,cis-DPhBPyP and PhTPyP.^ 99Figure 2.13B. Illustration of the chemical shifts of the pyrrole protonsof TPhPyP and some nitroporphyrins. ^  100Figure 2.14. Assignment of the pyrrole protons in the 1H NMR spectrumof cis-(NPh)DPhPyP (CDC13).^ 103Figure 2.15. 1H NMR spectrum of cis-(APh)PhBPyP in CDC13^ 104Figure 2.16. Resonance forms of an aminophenylporphyrin anda nitrophenylporphyrin^ 107Figure 3.1. Cell for anaerobic UV-visible spectroscopy^ 120Figure 3.2. 1H NMR spectra of {Coll[Tet(MPy)P]}C14 in D20^ 128Figure 3.3. 1H NMR spectra of Co(II) cationic porphyrins in DMSO-d6 under air ^ 131Figure 3.4. 1H NMR spectra of Colli[Tet(MPy)P] species in DMSO-d6^ 132Figure 3.5. 1H NMR spectra of Co[Tet(SPh)P] species in DMSO-d6 ^ 135Figure 3.6. 1H NMR spectra of cobalt complexes of tris-sulfonatoporphyrins inDMSO-d6.^ 136Figure 3.7. 1H NMR spectra of copper(II) complexes of cationic porphyrinsin DMSO-d6. ^ 138Figure 3.8. 1H NMR spectra of Co11[Tet(MPy)P]C14 in D20.^ 141Figure 3.9. 1H NMR spectra of Na4{Con[Tet(SPh)P]} in D20 143Figure 3.10. Mass spectrum of Na3{Colli[Tet(SPh)}P(OH2)}.^ 152Figure 3.11. The suggested molecular fragments for the major peaks in themass spectrum of Na3{Co111[Tet(SPh)P](OH2)).^ 153Figure 3.12. Paramagnetic Co(DEE) complexes^ 156Figure 3.13. UV-visible spectra of Con[Tet(MPy)P]. 160Figure 3.14. UV-visible spectra of Coll[T(SPh)P] in H20.^ 161Figure 3.15. 1H NMR spectra of Colll[T(MPy)13] in D20 atdifferent pD values.^  165Figure 3.16. 1H NMR spectra of Co111[Tet(SPh)P] in D20 atdifferent pD values.^  166Figure 3.17. The pKa measurement for Co111[Tet(MPy)1]0320)2. ^ 168Figure 3.18. The pKa measurement for Coul[Tet(SPh)P]0320)2 169Figure 4.1. The normalized spectra of Tet(SPh)P at various concentrationsin distilled water.^ 178Figure 4.2. Beer's law diagrams for Tet(SPh)P in distilled water.^ 179Figure 4.3. The normalized spectra of Tet(SPh)P at various concentrationsin a phosphate buffer.^ 180Figure 4.4. Deviation from Beer's law for Tet(SPh)P in a phosphatebuffer solution^ 180Figure 4.5. The normalized spectra of (APh)T(SPh)P at variousconcentrations in a phosphate buffer^ 182Figure 4.6. Beer's law deviation for PyT(SPh)P and (APh)T(SPh)P.^ 183Figure 4.7. The normalized spectra of trans-BPyB(SPh)P at variousconcentrations in distilled water^ 185Figure 4.8. The normalized spectra of trans-BPyB(SPh)P at variousconcentrations in a phosphate buffer^ 186Figure 4.9. The normalized spectra of cis-BPyB(SPh)P at variousconcentrations in a phosphate buffer^ 188Figure 4.10. The normalized spectra of cis-B(APh)B(SPh)P at variousconcentrations in a phosphate buffer^ 189Figure 4.11. The normalized spectra of trans-B(APh)B(SPh)P at variousconcentrations in a phosphate buffer^ 190Figure 4.12A. The normalized spectra of Tet(MPy)P chloride at variousconcentrations in distilled water^ 192Figure 4.12B. Plot of A/b vs. concentration for Tet(MPy)13 chloride indistilled water^ 193xivFigure 4.13. The normalized spectra of Tet(MPy)P in a buffer solution atvarious concentrations^ 194Figure 4.14.Figure 4.15.Figure 4.16.Figure 4.17.Figure 4.18.The normalized spectra of T(MPy)PhP at different concentrationsin a phosphate buffer.^ 195A plot of A/b vs. concentration for T(MPy)PhP in aphosphate buffer.^ 196The normalized spectra of cis-B(MPy)DPhP at variousconcentrations in a buffer solution.^  197Absorbance spectra of (APh)T(SPh)P in aqueous buffer/methanolmixtures.^  198Absorbance spectra of trans-BPyB(SPh)P inaqueous buffer/methanol mixtures.^ 200Figure 4.19A. Absorbance spectra of trans-B(APh)B(SPh)P inaqueous buffer/methanol mixtures.^ 201Figure 4.19B. Absorbance spectra of cis-B(APh)B(SPh)P inaqueous buffer/methanol mixture^ 202Figure 4.20. 1H NMR spectra of T(MPy)PhP in D20 and DMSO-d6^ 204Figure 4.21. Correlation between the 1H chemical shifts and theconcentration of T(MPy)PhP^ 206Figure 4.22. Aggregation models for T(MPy)PhP in water.^ 208Figure 4.23. 1H NMR spectra of PyT(SPh)P.^ 210Figure 4.24 Correlation between the 1H chemical shifts and theconcentration of T(SPh)PyP.^ 211Figure 4.25. Suggested aggregation model for PyT(SPh)P ^ 212Figure 4.26. Aggregation models for Tet(MPy)P ^ 217Figure 4.27. Aggregation models for trans-B(MPy)DPhP^ 220Figure 4.28A Best fit curves for the dimerization of Tet(SPh)P and PyT(SPh)Pin a buffer solution^ 223Figure 4.28B Best fit curves for the dimerization of (APh)T(SPh)P in a buffersolution and Tet(MPy)P in distilled water^ 224XVFigure 5.1 The vessel for toxicity assay. ^ 233Figure 5.2. Set up for the radiosensitization assay^ 235Figure 5.3. A representative example of survival curves (02 effect, ER — 3).^ 236Figure 5.4. Toxicities of compound 12 (100 gM) under hypoxicand oxic conditions. ^ 241Figure 5.5. Survival curves for radiosensitization by compound 18;a weak radiosensitizer. ^ 244Figure 5.6. Survival curves for effect of compound 3 with radiation;a weak protector.^ 245Figure 5.7. Results of drug accumulation in HT-29 cells.^ 246Figure 5.8. Photosensitization using 630 nm radiation bycompounds 1 (0.03 mM) and 9 (0.05 mM).^ 248xviList of tablesTable 2.1. Elemental analyses of the PhnPY(4-n)P PorPhYrins^ 40Table 2.2. 1H-NMR data for the PhnPyo_nr,P porphyrins 41Table 2.3. UV-visible data for the PhnPyo_nyP porphyrins^ 41Table 2.4. Elemental analyses for the nitroporphyrins 48Table 2.5. 1H NMR data for the nitroporphyrins^ 49Table 2.6. UV-visible data for the nitroporphyrins 50Table 2.7. Elemental analyses for the aminoporphyrins^ 52Table 2.8. 1H NMR data for the aminoporphyrins 53Table 2.9. UV-visible data for the aminoporphyrins^ 54Table 2.10. Elemental analyses for the sulfonated porphyrins^ 59Table 2.11. 1H NMR data for the sulfonatoporphyrins 60Table 2.12. UV-visible data for the sulfonatoporphyrins^ 61Table 2.13. Elemental analyses for the methylpyridiniumporphyrins^ 64Table 2.14. 1H NMR data for the methylpyridiniumporphyrins 65Table 2.15. UV-visible data for the methylpyridiniumporphyrins^ 66Table 2.16. Reports on elemental analysis of Tet(SPh)P 69Table 2.17. Calculated and observed chemical shifts of the nitrophenyl andaminophenyl protons^ 84Table 2.18. The assignments of the pyrrole protons^ 101Table 2.19. The chemical shifts of the H7 and H8 pyrrole protons^ 102Table 2.20. Soret bands of nitro and amino-porphyrins^ 107Table 2.21. Data from IR spectra^ 109Table 2.22. Data from CI mass spectra  111xviiTable 2.23. Cationic FAB mass spectra of some cationic porphyrins^ 111Table 2.24. Anionic FAB mass spectra of some anionic porphyrins^ 112Table 3.1. Elemental analyses and yields of copper-porphyrin complexes^ 126Table 3.2. Elemental analysis of zinc-porphyrin complexes^ 127Table 3.3. 1H NMR data of the Co cationic porphyrin complexes in DMSO-d6 ^ 134Table 3.4. 1H NMR data for Zn porphyrin complexes in DMSO-d6^ 140Table 3.5. Mass spectra of anionic Co-porphyrins^ 154Table 3.6. Mass spectral data for cobalt cationic porphyrins^ 154Table 3.7. Magnetic moments of Co-Porphyrins^ 155Table 3.8. Data of UV-visible spectra of Co-porphyrins 159Table 3.9. Data of UV-visible spectroscopy^ 163Table 3.10. The pKa values for cobalt porphyrins 167Table 4.1. 1H Chemical shifts (ppm) of T(MPy)PhP at concentrationsof 0.02 and 0.002 M^ 205Table 4.2. Chemical shifts in spectra of PyT(SPh)P ^ 209Table 4.3. Equilibrium K values for dimerization of porphyrins ^ 222Table 5.1. In vitro tests and compounds tested^ 230Table 5.2. Fluorescence maxima for excitation and emission^ 238Table 5.3. Radiation enhancement ratio for selected porphyrinsand metalloporphyrins^ 243xviiiLists of abbreviationsThe abbreviations are given in two parts. The commonly used abbreviations in thechemical and biological literature are listed in the first part, while the abbreviations for theporphyrins used in this thesis are listed in the second part.List of general abbreviationsII-1 NMR^Proton nuclear magnetic resonance2D^2-Dimensional ( NMR )A^Angstrom ( 10-8 cm )A^AbsorbanceAc0^Acetate ( CH3C00- )AIDS^The acquired immunodeficiency syndromeAPh^4-aminophenylBis ( in chemical names )Light-path length ( VU-visible spectroscopy )br^broad signal ( NMR )Concentrationoc^Degrees centigradeCHO^Chinese hamster ovary ( a cell line )cm^Centimeter ( 10-2 meter )Di ( in chemical names )Doublets ( NMR )DMF^N,N-dimethylformamideDMSO -d6 Deuterated dimethylsulfoxide ( a NMR solvent )DNA^Deoxyribonucleic acidDSS^Sodium-2,2-dimethy1-2-silapentane-5-sulphonate ( NMR reference )xixeqn^EquationER^Enhancement ratioESR^Electron spin resonanceFAB^Fast-atom bombardment ( mass spectroscopy )g^Gram(s)GY^Gray = 100 radsh Hour(s)IR^InfraredkiH^Coupling constant ( NMR )L Liter(s)M^Molarity ( moles per liter solution )m^Multiplet ( NMR )Me^Methylmg^milligram ( 10-3 gram )MHz^Megahertz ( 106 Hertz )min^Minute(s)mL^Milliliter(s)mol^Mole(s)MPy^4-Methylpyridiniumnm^Nanometer ( 10-9 meter )NMR^Nuclear magnetic resonance spectroscopyNPh^4-Nitrophey1OER^Oxygen enhancement ratioPBS^Phosphate buffer salinePE^Plating efficiencyPh^Phenylppm^Parts per million ( NMR )XXPy^Pyridylqt^Quartet (NMR)R^Alkyl or aryl groupRPM^Revolutions per minutes^Singlet ( NMR ); strong signal ( IR)SER^Sensitizer enhancement ratioSPh^4-SulfonatophenylT^Tris ( in chemical names )t^Triplet ( NMR )Tet^Tetrakis ( in chemical names )TMS^Tetramethylsilane ( NMR reference )UV^UltravioletV^Volume^ Volt6^Chemical shifte Extinction coefficientX^Wavelengthp.^Ionic strengthPIL^Microliter1-LM^MicromolarIx/List of Abbreviations of porphyrins131517porphyrin coreAbbreviations Full names^ Structurescis-(APh)DPhPyP^544-aminopheny1)-10,15-4ipheny1-20-(4-pyridy1)porphyrincis-(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)porphyrincis-B(APh)DPhP^5,10-12is(4-aminopheny1)-15,20-dipheny1porphyrintrans-B(APh)DPhP^5,15-bis(4-aminopheny1)-10,20-ilipheny1porphyrincis-B(APh)B(SPh)P^5,10-bis(4-aminoplieny1)-15,20-bis(4-su1fonatopheny1)poiphyrintrans-B(APh)B(SPh)P^5,15-bis(4-aminopleny1)-10,20-bis(4-sulfonatoaheny1)porphyrins03-trans-B(APh)PhPyP^5,15-bis(4-ilminoaleny1)-10-0eny1-20-(4-gyridy1)polphyrincis-B(MPy)B(NPh)P^5,10-bis(4-methy1pyridinhun)-15,20-bis(4-nitrogheny1)porphyrintrans-B(MPy)B(NPh)PN°25,15-bis(4-methylpyridinhun)-10,20-bis(4-nitrophenyl)polphyrinNO2NH N_+MeIQ 0 NMN HN xxivcis-B(MPy)DP1113^5, 10-bi s(4-methylpyridinium)-15,20-diphenylpcnphyrin+ MiNtrans-B(MPy)DPhP^5,15-bis(4-methy1pyridiniutn)-10,20-dipheny1porphyrin+ meecis-B(NPh)DPhP^5,10-bis(4-nitropheny1)-15,20-diaienylporphyrintrans-B(NPh)DPhP^5,15-bis(4-nitropheny1)-10,20-ilipheny1porphyrincis-B(NPh)BPyP^5,10-bis(4-nitropheny1)-15,20-his(4-p_yridy1)gorphyrintrans-B(NPh)BPyP^5,15-Igs(4-nitropheny1)-10,20-his(4-pyridyl)porphyrincis-B(NPh)PhPyP^5,10-12is(4-nitropheny1)-15-pheny1-2044-pyridy1Vorphyrintrans-B(NPh)PhPyP^5,15-bis(4-nitroaleny1)-10-phenyl-20-(4-pyridypporphyrincis-BPyB(SPh)P^5,10-12is(4-pytidy1)-15,20-his(4-su1fonatopheny1)otphytinStrans-BPyB(SPh)P^5,15-his(4-pyridy1)-10,20-his(4-fiu1fonatoplieny1)polphyrincis-DPhBPyP^5,104ipheny1-15,20-his(4-pyridy1)porphyrintrans-DPhBPyP^5,15-dipleny1-10,20-his(4-ayridyl)porphyrincis-(NPh)DPPyP^5-(4-nitropheny1)-10,15-dip1ieny1-20-(4-pyridy1)porphytincis-(NPh)PhBPyP^5-(4-nitropheny1)-10-plenyl-15,20-his(4-pyridypporphyrincis-(NPh)PyB(SPh)P^5-(4-nitropteny1)-10-(4-atidy1)-15,20-his(4-su1fonatopheny1)porphyrin\\ >--- N HNQ,..._\./5-(4-nitropheny1)-10,15,20-1risphenylomphyrin5-(4-nitropheny1)-10,15,20-tris(4-pyridyl)poiphyrin2,3,7,8,12,13,17,18-Qctagthy1porphyrin2,3,7,8,12,13,17,18-Qctaffiethy1porphyrin(NPIOTPhP(NPh)TPyPOEPOMPPhTPyP^5-pheny1-10,15,20-irsi(4-pyridyl)porphyrinPyT(SPh)P^5-(4-pyridy1)-10,15,20-tris(4-fiu1fonatopheny1)pozphyrinT(APh)PhP^5,10,15,/ris(4-aminopheny1)-20-plenylporphyrinTet(MPY)P^5,10,15,204etra1ds(4-methy1pyridinitun)porphyrinThis porphyrin is abbreviated as TMPyP in the literatureMEN0Tet(NPh)P^5,10,15,20-tetralcis-(4-nitrophenyl)porphyrinTetPhP^5,10,15,20-letraphenylporphyrinThis porphyrin is abbreviated as 7PP in the literature.TetPyP^5,10,15,20-Letrakis(4-pyridy1)porphyrinThis porphyrin is abbreviated as 7'PyP in the literatureTet(SPh)P^5,10,15,20-tetrakis-(4-sulfonatophenyl)porphyrinThis porphyrin is abbreviated as TPPS4 in the literatureT(MPy)(NPh)P^5,10,15-iris(4-methyloridiniurn)-20-(4-nitrophenyl)polphyrinT(MPy)PhPT(NPh)PhP5,10,15-vis(4-methy1pyridinium)-201teny1porphyrin5,10,15-vis(4-nitroaleny1)-20-pheny1porphyrincN5,10,15-iripheny1-20-(412.yridy1)porphyrinTPhPyPAcknowledgmentsMy gratitude and thanks go to both of my supervisors, Drs. Brian Jamesand Kirsten Skov, for their guidance, encouragement and patience during thecourse of this thesis work; and for their considerations for the difficulties I haveexperienced as a foreign student of chemistry, biology, English and Canadianculture.I am also grateful to my dear wife, Haibo, for her support, patience andsharing 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 goto Don Yapp and Chris Alexander for their help both in the ways of learningsciences and learning the words Chris uses the most. Going for coffee with allthese people has been an enjoyment.The help from support staff in chemistry department (especially PeterBorder, Marietta Austria and Lianne Diarg) is gratefully appreciated. Help from asummer 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 ofworking for this thesis enjoyable (at least sometimes).The opportunity to come to, live and study in this beautiful country, whichhas been made possible by my acceptance as a graduate student and the offerof finical support from the University of British Columbia, is highly appreciated.Chapter 1 IntroductionChapter 1 Introduction—The design of porphyrin compounds aspotential anti-cancer agents1.1 Porphyrin chemistryPorphyrin compounds (the free bases and metalloporphyrins) play essential roles inseveral biological systems, including human beings. Metalloporphyrins are involved in theprocesses of oxygen transportation (hemoglobin); oxygen storage (myoglobin); oxygenactivation (cytochrome P-450); and electron transport (the cytochromes).1 The importanceof these processes in biological systems, plus the challenges of porphyrin metabolismdisorders 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 porphyrincompounds.Porphyrin chemistry has been developing for more than a century since theisolation of hematoporphyrin in 1867.2 The early studies were mainly on natural products,particularly isolation and characterization of the porphyrins and metalloporphyrinscontained in biomolecules, especially iron protoporphyrin IX from hemoglobin andmyoglobin. The total synthesis of protoporphyrin in 1929,3 which proved the cyclictetrapyrrole 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 andOEP (2,3,7,8,12,13,17,18-octaethylporphyrin),6 are the corner stones of modernporphyrin chemistry. Porphyrins have been employed by chemists to mimic biologicalreactions both in the interests of understanding these reactions in biological systems and ofutilizing these reactions for chemical industry. Porphyrin compounds have been studied inclinical chemistry related to porphyrias, and as anti-cancer drugs for photodynamicIChapter 1 Introductiontherapy. The chemistry of porphyrins has been well reviewed in the seven-volume seriesentitled "The Porphyrins", edited by Dolphin.71.2 Nomenclature and abbreviations of porphyrins used in this thesisThe nomenclature of porphyrins is described by Bonnett.8 Figure 1.1 shows theporphyrin core and the numbers of the carbon atoms which indicate the substitutionpositions.1315172^20^18Figure 1.1. The porphyrin core.Synthetic porphyrins, such as TPP, OMP and OEP, are named according to thissystem. Besides the systematic numbering of the carbon atoms in a porphyrin core, thefour carbon atoms between the pyrrole structures, which are carbons 5,10;15, and 20 arealso said to be in the meso-positions.Abbreviations and the names of the porphyrins involved in this thesis are listed inthe List of Abbreviations. These abbreviations are derived according to the systematicnames, similarly to the way that TPP, OMP and OEP are derived, but in order todifferentiate phenyl group and porphyrin, Ph is used for phenyl, and P is used forporphyrin; to differentiate tri or tris and tetra or tetralds, T is used for tri or tris, and Tet isused for tetra or tetrakis. The other abbreviations used are B for bis, D for di.2Chapter 1 IntroductionAbbreviations used for the substituents, and the numbering in the substituents are listed inTable 1.1.Table 1.1. Abbreviations of porphyrin substituentsabbreviation name structure2 - 3APh 4-aminoaieny1 _ ,4 - NH2,\6 - 52 - 3MPy 4-methylpyridinium —1 CH3,6 -2 - 3NPh 4-nitrophenyl ,4 - NO2—1\6 - 52 - 3Py 4-pyridyl _ ,6 - 5_SPh 4-sulfonatophenyl —1 ,, — SO3-,6 -5Thus, 5,10,15,20-tetraphenylporphyrin (abbreviated as TPP in the literature) isabbreviated 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 asTet(MPy)PC14 or as Tet(MPy)P when the emphasis is on the porphyrin (as with the othercationic porphyrins). In this thesis, Tet(SPh)P and PhT(SPh)P are used to represent thetwo porphyrins, 5,10,15,20-tetralcis(4-sulfonatoaienypporphyrin, and 5-pheny1-10,15,20-tris(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 othersulfonated porphyrins). In the literature,9,1°,11 TPPS, TPPS-4, TPPS4 or TSPP have beenused for the tetra-sulfonated derivative, and TPPS-3 for the tri-sulfonated derivative.In this thesis, the abbreviations listed for the porphyrins represent the porphyrinfree-bases, but the abbreviations are also used as the porphyrin dianion for3Chapter I Introductionmetalloporphyrins. This is commonly done in the literature'," and is illustrated by thefollowing figure.TetPhP^M(TetPhP)Trivial names are used for natural porphyrins. Structures of two naturalporphyrins, protoporphyrin IX which is found in both hemoglobin and myoglobin, andhematoporphyrin, of which the derivatives are used as photo sensitizers for photodynamictherapy for cancer (Section 1.3), are shown below.H2C=CH^CH3^ HO-CHCH 3^CH3CH2I  ICH3 ^OHCH^CH3 ^ CHCH3CH3CH2CH2C 02^C H2 C H2 C 02NH\^/HNC H3^\ \ / / CH3C H2 C H2 C 02^C H2 C H2 C 02protoporphyrin IX^hematoporphyrin4Chapter 1 Introduction1.3 Porphyrin and cancer1.3.1 Tumor localization of porphyrinsAbout half a century ago, Auler and Banzer12 examined the in vivo distribution ofporphyrins in tumor-bearing animals and demonstrated accumulation of the porphyrins intumors by fluorescence. Lipson et al. synthesized hematoporphyrin derivatives (HPD) in1961 and initiated the use of this drug in photodynamic therapy (PDT).13 The selectiveaccumulation of this porphyrin at the tumor became widely accepted and has been studiedby many different experimental techniques." These experiments have shown that in animalmodels selectivity toward some tumors, compared to surrounding muscle tissue, doesexist, although the uptake of this porphyrin product in tumor is lower than the uptake insome tissues including the kidney and the liver.14a,15 The high uptakes by kidney and liveris considered not crucial for the therapy because light received by these tissues isnegligible, and the drug is basically non-toxic in the dark.16 The mechanism of the selectiveretention of this porphyrin product in different tissues, especially in tumors, is still notclear. 16Synthetic porphyrin compounds have also been studied from the aspect of selectivetumor accumulation. For example, it has been shown that Tet(SPh)P has an approximately50-fold superiority over HPD as a tumor localizer." Other porphyrins, including5, 10,15,20-tetralds(3-hydroxyphenyl)porphyrin and 5, 10,15,20-tetralds(4-hydroxyphenyl)porphyrin (structures are shown later in Section 1.6.2) have been shown tohave a good selectivity for tumor relative to muscle and skin." Levels of radiocopper-labeled 5,10,14,20-tetralds(4-carboxyphenyl)porphyrinatocopper(11) were reported to be 2to 3 times higher in tumor than in skin, blood, fat and muscle.195Chapter I Introduction1.3.2 Porphyrins used in PDT (photodynamic therapy)PDT, a therapeutic method to treat tumors, requires a photosensitizing drug and asource of drug activation using light." The main toxic species is believed to be 102produced by the light in the presence of the drug.21Photofrin II, a purified form of hematoporphyrin derivatives (HPD), is currentlythe most widely used photosensitizer in PDT.22 The drug is a mixture of a few porphyrincompounds, the structures of which are still not clear.22HPD has been studied as a photosensitizer for more than 15 years. PDT, whichinitially 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 thetumor), and the complex composition of the drug make the application limited.22 Manynew drugs, based on benzoporphyrin derivatives (BPD), sulfonated derivatives ofphthalocyanines and chlorin compounds, are under development.22 The attractiveproperties of these compounds are localization at the tumor, low toxicities, fluorescence,and photodynamic activities.22Although work in developing new photosensitizers has been directed mainly atimprovement of the absorption of light at longer wave lengths, other factors, especiallyhigh selectivities and efficacy, are also important and should also be considered.23 Inanimal models, a synthetic porphyrin Tet(SPh)P has been reported to have preferentialuptake by some tumors;17 another synthetic porphyrin has been reported to be 25-30 timesmore efficient than HPD." Thus, a porphyrin designed with desired functional groups,charge and symmetry, could exhibit better photosensitizing abilities and accumulation attumor sites than I-IPD, and these are the rationales for studying the chemistry ofporphyrins as photosensitizers. In addition, chlorin compounds, which strongly absorb redlight, can be easily made from porphyrins.236Chapter 1 Introduction1.3.3 Porphyrins used in tumor detectionAlthough HPD shows some promise for the detection of tumors based on thegreater uptake in tumors and fluorescent properties, the phototoxicity of this drug is aproblem in diagnosis. A non-toxic, less phototoxic, and more fluorescent drug would bepreferable.24 The development of such new drugs, probably porphyrins or relatedcompounds, is likely to depend on synthesis of new porphyrins.Motivated by the accumulation of porphyrin compounds at tumors, researchershave used manganese(III) complexes of porphyrins as contrast agents for magneticresonance imaging (MRI), by employing the paramagnetic properties, the lowphototoxicity (transition-metalloporphyrins are usually not photoactive), and tumorlocalization of these compounds. The most used compound in these studies isMn[Tet(SPh)P].25 With this agent, the detection of tumors by MRI can be effectivelyenhanced.25 The Mn[PhT(SPh)P] complex is reported to be a better contrast agent thanMn[Tet(SPh)P].261.3.4 Porphyrins used in chemotherapyPorphyrins are also of interest as tumor localizing agents for boron neutroncapture therapy (BNCT) and chemotherapy. BNCT is a potential therapeutic technique forcancer treatment using a 1°B-containing drug which can accumulate in a tumor and the 1°Bcan be "activated" by thermal neutron radiation. The substantial advantage of BNCT overPDT is that neutrons have far greater penetrability than photons and thus can be used totreat deep-seated malignant cells.27 Boronated porphyrins referred to as SBK-II andBOPP (both contain a carborane linked to natural porphyrins) appear to be promisingdrugs.287(CH2)40^\\/—N+h\\^H3CCH3/—\\N+ —CH3—/Chapter! IntroducdonPorphyrins containing the well-known chemotherapeutic drugs, 5-fluorouracilFigure 1.2a),29 dichloroethylenediamineplatinum(II) (Figure 1.2b)29 and bleomycin (Figure1 .2c shows one example of a related compound)3° have been synthesized for use asanticancer agents.a^ bNa03S Na0 SSO Na^SO NaC, CH3/ N4'OAcFigure 1.2. Porphyrins containing anti-cancer agents.1.3.5 Porphyrins as radiosensitizersCompared to use in PDT, porphyrins have not been as well studied asradiosensitizers. Although the tumor selectivity of porphyrins has drawn the attention ofradiobiologists, early studies on natural porphyrin free-bases suggested their use asradiosensitizers was not promising.313328Chapter 1 IntroductionBased on the findings in various laboratories on the importance of the presence ofa nitro group in a radiosensitizer (Section 1.4), the present thesis work focusing onsynthesis of nitro-substituted porphyrins was initiated. During this thesis work, someencouraging results of synthetic porphyrins as radiosensitizers were published by O'Haraand coworkers.1° In this report, synthetic instead of natural porphyrins andmetalloporphyrins were tested. The compounds tested were either cationic or anionicporphyrin 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(MPOPTet(tMAPh)PTet(SPh)PTet(CPh)PSOf^Tel(SPhPOPtMAPh = 4-trimethylammoniumRhenylCPh = 4-carboxylatoRhenylSPhPh = 4-(4-sulfonatoRhenyl)RhenylFigure 1.3. Structures of some water-soluble porphyrins.9Chapter 1 IntroductionAssessment as radiosensitizers was done in Chinese hamster fibroblast (V79N)cells. All the free-base porphyrins tested had essentially no sensitization in this system. Thegreatest 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.331.4 RadiosensitizersRadiotherapy is an important form of treatment for cancer. The radioresistance ofsome tumor cells is the main limitation of this therapy. The very efficient cell proliferationassociated with malignant tumor growth reduces oxygen concentration, especially in theareas away from vascular capillaries, and this is believed to be one cause of theradioresistance.34 One approach for overcoming radioresistance of hypoxic (02-deficient)cells is the use of chemical radiosensitizers. A radiosensitizer is a drug which can enhanceradiation kill of the tumor, especially for hypoxic cells.There are findings that sensitizers operate via several mechanisms besides theoxygen-mimicking mechanism which is derived from early thoughts about oxygensensitization (see below). Thiol suppression, DNA-repair suppression and effects on DNAconformation 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 cellsprotects them from radiation. Drugs bound to glutathione or inhibiting its synthesis canenhance the killing effect of radiation.36,37 DNA molecules damaged by radiation can berepaired in the cells. Drugs such as platinum complexes, which can bind to DNA and mayinhibit such repair processes, can also sensitize cells, particularly at low doses ofradiation.38 This thesis work focuses upon oxygen-mimicking radiosensitization, which isdescribed below (Sections 1.4.1-1.4.4).1 0irradiationsensitizationchapter 1 Introduction1.4.1 MechanismsWhen irradiation is applied to a biological molecule such as DNA, a series ofreactions can occur, as shown in Figure 1.4.39,413 The radiation produces a chargeseparation, and the electron migrates to an "electron-affinic" site on the molecule andforms a dipolar molecule. The degree of damage due to radiation is related to thecompetition between ionization and charge recombination of the dipole molecule. Thedamage is fixed by the process where a proton dissociates from the cation to give a freeradical. If a sensitizer S, which is an "electron-affinic" compound (dioxygen or itsmimicking compound) is present, it can react with the dipolar molecule to give a cationicradical, and this increases the damage. This "free radical mechanism" proposed by Adam'sgroup39'4° is supported by the results of ESR studies on the free radicals generated by fastmixing pulse radiolysis.41,42 There are other possible mechanisms, such as the indirectmechanism, 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 availableto react with the target molecules or enhances the damage produced by the radicals.43 Thesensitizer (e.g. 02) is also possibly involved in reaction with the biological free radicalsproduced by radiation (either through direct or indirect mechanism), and this can furtherfix the damage.44chargerecombinationionizationS + e— (og)H (aq)free radicalFigure 1.4. A proposed sensitization mechanism (direct, S = radiosensitizer).11Chapter 1 Introduction1.4.2 Properties of radiosensitizersOne of the criteria for effectiveness of radio sensitizers is the SER value (sensitizerenhancement ratio), which is defined as the ratio of radiation doses required to produce agiven effect in the absence and presence of the drug:SER — dose without drug , for equal effect.dose with drugThe SER for dioxygen is called the OER (oxygen enhancement ratio). A high SER for adrug at a clinically achievable dose is desirable, but it does not have to be as high as theOER value (typically about 3 for mammalian cell lines at 1% survival).45"Electron affinity", referring to the ability of a compound to mimic the sensitizingaction of oxygen by accepting electrons, is an important property of a radiosensitizer asshowed by the proposed sensitization mechanism in Section 1.4.1.46 Of theradiosensitizers developed, the majority are nitro-aromatic compounds which do havesuch electron-accepting properties.46The ability to accumulate in a tumor, although not essential for a radiosensitizer, isa property which should be considered in the development of a new drug.35 It willobviously help to increase tumor kill and reduce side-effects such as toxicity andsensitization of normal tissues. The partition property of a drug, related to lipophilicity andsolubility in water, can affect the distribution of the drug in the body, tumor selectivity, theability to penetrate into the center of the tumor, and the ability to enter the cells. Theseproperties in turn can affect the sensitivity to radiation and toxicity. 47,48 Water-solubledrugs 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 inthe area adjacent to the target molecules. If a drug can be targeted to the required12Chapter 1 Introductionmolecules, then a lower drug dose can be used. Thus side-effects can be reduced. Metalcomplexes have been used as the carriers to target radiosensitizers to DNA.49,5° Besidesthe use of metal complexes to target DNA, metal-containing drugs which have cross-linking abilities and possibly inhibit the DNA repair, may also be of clinical benefit.38Although there are insufficient data to show how molecular symmetry of a drug isrelated to the drug's ability to associate with a specific biomolecule (which is alwaysasymmetric) and to the drug's selectivities to types of tumors, symmetry must play a rolein the selectivity of a drug toward biological targets.Other properties such as low toxicity, stability under clinical conditions, and easeof synthesis, are also obviously important.1.4.3 Nitroimidazole radiosensitizersOne of the first drugs used in clinical trials was misonidazole (Figure 1.5), whichwas found to be an effective radiosensitizer,51 but results have shown the use of this drugis limited by its neurotoxicity.35 This may be related to the lipophilic properties of themateria1.52,53 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 lesstoxic than misonidazole.54 A promising aspect of this drug is that it shows a significantdegree of selectivity toward human tumor tissues due to accumulation in low pHenvironments.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, anddecrease neurotoxicity, is showing some promising effects in on-going clinical trials.57Related to the development of radiosensitizers, drugs aiming to kill hypoxic cells intumors are also under study. Recently, many such bioreductive drugs (more toxic in13^A ?HNCH2CHCH2N-^\^^\ OHNCH2CHCH2OCH3Chapter 1 Introductionhypoxic conditions than in oxic conditions) have been developed. These drugs are reducedinto more toxic forms via metabolic processes in hypoxic cells."Some dual functional radiosensitizers have also been developed. These combineradiation sensitizing and alkylating properties in the same structure. One example of thiscategory is RSU 1069, which combines a 2-nitroimidazole with an aziridine group; thelatter reacts with DNA, phosphate or glutathione to form ring-opened products in theside-chain.59,60 In vitro and in vivo studies show that this drug is more toxic to hypoxiccells than to oxic cells (a good bioreductive agent), and more efficient than misonidazoleas a radiosensitizer.6° Unfortunately, this drug is too toxic for clinical use, but a secondgeneration "prodrug" (RSU-6145, an analog with the aziridine ring open) is promising.61NO2^ NO2MisonidazoleI^1^0INC H 2C N HC H 2C H 20HNO2Etanidazole(SR-2508)Pimonidazole(Ro-03-8799)OH ^\^OHN 02RSU-1069^ RSU4145NNO2Figure 1.5. Structures of some radiosensitizers.All the drugs mentioned thus far in this section are nitroimidazole compounds, andmost radiosensitizers fall into the category of nitro-aromatics. This class of compounds, asmentioned above, is also being evaluated as hypoxic cytotoxins (bioreductive agents).62Besides nitro-aromatics, metal-containing complexes as radiosensitizers are ofspecial interest to this thesis work. Cis-DDP [cis-diamminedichloroplatinum(11)] has been14Nstructure of nitracrineChapter 1 Introductionknown since 1969 to have anti-tumor activity,63 and it was shown(e.g.64) that platinumcomplexes bind to DNA. The DNA binding abilities of platinum complexes suggested arationale for the use of such species as radiosensitizers, and indeed Richmond and Powersreported in 1978 that cis-DDP sensitized the spores of Bacillus megaterium to radiation.65Since this time, many platinum complexes have been studied as radiosensitizers withdifferent systems.38,48 The mechanism of the sensitization is still not clear: it could occurby inhibition of DNA repair through the binding of the complexes to DNA,66 or by thereaction of the complexes with radiation protectors present in biological systems.67Another example of a metal-containing sensitizer is cis,cis,cis-RuC12(DMS0)2(4-nitroimidazole)2, which was studied with the rationale of using the DNA binding abilitiesof such a metal complex to lead a radiosensitizer to a DNA site, and thereby increase theefficiency.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 agentsAs described in the last section, some nitro-imidazoles have been found to beselectively toxic to hypoxic cells. Other nitro-aromatics have also been found to havesimilar properties. An example of this class of compounds is nitracrine (figure below),62 anitro 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 asbioreductive reagent. It has been found to be selectively toxic to hypoxic cells68 andsensitizing hypoxic cells to radiation.69/- \(H3C)2N^NH NO215Chapter 1 Introduction1.6 The design of porphyrins as anti-cancer agentsMost 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 inside-effects or even induce secondary malignancies.72 Solutions may lie in the use of drugswhich preferentially accumulate in tumors (or hypoxic cells in tumors), and then sensitizetumor cells to radiation efficiently (as radiosensitizers) or are toxic to these cells (hypo)dccytotoxins). There is a strong motivation to develop such drugs using porphyrincompounds because these compounds are known to have low toxicity, 10,72 to accumulatein tumor,9,24,17 and to sensitize cells to ionizing radiationlo (Section 1.3). Besides theseproperties, porphyrin compounds bind to DNA,73,74 and can be modified into nitro-aromatic compounds which are found to be radiosensitizers and bioreductive agents(Sections 1.4 and 1.5). Porphyrins offer considerable flexibility in their organic andinorganic chemistry so that they can be modified in order to build-in some desirablefunctionalities. The idea for this thesis project was that specially designed porphyrincompounds could have useful anti-cancer properties. To design porphyrin compounds asradiosensitizers, several factors have to be considered, and these are discussed below.Although the design of porphyrin radiosensitizers is the major topic of this chapter, mostof these considerations would also be relevant to the design of bioreductive agents andother anticancer agents.1.6.1 Electron affinityResearchers who are developing radiosensitizers and bioreductive agents based onnitro-aromatics, especially nitro-heterocyclic compounds, consider that such compoundshave high electron affinities. 46 In this respect then, nitro groups could be introduced intothe porphyrin structures (which are heterocyclic aromatics) in order to contribute a16Chapter 1 Introductionreducible 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 anobvious aim was to synthesize water-soluble porphyrins containing nitro groups.Introducing metal ions into porphyrin systems is another way to introduce areducible function in order to increase electron affinity.10 In this case, transition-metal ionswith higher oxidation states should be considered, e.g. Co(III), Fe(III) and Mn(III), butother metal ions could also be considered; e.g., Zn[Tet(MPy)P] has been reported to havea relatively high SER.101.6.2 Selectivity toward tumorsChanges in porphyrin structures can affect the tumor-accumulating property of thedrugs. This was first demonstrated by Winkelman17 with the comparison of the tumor-accumulation abilities of HPD and Tet(SPh)P. It was found that the ratio of HPDconcentration in tumor and in muscle was less than 2, and that ratio of Tet(SPh)P wasabout 7. Studies on tetrahydroxophenylporphyrins (Figure 1.6)1' also demonstrated theeffect of changes in porphyrin structures on tumor-accumulating property. The para- andmeta-derivatives were found to have better selectivity than HPD (for accumulation attumor vs. skin, which is very important for PDT therapy). The meta-derivatives were alsofound to be much more potent than HPD.18 Another example of the effects of porphyrinstructure on drug distribution was reported in 1987 by Kessel and coworkers9, whodetermined, with in vitro studies, that sulfonated derivatives of TetPhP [TetP(SPh)P, cis-DPhB(SPh)P, trans-DPhB(SPh)P, PhT(SPh)P, Tet(SPh)P] (the structures are shown inFigure 1.7), had different sites of photosensitization. The most lipophilic drug of the fivederivatives, TPh(SPh)P, catalyzed lethal photodatnage mainly at intracellular loci, whilethe major phototoxic effect of the other drugs occurred at cell membranes. Studies oftumor localization of these drugs in vivo showed that they all had tumor preference, but17R =OH^para-derivativeHOFigure 1.6. Structures of tetrahydroxophenylporphyrins.NH^NOHmeta-derivativeortho-derivativeR 1 0NH^NR5HNR 20Chapter 1 IntroductionPhT(SPh)P and cis-DPhB(SPh)P were partitioned into neoplastic cells while the otherswere partitioned into tumor stroma. The efficiencies of photosensitization were alsodifferent for various porphyrins. Similar phenomena were found to be the case forsulfonated derivatives of phthalocyanines.77 The existing data are insufficient todemonstrate which structural changes would result in better selectivity, but the data haveindicated generally that selectivity changes on modification of the structures. A balance ofhydrophilic and lipophilic character, the symmetry of the structure, and the properties ofthe substituents, are all considered to play a role in selectivity.R5 = RI 0 = Ri5 = Ph^R20 = SPh^ TPh(SPh)PR5 = R10 = Ph^R15 = R20 = SPh^cis-DPhB(SPh)PR5 = R15 = Ph R10 = R20 = SPh trans-DPhB(SPh)PR5 = Ph^Rio = Ri5 = R20 = SPh^PhT(SPh)PR5 = R10 = R15 = R20 = SPh^Tet(SPh)PFigure 1.7. Structures of sulfonated derivatives of TetPhP.18Chapter 1 Introduction1.6.3 Cell uptakeIt is generally believed that radiosensitizers have to enter cells in order to beeffective.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 bevaried by introduction of substituents with charges or higher polarity (vs. non-polarsubstutuents).1.6.4 DNA-bindingDNA molecules are the main targets of radiation damage,78 and thus the efficiencyof radiosensitizers could be increased by improving their DNA-binding abilities.Poiphyrins are known to be DNA binders, and the binding models and the relationships ofstructures with the binding models have been reviewed.73,79 In the design of porphyrinswith improved DNA-binding abilities, molecular symmetry and presence of groups withcapacity to form a hydrogen-bond, such as -NH2, -NO2, -OH and pyridyl, should beconsidered.1.6.5 Solubility and lipophilicityThe porphyrin core under neutral conditions is essentially a lipophilic structure. Tomake water-soluble porphyrins, ionic substituents have to be introduced, and the ones thatcan be easily introduced are methylpyridinium, trimethylphenylaminium, phenylsulfonate,and phenylcarbonate. When increasing water-solubility of a porphyrin structure, itslipophilicity has also to be considered, because this plays a key role in cell uptake.9 Thusthe water-solubility and lipophicility should be "balanced". From this point of view, thelipophicilities 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 ionic19Chapter 1 Introductiongroups. Fewer than four ionic groups in a structure of a porphyrin is suggested to be oneof the criteria for design of porphyrins as radiosensitizers.1.6.6 Multiple functionsTo synthesize porphyrins for use in radiosensitization, all of the factors discussedabove and some others (such as stability, toxicity, economy, etc.) have to be considered.One potential approach was to introduce several different functional groups into onestructure, in which the functions might be "balanced" (e.g water solubility vs.lipophilicity), although it was not clear which structure would be most beneficial. Thissuggested an approach in which a large number of porphyrins was to be synthesized inorder to find an effective drug. Thus the methodology employed to synthesize theporphyrins had to be simple yet powerful enough to make a large variety of products.1.7 Porphyrin synthesesThe synthesis of porphyrin free-bases has been well reviewed in variouspublications.80,81 The most convenient synthesis pathway is the monopyrrole-condensationmethod, which often gives symmetric products. The synthesis of TetPhP5 is an example: CHO+43/202+ 7H20Many other symmetric porphyrins with four substituents of the same kind are also madewith this method,82 including the so called "picket fence" porphyrins which were designedto mimic the bulky protein structure surrounding the heme active center (Figure 1 .8a).8320Chapter 1 IntroducdonTo make a porphyrin with two different aryl substituents at the four meso-positions with this method requires the use of two different aldehydes. Such a synthesiscan lead to 6 different porphyrin products. This so called mixed aldehyde approach wasfirst developed by Little et al." Since then (1975), many interesting unsymmetricporphyrins have been made.85,86 These include: the "looping over" porphyrins with a side-arm providing a fifth intramolecular coordinating ligand (Figure 1.8b), in order to mimicdeoxy-hemoglobin and -myoglobin in which iron has a fifth coordination site occupied byan imidazole of a histidine amino acid residue of the protein chain," a Zn complex of5, 1 0, 1 5-tris(4-carboxylatopheny1)-20-(44 1 -bromobutylpyridinium))porphyrin (Figure1.8c) which has been used in a study of redox systems for photochemical utilization ofsolar energy;88 a bifunctional metalloporphyrin (Figure 1.8d) which has been made from5,10,15-tris(4-toly1)-20-(4-pyridyl)porphyrin, with the pyridyl coordinated to a Rucomplex moiety in order to give a second redox-active center.89Sari et al., using column chromatography, separated the 6 porphyrins shown inFigure 1.8e which were formed by condensation from a mixture of pyrrole, benzaldehydeand 4-pyridylcarboxaldehyde; the pyridyl nitrogen atoms were then methylated to makethe porphyrins water-soluble in order to do DNA-binding studies." The details of thiswork were published during the course of this thesis work."The condensation of three different aldehydes with a pyrrole, in principle, couldgive a mixture of 21 porphyrins. The difficulty in the separation of such a mixture makesthe method of little use for the synthesis of porphyrins with more than two different mesosubstituents. In fact, no porphyrins with three different meso substituents had been madeprior to this thesis work.21C4 H8 BrC 0 2CH3CH3H3C\^(-1.4H3c ci H3^CNH^_,O H3C`;, CH3CO c-- 3 \VICO\ I^CHNH3cy-13c, C H3NH^Ico^/ /NHRiu (N H 3 )5 3 4"CH3Chapter 1 Introductiona1R= R10 = R15 = R70 = PhR5 =Rin=1115 =PhR5 = R10 = PhR5 =R15 =PhR5 = PhR70 = PyR15 =R7.0 =PyR10 = R70 = PyR10 =R15 =R20=PyR5=R10=R15=R20=PyTetPhPTPhPyPcis-DPILBPyPtrans-DPhBPyPPhTPyPTetPyPFigure 1.8. Structure of some synthetic porphyrins.22Chapter I IntroductionOther methods of porphyrin synthesis, such as the coupling of dipyrroles orcyclization of a tetrapyrrole, are for making porphyrins with more complicatedstructures," but the difficulties in preparing the intermediates make the methods generallyless useful for the concerned purpose.To make a large variety of porphyrins especially with different substituents, twomethods are used in this thesis work: (a) monopyffole-condensation and (b) directmodification on an isolated porphyrin. The latter method introduces a functional groupinto a synthesized porphyrin structure. Sulfonation of TetPhP is an example of amodification, which converts a non water-insoluble porphyrin (TetPhP) into a water-soluble one [Tet(SPh)P].92 Modification can be done in a controlled manner, and differentporphyrins can be synthesized at different stages of modification. For example, thesulfonation of TetPhP can be stopped at one, two, three or four sulfonations; with properpurification 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 bemade, and the 5 porphyrins indeed have different biological activities.9 Nitration is anotherexample of such modification. Although TetPhP has been known for many years, itsnitration was reported only recently.76 According to this report, nitration of TetPhPyielded (NPh)TPhP as the main product. Reduction of the nitro group into amine, as asecond modification, can be done after nitration, in order to synthesize a porphyrin with aperipheral amine.76,93 A third modification, such as sulfonation, can then be undertaken tomake the amine-containing, water-soluble porphyrin (Figure 1.9).76Although there are some examples of modifications on synthetic porphyrins asmentioned above, modification as a method to produce a large variety of porphyrins hadnot been well developed before this project. Many more porphyrins can been synthesizedusing the methodology of combining monopyrrole-condensation with subsequent23N H 2H2 SO4\^HN\^/0NH^N0y^(APh)TPhPNH2NH^NN^HN/0-,,--^TetPhP/Chapter 1 Introductionmodification, as described herein. A small fraction of possibilities was chosen as the focusof this thesis.Modifications of porphyrins from many possible starting materials, such as the 6compounds made from the condensation of pyrrole, benzaldehyde and 4-pyridylcarboxaldehyde, plus metallation can make a large variety of porphyrins withdifferent "functional groups", and a major part of this project addresses the syntheses andcharacterizations of these porphyrins and metalloporphyrins. Figure 1.10 shows some ofthe possible modification schemes for one of the the six porphyrins addressed above,5,10,15-tripheny1-20-(4-pyridyl)porphyrin (TPhPyP). Nitrations can been performedFigure 1.9. Modifications of TetPhP.24Chapter 1 Introductionalso on two or all three of the phenyl groups of this porphyrin; these nitrations and thefollowing reduction, sulfonation and methylation are not shown. A complete scheme of themodifications of the six porphyrins performed in this thesis work is presented in Figure2.1, in Chapter 2.Metallation of these porphyrins is another area included in this project. Theimportance of introducing metals into radiosensitizers was previously discussed inSections 1.3 and 1.4. The chemistry of some metalloporphyrins is discussed in Chapter 3.1.8 In vitro studies on synthetic porphyrinsThe term "in vitro" refers to "in cells" and the term "in vivo" refers "in animals" inthis 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 vivostudies, and clinical trials. In vitro studies are usually sensitive, rapid and economical, andthey can be used to predict some properties of drugs. Prior to in vivo studies and clinicaltrials, which give better predictions for success but are more difficult to do and moreexpensive, it is useful to know how toxic a compound is toward a particular cell line, andhow effectively the compound sensitizes these cells to radiation. The proliferative capacityof a cell is examined using survival curves are generally used to screen new compounds asbioreductive agents, radiosensitizers or photosensitizers. The principles and themethodology of using these assays to evaluate the toxicity (in oxic or hypoxic conditions),radiosensitization and photosensitization ability of a compound are introduced inChapter 5.25HNO3, N.......,,, ...,7,-... .„...,'.1■11-1^N/--- N^HN---\\ 1[H ]N °2NMe3+cis-(MAP)(MPy)DPhPt\_- NH N\H2NH^N\7---- N^HNCHChapter 1 Introductioncis-(NPh)DPhPyPNH^N---/-03S/^NO2\^HNr--- NI\ //0^cis-(NPh)PyB(SP)PSO3---■cis-(APh)DPhPyPikH204N.0^cis-(AP)PyB(SPh)PYSO3-Figure 1.10. Some possible modifications of TPhPyP.26Chapter 1 Introduction1.9 The objectives of this thesis workA limitation of radiation therapy is the lack of selectivity toward tumors, especiallytoward the hypoxic cells in a tumor. PDT has good selectivity toward tumors but has alimitation from poor tissue penetration of the light. The purpose of this project was toexploit the possibility of synthetic porphyrin drugs which accumulate in tumors andsensitize cells to radiation. Using this kind of drug, some limitations of radiation therapyand PDT can potentially be overcome. The synthetic chemistry and the porphyrinssynthesized also may be beneficial to the eventual discovery of new drugs asphotosensitizers, hypoxic cytotoxins or tumor imaging agents. This thesis work focusesmainly on the synthesis and characterization of some designed porphyrins andmetalloporphyrins, although some preliminary in vitro studies of these compounds asradiosensitizers or photosensitizers are also presented. The work is described within thefollowing 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 andcopper) (Chapter 3).3. Behavior of the synthesized porphyrins in aqueous solutions (Chapter 4).4. In vitro studies: radiosensitization, photosensitization, and toxicity of thesynthesized porphyrins and metalloporphyrins (Chapter 5).5. General conclusions and recommendation for future work (Chapter 6).27Chapter I IntroductionReferences-Chapter 11^"The Porphyrins", Vols. VI and VII, D. Dolphin, ed., Academic Press, New York, 1978.2^D.L. Drabkin, in 'The Porphyrins", Vol. 1, D. Dolphin, ed., Academic Press, New York,1978, p.35.3^H. Fischer and K. Zeike, Justus Liebigs Ann. Chem., 468, 98 (1929).4^H. Fischer and B. Walach, Justus Liebigs Ann. Chem., 450, 164 (1926).5^P. Rothemund, J. Am. Chem. Soc., 61, 2912 (1939).P. Rothemund and A.R. Menotti, J. Am. Chem. Soc., 63, 267 (1941).6^H.H. Inhoffin, J.H. Fuhrhop, H. Voigt and H. Brocicmarm, Jr., Justus Liebigs Ann. Chem,695, 133, (1966).7^"The Porphyrins", Vols. I-VII, D. Dolphin, ed., Academic Press, New York, 1978.8^R. Bonnett, in "The Porphyrins" Vol. I, D. Dolphin, ed., Academic Press, New York, 1978,p.2.9^D. Kessel, P. Thompson, K. Saatio and K.D. Nantwi, Photochem. Photobiol., 45, 787 (1987).10^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).11^A. Harriman and G. Porter, J. Chem. Soc. Faraday Trans. 2, 75, 1532 (1979).R.F. Pastemack, P.R. Huber, G. Engasser, L. Francesconi, E. Gibbs, P. Fasella, G.C.Venturo and L. deC. Hinds, J. Am. Chem. Soc., 94, 4511 (1972).12^H. Auler and G. Banzer, Z Krebsforsch, 53, 65 (1942).13^R.L. Lipson, E.J. Baldes and A.M. Olsen, J. Natl. Cancer Inst., 26, 1(1961).14^(a) D.A. Bellnier and B.W. Henderson, in " Photodynamic Therapy-Basic Principles andClinical Applications", B.W. Henderson and T.J. Doughty, eds., Marcel Dekker, NewYork, 1992, p 117.(b) J.R. Shulok, M.H. Wade and C. Lin, Phoyochem. Photobiol.,51, 451, (1990).(c) M.R Quastel, A.M. Richter and J.G. Levy, Br. J. Cancer, 61, 687 (1990).(d) D.A. Bellnier, Y. Ho, R.K. Pander, J.R. Misser and T.J. Dougherty, Phoyochem.Photobiol.,50, 221, (1989).(e) P.J. Bugelski, C.W. Porter and T.J. Doughty, Cancer Res., 41, 4606 (1981).28Chapter I Introduction(f) C.J. Gomer and T.J. Dougherty, Cancer Res., 40, 146 (1979).15^Q. Peng, J. Moan, M. Kongshaug, J.F. Evensen, H. Anholt and C. Rimington, Int. I Cancer,48, 258 (1991).16^J. Moan and K. Berg, Phoyochem. Photobiol.,55, 931, (1992).17^J.W. Winkelman, in "Methods in Porphyrin Photosensitization", D. Kessel, ed., Plenum Press,New York, 1985, p.91.18^M.D. Berenbaum, S.L. Akande, R Bonnett, H. Kaur, S. Ioannou, RD. White andU.J. Winfield, Br. J. Cancer, 54, 717 (1986).19^J.A. Mercer-Smith, D.A. Cole, J.C. Roberts, D. Lewis, M.J. Behr and D.K. Lavallee,Advances In Experimental Medicine and Biology, 258, 103 (1989).20^R.K. Pondey, D.F Majchrzycki, K.M. Smith and T.J. Dougherty, in "PhotodynamicTherapy: Mechanisms", T.J. Dougherty, ed., SPIE, Bellingham, 1989, p.164.21^R. Pottier and T.G. Truscott, Int. J. Radiat. Biol., 50, 421 (1986).22^T.J. Dougherty in "Advances in Photochemotherapy", T. Hasan, ed., SPIE, Bellingham,1988, p.2.23^Bonnett and M. Berenhaum, in "Photosensitizing Compounds: Their Chemistry,Biology and Clinical Use", John Wiley & Sons, Chichester, 1989, p.40.24^A.E. Profio and O.J. Balchum, in"Methods in Porphyrin Sensitization", D.Kessel, ed.Plenum Press, New York, 1984, p.43.25^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).Emestus, L.J. Wilmas and M. Hoehem-Berlage, Clin. Exp. Metastasis, 10, 345 (1992).D.A. Place, Pi. Faustino, K.K. Berglunans, P.C.M. van Zijl, A.S. Chesnick and J.S. Cohem,Investigation Radiology, 25, S69 (1990).26^RJ. Fiel, D.A. Musser, E.H. Mark, R Mazurchuk and J.J. Alletto, Magn. Reson. Imaging,8, 255 (1990).27^S.B. Kahl, M. Koo, B.H. Laster and R.G. Fairchild, Strahlenther. Onkol. 165,134, (1989).28^J.S. Hill, S.B. Kahl, A.H. Kaye, S.S. Stylli, M. Koo, M.F. Gonzales, N.J. Vardaxis and29Chapter 1 IntroductionC.I. Johnson, Proc. Natl. Acad. Sci. USA, 89, 1785 (1992)29^Y.Sun, A.E. Martell, D. Chen, RD. Macgarlane and C.J. McNeal, J. Heterocyclic Chem.,23, 565 (1986).30^L. Ding, G. Etemad-Moghadam S. Cros, C. Auclair and B. Meunier, J. Med. Chem., 34,900 (1991).L. Ding, C. Casas, G. Etemad-Moghadam and B. Meunier, New J. Chem, 14, 421 (1990).L. Ding, G. Etemad-Moghadam and B. Meunier, Biochemistry, 29, 7868 (1990).G. Etemad-Moghadam, L. Ding, F. Tadj and B. Meunier, Tetrahedron, 45, 2641 (1989).31^J. Moan and E.O. Pettersen, Int. J. Radial. Biol., 40, 107 (1981).H.P. Mack, W.K. Diehl, G.C. Peck and F.H.J. Figge, Cancer, 10, 529 (1957).32^S. Schwartz, M. Keprios, J. Modelevslcy, H. Freyholiz, R. Walters and L. Larson, in"Diagnosis and Therapy of Porphrias and Lead Intoxication", M. Doss, ed.,Springer-Verlag, Berlin, 1978, p.227.33^J. North, R. Coombs and J. Levy, in "Photodynamic Therapy and Biomedical Lasers",P.Spinelli, M. Dal Frante and R. Marchesini, eds., Elsevier Science Publishers, 1992, p.103.D.W. Dixon, L.G. Marzilli and R. Schinazi, Ann N. Y. Acad. Sc., 616, 511 (1990).34^L.H. Gray, A.D. Conger, M. Ebert, S. Hornsey and O.C.A. Scott, Br. J. Radio!., 26,638 (1953).35^G. Adams and I.J. Stratford, in "Radiobiology in Radiotherapy", N.M. Bleehen, ed.,Springer-Verlag, London, 1988, p.153.36^J.E. Biag,low, M.E. Varnes, E.P. Clark and E.R. Epp, Radiat. Res., 95, 437 (1983).37^K.A. Skov and S.MacPhail, Int. J. Radiat °mot Biol. Phys., 22, 533 (1992).38^K.A. Skov and S. MacPhail, int J. Radiat. Oncol. Biol. Phys., 20, 221 (1991).K.A. Skov, M. Korbelik and B. Palcic, Int J. Radiat. Oncol. Biol. Phys., 16, 1281 (1989).M. Korbelik and K.A. Skov, Radiat Res., 119, 145 (1989).N.P. Farrell and K.A. Skov, J. Chem. Soc. Chem. Commun., 1987, 1043;39^G.E. Adams and M.S. Cooke, int. .1. Radiat. Biol., 15, 457 (1969).40^G.E. Adams, in " Radiation Protection and Sensitization", H.L. Moroson and30Chapter 1 IntroductionM. Quintiliani, eds., Taylor and Francis, London, 1970, p.1.41^G.E. Adams and B.D. Michael, in "Energetics and Mechanisms in Radiation Biology",G.O. Phillips, ed., Academic Press, New York, 1968, p.333.42^Radiello and M. Tamba, in "Radiosensitizers of Hypoxic Cells", A. Breccia, C. Rimondiand G.E. Adams, eds., Elsevier/North-Holland Biomedical Press, Amsterdam, 1978, p.75.43^L.H. Gray, Radiat. Res., 1, 189 (1954).44^T. Alper, Radiat. Res., 5, 573 (1956).45^B. Palcic, J.W. Brosing and L.D.Skarsgard, Br. J. Caner, 46, 980 (1982).46^G.E. Adams, E.D. Clarke, I.R. Flocichart, R.S. Jacobs, D.S. Seluni, I.J. Stratford,P. Wardman, M.E. Watts, J. Parrick, R.G. Wallace and C.E. Smithen,Int. J. Radiat. Biol., 35, 133 (1979).47^"Absorption and Distribution of Drugs", T.B. Binns, ed., Livingstone, London, 1964.48^S.H. Curry, "Drug Disposition and Phannacokinetics", Blackwell, Oxford, 1977.49^N. P. Farrell, "Transition Metal Complexes as Drugs and Chemotherapeutic Agents",Kluwer Academic Publishers, Dordrecht, 1989.50^P.K.L. Chan, BR James, D.C.Frost, P.K.H. Chan, H.L. Hu and K.A. Skov, Can. J. Chem.,67, 508 (1989).51^J.C. Asquith, M.L. Watts, K.B. Pattel, C.E. Smithen, G.E. Adams, Radiat. Res., 60,108 (1974).52^J.M. Brown and P. Workman, Radiat. Res. 82, 171 (1980).53^C. Clarke, KB. Dawson, P.W. Sheldon and I. Ahmed, Mt. J. Radiat. Oncol. Biol. Phys.,8, 787 (1982).54^M.V. Williams, J. Denekamp, A.I. Minchinton and M.R.L. Stratford, Br. J. Cancer,46, 127 (1982).55^J.T. Roberts, N.M. Bleehen, P. Workman and M.I. Walton, Mt. J. Radiat. Oncol. Biol. Phys.,10, 1755 (1984).56^S. Dische, M.I. Daunders, M.H. Bennett, E.P. Dunphy, C. Des Rochers, M.RL. Stratford,A.I. Minchinton and P. Wardman, Br. J. Radio!., 59, 911 (1986).57^D. Chassagne, I. Charreau, H. Sancho-Gamier, F. Eschwege and E.P. Malaise, hit. J. Radiat.31Chapter 1 IntroductionOncoL Biol. Phys., 22, 581 (1992).58^G. Adams, I.J. Stratford, E.M. Fielden and M.A. Naylor, in "Radiation Research:A Twentieth-Century Perspective" J.D. Chapman, W.C. Dewey and G.F. whitmore,eds., Academic Press, San Diego, 1991, p.76.59^G.E. Adams, I. Ahmet, P.W. Sheldon and I.J. Stratford, Br. J. Cancer, 49, 571 (1984).60^G.E. Adams, I. Alunet and P.W. Sheldon, I.J. Strafford, Int. J. RadiaL OncoL Biol. Phys.,10, 1653 (1984).I.J. Stratford, P. O'Neill, P. W. Sheldon, A.R.J. Silver, J.M. Walling and G.E. Adams,Biochem. PharmacoL, 35, 105 (1986).61^T.0 Jenkins in "The Chemistry of Antitumour Agents", D.E.V. Wilma'', ed., Blacicie,Glasgow, 1990, p.342.62^W.A. Denny in "The Chemistry of Antitumour Agents", D.E.V. Wilman, ed., Blacicie,Glasgow, 1990, p.l.63^B. Rosenberg, L. VanCamp, J.E. Trosko and V.H. Mansour, Nature, 222, 385 (1969).64^S.E. Sherman, D. Gibson, A.H.J. Wang and S.J. Lippard, Science, 230, 412 (1985), andreferences therein.65^R.C. Richmond and E.L. Powers, Radiat. Res., 68, 215 (1978).66^K. Lindquist, E.B. Douple and R.C. Richmond, Radiat. Res., 91, 408 (1982).67^E. Smith and W.D.B. Johnson, Int. J. RadiaL OncoL Biol. Phys., 10, 1803 (1984).68^W.R. Wilson, W.A. Denny, S.J. Twigden, B.C. Baguley and J.C. Probert, Br. J. Cancer, 49,215 (1984).69^W.R. Wilson, W.A. Denny, G.M. Steward, A. Fenn and J.C. Probert, Int. I Radiat. OncoLBiol. Phys., 12, 1235 (1986).70^S. Dische, in "Radiobiology in Radiotherapy", N.M. Bleehen, ed., Springer-Verlag,London, 1988,p.165.71^H. Bartelink, A.C. Begg, L. Dewit and F.A. Stewart, in "Radiobiology in Radiotherapy",N.M. Bleehen, ed., Springer-Verlag, London, 1988, p.177.72^T.J. Dougherty, Photochem. Photobiol., 45, 879 (1987).32Chapter 1 Introduction73^R.J. Fiel, J. Biomol. Struct & Dynamics, 6, 1259 (1989).74^J.A. Strickland, L.G. Marzilli, K.M. Gay and W.D. Wilson, Biochemistry, 27, 8870 (1988).R.F. Pastemack, E.J. Gibbs and J.J. Villafranca, Biochemistry, 22, 2406 (1983).75^L. Gong and D. Dolphin, Can. J. Chem., 63, 401 (1985).76^W.J. 1Cruper, Jr., T.A. Chamberlin and M. Kochanny, J. Org. Chem., 54, 2753 (1989).77^J.E. van Lier and J.D. Spikes, in "Photosensitizing Compounds: Their Chemistry, Biologyand Clinical Use", John Wiley & Sons, Chichester, 1989, p.17.78^"Radiobiology for the Radiologists", J.B. Lippincott, ed., Pholadelphia, 1988.K.A. Skov and N.P. Farrell, Int. J. Radiat Biol., 57, 947 (1990).79^R.F. Pastemack and E.J. Gibbs, in "Metal-DNA Chemistry", T.D. Tullins, ed., Am. Chem.Soc., 1989, p.59.80^"The Porphyrins", Vol. I, Chap. 3-5, D. Dolphin, ed., Academic Press, New York, 1978.T.P. Wijesekera and S. David, J.B. Paine DI, BR James and D. Dolphin, Can. J. Chem.,66, 2063 (1988).81^T.P. Wijesekera and D. Dolphin, in "Methods in Porphyrin Photosensitization", D. Kessel,ed., Plenum Press, New York, 1984, p.229.82^J.L. Nian, L. Min and H.A. Kong, Inorg. Chim. Acta, 178, 59 (1990).L.S. Lindsey and R.W. Wagner, J. Org. Chem., 54, 828 (1989).D.W. Thomas and A.E. Martell, J. Am. Chem. Soc., 78, 1335 (1956).83^J.P. Collman, 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).84^R.G. Little, J.A. Anton, P.A. Loach and J.A. Ibers, J. Heterocyclic Chem., 12, 343, (1975).R.G. Little, J. Heterocyclic Chem., 18, 129, (1981).85^J.A. Anton and P.A. Loach, J. Heterocyclic Chem.,12, 573 (1975).J.A. Anton, J. Kwong and P.A. Loach, J. Heterocyclic Chem.,13, 718 (1976).33Chapter 1 Introduelion86^Y. Sun, A.E. Martell and M. Tsutsui, J. Heterocyclic Chem., 23, 561 (1986).87^L. Salmon, C. Bied-Charreton, A. Goudemer, P. Moisy, F. Beclioui and J. Devynck,Inorg. Chem., 29, 2734 (1990).88^Y. Kinumi and I. Okura, Inorg. Chim. Acta, 153, 77 (1988).89^D. Franco and G. McLendon, Inorg. 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).34Chapter 2 Synthesis of porphyrinsChapter 2 The synthesis of the designed porphyrins2.1 IntroductionThe synthesis and characterization of porphyrin free-bases are the main subject ofthis chapter. Porphyrins are synthesized with the monopyrrole-condensation method andsubsequent modifications as discussed in Section 1.7. Condensation of pyrrole,benzaldehyde and 4-pyridinecarboxaldehyde gives a mixture of six porphyrins with generalformula of PhnPyo_roP. The mixture is separated by column chromatography. Subsequentmodifications as required by the design of the structures (Chapter 1) of the resultantporphyrins are: nitration of phenyls; reduction of the nitro groups to form amines;sulfonation of phenyls; and methylation of pyridyls. The synthetic schemes of the designedpoiphyrins are shown in Figure 2.1. The full names and the corresponding structures forthe abbreviations given in Figure 2.1 can be found in the List of Abbreviations. In thischapter, only the porphyrin free-bases are involved, so the abbreviations of porphyrins areused to represent the porphyrin free-bases.Purification of non-water-soluble porphyrins was mainly performed with columnchromatography. For water-soluble porphyrins, dialysis was employed as the majorpurification process. Details can be found in the experimental section.Proton NMR spectroscopy was the main method used for characterization ofporphyrins, especially for distinguishing isomers. Elemental analysis and UV-visiblespectroscopy were also used to characterize all the porphyrins. Further characterizationwas carried out using mass and infrared spectroscopies for some of the porphyrins, andthe data are presented in Sections 2.3.4 and 2.3.5. Aggregation has been found to be ageneral property of all water-soluble porphyrins, and is discussed in Chapter 4.35(NPh)WyPPhTPyPChapter 2 Syntheds of porphyrinsTetPhP ^• (NPh)TPhP^(APh)TPhP^PPOT(SPOP^ c-B(NPh)DPhP- -111- c-B(APh)DPhP^c-BPPWSPOPt-B(NPh)DPhP-^t-F3(APh)DPhP^1-09910SPh,^ T(NPh)PhP^T(APh)PhPI"" Tel(SPOPTPhPyP c-(NPh)DPhPyP^c-(APh)DPhPyPc-B(NPh)PhPyP I c (/P03,9(5P47t-B(NPh)PhPyP^t-B(APh)PhPyP T(NPh)PyP PyISPOPc-DPhBPyPt-DPh8PyP ^c-(NPh)Ph3PyP^c-(APh)PhRPyPc-B(NPh)BPyPt-B(NPh)RPyP^ c-B(44)8(VPOPc-85,8(SPOP^ c-8400,0P4I)1-8(4110,90POP1-8Py81SPO9^ /-80/9)DPhP1404,02.h.,1°Iriii/9/)PhPTetPyP ^ • 7009y)PFigure 2.1. Synthetic schemes for designed porphyrins(c = cis, t = trans, new_p_om1=,1^water-soluble porphyrin)36Chapter 2 Synthesis of porphyrins2.2 Experimental2.2.1 Materials and methodPyrrole, benzaldehyde, 4-pyridinecarboxadehyde, methyl p-toluene sulfonate werepurchased from Aldrich Chemical Company. Other chemicals (including, nitric acid,sulfuric acid, etc.) were purchased as analytical grade, and used without furtherpurification. Anion (CO exchange resin (Amberlite IRA-402) was purchased from BDH.Molecular porous membrane tubing (molecular weight cut off = 1,000) was purchasedfrom 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 withmoisture and had lower activity, approximately activity HI) and silica gel were purchasedfrom BDH.NMR spectra were recorded on a Varian XL-300 or a Bruker WH-400 instrumentoperating 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 552Aspectrometer, with quartz cells (0.100 or 1.00 cm). Mass spectroscopy (El ; NH3 chemicalionization 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 byMr. 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 molecularporous membrane tubing (3 cm wide) was rinsed with distilled water, then one end of it37Chapter 2 Synthesis of porphyrinswas closed with a piece of string. The porphyrin sample was dissolved in a minimumamount of distilled water (2 - 10 mL), and the resultant solution was transferred into thetubing; the other end of the tubing was then closed with string. This dialysis "bag" wasthen put into the beaker containing distilled water (-800 mL) and the contents were stirredwith a magnetic stir bar. The water outside the dialysis bag was changed to distilled water2 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 aRotovap. Acetone was then added to the residue, which was then scraped off the flaskwall; the suspension was filtered and the solid was collected and 6r-dried.2.2.2 The porphyrins with a general formula of PhnPyo-oPThese poiphyrins, comprising TetPhP, T'PhPyP, cis-DPhBPyP, trans-DPhBPyP,PhTPyP and TetPyP (see Figure 2.1), have four meso substituents which are phenyl and/orpyridyl. The porphyrins were synthesized from condensation of pyrrole, benzaldehyde and4-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 hotpropionic acid (250 mL) in a 500 mL round-bottom flask, and the mixture was refluxedfor 1 h. A precipitate (0.55 g) was filtered off from the cooled down reaction mixture. Thefiltrate was concentrated on a Rotovap to about 50 mL and then acetone (500 mL) wasadded with stirring; the mixture was filtered to yield another 1.78 g precipitate, which wascombined 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 ona column (5 x 30 cm) of silica gel. The solution of TetPhP and TPITyP was eluted offwith CHC13 (— 1.5 L) successively; the mixture of the other four porphyrins was eluted offfrom this column using a mixed solvent (CHCI3 and methanol 9:1). This solution of theporphyrin mixture was evaporated to dryness, and then the residue was dissolved with a38Chapter 2 Synthesis of porphyrinsminimum volume of CHC13 (— 150 mL), loaded on a column (3 x 70 cm) of aluminaactivity 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 wasforced 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 aluminacontaining porphyrin were washed on fret filters using a mixed solvent to dissolve theporphyrins (10% Me0H in CHC13 , ca. 0.2 L for each portion) . The bands in order fromtop to bottom of the column contained TetPyP, PhTPyP and cis-DPhBPyP. All of thesolutions of porphyrins were washed with 5% Na2CO3 solution and then twice withwater; the organic phases were evaporated to dryness on a Rotovap. Acetone was addedto the residues, and these were filtered and air-dried.TetPyP was also synthesized by the condensation of pyrrole and4-pyridinecarboxaldehyde (1:1) in propionic acid as in the procedure described above. Themain portion of the propionic acid having been evaporated off first, then the porphyrinproduct was precipitated by adding acetone; this precipitate was then filtered, washed withacetone 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. Datafrom NMR and UV-visible spectra are shown in Tables 2.2 and 2.3. The hydration ofsome of the porphyrins is discussed in Section 2.3.1.1; the assignments of the NMRsignals are discussed in Section 2.3.2. Spectral data from IR and mass spectroscopies arepresented in Sections 2.3.4 and 2.3.5, respectively.39Chapter 2 Synthesis of porphyrinsTable 2.1. Elemental analyses of the PhnPy(4-n)P porphyrinsC (%) H (%) N (%) Yield (%)TetPhP expected 85.99 4.89 9.12 7.5found 85.86 4.82 8.95TPhPyP expected 83.90 4.72 11.38 9.0found 83.81 4.69 11.34trans-DPhBPyP.H20 expected 79.48 4.76 13.23 1.6found 79.66 4.64 13.12cis-DPhBPyP^a expected 81.84 4.55 13.64 4.3found 78.40 4.70 12.74PhTPyP^a expected 79.73 4.40 15.87 2.0found 77.13 4.38 15.09TetPyP^a expected 77.67 4.23 18.12 0.7found 76.55 4.88 16.45TetPyP.1/2H20^b expected 76.56 4.56 17.67 25.0found 76.56 4.34 17.86a: 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 mLof mixed solvent was passed through this column, and the elute was evaporated to dryness in a flask on aRotovap. A white residue was observed on the wall of the flask.b: Sample was synthesized from condensation of pyrrole and 4-pyridinecarboxaldehyde.40Chapter 2 Synthesis of porphyrinsTable 2.2. 11I-NMR data for the PhnPy(4-n)P porphyrins (a)pyrrole 3,4,5-Ph 2,6-Ph 3,5-Py 2,6-Py N-pyrroleTetPhP 8.85 s(8) 7.78 m(12) 8.23 m(8) - - -2.78 s(2)TPhPyP 8.90 d; 8.87 s 7.78 m(9) 8.23 m(6) 9.04 q(2) 8.18 q(2) -2.80 s(2)8.81 d (2,4,2)trans- 8.91^d;^8.81d 7.80m(6) 8.22m(4) 9.05q(4) 8.18q(4) -2.84s(2)DPhBPyP (4,4)cis- 8.92 d; 8.88 s 7.80m(6) 8.22m(4) 9.05q(4) 8.18q(4) -2.84s(2)DPhBPyP 8.85 s; 8.81 d(2,2,2,2)PhTPyP 8.93 d; 8.86 s 7.81 m(3) 8.22 m(2) 9.06 m(6) 8.18 m(6) -2.88 s(2)8.83 d (2,4,2)TetPyP 8.88s(8) - - 9.08 q (8) 8.19q(8) -2.92s(2)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 4TetPhP 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.41Chapter 2 Synthesis of porphyrins2.2.3 NitrationsThe nitration reactions were performed with concentrated nitric acid in Schlenktubes. 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 theSchlenk tube was stoppered; the stopcock on the side-arm was left open to avoid build-upof pressure, and the water-bath was removed. The color of the reaction mixture changedfrom purple to green when either sulfuric acid or nitric acid was added. The ratio of thereactant chemicals used depends on the porphyrin and the intended degree of nitration.The reaction time depends on the porphyrin and the degree of nitration. Thenitration was monitored by TLC (silica gel or alumina).Samples for TLC taken from the reaction mixture werefirst neutralized with a solution of Na2CO3 and extractedwith CHC13; then the CHC13 solutions were introducedonto TLC plates, which were developed using CHC13. Theretention time of a porphyrin increased with the number ofnitro groups.When the intended nitration was completed, coldwater (about the same amount as the reaction mixture)was added slowly to quench the reaction (heat wasproduced when water was added). The hot mixture wascool by an ice-bath and neutralized firstly withconcentrated 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).42Chapter 2 Synthesis of porphyrinsThe product was extracted with CHC13 (100 - 200 mL), the CHC13 portion was washedwith water three times (— 100 mL each time), and the CHC13 layers was evaporated todryness on a Rotovap. The residues was washed with acetone (— 50 mL), filtered anddried.Nitration usually gave a mixture of products, and column chromatography wasused to separate them. The compositions of the reaction mixtures, reaction times, detailsabout column chromatography and other experimental conditions for each porphyrin aredescribed below.(NPh)TPhP [5-(4-nitropheny1)-10,15,20-triphenylporphyrin]. TetPhP (0.5 g) wasdissolved in acetic acid (50 mL), and concentrated nitric acid (20 mL) was added withstirring. Reaction was monitored with TLC (silica gel, 0.2 cm). When no TetPhP could bedetected on a TLC plate (reaction time was 1 h), the reaction was stopped and the crudeproduct was collected as described above. This product was passed through a column ofalumina (activity IR 3 x 10 cm) with CHC13 as eluant to separate out some of the non-porphyrin materials, which remained on the top of the column. The eluted solution wasevaporated to dryness on a Rotovap, and 0.51 g crude product was collected; this wasloaded onto a column of alumina (activity I, 3 x 30 cm), eluted with CHC13, when threebrown bands were observed. After a trace of TetPhP was eluted, solutions of (NPh)TPhPand B(NPh)DPIT were collected successively. The solutions were evaporated on aRotovap 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 and5,15-bis(4-nitropheny1)-10,20-diphenylporphyrin]. All the conditions were the same asabove, except that the reaction was stopped when no (NPh)TPhP could be detected byTLC on silica gel (-5 h reaction time). After a trace of (NPh)TPhP was eluted,43Chapter 2 Synthesis of porphyrinsB(NPh)DPhP was eluted from a column of alumina (activity I, 3 x 30 cm). The bis-nitroproduct (0.40 g, 70% yield) was collected. Nitration of two of the phenyl groups inTetPhP gave a mixture of cis and trans products, as judged by 1H NMR (see Section2.3.2). 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 conditionswere the same as described above for the synthesis of (NPh)TPhP, but the reactionmixture 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% yieldvia the major eluted band from an alumina column (activity I, 3 x 30 cm). Traces ofB(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 for7 days under the conditions used for the synthesis of (NPh)TPhP) damaged the porphyrinstructure. Purification of this porphyrin, isolated as by-product from the synthesis ofT(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, pyrroleprotons; doublets at 8.63 and 8.38 ppm for nitrophenyl protons; and singlet at -2.84 ppmfor 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 thereaction 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 waseluted by CHC13 from a column of alumina (activity DI, 3 x 30 cm); the solution of cis-44Chapter 2 Synthesis of porphyrins(NPh)DPhPyP was collected, washed with water, and the CHC13 layer was separated andevaporated 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-(4-pyridypporphyrin] and trans-B (NP)PhPyP [5,15-bis(4-nitropheny1)-10-pheny1-20-(4-pyridyl)porphyrin]. All the conditions and precursor were the same as described above forthe 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)PyPwere eluted from a column of alumina (activity lg 3 x 30 cm). The cis and trans isomerswere observed as the two major bands on the column, but were collected together becausepoor separation (ca. 60% yield). A smaller amount (20 mg) of this mixture was separatedusing a longer column (alumina active a 2 x 40 cm), the trans isomer being eluted beforethe 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 reactionconditions and the precursor were again the same as described above for the synthesis ofcis-(NPh)DPhPyP, but the reaction was stopped when a significant amount of T(NPh)PyPcould be detected by alumina TLC (40 h reaction time). After traces of. cis and trans-B(NPh)PhPyP were eluted by CHC13, T(NPh)PyP was eluted out with 10% Me0H inCHC13 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 thisporphyrin 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 stoppedwhen no trans-DPhBPyP and no mono-nitration product could be detected by alumina45Chapter 2 Synthesis of porphyrinsTLC (24 h reaction time). The solubilities of the crude product (70% yield) in CHC13 orCH2C12 were low. The crude product looked fairly pure by TLC and 111 NMR data. Aportion of this crude product (20 mg) was purified on a column of alumina activity IV (2 x40 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 trans-B(NPh)BPyP, except that cis-DPhBPyP was used as the precursor and no sulfuric acidwas used (i.e. 0.20 g porphyrin, 30 mL acetic acid and 10 mL nitric acid). The reactionwas stopped when no cis-DPhBPyP could be detected by alumina TLC (20 h reactiontime). The crude product, isolated from the reaction mixture, was separated with a columnof 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] . Theconditions were exactly the same as the ones given for the synthesis of trans-B(NPh)BPyP, but cis-DPhBPyP was used as precursor. The reaction was stopped whenno cis-(NPh)PhBPyP could be detected by alumina TLC (24 h reaction time). The crudeproduct was separated with a column of alumina (activity IV, 2 x 40 cm). After a trace ofcis-(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 theconditions were the same as given above for the synthesis of trans-B(NPh)BPyP, butPhTPyP was used as precursor. The reaction was stopped when no PTPyP could bedetected by alumina TLC (2 days reaction time). The pure product (-60% yield) wasseparated with a column of alumina (activity IV, 2 x 40 cm) from a green impurity.46Chapter 2 Synthesis of porphyrinsElemental analyses (for samples stored and weighed out in air) of the nitratedporphyrins are shown in Table 2.4. The 1H NMR and UV-visible data are given in Tables2.5 and 2.6, respectively. Spectral data from IR and mass spectroscopies are presented inSections 2.3.4 and 2.3.5, respectively. The hydration of some of the porphyrins isdiscussed in Section 2.3.1.1; the assignments of the NMR signals are discussed in Section2.3.2.2.2.4 Reductions of the nitro-porphyrinsReductions were performed according to the method reported in the literature.2,3,4Generally, the porphyrin (0.1 g) was dissolved in CHC13 (20 mL) in a 250 inL round-bottom flask with magnetic stirring; acetic acid (30 mL) and then a solution of SnC12 (0.3g in 30 mL concentrated HCI) were added. The mixture was stirred and refluxedovernight, and then was neutralized with concentrated NaOH and dilute Na2CO3 solutionsto 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 (100inL) 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 crudeproduct was purified on a column of alumina (activity IV, 2 x 30 cm), using —250 mLCHCI3 or a solvent mixture of Me0H and CHC13 as eluant. The yield was above 90% forall the porphyrins.To synthesize cis and trans-B(APh)DPIIP [5,10-bis(4-aminopheny1)-15,20-diphenylporphyrin and 5,15-bis(4-aminopheny1)-10,20-diphenylporphyrin], a mixture ofcis and trcms-B(NPh)DPhP was used as the precursor, because this mixture had not beenseparated (see Section 2.2.3). A separation of the two reduced isomers on a column of47Chapter 2 Synthesis of porphyriesTable 2.4. Elemental analyses for the nitroporphyrinsC^% H^% N^%(NPh)TPIT expected 80.12 4.40 10.62found 80.03 4.46 10.63B(NPh)DPhP (mixture) expected 75.00 3.98 11.93found 74.96 3.95 11.79T(NPh)PhP.1/2 H20 expected 69.66 3.69 12.93found 69.51 3.85 12.82cis-(NPh)DPhPy13-1/2 H20 expected 77.20 4.33 12.56found 77.46 4.22 12.58cis-B(NPh)PhPyP.H20 expected 71.37 4.01 13.55found 71.51 3.90 13.61trans-B(NPh)PhPyP-1-120 expected 71.37 4.01 13.55found 71.74 3.80 13.61T(NPh)PyP^a expected 68.25 3.44 15.61found 63.95 4.35 11.45trans-B(NPh)BPyP expected 71.38 3.70 15.85found 71.19 4.00 15.52cis-(NPh)PhBPyP 1/2.H20 expected 75.66 4.18 14.40found 75.22 4.21 14.63cis-B(NPh)BPyP•H20 expected 69.61 3.87 15.48found 69.49 3.72 15.18(NPh)TPyP 1/2•20 expected 73.33 4.05 16.68found 73.20 4.09 16.78a: Sample was contaminated by alumina (see footnote a under Table 2.1).48Chapter 2 Synthesis of porphyrinsTable 2.5. 1H NMR data for the nitroporphyrins (a)pyrrole 2,6-Ph3,4,5-Ph3,5-Py2,6-Py3,5-NPh2,6-NPhN-pyrrole(NPh)TPhP 8.90 d; 8.87 s; 8.74 d 8.22 m (6) - 8.64 d (2) -2.80 s (2)(2,4,2) 7.78 m (9) 8.41 d (2)B(NPh)DPhP h 8.90 d; 8.86 s; 8.77 s; 8.73 t 8.21 m (4) - 8.64 m (4) -2.83 s (2)(5/2, 3/2, 3/2, 5/2) 7.80 m (6) 8.38 m (4)T(NPh)PhP 8.91 d; 8.79 s; 8.75 d 8.19 m (2) - 8.63 d (6) -2.83 s (2)(2, 4, 2) 7.99 m (3) 8.38 d (6)cis- 8.89 d; 8.86 s; 8.82 d 8.20 m (4) 9.04 q (2) 8.62 d (2) -2.88 s (2)(NPh)DPhPyP 8.79 d; 8.76 d; 8.73 d 7.77 m (6) 8.15 q (2) 8.37 d (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) -2.87 s (2)B(NPh)PhPyP 8.79 d; 8.78 s; 8.75 d 7.78 m (3) 8.14 q (2) 8.37 m (4)(2, 1, 1, 1, 2, 1)trans- 8.92 d; 8.84 d; 8.79 d; 8.75 8.19 m (2) 9.04 d (2) 8.63 m (4) -2.85 s (2)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) -2.87 s (2)8.15 d (2) 8.38 d (6)trans-^ c 8.86 d;^8.80 d - 9.05 m 8.65 m -2.90 sB(NPh)BPyP 8.14 m 8.38 mcis- 8.90 d; 8.83 d; 8.83 s 8.19 q (2) 9.04 q (4) 8.63 m (2) -2.84 s (2)(NPh)PhBPyP 8.80 d; 8.78 d; 8.74 d 7.77 m (3) 8.14 q (4) 8.38 m (2)(2, 1, 2, 1, 1, 1)cis- 8.86 m; 8.80 m (4,4) - 9.05 d (4) 8.65 d (4) -2.90 s (2)B(NPh)BPyP 8.14 d (4) 8.38 d (4)(NPh)TPyP 8.82 m; 8.77 d (6,2) - 9.02 d (4) 8.61 d (2) -2.99 s (2)8.10 d (4) 8.34 d (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.49Chapter 2 Synthesis of porphyrinsTable 2.6. UV-visible data for the nitroporphyrins (a)Soret visible 1 visible 2 visible 3 visible 4(NPh)TPhP 418.0 (418) 515.3 (17.8) 550.5 (8.7) 589.3 (6.4) 646.6 (5.3)B(NPh)DPIT 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)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 minimumones.50Chapter 2 Synthesb of porphyrinsalumina (CHC13 as eluant) was achieved. The syntheses of the isomers of cis and trans-B(APh)PhPyP also were started with a mixture of cis and trans-B(NPh)PhPyP as theprecursor, and the isomers were again separated after reduction to the amino derivativesusing a column of alumina (CHC13 as eluant). The separation of the reduced isomers waseasier than the separation of the precursor bis(nitrophenyl) isomers. The trans isomer waseluted from the column before the cis one in both cases.Samples of cis-(APh)Phl3PyP was purified on a column of alumina using a mixedsolvent as eluant (10% Me0H in CHC13), because these porphyrins having more polarsubstituents (two pyridyls and one or two aminophenyls) could not be eluted off usingCHC13. All the other amino porphyrins were purified on an alumina column using CHC13as eluent.The (APh)TPyP compound was synthesized from (NPh)TPyP, but only the 'IINMR spectrum was recorded. A pure sample of this compound was not obtained becauseits 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 andUV-visible data are shown in Tables 2.7, 2.8 and 2.9, respectively. The hydration of someof the porphyrins is discussed in Section 2.3.1.1; the assignments of the NMR signals arediscussed in Section 2.3.2. The abbreviations are derived as described in Section 1.2 fromthe full names which can be found in the List of abbreviations.51Chapter 2 Synthesis of porphyrinsTable 2.7. Elemental analyses for the aminoporphyrinsC^% H^% N^%(APh)TPhP expected 83.94 4.92 11.13found 83.81 4.83 10.98cis-B(APh)DPhP.H20 expected 80.86 5.05 12.86found 80.79 5.13 12.87trans-B(APh)DPhP•H20 expected 80.86 5.05 12.86found 81.06 5.13 12.60T(APh)Ph1P.1/2 H20 expected 79.04 5.09 14.67found 79.34 5.23 14.58cis-(APh)DPhPyP.H20 expected 79.63 4.94 12.96found 79.63 4.91 12.59trans-B(APh)PhPyP expected 77.83 4.98 14.78found 77.89 5.03 14.46cis-(APh)PhBPyP^a expected 79.87 4.60 15.53found 75.78 4.49 14.56a: Sample contaminated by alumina (see footnote a under Table 2.1).52Chapter 2 Synthesis of porphyrinsTable 2.8. 111 NMR data for the aminoporphyrins (a)pyrrole 2,6-Ph3,4,5-Ph3,5-Py2,6-Py2,6-APh3,5-APh-NH2 N-pyrrole(APh)TPhP 8.93 d(2); 8.82 m(6) 8.25 m(6) - 7.98 d(2) 4.00 s(2) -2.78 s(2)7.74m(9) 7.05d(2)cis- 8.91 m(4); 8.80 m(4) 8.20q(4) - 7.98d(4) 4.00 s(4) -2.74s(2)B(APh)DPhP 7.74 m(6) 7.04 d(4)trans- 8.91d(4); 8.81d(4) 8.20q(4) - 7.98d(4) 4.00 s(4) -2.73s(2)B(APh)DPhP 7.75 m(6) 7.04 d(4)T(APh)PhP 8.89s(6); 8.77d(2) 8.19q(2) - 7.97d(6) 4.01 s(6) -2.73s(2)7.73m(3) 7.04d(6)cis- 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)8.83 d(1); 8.78 d(2)trans- 8.98-8.67m(8) 8.19d(4) 9.01 d(2) 7.97d(4) 4.05s(4) -2.80s(2)B(APh)PhPyP 7.75 m(6) 8.15 d(2) 7.06 d(4)cis- 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)8.81 s(2); 8.77 d(2)(APh)TPyP12 9.00 d; 8.81 s - 9.03 m 7.97 d 4.05 s -2.85 s8.79d 8.14m 7.08da: In CDC13 at room temperature; chemical shift in ppm signal pattern (number of protons).b: A dilute solution sample resulting from low solubility.53Chapter 2 Synthesis of porphyrinsTable 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)cis-(APh)PhBPyP b 419.8 (308) 515.4 (16.4) 552.7 (8.4) 590.0 (5.6) 648.3 (3.8)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 theminimum ones.54Chapter 2 Synthesis of porphyrins2.2.5 SulfonationsSulfonation reactions were performed using a modified literature procedure.s Thesynthesis procedure for each of the porphyrins used in this work was about the same, andis described generally here as shown by the following scheme:Porphyrin + H2SO4green paste100°c(4-12h)(”i H 2 0green solutionNaOH(2) Na 2 CO 3purple solution(3)evaporated"t cooledpurple solution + purple precipitate(4) filteredpurple solution^purple precipitate\ombined washed with Me0H\rrple solution^yellow precipitate(inorganic salts)purple solution + precipitate(6)  ifilteredpurple solution^purple precipitateto dryness^b\combined^washed with Me0H(7) evaporatedpurple solution^yellow precipitate(inorganic salts)purple solutionevaporated(9) to drynesscrude productyellow precipitate(inorganic salts)(5) Me0H addedpurple residue(porphyrin + inorganic salts)(8) washed with Me0H55Chapter 2 Synthesis of porphyrinsThe porphyrin (-0.50 g) was mixed well with concentrated sulfuric acid (10 mL) ina 50 mL round-bottom flask. (1) The flask was stoppered and heated in a oil-bath at 100-110°C for 4-12 h (see below). After the reaction the mixture cooled down to roomtemperature, ice cold water (50 mL) was added slowly. (2) The mixture was cooled by anice-bath and carefully neutralized with concentrated NaOH and dilute Na2CO3 to pH = 8-9. (3) After being concentrated by evaporation to about 20 inL, the solution was cooled inan ice-bath, when a precipitate of inorganic salts (mainly Na2SO4) formed. (4) Theprecipitate was filtered off and washed with methanol (50 inL). (5) To the filtrate(aqueous solution and methanol solution combined), 100 mL methanol was added, whenmore inorganic salt precipitated. (6) This mixture again was filtered (the precipitate wasdiscarded) and then (7) the filtrate was evaporated to dryness on a Rotovap. (8) Thepurple residue (which was a mixture of porphyrin product and inorganic salt) was washedwith methanol until only a light yellow solid remained. (If the purple color of the residuecould not be washed out by methanol, then the residue was dissolved into a minimumamount of hot water, reprecipitated out with methanol, and then filtered.) (9) The purplemethanol solution was evaporated to dryness on a Rotovap, and the purple residue wascollected 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), andthe solution was filtered to remove some insoluble impurity. To the filtrate, acetone (-200inL) was added with stirring; a brown precipitate formed which was filtered off and air-dried. 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 andvery hygroscopic. Another form of the product was made by evaporation of aqueoussolutions of the brown material to dryness. Bright purple flakes resulted and there werecollected; this material was essentially not hygroscopic.56Chapter 2 Synthesis of porphyrinsFor the other sulfonated porphyrins, the crude products were either purified bydialysis or by repetitive methanol-acetone reprecipitation (3 times). Products purified bydialysis were collected by evaporation of the aqueous solutions and were essentially nothygroscopic. The actual purification method used for each porphyrin is indicated in Table2.10. The overall yields of the reactions following purification of the products were 70-80%.Reaction of the nitro precursor (NPh)TPhP with hot sulfuric acid for 30 min gavethe amino product Na3(APh)T(SPh)P instead of the expected nitro productNa3(NPh)T(SPh)P; the yield of this porphyrin product was about —40%, which wasrelatively low compared to the yield of sulfonation of (APh)TPIT (80%). The amineproduct was identified by its 1H NMR spectrum (identification of the porphyrins by 1HNMR 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 trans-B(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 wereidentified by 1H NMR as B(NPh)B(SPh)P (the major product), (APh)(NPh)B(SPh)P andB(APh)B(SPh)P (the 1H NMR data are not listed in Table 2.11; the spectra of the threecompounds are shown in Figure 2.2, p.'77, instead). The separation of the cis and trans-isomers of these three products using silica gel columns failed. These products were onlyisolated in sufficient amounts for samples for NMR spectra, because of the low yield andthe difficulty of their separation.However, sulfonation of cis-(NPh)DPhPyP, another nitro porphyrin, for a 6 hreaction time, gave the sulfonated nitro porphyrin Na2[cis-(NPh)PyB(SPh)13] as the mainproduct, although product distribution varied with reaction time (Section 3.3.1). The57Chapter 2 Synthesis of porphyrtoscrude product from this sulfonation reaction was dissolved in methanol (-30 mL), loadedon a column of silica gel (3 x 30 cm), and the column was eluted by a mixture of CH2C12and 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)Pwere synthesized via the porphyrin precursors (APh)TPhP, cis and trans-B(APh)DPhP inhigher 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 befound in the List of Abbreviations. The naming of the counterion (Na+) is omitted in thisthesis when the emphasis is on the porphyrin structure. The hydration of the porphyrins isdiscussed in Section 2.3.1; the assignments of the NMR signals are discussed in Section2.3.2 and the 1H NMR data are given in Table 2.11.The UV-visible spectra of the sulfonated porphyrins in aqueous solution arecomplicated because of aggregation of the porphyrins, which is more fully discussed inChapter 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 2.3.5.58Chapter 2 Synthesis of porphyrinsTable 2.10. Elemental analyses for the sulfonated porphyrinsC % H % N% S%Na3[(APh)T(SPh)P].2H20^A expected 54.31 3.30 7.20found 54.51 3.06 7.40Na2[cis-B(APh)B(SPh)P].2H20^A expected 59.73 4.10 9.41found 59.87 3.85 9.50Na2[trans-B(APh)B(SPh)P).3H20^A expected 58.93 4.02 9.38found 59.13 4.31 9.43Na4[Tet(SPh)P].10H20^h expected 43.93 3.83 4.66found 43.65 3.70 4.39Na2[cis-(NPh)PyB(SPh)P].4H20^h expected 55.31 3.64 9.00found 55.60 3.95 8.76Na3[PyT(SPh)P].2H20^A expected 53.91 3.13 7.31found 53.84 3.36 7.20Na1[PyT(SPh)P].11H20^h expected 46.87 4.36 6.36found 46.63 4.21 6.23Na3[PyT(SPh)P].2H20^h and C. expected 53.91 3.13 7.31 10.03found 53.91 2.92 7.22 10.10Na2[cis-BPyB(SPh)P] - 1 OH20^h expected 50.40 4.60 8.40 6.40found 50.04 4.45 8.41 6.70Na2 [trans-BPyB(SPh)11.7H20^h expected 53.28 4.23 8.88 6.77found 53.45 4.25 8.66 6.65a: 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 undernitrogen.59Chapter 2 Synthesis of porphyrinsTable 2.11. 1H NMR data for the sulfonatoporphyrins (a)pyrrole 2,6-SPh3,5-SPh3,5-Py2,6-Py2,6-APh3,5-APh-NFI7 N-pyrroleNal RAPh)T(SPh)P] 8.99 d(2); 8.85 s(6) 8.18 d(6) - 7.90 d(2) 5.61 s(2) -2.80 s(2)8.06 d(6) 7.04 d(2)No [cis- 8.92 s(2), 8.92 d(2) 8.17 d(4) - 7.87 d(4) 5.55 s(4) -2.80 s(2)B(APh)B(SPh)131 8.79 s(2), 8.78 d(2) i8.05 d(4) 7.00 d(4)Na,[trans- 8.94 d(4); 8.79 d(4) 8.17 d(4) - 7.86 d(4) 5.57 s(4) -2.84 s(2)B(APh)B(SPh)P] 8.03 d(4) 7.00 d(4)Na4[Tet(SPh)P] 8.84 s(8) 8.18 d(8) - - - -2.95 s(2)8.04 d(8)No [cis- 8.87 sb(8) 8.16 d(4) 9.02 d(2) 8.66 d(2) h - -3.01 s(2)(NPh)PyB(SPh)Pl 8.03 d(4) 8.25 d(2) 8.49 d(2)12Nal [PyT(SPh)13] 8.86 m(8) 8.19 m(6) 9.03 d(2) - - -2.97 s(2)8.07 d(6) 8.27 m(2)Na,[cis-BPyB(SPh)P] 8.89 m (8) 8.18 m(4) 9.04 d(4) - - -2.98 s(2)8.05 m(4) 8.28 d(4)No [trans- 8.89 m (8) 8.18 d(4) 9.04 d(4) - - -2.98 s(2)BPyB(SPh)P] 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.60Chapter 2 Synthesis of porphyrinsTable 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) - - - 665 (13.9)trans-B(APh)B(SPh)P 413 (61.7) 491 (65.9) - - 700 (23.0)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)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).61Chapter 2 Synthesis of porphyrins2.2.6 MethylationsThe methylation reaction of TetPyP was carried out according to the literatureprocedure.6 Briefly, TetPyP (0.50 g) was mixed with 50 inL DMF, in a 100 mL round-bottom flask with a magnetic stir bar, and the mixture was heated to almost boiling (whenthe porphyrin dissolved); methyl p-toluene sulfonate (— 2 mL) was added with stirring andthe 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; thisprecipitate was filtered, washed with acetone and air-dried. 95% yield.The porphyrins PhTPyP, (NhP)TPyP, cis and trans-DP1BPyP, and cis and trans-B(NPh)BPyP were methylated under conditions similar to those described above (0.20 gporphyrin, 20 mL DMF, 1 mL methyl p-toluene sulfonate, 4 h). However, the tosylate saltof 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 —10mL; acetone (-100 mL) was then added to the mixture with stirring, and the resultingmixture was filtered; the precipitated purple, tosylate salt of the methylated porphyrin waswashed with acetone (— 20 mL) and dried under vacuum at room temperature. Thesimilarly formed methylated products from cis and trans-DPhBPyP, and cis and trans-B(NPh)BPyP were washed with cold water (— 20 mL) on filtration funnels; and then driedunder vacuum at room temperature.All the tosylate salts were converted to the corresponding chloride salts by passingan aqueous solution of the porphyrin through an anion exchange column (Cl). Theaqueous solutions of the tosylate salts of Tet(MPy)P, T(MPy)PhP and (NPh)TPhP wereobtained 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 trans-B(MPy)B(NPh)P were dissolved in water by stirring these products with hot water (4062Chapter 2 Synthesis of porphyrinsmL for 0.2 g porphyrin, 50-60 °C) with the presence of the Ci ion exchange resin (-2 mLfor 0.2 g porphyrin). The purple aqueous solutions from the anion exchange material wereevaporated to dryness. Acetone (— 10 mL) were added to the purple residues; these werescraped off the wall of the flasks, filtered off and dried under vacuum at 100°C. Theoverall 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 werefurther purified by dialysis because the presence of impurities caused low C, H and Ncontents in the elemental analyses. Analysis of trans-[B(MPy)B(NPh)11C12 gave low C, Hand N content, even after an attempt to purify this porphyrin by dialysis; this failed mainlybecause of the low solubility of this porphyrin in water.The elemental analyses (for samples stored and weighed out in air) and data fromNMR spectra are shown in Tables 2.13 and 2.14, respectively. Discussions about thehydration of the compounds, and the assignments of the 1H NMR signals, are presented inSections 2.3.1.1 and 2.3.2, respectively.The UV-visible spectra of these porphyrins again are complicated because ofaggregation of porphyrins in aqueous solutions, and this is discussed in Chapter 4. Thedata of UV-visible spectra for these porphyrins at the concentration of 1.0 x 10-5 M arepresented 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 nameswhich can be found in the List of abbreviations. All the porphyrins are isolated in the formof a chloride salt.63Chapter 2 Synthesis of porphyrinsTable 2.13. Elemental analyses for the methylpyridiniumporphyrinsC% H% N%[cis-B(MPy)DPhI]C12•5.5H20^a expected 64.73 5.52 10.30found 64.91 5.32 10.14[cis-B(MPy)B(NPh)P]C12•4H20^a expected 60.07 4.55 12.74found 59.86 4.80 12.50[trans-B(MPy)DPhP]C12.4.5H20^a expected 66.17 5.38 10.52found 66.21 5.22 10.55[trans-B(MPy)B(NPh)P]C12-4H20^a expected 60.07 4.55 12.74found 53.50 5.22 10.18[T(MPy)PhIlC13•4H20 expected 62.82 5.23 11.86found 63.15 5.23 11.66[T(MPy)(NPh)P]C13.4H20 expected 59.63 4.86 12.65found 59.72 5.00 12.47[Tet(MPy)P]C14•4H20 expected 59.21 5.16 12.56found 59.52 5.36 12.55a: Sample purified by dialysis.64Chapter 2 Synthesis of porphyrinsTable 2.14. 1H NMR data for the methylpyridiniumporphyrins (!)pyrrole 3,5-NPh2,6-NPh3,5-MPy2,6-MPy2,6-Ph3,4,5,-Ph-CH3 N-pyrrole[cis- 9.12 s(2) - 9.46 d (4) 8.21 d (4) 4.73 s(6) -2.95 s (2)B(MPy)DPhP]C12^129.00 m(4) 9.00 d (4) 7.89 m(6)8.91 s(2)[cis- 9.0 m (8) 8.71 d (4) 9.49 d (4) - 4.72 s(6) -3.02 s (2)B(MPy)B(NPh)P]C12^12 8.51 d (4) 9.00 d (4)[trans- 9.0 m (8) - 9.48 d (4) 8.23 d (4) 4.72 s(6) -2.99 s (2)B(MPy)DPIT]C12^12. 9.03 d (4) 7.90 m(6)[trans- 9.0 m (8) g 8.72 d (4) 9.46 d (4) - 4.70 s(6) -3.02 s (2)B(MPy)B(NPh)11C1?^12 8.50 d (4) 9.02(4) g[T(MPy)PhI9C13 9.00 q (4) - 9.50 d (2) 8.23 d (2) 4.71 s(9) -3.04 s (2)9.20 s (4) 9.03 m(2) 7.90 m(3)[T(MPy)(NPh)1103 9.1 m(8) 8.74 d (2) 9.51 d (6) - 4.74 s(9) -3.06 s (2)8.52 d (2) 9.00 d (6)[Tet(MPy)I1C14 9.2 s(8) - 9.55 d (8) - 4.77 s(12) -3.10 s (2)9.00 d (8) .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.65Chapter 2 Synthesis of porphyrinsTable 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).66Chapter 2 Synthesis of porphytins2.3 Results and discussionThe major goal of the work described in this chapter was to synthesis water-soluble porphyrins. A method of monopyrrole-aldehyde condensation followed bymodification was developed and used in this work. Use of this method allows for thepossible synthesis of many more porphyrins, especially water-soluble ones. Some of thesepossibilities 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 impuritywas found to be alumina from column chromatography used for the porphyrin separations.Further purification on these potphyrins was generally not carried out because theseporphyrins were used as precursors for the synthesis of the water-soluble porphyrins.1H NMR spectroscopy was used as the major method for characterization of theporphyrins. UV-visible, infrared and mass spectroscopies were also recorded. Howeverthe IR and MS techniques were only applied to a limited number of compounds because ofthe limitation in time. The 1H NMR and UV-visible data have been presented in Section2.2 together with the synthesis procedures. The assignments and other features of the 1HNMR signals are discussed in Section 2.3.2. The UV-visible spectra are discussed inSection 2.3.3, while the infrared and mass spectral data are presented and discussed inSections 2.3.4 and 2.3.5, respectively.The retention times of the TLC of the porphyrins are not reported becauseirreproducible 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 thealumina TLC data.67Chapter 2 Synthesis of porphyrins2.3.1 Synthesis2.3.1.1 Hydration of porphyrinsAssociated water molecules (hydration) were generally found in samples of theporphyrin free-bases. For the non-ionic porphyrins, samples were only dried under vacuumat room temperature, and elemental analyses were carried out by handling the compoundsin air. Different degrees of hydration were found in these samples as judged by elementalanalysis and the intensity of the water signals in '1.1 NMR spectra. The hydration isprobably related to the possibility of hydrogen-bonding between associated watermolecule(s) and the pyrrole-N, pyrrole-NH, -NH2, -NO2, or pyridyl moieties in theporphyrin 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. Theporphyrins TPhPyP7 and (NPh)TPyr have been reported in the literature as hydrated withone-half and one water molecule, respectively, while the present work supports zero andone-half water molecule, respectively. Comparison of the elemental analysis data for theother potphyrins to the literature can not be made because elemental analyses of therelevant compounds are not reported in the literature, although (APh)TPyP, T(APh)PhP,T(NPh)PhP and the porphyrins of general formula Ph Pv4-- (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 thoughdifferent purification methods were used by different authors, hydration molecules werealways present except when a "strong" drying process was used. Attempts to remove thehydrated water completely were not made because the handling of the hygroscopic, drysamples in biological experiments was thought to be inconvenient.68Chapter 2 Synthesis of porphyrinsTable 2.16. Reports on elemental analysis of Tet(SPh)P(0)C % H % N % S % Purificationreference.C44H26N4012S4Na4.12H20 42.66 4.04 4.50 10.29 Et0H-1170 recrystallization and(4.54) (10.55) drying at 100 °C 5C44H26N4012S4(NH4)4.9H20 44.23 4.17 9.66 10.80 Me0H-acetone^reprecipitation(44.22) (4.52) (9.38) (10.72) (6 times)1°C44H26N4012S4Na4 51.62 2.54 5.48 12.52 dialysis, and drying at 150°C in(5.17) (12.29) vacuo 11C44H26N4012S4(NH4)4.9H20 44.23 4.17 9.66 10.75 Celite column (pyridine-water-(44.12) (4.09) (9.74) CHC13 eluent) 12C44H26N4012S4Na4.10H20 43.93 3.83 4.66 10.67 dialysis, and drying at 100°C in(43.65) (3.70) (4.39) vacuothis worka: Calculated (found).Hydration was also apparent from the 41 NMR spectra of the compounds. Thewater content in a sample of Na3PyT(SPh)P was measured by 111 NMR spectroscopy inDMSO-d6 as described below.Two NMR tubes, one with and one without the sample (— 2 mg) were sealedunder N2 with septa, and then placed into a large Schlenk tube (3 x 20 cm, see Section2.2.3) with a wide mouth, through which N2 flowed. DMSO-d6 was then injected into theNMR tubes from a syringe under N2, and the NMR spectra of the two samples wererecorded. 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 andsulfonatophenyl (111, 24 protons); and peak of the N-pyrrole protons (H",2 protons).In the spectrum of neat solvent:69Chapter 2 Synthesis of porphyrinswater peak (w); and the solvent peak (s).Then the value of W = W – —w • S represents the relative amount of water in the porphyrinsample. The number of water molecules associated with a porphyrin molecule wascalculated 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 11estimated by elemental analysis (see Table 2.10).The addition of DMSO-d6 into the NMR tubes under N2 was necessary to obtain areproducible result because this solvent absorbed moisture strongly. Errors in thismeasurement could arise also from the low accuracy of the integrations of the fairly widelyspaced peaks; and from the possible differences in the sensitivities of the various protonsbecause of possible differences in relaxation times.For Na3[PyT(SPh)13], two associated water molecules were present in a sampleprecipitated from Me0H by adding acetone, and in a sample formed by evaporation ofaqueous solution with subsequent drying at 100°C under vacuum (Table 2.10). This mayindicate that these two water molecules associate to the porphyrin molecule differentlyfrom the other nine water molecules present in a sample collected from evaporation of anaqueous solution (11 associated water molecules were estimated in this. sample, Table2.10). The two water molecules may be associated with the porphyrin molecule by stronghydrogen-bonding via the pyrrole-N, the pyrrole-NH and/or the pyridyl-N.2.3.1.2 Synthesis of the porphyrins with general formula Phn,,Pv(4)1'The synthesis of these porphyrins have been reported,9 following the proceduredescribed by Little et al.' In the present work, different from the literature procedure, asignificant amount of porphyrin product, besides that precipitated directly from the70Chapter 2 Synthesis of porphyrinsreaction mixture, was collected by precipitation from the concentrated condensation-reaction mixture using acetone, and therefore the yield was higher. For the separation ofthe 6 porphyrins, alumina (activity III) and silica gel chromatographies with CHC13 aseluent were used instead of the reported method, which used silica gel columnchromatography with a mixed solvent of CHC13, acetone and methanol as eluent. All the 6porphyrins could be separated on a single column of the alumina activity M. However, useof silica sel to first separate TetPhP and TPhPyP from the other 4 porphyrins, followed byuse of the alumina column to separate the other 4 porphyrin, was more efficient. There isan advantage of using neat solvent because the solvent can be readily recovered bydistillation and drying.Mass, 111 NMR (in CDC13) and UV-visible (in CH2C12) spectroscopy data of thesepoiphyrins have been reported recently.9 The data obtained from MS (Section 2.3.5) and111 NMR spectroscopy (Table 2.2, p.41) are in agreement with the reported data. Therewere some deviations of the UV-visible data from the reported data; e.g., the extinctioncoefficients 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 thereported values.9 The impurity in samples of PhTPyP and cis-DPhBPyP might result in thelow e values, although some difference may also result from the difference of the solventused (CHC13 was used in this work, while CH2C12 was used in the literature work). TheUV-visible (in CHC13) and Ill NMR (CDC13) spectra of TPhPyP have also been reportedby another group;7 and data obtained in the present work generally agree with those in thisreport, though there is some disagreement about the assignments of the NMR signals (seeSection 2.3.2).2.3.1.3 Synthesis of nitro-porphyrins71Chapter 2 Synthesis of porphyrinsThe nitration reagents used for the nitrations of the porphyrins varied according tothe structures of the porphyrins. Generally, the more pyridyl and nitrophenyl groupspresent, the stronger the reagent needed. For example, the nitration of TPhPyP is moredifficult than that of TPhP. The protonated pyridyl in the reaction medium probablyreduces the activity of the porphyrin for the electronphilic substitution. The first nitrationof TPhPyP gives almost solely the cis-isomer (as judged by 1 NMR, Section 2.3.2), whichmeans that the phenyl trans to the pyridyl is more inert.In the course of this work, the reaction times for nitration were found to dependsignificantly on temperature, which was not controlled (room temperature), and so thereported reaction time varied with room temperature. It was more reliable to monitor thereaction by TLC than to control the reaction time.Strong nitration reagents have been used to nitrate porphyrins in some literaturereports. NO2 in acetone and nitronium tetrafluoroborate were used for the nitration ofOEP, 13514 and fuming nitric acid was used for the nitration of TetPhP.2 All these reagentsare strong oxidants and can probably destroy the porphyrin ring structure, thus giving lownitration yields. Under the reaction conditions used here, NO2 was produced gradually inthe reaction mixture, by using nitric acid mixed with a dehydration reagent such as aceticacid or a mixture of acetic acid and sulfuric acid; and thus relatively mild reactionconditions were provided leading to 60-85% yield from porphyrin precursors.The (NPh)TPIT poiphyrin has been synthesized by direct condensation ofbenzaldehyde, 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 reagentswith 7% yield.8 These low yields probably result from the high activity of 4-nitrobenzaldehyde.15 The (NPh)TPhP compound (55% yield from TetPhP), a mixture ofcis- and trans-B(NPh)DPhP (28% yield) and T(NP)PhP (no yield reported) have also been72Chapter 2 Synthesis of porphyrinssynthesized by nitration of TetPhP using fuming nitric acid.2 The methods used in thisthesis 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 beenreported before.2,3 The spectral data obtained in this thesis work agree with those reportedexcept that the extinction coefficient for the Soret band obtained here (in CHC13) wasabout 25% lower than the reported value (in CH2C12).2 The 'H NMR, UV-visible andmass spectra of the mixture of cis and trans-B(NPh)DPhP have also been reported by thesame group,2 and the thesis data agree well. The synthesis, and 1H NMR and UV-visiblespectra of (NPh)TPyP have been reported recently by another group;8 the extinctioncoefficients 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 (seeSection 2.3.2).2.3.1.4 Synthesis of aminophenylporphyrinsThe (APh)TPIT porphyrin has been synthesized and characterized by elementalanalysis, 1H NMR and mass spectroscopy.2.3 The data for this porphyrin obtained hereagree with those the literature. The other 6 aminophenylporphyrins (see Figure 2.1, p.36)are new compounds.2.3.1.5 Synthesis of sulfonated porphyrinsSeveral reports about the sulfonation of TetPhP to synthesize Tet(SPh)P can befound in the literature,5, 10,11 and the sulfonation procedures reported here are essentiallythe 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 outin this thesis work. Samples of the reaction mixture at different reaction times were taken;73Chapter 2 Synthesis of porphyrinsthe porphyrin samples then were isolated (in the same way as described in the synthesisprocedure), and Ill NMR spectra of these porphyrin samples were recorded. The degreeof sulfonation was calculated by the integration ratio of the sulfonatophenyl peak to thephenyl (unsulfonated) peak. It is observed that the more pyridyl groups present, the longerreaction 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 timerequired for TetPhP (4 h).In the literature,5,10,11 there are major differences reported for purification of thesulfonated product. Following the initial studies in 1962 by Winkelman16 on thelocalization of sulfonated TetPhP in tumors, Fleischer et al.1° developed a syntheticmethod 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 excesssulfiiric acid with formation of a precipitate of calcium sulfate from the sulfonation-reaction mixture, leaving the Na4Tet(SPh)P in the solution; the residue from theevaporation 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 purifyNa4Tet(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 SephadexG-10 column chromatography used to purify (NH4)3[(APh)T(SPh)13].2As part of this thesis work, Srivastava and Tsutui's method using lime to neutralizethe H2SO4 was followed, but low contents of C, H, and N were found in the purifiedsample by elemental analysis; this probably means that inorganic salt was present asimpurities, 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 porphyrin74Chapter 2 Syntheab of porphyrinsresulted 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 ofthe porphyrin by the stationary phase. Dialysis proved to be a good method forpurification 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 thesulfonated porphyrins in the dialysis process are probably related to the differentaggregation 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 topurify Na4Tet(SPh)P in this work.The 1H NMR spectra of the tetrasodium salt of Tet(SPh)P in D20 and in DMSO-d6 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 NMRspectral data (in DMSO-d6) of the sodium salt of the porphyrin diacid and of theammonium salt of the porphyrin free-base, and the UV-visible spectrum of the ammoniumsalt in 0.1 M ammonium carbonate were reported in this paper.2 The 1H NMR spectraldata in DMSO-d6 reported here (Table 2.11, p.60) were obtained from the sodium salt ofthe porphyrin free-base, and the UV-visible spectrum was obtained from the sodium salt indistilled water at 1.0 x 10-5 M. The data are not comparable because of the differences inthe 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 ofunsulfonated phenyl group(s), which was detected by proton-NMR spectra, even if the75Chapter 2 Synthesis of porphyrinsreaction time was prolonged by two or three times. This observation tends to support thereversibility of the sulfonation reactions, which is common in organic chemistry."Sulfonation of (NPh)TPIIP gives an interesting reaction, in which the nitro group isreduced to an amine group, probably by an intermediate(s) formed by destruction of theporphyrin 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 sulfonatednitro porphyrin product. The 11-1 NMR spectrum of the product in DMSO-d6 is essentiallythe same as the spectrum of the product from sulfonation of (APh)TP1113 which gives thecharacteristic signals for aminophenyl at 7.90 ppm (doublet), 7.04 ppm (doublet) and 5.61ppm (singlet) (see Table 2.11 for assignments).From sulfonation of B(NPh)DPhP (a mixture of cis and trans-isomers), threeproducts 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 thebis(nitro)porphyrin is the major product. These products are presumably mixtures of thecis and trans-isomers because the starting material is a mixture of the cis and trans-isomers. The two isomers could not be identified by the spectra as described in Section2.3.2.3 because of the overlapping of the signals of the pyrrole protons. The separationprocedure is not capable of separating the cis and trans isomers. The spectra of theseproducts are shown in Figure 2.2, in which the structures of cis-isomers are used torepresent 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 speciesare also not shown. The porphyrins are not listed in Figure 2.1 (p.36) because purifiedsamples were not isolated. Further studies on these products were abandoned because ofthe low yield of the synthesis reaction and the difficulty in the separation procedure.76Cluipter 2 Synthesis of porphyrins•9.0 8.0•7.0Figure 2.2. 1H NMR spectra of B(NPh)B(SPh)P, (APh)(NPh)B(SPh)P andB(APh)B(SPh)P in DMSO-d6.Assignment (which are essentially identical for each species):chem. shift(ppm) 8.8-9.1 8.70 8.55 8.20 8.08 7.90 7.04pattern m d d dorm dorm d dassignment. pyrrole 3,5-NPh 2,6-NPh 2,6-SPh 3,5-SPh 2,6-APh 3,5-APh77Chapter 2 Synthesis of porphyrinsSulfonation of cis-(NPh)DPITyP in a 6 h reaction time gives the unreducedporphyrin 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 Figure2.3. Compared to (NPh)TPhP, the presence of the protonated pyridyl on cis-(NPh)DPhPyP in acidic solution probably stabilizes this porphyrin structure fromnucleophilic attack and therefore limits the formation of the reducing intermediateprobably formed from degradation of the porphyrin structure. A reaction time of 6 h wasoptimal for synthesis because more amino porphyrin was formed with a longer reactiontime, 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 pyrroleprotons appears only as a broad singlet. This does not give enough information about thesymmetry of the product (cis-isomer vs. trans-isomer; details about this identification arediscussed in Section 2.3.2.3). However, the product is considered to be the cis-isomerbecause it is made from cis-(NPh)DPhPyP which is well defined by the pyrrole protonsignals in the 1H NMR spectrum (see Figure 2.14, Section 2.3.2).2.3.1.6 Synthesis of methylpyridiniumporphyrinsMethylation of the pyridyl moieties on a porphyrin can be easily performed withmethyl 4-toluenesulfonate in DMF with a high yield, as described in the literature.6 Areaction time of 4 h was adequate for this reaction instead of overnight as reported, andprolonged reaction times caused complications in isolation of the product. The tosylatesalts of the dicationic porphyrins are not soluble in cold water or acetone, but are solublein the mixture of these two solvents. This probably results from the fact that organicsolvents break up the aggregation of these porphyrins (Chapter 4) and therefore increasetheir solubility in water. A more detailed discussion about the effect of organic solvents on789.2^9.0^9.8^8.6^8. 4^8.2^8.0^7 8 PPMB.I I^I^1^I^I^IIaChapter 2 Synthesis of porphyrins'0 N S 03Figure 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) 9.02 8.87 8.66 8.49 8.25 8.16 8.03pattern d s(broad) d d d d dassignment 3,5-Py pyrrole 3,5-NPh 2,6-NPh 2,6-Py 2,6-SPh 3,5-SPh79Chapter 2 Synthesis of porphyrinsporphyrin aggregation can be found in Chapter 4. In the process of synthesis, some water-soluble impurities in these dicationic products can be washed off using cold water, butacetone, which is used to wash away the reaction solvent and organic impurities, has to becompletely removed first before the washing with H20 in order to avoid losing theproduct.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 byHambright and Fleischer in 1970.19 The tosylate salt of this porphyrin was synthesized byPasternack et al. in 19726 and characterized by elemental analysis and UV-visible spectrain aqueous solutions. The chloride salt has been noted later in the fiterature,2° butcharacterization 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, havebeen synthesized before.9 However, elemental analyses were not reported, while the 111NMR spectra were reported in D20, CD3OD or CD30D-CD2C12 without specificreference to the solvent used for each compound, and the UV-visible spectra in aqueoussolutions 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 indistilled water were found to be concentration dependent (Chapter 4) and are recorded at1.0 x 10-5 M in this work (Table 2.15, p.66). These differences in the experimentalconditions make the spectral data not easily comparable with the literature data.Dialysis was also employed in purification of the cationic methylpyridinium-porphyrins, but a major loss of porphyrin product was observed from the dialysis ofT(MPy)PC14; this probably results from different aggregation properties of this compoundin comparison to the other cationic porphyrins (Chapter 4).80Chapter 2 Synthesis of porphyrins2.3.2 Proton-NMR spectraThe chemical shifts and patterns of the 1H NMR signals, the signal-intensity ratioof the substituents, plus the information from the syntheses were the main data used in thisthesis to assign a structure to a synthesized porphyrin.The 1H NMR spectra of water-soluble porphyrins were measured in DMSO-d6solvent. Measurements in D20 often gave complicated spectra which may result fromaggregation of the porphyrins (Chapter 4). For non water-soluble porphyrins, CDC13 wasused.The nitration and sulfonation always occur at the para positions of the meso-phenyls. This was evident by the characteristic two doublets of the NMR spectrumcorresponding to the meta and ortho protons of a 4-nitrophenyl or 4-sulfonatophenylgroup. The formation of a 4-aminophenyl derivative was confirmed by the two doubletsfor the meta and ortho protons of aminophenyl and the appearance of the signal for theamine protons at 4.00-4.05 ppm in CDC13, which disappeared when D20 was added tothe NMR samples.For the following discussion, a pyrrole proton refers to a proton bonded to acarbon atom of a pyrrole ring, while the proton associated with the pyrrole nitrogen isreferred to as an N-pyrrole proton.2.3.2.1 Identification of substituents from Ill NMR spectraAll the substituents at the meso positions have characteristic chemical shifts(ppm) which are listed in the following table with associated JHH coupling constants ( inHz). The detailed assignments are considered in Section 2.3.2.2.81Chapter 2 Synthesis of porphyrinssolvent -NH2 APh MPy NPh 2,6-Ph,3,4,5-.PhPy SPh N-pyrroleCDC1/ 4.0 8.0, 7.1(8.1)-4- 8.6, 8.4(8.6)18.2 7.8 9.1, 8.2 - -2.8— -3.1DMSO-d6 5.6 7.9, 7.0(8.2)19.5, 9.0(5.7)!8.7, 8.5(8.5)18.1(7.5)!7.9 9.0, 8.3(6.3)a8.2, 8.1(8.0)1-2.8— -3.0a^Coupling constant between ortho- and meta-protons.The coupling constant for the pyrrole protons is 4.7 Hz in CDC13 and 4.3 Hz inDMSO-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, thespectra of APh, MPy, NPh and SPh appear as AB quartets, which means the couplingconstants between the 2 and 5, 2 and 6, and 3 and 5 protons are all small, so the systemcan be treated approximately as an AB system (i.e.,only the coupling of the 2 and 3 or 5and 6 protons are considered). The coupling constants listed in the above table areobtained in this manner. The same approximation cannot be applied to the 4-Py and Phsystems 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 bycomparisons with related spectra. For instance, a comparison of the spectrum of TetPhPto 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 inintensity 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 the824/\ /\I —Porphyrin5 6+0.33ON-0.75 -0.24-0.75 -0.24-0.63+0.95 +0.17+0.95 +0.17H NChapter 2 Synthesis of porphyrinsrelevant spectra together with information from the syntheses (see the synthesis scheme inFigure 2.1, p.36).2.3.2.2 Assignments of signals to the protons of the meso -substituentThe assignment of each signal to corresponding protons of the substituentspresents a reasonably straightforward problem. The signals for the phenyl protons in thespectrum of TetPhP are easily assigned (as 7.78 ppm for the 3,4,5-Ph protons and 8.23ppm for the 2,6-Ph protons) by consideration of the distances from the protons to theporphyrin ring, and the deshielding effect on the phenyl protons. These assignments haveappeared in the literature.22 When the 4-phenyl proton is replaced by a nitro or an aminogroup, the chemical shifts can be calculated according to the shielding parameters of thenitro or amino group within aromatic compounds (figures shown below);23 these havebeen used by Sun et al.24 to calculate the chemical shifts for the protons of the 3-nitrophenyl group of 5-(3-nitropheny1)-10,15,20-triphenylporphyrin. Table 2.17 presentsthe results of calculated and experimental chemical shifts (the numbering system ispresented in Section 1.2, p.2, ans drawn again below).83Chapter 2 Synthesis of porphyrinsTable 2.17. Calculated and observed chemical shifts of the nitrophenyl andaminophenyl protons2,6-nitrophenyl 3,5-nitrophenyl 2,6-aminophenyl 3,5-aminophenylcalculated 8.23A+0.17=8.40 7.78A-1-0.95=8.73 8.231-0.24=7.99 7.78A-0.75=7.03observedk 8.41 8.64 7.98 7.0511: 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 thenitrophenyl or aminophenyl group; the 'least satisfactory' calculated value (for the 3,5-nitrophenyl protons) is 0.09 ppm greater than the observed value. Figure 2.4 shows thespectrum of (NPh)TPhP and the assignments of the signals.The 1H NMR spectra of the sulfonated porphyrins are recorded in DMSO-d6. The1H NMR spectrum of TetPhP in DMSO-d6 is not available (because of low solubility ofthis compound), and neither are the shielding parameters for the sulfonato group within aphenyl ring. However, the 1H NMR spectrum of T(MPy)PhP in DMSO-d6 gives thechemical 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 sulfonatedporphyrins show two signals at 8.20 and 8.03 ppm in DMSO-d6 (Table 2.11, p.60). Whenthe 4-phenyl proton is replaced by sulfonate, an electron withdrawing group, the chemicalshifts of both of the phenyl signals (2,6- and 3,5-protons) should increase,23 whichsuggests that the 2,6-sulfonatophenyl protons resonate at 8.20 ppm (a shielding parameterof +0.07 ppm), and that the 3,5-sulfonatophenyl protons resonate at 8.03 ppm (a shieldingparameter of +0.24 ppm). The opposite assumption would make the shielding parameters84N-pyrrole..A,. ,,,,, ..A.. ....... • • ' " • " 4 ' " ' "^MS" • • • "H20Chapter 2 Syntheab of porphythasfor sulfonato to be abnormal (-0.10 for 2,6-protons and +0.31 for 3,5-protons).23 Anexample of the assignments for a sulfonated porphyrin is shown in Figure 2.5.Figure 2.4. 1H NMR spectrum of (NPh)TPhP in CDCI3.Assignments:Chem. shift (ppm)^8.7-9.01^8.64^8.41^8.22^7.78pattern^d-s-dassignment^pyrrole^3,5-NPh^2,6-NPh^2,6-Ph^3,5-Ph1 Assigned individually later in Section 2.3.2.4.85sove'N-pyrrole —111!1■!4^S.2^1.1 • .;.• PPM4,2sq.Figure 2.5. 1H NMR spectrum of trans-BPyB(SPh)P in DMSO-d6.Assignments:Chem. shift (ppm) 9.04 8.89 8.25 8.18 8.05pattern d m d d dassignment 3,5-Py pyrrole 2,6-Py 2,6-SPh 3,5-SPh868.55^7.21/r-Th\j\+1.28 -0.06/\^+0.339.06^8.17/N^\ porphyrin7.60Chapter 2 Synthesis of porphyrinsThe shielding parameter for the N atom within a pyridine is not listed in thefiterature.23 The spectrum of pyridine in CDC13 was recorded, and the shieldingparameters for the pyridine nucleus were calculated using 7.27 ppm as the chemical shiftfor benzene.23 Then the chemical shifts of the 4-pyridyl on a porphyrin structure can becalculated using the chemical shifts for the phenyl groups of TetPhP (in the same way asshown in Table 2.17); the values are shown below:chemical shifts shielding parameters calculated chemical shiftsThese calculated data agree very well with the observed data, which show average valuesof 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 arerecorded in DMSO-d6, can be assigned with these data although these signals appear atslightly different positions (9.04, 8.25 ppm, Figure 2.5), presumably because of thedifference in the solvent.The chemical shifts of the pyridyl protons of PyT(SPh)P in DMSO-d6, which are9.03 and 8.27 ppm, are used generally here as those for pyridyl protons on a porphyrin inthis solvent. These chemical shifts increase to 9.50 and 9.02 ppm when methylationoccurs; the methylation of the pyridyl groups is expected to make both signals shift tolower field. Therefore the signal at 9.50 ppm is assigned to the 3,5 -MPy protons and thesignal at 9.02 ppm to the 2,6-MPy protons. An example of the assignments in a spectrumof a cationic methylpyridiniumporphyrins is shown in Figure 2.6.87Chapter 2 Synthesis of porphyrinsThe assignments of the signals of the protons within the porphyrin substituents arelisted in Tables 2.2 (p.41), 2.5 (p.49), 2.8 (p.53), 2.11 (p.60) and 2.14 (p.65).MeNO2Figure 2.6. 1H NMR spectrum of (NPh)T(MPy)P in CDCI3.Assignments:Chem. shift 9.50 9.17-9.00 9.00 8.74 8.52 4.75pattern d m d d d sassignment 3,5-MPy pyrrole 2,6-MPy 3,5-NPh 2,6-NPh Clii88Chapter 2 Synthesis of porphyrinsThe signals of the pyridyl protons within pyridylporphyrins have been assigned in afew literature reports. Williams et al.7 assigned signals at 9.01 and 8.22 ppm to the 2,6-and 3,5-Py protons of TPhPyP, respectively, and Ding et al. assigned signals at 9.06 and8.15 ppm to the 2,6- and 3,5-Py protons of (NPh)TPyP, respectively.8 Differently, Sari etal.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 givenwithout reasoning. The assignments given in the present work agree with the report ofSari et al. and not with the other two earlier reports.Ding et a!.8 also assigned the signals of the nitrophenyl protons within (NPh)TPyPas 8.66 ppm for the 2,6-NPh protons and 8.38 ppm for the 3,5-NPh protons. Theassignments given here for the nitrophenyl signals are opposite to these, i.e., 8.61 ppm forthe 3,5-NPh protons and 8.34 ppm for the 2,6-NPh protons, and these agree with theassignments for a 3-nitrophenyl porphyrin given by Sun et a!.24The signals of the aminophenyl protons within (APh)TPyP are also assigned byDing et al.,8 and are consistent with those given here.The assignments within some methylpyridinium porphyrins have also appeared inthe literature.8,9 The assignments given in this thesis work agree with these reports in thatthe 3,5-MPy protons resonate at lower fields than the 2,6-MPy protons, although theNMR spectra are taken in different solvents.The two doublets of the sulfonatophenyl protons in the spectrum of Tet(SPh)P inDMSO-d6 have been assigned by using partial deuteration at the 2,6-position,17 and theassignments given here agree with the reported ones.It is more challenging to assign the split pyrrole signals to individual protons, andthis is discussed later in Section 2.3.2.4.8912 12\ \1313B^A17^3^\._..-- NH^// A C 2N^H N1„ \„'C22 2cis-isomer^ trans-isomerChapter 2 Synthesis of porphyrins2.3.2.3 Identification of isomersAnother interesting feature of the 111 NMR spectra of the pyrrole protons is theinformation provided for structure assignments, especially for distinguishing between cis-and trans-isomers. The following figure shows some of the symmetry elements of the twoisomers of the A2B2P type; the treatment ignores the N-pyrrole protons because there isfast tautomer inter-conversion at room temperature in organic solvents."iiIt is clear that for the cis-isomer, there are four types of pyrrole protons, while forthe trans-isomer there are only two types of pyrrole protons. Two identical protons on thesame pyrrole ring within a porphyrin give rise to a singlet, and two different protons onthe same pyrrole ring give two coupled doublets. So, two singlets and two doublets areexpected for the cis-isomer and two doublets are expected for the trans-isomer in the 1HNMR spectra of pyffole protons. Figure 2.7 shows the spectra of cis- and trans-DPhBPyP. The trans isomer gives two doublets, and the cis one gives two doublets andtwo singlets. Thus, the two isomers can be identified easily.90Chapter 2 Syntheah of porphyrins9 0^8 8 8 . 6^8 . 4 8 . 2^8 . 0 7.8^7. 6PPM d^d^JL^111{!1111‘^9 0^88^8.6^84^82^80^7. 8 ppm 7 6Figure 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. Thereare two doublets and two singlets for the cis-isomer and two doublets for the trans-isomer. For theassignments of these peaks, see Table 2.18, p.101; for the assignments of the other peaks, see Table 2.2,p.41.91H7R5H3#1 R5=R1 0R1 5=R20#2 R5; R10=R15=R20#3 R5=R10; R15=R20#4 R5=R15; R10=R20#5 R5=R10; R15; R20#6 R5=R15; R10; R20I I^IIHIll'''^H^II^H Chapter 2 Synthesis of porphyrinsAn observation that the two protons on the pyrrole ring appear identical in the 111NMR spectrum, as long as the two adjacent meso substituents are identical, has beenfound to be true for all of the porphyrins studied in this thesis work. An example of thisobservation is the spectrum of (NPh)TPhP (Figure 2.4); in this compound the mesopositions 5 and 20 have different substituents, but H12 and H13 give a singlet because thesubstituents at meso positions 10 and 15 are identical. Using this observation, thefollowing patterns for the 1H NMR spectra of the pyrrole protons of porphyrins ( in the8.6 - 9.0 ppm region), with different types of meso-substituents, may be drawn (Figure2.8, relative intensities are also shown):s [8]d[2]d[2]d[4]d[1]d[2]s[4]s[2]d[4]d[1]d[2]d[2]s[2]d[1]d[2]s[2]s[2]d[2]d[1] d[1] d[1]*H13R15H17* The relative position of the singlet can vary.Figure 2.8. The patterns for the 1H NMR spectra of the pyrrole protons.92Chapter 2 Synthesis of porphyrinsThe spectra of all the porphyrins synthesized in this thesis work are consistent withthese 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), asinglet (4 protons) and a doublet (2 protons), but the spectrum appears to consist of adoublet (2H) and a broad singlet (6 protons); the latter arises from the overlapping of thesinglet (4 protons) and one of the doublets (2 protons) (see Table 2.8, p.53). The spectraof the water-soluble porphyrins usually give broad and less informative signals (see Tables2.11, p.60 and 2.14, p.65; Figures 2.5 and 2.6), but the cis- and trans-isomers can beassigned according to the spectra of the precursors (see Figure 2.1), whose isomers canbe identified by the peaks of the pyrrole protons.More examples of the spectra of the pyrrole protons of cis- and trans- isomers aregiven in Figures 2.9 and 2.10. Figure 2.9 shows the spectra of the pyrrole protons of cis-and trans-B(NPh)BPyP. In the spectrum of the cis- isomer, the two singlets overlap withthe two doublets (see the inset), but there is no doubt about the identifications of the cis-and 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-isomeras shown by pattern #6 in Figure 2.8, but for the cis-isomer, the #5 pattern can been seenonly with careful inspection. Nevertheless, the identification of the two isomers can bereadily achieved using these spectra.The pyrrole protons of the mixture of cis- and trans-B(NPh)DPhP give a spectrumof two doublets and two singlets, the pattern expected for the cis-isomer, but the intensityratio of the signals is 5/2:3/2:3/2:5/2 (see Table 2.5, p.49) instead of the required 2:2:2:2for the cis-isomer (see # 3 in Figure 2.8). The observed ratio results from the overlappingof the signals for the cis- and trans-isomers. From the ratio, the isomer composition canbe calculated:93H N N H2N H2Chapter 2 Synthesis of porphyrins9.0^8.0^7.0^6.0^5.0^4.0 ppmFigure 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 twosinglets in the spectrum of the cis-isomer overlap, the #3 pattern can still be seen. The trans-isomer hasthe #4 pattern as expected.940 N‘.0•1fr^:51.!1;.1.11111^.5.1:, 11;1 1 • .1!5.,;;m^,),,I i1i.,,I^SillChapter 2 Synthesis of porphyrinsN 0 21.0^1.8^1.11^1.4^1.2^1.0^7.1 PPMFigure 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 givea more intense doublet (2H) at 8.90 ppm, and the singlet overlaps with two of the other doublets; this canbe seen when the spectrum is expanded. The trans-isomer gives four doublets (pattern #6) as expected.95Chapter 2 Synthesis of porphyrinsIf X is the molar portion of the cis-isomer, then 2X = 3/2, and X = 75%, and therefore themolar portion of the trans-isomer is 25%.X [cis- isomer (2:2:2:2)]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 protonsThe following discussion is devoted to the assignment of the 1H NMR signals toindividual pyrrole protons. The numbering scheme is as shown in Figure 2.8. Thesubstituents are always arranged alphabetically with respect to the numbered mesopositions; i.e., the lowest numbered 5 -meso position always accommodates theaminophenyl 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 theassumption that C2 and Cg are more electron deficient than C3 and C7, the doublet at lowfield (8.90 ppm) is assigned to H2 and Hg, and doublet at higher field (8.74 ppm) to H3and H7. The assumption is supported by the electronic distribution on 2-nitronaphthalene,as demonstrated by electronphilic substitution reactions," considering the wholenitrophenyl moiety as an electron-withdrawing group and the similarity of the relevantparts of 2-substituted naphthalene and the porphyrin (Figure 2.11 a). The assumption isalso supported by the resonance forms of this nitroporphryin (Figure 2.11 b), and can beexpanded to suggest that the pyrrole proton adjacent to an electron-withdrawing meso196ON /--0 Nmajor productNO2+Chapter 2 Synthesis of porphyrinssubstituent (including 4-Py, 4-MPy, 4-NPh, and 4-SPh) resonates at a higher field than theother proton on the same pyrrole ring.aNph.^ j, NH^N::-_-\ i^/L 7-ìN HNb--.1,...,^\^H7^NH^N____0 -N/ \//^\--- ---^\/----- N^HN^0 H3H2^•-...,. ....^---,...„0-\\ ^Nz.::^ NH^No-^NHN 4--/ -ave. \N/ ---"\^/ ---\^N^HN /^•^-...^----N^HN0 -^H3 —\ ...i ^0 - H31\H2 H2Figure 2.11. Resonance structures of a nitrophenylporphyrin.In contrast to the (NPh)TPIT example, the resonance forms of (APh)TP1113 areshown in Figure 2.12. The assumption is now that the pyrrole proton adjacent to anelectron-donating meso substituent (4-APh) resonates at a lower field than the otherproton on the same pyrrole ring, i.e., H3 and H7 resonate at lower field (at 8.93 ppm) thanH2 and H8 (at 8.82 ppm) in the case of (APh)TPhP (Table 2.8, p.53).97HaH ^ H7^NH N____/,..,---,.r.-/ NN HNH^H3 \^L_/H 1...-...- N +—/^ /^/ — \N HNHH3^H_^---H 1H 1--y.H^H7^1^H\ NH N__ \N --/— ^Chapter 2 Synthesis of porphyrinsFigure 2.12. Resonance structures of an aminophenylporphyrin.The signals of patterns # 2 and # 4 in Figure 2.8 can be assigned using these twoassumptions, and so can the two doublets of pattern # 3. For the assignments of the twosinglets in pattern #3, the following observations are employed:As shown schematically in Figure 2.13A, in the spectrum of TPhPyP, the singletwhich corresponds to the pyrrole protons between two phenyls appears closer to thedoublet at lower field; however, in the spectrum of PhTPyP, the singlet corresponding tothe pyrrole protons between two pyridyls appears closer to the doublet at higher field. Aconclusion made from these observations is that in the spectrum of cis-DPIA3PyP, thesinglet at lower field for the pyrrole protons corresponds to the protons between the twophenyls, 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 pyrroleprotons situated between two meso electron-withdrawing substituents resonate at lowerfields than the protons situated between two relatively electron-donating substituents.Another example is shown in Figure 2.13B. Figure 2.13B a shows the chemicalshifts 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 thespectrum of cis-B(NPh)DPhP the singlet at 8.86 ppm corresponds to H17 and H18, andthe singlet at 8.77 ppm corresponds to H7 and Hg, as shown in Figure 2.13B b.98Chapter 2 Synthesis of porphyrins8.928.88 NH ft-/881ON\ N HN\^L../8 .85Figure 2.13A. A comparison of the schematic spectra of TPhPyP, cis-DPhBPyPand 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 resonancestructures demonstrated in Figures 2.11 and 2.12 and using comparisons similar to the onedemonstrated in Figure 2.13B. The results are included in Table 2.18.990 NNO208.79NH N\ N HN \ //0 NaChapter 2 Synthesis of porphyrinsFigure 2.13B. Illustration of the chemical shifts of the pyrroleprotons of TPhPyP and some nitroporphyrins.100Chapter 2 Synthesis of porphyrinsTable 2.18. The assignments of the pyrrole protons®1-17 H3 H7 Hit 111 H13 H17 Hi gTPhPyP 8.81 d 8.90 d 8.87 s 8.87 s 8.87 s 8.87 s 8.90 d 8.81 dtrans-DPhl3PyP 8.81 d 8.91 d 8.91 d 8.81 d 8.81 d 8.91 d 8.91 d 8.81 dcis-DPhBPyP 8.81 d 8.92 d 8.88 s 8.88 s 8.92 d 8.81 d 8.85 s 8.85 sPhTPyP 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 scis-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 strans-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 dT(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 dcis-(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 dcis-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 dtrans-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 dT(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 dtrans-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 dcis-(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 scis-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 scis-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 strans-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 dT(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 dcis-(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 dcis-(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 scis-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 strans-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 dcis-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 sa: Chemical shift in ppm, peak pattern; in CDC13 unless stated otherwise.b: In DMSO-d6.101Chapter 2 Synthesis of porphyrinsFor the porphyrins having three different substituents, the pattern for the pyrrolesignals 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 thesesignals 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 theseA values for examples of various porphyrins. The actual chemical shifts of the coupleddoublets 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 H8trans-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 forthe 'inside' signals, this resulting from second order coupling. Using this information andthe data in Table 2.19, the coupled doublets can be selected and assigned to a particularpyrrole ring. The two doublets corresponding to a pyrrole ring can be assigned using theassumptions 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.102H8 H12 H2 H18 H3 H7I1H17^H13IChapter 2 Synthesis of porphyrinsFigure 2.14. Assignment of the pyrrole protons in the 11-1 NMR spectrum ofcis-(NPh)DPhPyP (CDCI3).The doublet (2H) at 8.89 ppm is coupled to both of the doublets at 8.79 ppm (A= 0.10ppm) and 8.73 ppm (A= 0.16 ppm), as indicated by two dimensional NMR. The other coupleddoublet is also shown above. Assignments are achieved as described in the text.103IH2N. . i 11111111^1 .9 . 0II I^I H3 H12u111111111118 . 9^8 . 8II IiH7 H13 H17 H2H18 H8ioA pyridyl protonsV phenyl protons• aminophenyl protons0 amine protons0 solvent1111-111111111111111111111111111118^6^4Chapter 2 Synthesis of porphyrtnaFigure 2.15. 1H NMR spectrum of cis-(APh)PhBPyP in CDCI3.104Chapter 2 Synthesis of porphyrinsResults from two dimensional NMEt of cis-(NPh)DPhPyP and cis-(APh)PhBPyPshow which doublets couple to each other, and support the given assignments.The assignments of the pyrrole signals for all the porphyrins are presented in Table2.18. 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,1k2.7,8,9but only the pyrrole signals for 5-(2-nitro-5-hydroxylpheny1)-10,15,20-tris(4-tolypporphyrin have been assigned; Little" suggested that H3 and H7 resonate at higherfields than H2 and Hg, which agrees with the assignments of the nitroporphyrins presentedhere.2.3.3 UV-visible spectraThe intense absorbance of hemoglobin at 400 nm was discovered in 1883 bySoret,26 and this band was later observed in porphyrins.25 The optical spectra ofporphyrins and related compounds have been reviewed in the literature.25,27 For porphyrinfree-bases, there is a strong band in the near UV (around 400 nm), referred to as the Soretband, and four visible bands (from 500 to 650 tun) labeled as I, H, III, and IV on goingfrom 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 visiblebands.25 The porphyrins synthesized in this thesis work generally give the typical etiospectra (intensities of the visible bands: IV>IH>II>I). However, there are a fewexceptions. 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 ofasymmetry in the it electron cloud;25 however, the fact that TPliPyP has an etio-typespectrum and PhTPyP a phyllo-type suggests that other factors are involved, because both105Chapter 2 Synthesis of porphyrinsthese porphyrins have the same symmetry. Cis-(APh)DPhPyP and trans-B(APh)PhPyP arealso unusual in that the intensity of band I is greater than that of band II. This type ofspectra does not fall into any of the four standard types. A third exception is apparent withthe water-soluble porphyrins cis- and trans-B(APh)B(SPh)P in aqueous solution, thespectra of which have basically lost some of the characteristics of porphyrin structures: theintensity of the Soret bands is dramatically reduced from typically 3-4 x 105 M-'cm' to <1x 105 M-'cm-1, while the intensities and wavelengths of the visible bands changedramatically from those of a normal porphyrin spectrum (Table 2.12, p.61). No theory toexplain 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 aqueoussolution, which is discussed in Chapter 4.A considerable amount of work in the literature have been devoted into developingmodels to explain the optical spectra of porphyrins and metalloporphyrins. Reviews on thisaspect can be found in the literature,27,28 but discussion on the models is beyond the scopeof this thesis.The Soret band and the four visible bands of a porphyrin generally shifts to longerwavelengths after nitration of the porphyrins and, when the nitro group is reduced to anamine, the bands shift to even longer wavelengths. The wavelengths of the Soret bands ofTetPhP, TPhPyP and their nitro and amino derivatives are listed in Table 2.20. The redshifting of the bands on alkylation of pyrrole hydrogens on going from porphin (theporphyrin core) to octa-alkylporphyrin has been explained by the electron-donating effectsof the substituents.27 However, the red shifts noted in Table 2.20 cannot be explainedsimilarly because both electron-withdrawing and -donating substituents give the sameeffect. Another approach to explain these red shifts is to consider the effect of increasedconjugation in the systems when a nitro or amine group is introduced into the106NH N =f--)7/—\\N^N 020NH N ^\\/ _N H N z/0-NChapter 2 Synthesis of porphyrinsstructure.15,29 The resonance forms of the aminophenyl- and nitrophenylporphyrin areshown in Figure 2.16 a; there are planar forms, and these are more stable, thereforecontribute more, than the similar resonance forms for TetPhP (Figure 2.16 b). This willtherefore increase the conjugation in the system, and lower the excitation energies. It isknown that the phenyl ring is essentially perpendicular to the porphyrin ring in the case ofTetPhP in the solid state.3°Table 2.20 Soret bands of nitro and amino -porphyrinsAmax (tun) ?.max (nm)TetPhP 416.7(NPh)TPhP 418.0 (APh)TP1113 419.5B(NPh)DPIT 419.5 cis-B(APh)DPhPtrans-B(APh)DPhP421.5421.0T(NPh)PhP 421.3 T(APh)PhP, 423.8TPhPyP 416.5cis-(NPh)DPhPyP 417.6 cis-(APh)DPhPyP 418.8cis-B('NPh)PhPyP 418.6 cis-B(APh)PhPyP 421.5trans-B(NPh)PhPyP 419.0 trans-B(APh)PhPyP 422.0T(NPh)PyP 419.4a NH NrN HN^—^H%7)NN H N^/\^_^KVfrN HN^—^HN H N11)1NT-I 1\7-// ^ /rN HNNN ^H _*)\Figure 2.16. Resonance forms of an aminophenylporphyrin and anitrophenylporphyrin107Chapter 2 Synthesis of porphyrituSimilar red shifts have also been observed with substitution of the para-protons ofthe four phenyls by -OCH3, -CH3, -Cl and -NO2, and these shifts were rationalized in theterms of greater conjugation.15,292.3.4 Infrared spectraThe infrared spectra of TetPhP and some of its symmetric p-phenyl substitutedderivatives have been reported,29 and the infrared spectra of some other porphyrins andmetalloporphyrins have been reviewed.31 Table 2.21 lists the data obtained from IRmeasurements in the current studies. The assignments of the common peaks of theporphyrin ring structure can be found in the literature.29,31The spectrum of TetPhP recorded is in general agreement with the literaturedata.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 NO2groups) within the nitro porphyrins. Peaks at around 1618 cm-1 (8NH)3 were detected forthe amino porphyrins. The VcN of the pyrrole and pyridyl rings32 at —1595 cm-1 becomesmore intense for the porphyrins having more pyridyl substituents.2.3.5 Mass spectraThe mass spectra of porphyrins have been reviewed in the literature.33 Theporphyrins 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 chemicalionization (NI-13) technique are shown in Table 2.22, which lists the peaks with relativeintensity higher than 20 %. The peaks are basically M+, (M+1)+ and (M+2)+. For theamino-porphyrin, (M+NH)+ and (M+NH2)+ are also relatively strong peaks; these mayresult from the affinity of the atninophenyl group for the fragments from the iodizingchemical (NH3).108Chapter 2 Synthesis of porphyrinsTable 2.21. Data from IR spectraTetPhP TPhPyPtrans-DPhBPyPcis-DPhBPyP PhTPyP TetPyPcis-(NPh)PhBPyPcis-B(NPh)BPyPcis-(APh)PhBPyP T(APh)PhP1623w 1618s1607w 1604s1560w1597w 1598s 1594vs 1594vs 1592vs 1593vs 1592s 1592vs 1593s1575w 1575w 1580w 1548m 1545w 1546w 1563 1564vwbr br1510m1517m 1516vs1508w1499w1444w 1444w 1444w 1444w1404m 1408s 1405m 1400w 1401w 1402w 1405m 1405m1350m 1351m 1352m 1349w 1350w 1351w 1349w 1351m1348m 1349vs1310w 1310m 1307w 1309w 1293m br1286w 1281s br1251w 1252w 1255w 1255vw 1253w1222w 1224w 1226w 1225w 1229w 1226w 1228w1213w 1217w 1221w 1216w 1210w 1213w 1220w 1214vw 1217w1189m 1190w 1188w 1188w 1188w 1190w 1188w 1187w1178m 1179w 1178w 1177m 1179s1155m 1155m 1155m 1157w 1156 1158w 1155w 1158m 1159w 1160m1143w1082w 1082w 1078w 1081w 1111w 1110w1069m 1069m 1069w1031m 1033w 1032vw 1032w 1020w 1125w1001m 1002m 1001w 1003m 1003m 1004w 1002w 998w 1002w980m 979m 981m 980m 981m 980m 980m 980m 981w 984w966s 966s 968s 970s 970s 971s 969s 969s 968s 967s109Chapter 2 Synthesis of porphyrinsTable 2.21 continuedTetPliP TPhPyPtrans-DPhBPyPcis-DPhBPyP PhTPyP TetPyPcis-(NPh)PhBPyPcis-B(NPh)BPyPcis-(APh)PhBPyP T(APh)PhP891w 893w 892w 892vw875m 878m 881m 880m 881m 882sbr 881m 882m 880w 879w867m 866m872w 872m850m 852m 853w 853vw 850w 849m 849s 848w 846m844w 844w 843w 841w812m 817w 822w 810w799vs 796vs 801vs 795vs 796s 799s 796s 800s 799s 805sbr br br br br br br786m 787m 794m 784s 786s 786s759m 759s748m 752w 753s 751m 749w 749w 751vw 755s744w 745s 743w739s 739m 738s705m 705m 713m 703w 710m 704vw 703w699vs 699vs 706s 698m 698m673m 673m 675m 673vw 673vw668w 664w656m 656m 656m 658m 658s 659m 657w 659vw646w640m 640m 639w 638w 639w 639w 640w 641w 641vw br631w619w 619w 622w 622vw560w 563w 564m 565w 565w 565w 564w 565w 560w 560w552w 554w 553vw 553w 553w 554w 554w 553w 552w 552w516w 518w 521w 527w 530w 531wbr519w 519w 517w 517ww = weak; s = strong; m = medium; br = broad, vs = very strong, and vw = very weak.110Chapter 2 Synthesis of porphyrinsTable 2.22. Data from CI mass spectraNi+ (!) (vi+1)+co (m+2)+4) (m+NH)(A) (m+NH0+(!)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) 646 (20) 647 (28)a: Intensity as a percentage, relative to the major peak as 100 %.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 mainpeaks in an El spectrum of [PhT(MPy)P]C13 were at 618 ([1■4-3C1-3CH3], 100%) and619 (43%).Table 2.23 lists the data from cationic FAB (3-nitrobenzylalcohol matrix) spectraof some cationic porphyrins. The major peak in these spectra is that of the porphyrinfragment without the counterions.Table 2.24 lists the data from anionic FAB (3-nitrobenzylalcohol matrix) spectra ofsome anionic porphyrins. The major peak is [M-Nal- or [M-2Na+Hr, ignoring the matrixpeaks.Table 2.23. Cationic FAB mass spectra of some cationic porphyrinsTet(MPy)PC14 6781 663 648 633 69411 713M-4C1 M-4C1-CH3 M-4C1-2CH3 M-4C1-3CH3 M-3C1T(MPy)PhPC13 719! 704 689 735k 754M-3C1 M-30-CH3 M-3C1-2CH3 M-2C1T(N1Py)(NPh)PC13 7071 692 677 723kM-3CI M-3C1-CH3 M-3C1-2CH3cis- 7361 721 75211B(N1Py)B(NPh)PC11 M-2C1 M-2C1-CH3cis-B(MPy)DPhPC12 6461 631 66112M-2C1 M-2C1-CH3A: The major peak (ignoring the matrix peaks).Peak not identified.111Chapter 2 Synthesis of porphyrhtsTable 2.24. Anionic FAB mass spectra of some anionic porphyrinsNa2[cis-B(APh)B(SPh)13] 8251M-Na803M-2Na+HNa2[trans-B(APh)B(SPh)11 8253M-Na803M-2Na+HNa2[cis-BPyB(SPh)P] 7973M-Na775M-2Na+HNa2[cis-(NPh)PyB(SPh)11 8191M-2Na+H841M-Na3: The major peak.The cationic spectrum (3-nitrobenzyl alcohol matrix, thioglycerol) of cis-BPyB(SPh)P was also recorded, but it was of little use for characterization.112Chapter 2 Synthesis of porphyrinsReferences1^(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^W.J. Kruper, Jr., T.A. Chamberlin and M. Kochanny, J. Org. Chem., 54, 2753 (1989).3^E. Tsuchida, E. Hasegawa and T. Kanayama, Macromolecules, 11, 947 (1978).4^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).113Chapter 2 Synthesis of porphyrins13^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).114Chapter 2 Synthesis of porphyrins25^K.M. Smith, in "Porphyrins and Metalloporphyrins", H.E. Falk, ed., Elsevier ScientificPublishing 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.395.115Chapter 3 Water-soluble metalloporphyrinsChapter 3 Water-soluble metalloporphyrins3.1 IntroductionA porphyrin can coordinate to almost any transition metal.' Due to the limitationof time and the interests of this project, only complexes of cobalt, copper and zinccomplexes of the designed porphyrins were studied in this thesis work.3.1.1 Cobalt complexes of water-soluble porphyrinsCobalt complexes of water-soluble porphyrins were reported to have the besteffects among the metalloporphyrins of eight metals tested for radiosensitizers.2 Becauseof these findings, the cobalt complexes of the water-soluble porphyrins synthesized in thisthesis work (Chapter 2) became of interest for potential use as radiosensitizers.The cobalt complexes of some water-soluble porphyrins, in particular ofTet(MPy)P and Tet(SPh)P have long been of interest in chemistry and biochemistry. Theelectrochemical properties,3,4 redox properties,5,6,7 interactions with dioxygen,8,9association with DNA and amino acids,1°,Il and their potential as anti-cancer agents,2 haveall been addressed in the literature. The increased lability of axial ligands ofCom[Tet(MPy)P], relative to those of the usually inert Com complexes, was alsostudied12,13,"The complex, formulated as Co111[Tet(MPy)P]C15.(H20)2, has been synthesizedand characterized by Pastemack et al.12ab However, Neta5 found later that when a solutionof this complex was evaporated to dryness, part of this material was reduced toCo11[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 notassure a cobalt(I11) product. Abwao-Konya et al." used Hg(NO3)2 to oxidize Co" to Com,116Chapter 3 Water-soluble Inetalloporphytiaabut there was no evidence to support a Co" formulation in their report. A subsequentsynthesis of Co111[Tet(MPy)P](C104)5.2H20 was reported by Ashley et a1.13 in whichCo(C104)2 and [Tet(MPy)P](CI04)4 were reacted in DMF, followed by precipitation withethanol; 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 inertatmosphere.8,16 The complex was also isolated by Hambright and Fleisher from anaqueous solution as CollfTet(MPy)PNC104)4;17 however, these authors presented nophysical data. Synthesis of Coll[Tet(MPy)P](PF6)4 has also been addressed in theliterature.18The compound Na4Co11[Tet(SPh)19.8H20 was first prepared in 197619 by refluxingcobalt(II) acetate and the sodium salt of Tet(SPh)P in water. The resultant material waspurified by recrystallization from ethanol-water, and characterized by elemental analysisand ESR. The synthesis of this complex has also been reported by other authors. 20,21Although Com[Tet(SPh)P] has been used in various studies,2,21,22 the synthesis ofthis complex has not been reported in detail. In an earlier report,23 this complex wasclaimed to be synthesized by reaction of CoSO4 and Na4[Tet(SPh)13] in DMF, followed bywork-up of the DMF solution on an alumina column (eluted with dilute aqueous NaOHsolution), and work-up of the aqueous solution on a Sephadex column; however, theelemental analysis quoted value for carbon was low by 3.23% and for hydrogen was highby 0.48% from calculated values for Na3Com[T(SPh)11(H20)2. Hambright and Langley14reported the synthesis of Na3ColiT(SPh)11.12H20 by reaction of the porphyrin withCoO, however the formulation of a Con complex fit better for the reported elementalanalysis data.117Chapter 3 Water-soluble metalloporphyrksAs can been seen from this brief overview, a detailed procedure for isolation of aspecific oxidation state, cobalt water-soluble porphyrin complex has yet to be reported. Inthis chapter, the synthesis and characterization of the Coll and Com complexes ofTet(MPy)P and Tet(SPh)P, and some results on their solution chemistry, are presented. Inaddition, the synthesis and characterization of the novel cobalt (II and/or III) complexes ofthe other water-soluble porphyrins addressed in the previous chapter, includingT(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 porphyrinsCopper and zinc complexes of water-soluble porphyrins are also reported to haverelative 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," orCu(Ac0)219 with the porphyrins in water. The aggregation properties of these complexeshave also been studied.24,25 In this thesis work, the copper complexes of 6 water-solubleporphyrins 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 withZnO in aqueous solution. The Zn(II) complexes of these two porphyrins and of three othernew porphyrins are synthesized in this thesis work.3.2 Experimental3.2.1 Materials and methodsDescription is limited to material not covered in Section 2.2 in Chapter 2.118Chapter 3 Water-soluble metalloporphyrinsThe chloride and tosylate salts of the cationic porphyrins and the sodium salts ofthe anionic porphyrins were synthesized as previously described in Chapter 2. All otherchemicals were analytical grade and were used without further purification. The cationexchange 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 testpaper. The pH values for pKa measurements were measured with a pH meter (RadiometerCopenhagen PHM25) using a glass combination electrode (Cole-Parmer Chemical Co.).The general dialysis procedure described in Section 2.2 was followed, somemodifications 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 undervacuum were made by vacuum transfer of degassed solvent, and the NMR tube wasflame-sealed. NMR samples under N2 were made by adding degassed solvent to solidsamples degassed under N2.Anaerobic UV-visible spectra were measured with a specially designed cell (Figure3.1). Samples were weighed into the cell, and a known amount of distilled water wasadded to the side-arm flask. Water was degassed three times by a freeze-pump-thawprocess, then mixed with the complex. A spectrum was then taken. To observe thereactions of the complex with 02, the gas at 1 atm was bubbled through the sample for20 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 magneticsusceptibility balance (Johnson Matthey Fabricated Equipment). The diameter of thesample tube was 0.30 cm, and the length of sample was about 1.5 cm (-0.04 g). Theactual sample length and weight in the tube were measured using a ruler and an analyticalbalance.119Chapter 3 Water-solahle metalloporphyrlasAll elemental analyses were done on samples handled under N2, unless statedotherwise.Figure 3.1. Cell for anaerobic UV-visible spectroscopy..3.2.2 {Con[Tet(MPy)PJ)C14.2H20A solution of CoC12.6H20 (500 mg, 2.1 mmol, in 20 mL water) was added to asolution of the Tet(MPy)P tosylate salt (500 mg, 0.35 nunol, in 50 mL water), and themixture was refluxed for 45 min. The reaction mixture was cooled and 10 rriL saturatedNaC104 solution was added, resulting in the formation of a brown precipitate, presumablya perchlorate salt. The precipitate was filtered, washed twice with cold water (-5 mL each120Chapter 3 Water-soluble metalloporphyrinsand passed through a column (2.5 x 25 cm) of Cl" ion exchange resin, which wasprewashed with distilled water. The purple eluent was evaporated to dryness on a Rotovapat 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•xH20The brown precipitate obtained by adding NaC104 to the reaction solution asdescribed in Section 3.2.2 was dissolved in warm (-50°C) 0.001 M perchloric acidsolution (50 mL) and the solution stirred under air for 1 h; this solution was thenlyophilized to dryness (-0.25 g product).3.2.4 {Comffet(MPy)PROHDC14-2H20Co11[T(MPy)11C14-2H20 (200 mg, 0.22 mmol) was dissolved in 20 mL of 0.001 MHC1 and stirred in air for 1 h; the solution was dialyzed in 1 L water for 1 h, and then thesolution in the dialysis bag was rotary evaporated at 25-30 °C to dryness. The resultantbrown residue was dried at 100°C under vacuum (< 0.1 ton) overnight. Elementalanalysis: Found: C, 56.78; H, 4.13; N, 12.00; Cl, 15.37 %; calculated forC44H4104CoN803: 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 distilledwater and dialyzed in 1 L water for 1 h. Then the solution in the dialysis bag was rotaryevaporated 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 undervacuum. The resultant purple material was dried at 100°C under vacuum (< 0.1 ton)121Chapter 3 Water-soluble metalloporphyrinsovernight. Elemental analysis: Found: C, 54.58; H, 4.44; N, 11.92; Cl, 18.06 %; calculatedfor C44H42C15CoN803: C, 54.72; H, 4.35; N, 11.61; Cl, 18.39 %. Yield 75%.3.2.6 (Con[T(MPy)PhPDC13.1/2H20T(MPy)PhP trichloride (200 mg, 0.24 mmol) and CoC12-6H20 (200 mg, 0.84mmol) 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 precipitateformed; this was filtered and washed with cold water twice (-5 mL each). The brownpowder was dissolved in hot water, and passed through a column (3.5 cm x 30 cm) of C1exchange resin. The solution was evaporated to dryness with rotary evaporation at 20-30°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•2H20Co11[T(MPy)PhP]C13.1/21120 (100 mg, 0.11 mol) was dissolved in 0.001 M HC1solution (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. Elementalanalysis: 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))C14.1120A cobalt(11) complex was first made using the same procedure as that used for thesynthesis 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 thenfollowed. The title compound was collected with a yield of 90%. Elemental analysis:122Chapter 3 Water-soluble metalloporphyrinsFound: 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))C13.2H20A solution made by dissolving cis-[B(MPy)B(NPh)11C12 (200 mg, 0.22 mmol) andCoC12.6H20 (200 mg, 0.84 mmol) in 50 mL distilled water was refluxed for 5 h. Then tothis reaction mixture, 30 niL saturated NaC104 was added; the resultant precipitate wascollected by filtration, washed with distilled water (10 niL x 3) and air-dried. The brownpowder and -20 inL prewashed Cl- exchange resin were suspended in 150 mL distilledwater, and stirred at -50°C for 4 h. This mixture was filtered and the purple filtrate wasloaded on a prewashed column (3 x 15 cm) of Cl- exchange resin. The purple eluant wasconcentrated to -10 inL by roto-evaporation, and the resultant solution was transferredinto a dialysis bag and dialyzed for 2 h in 1 L distilled water. The solution in the dialysisbag was then evaporated to dryness. To this residue, 0.001 M HC1 solution (10 inL) wasadded, and 02 was bubbled through the resultant solution for 0.5 h. This solution waslyophilized to dryness, and the residue was dried under vacuum at 100°C. Elementalanalysis: Found: C, 55.22; H, 3.76, N, 11.90; Cl, 10.89%; calculated forC44H36C12CoN807: 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 addedto a solution of Na4[Tet(SPh)P].10H20 (500 mg, 0.42 mmol, in 50 mL water). The mixedsolution was refluxed in air for 60 min, cooled and passed through a Na+ ion exchangecolumn (2 x 20 cm). The eluant was evaporated to dryness, and the resultant purpleresidue was redissolved in 50 friL methanol; the methanol 'solution was filtered and thefiltrate 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 collectedby vacuum filtration. This methanol-acetone reprecipitation procedure was repeated twice123Chapter 3 Water-aohtble metalloporphyrinsmore 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 forC44H32CoN4Na4016S4 (n=4): C, 46.01; H, 2.51; N, 5.11%. Yield 85%. This product wasexposed to air for a week, and then a second elemental analysis was carried out (samplewas 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 asolution of Co(acetate)2.6H20 (500 mg, 1.8 mmol in 10 mL water) were mixed andrefluxed in air for 60 min. The reaction mixture was passed through a Na+ ion exchangecolumn (2 x 20 cm), and the eluant was evaporated to dryness. The ion exchange colunmwas used to ensure that any Co2+ as the counterions for the porphyrin was removed, whichis proved by mass spectral data (Section 3.3.3.1). The resultant residue was dissolved in0.01 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 adialysis bag surrounded by 1 L distilled water for 2 h. The solution recovered from thedialysis 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 forC44H54CoN4Na3027S4 (n = 14): C, 39.81; H, 3.91; N, 4.17%. Yield —50%. This samplewas then dried at 100°C under vacuum (<0.1 ton) overnight; elemental analysis of thedried 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 forNa3{ConfTet(SPh)PK0H2)).3H20 (Section 3.2.11). Elemental analysis: Found: C, 54.10;124Chapter 3 Water-soluble metalloporphyrinsH, 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 forNa3{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)}.2H20This complex was synthesized by following the synthetic procedure used forNa3{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 complexesCopper complexes of cationic water-soluble porphyrins were synthesized with aprocedure 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 indistilled water (-10 mL); the resultant solution was transferred into a dialysis bag, anddialyzed in 1 L distilled water for 2 h. The solution in the dialysis bag was roto-evaporatedto 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 inTable 3.1.125Chapter 3 Water-soluble metalloporphyrinsThe copper complexes of anionic porphyrins were synthesized by reaction of theporphyrins with CuC12. Briefly, the porphyrin (200 mg, -0.2 mmol) and CuC12•6H20 (200mg, 0.69 nunol) were dissolved in distilled water (50 mL), and this solution was refluxedfor 1 h. The reaction mixture was evaporated to dryness, and the residue was washed withacetone (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 thepurple 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 aRotovap, 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 inTable 3.1.3.2.16 Zinc porphyrin complexesZinc complexes were synthesized using the procedures as described for thesyntheses of the copper complexes. OH' or Cl- was found to be the axial ligand accordingto the elemental analyses as listed the Table 3.2, but the OH' was not detected by IRspectroscopy. Yields for the syntheses were 80-90%.Table 3.1. Elemental analyses and yields of copper-porphyrin complexesC% H% N% yield %{ Cu[Tet(MPy)P]} C14. 10H20 expected 49.74 5.28 10.55 78found 49.47 5.00 10.42{ Cu [T(MPy)PhP] } C13- 10H20 expected 52.27 5.35 9.70 90found 52.56 5.24 9.54{ Cu [T(MPy)(NPh)P] }C13•6H20 expected 53.71 4.93 11.39 79found 53.67 4.56 11.01Na4{Cu[Tet(SPh)P] } • 10H20 expected 41.75 3.63 4.43 81found 41.57 3.40 4.37Na3 { Cu [(APh)T(SPh)P] } • 9H20 expected 45.58 3.80 6.04 90found 45.81 3.85 5.99Na3{Cu[PyT(SPh)P]) •9H20 expected 45.32 3.69 6.15 92found 45.57 3.68 6.02126Chapter 3 Water-soluble metalloporphyrinsTable 3.2. elemental analysis of zinc-porphyrin complexesC% H% N%Zn112 {Zn(C1)[Tet(MPy)P] } Cl4. 9H20 expected 45.29 4.63 9.61found 45.59 5.67 9.63Znin{Zn(CD[T(MPy)PhP] } C13. 7H20 expected 51.36 4.86 9.53found 51.43 4.78 9.10Na5{Zn(OH)[Tet(SPh)P] } .4H20 expected 44.02 2.92 4.67found 44.16 3.34 4.38Na4{Zn(OH)[(APh)T(SPh)P] } .9H20 expected 43.99 3.75 5.52found 44.01 3.60 5.83Na4{Zn(OH)[PyT(SPh)P] } .10H20 expected 42.37 3.84 5.73found 42.21 3.89 5.513.3 Results and Discussion3.3.1 111 NMR spectra of the metalloporphyrinsIn this thesis work, both Co" and Coll complexes are found to be air-stable inDMSO-D6 and the species do not aggregate, as judged by the concentration independenceof the 1H NMR spectra; 1H NMR spectroscopy in DMSO-d6 is very useful forcharacterizing these complexes, although 1H NMR data in the literature are rare. The 1HNMR spectra of some of these complexes in D20 are also presented.3.3.1.1 1H NMR spectra of Co complexes of cationic-porphyrinsFigure 3.2 shows 1H NMR spectra of {Co11[Tet(MPy)P])C14 in D20 in air andunder vacuum. Spectrum 3.2a was measured 24 h after the sample was made by dissolving2 mg the complex in 0.5 mL D20 (c2', 4 x 10-3 M) in air; oxidation to a diamagneticCo(III) species has clearly taken place. In this spectrum, the pyrrole protons give a singletat 9.11 ppm, which overlaps with a doublet corresponding to the 3,5-MPy protons at 9.13ppm (JHH = 6.6 Hz), a doublet for the 2,6-MPy protons appears at 8.80 ppm (JHH = 6.6127abilmilmilininlymp ill^ni i^1 ni9.2^2.1^9.0^U.S^CO PF911.7Chapter 3 Water-soluble metalloporphirinsHz), and the signal of the methyl protons overlaps with the water peak at 4.63 ppm. Thisis considered to be a spectrum of {Co111[Tet(Mr.Py)P](H20)(OH))04 (although a peroxide-bridged dimer cannot be ruled out completely, see Section 3.3.5), mainly because thespectrum of this porphyrin is pD-dependent (Section 3.3.6); the pD value of this solutionis about 8 according to Figure 3.17, as determined by the chemical shift of the pyrroleprotons (Section 3.3.6).liiiilitilitilittitimillittitittitI 1111114^ 12^ 10111111111111116^ 4 PPMI^I14^ 12^ 10-v1111111111^1:^11,111PPMFigure 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 D20.128Chapter 3 Water-soluble metalloporphyrinsSimilarly, 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 casesfor the porphyrin free-bases (Section 2.3.2, p.81), the singlet and the two doublets can beassigned to the 7, 8, 12, 13 and, to 2, 3, 17, 18 pyrrole protons, respectively. The othersignals can also be assigned by comparing the spectra to those for the porphyrin free-bases. The 1H NMR data and assignments for the two complexes at pD-7 are listed in thetable below:7,8,12,13pyrrole2,3,17,18pyrrole3,5-MPy 2,6-MPy 3,4,5-Ph or3,5-NPh2,6-Ph or2,6-NPh{C,olll[T(MPy)Ph11}C14 9.12 s 9.23 d '9.04 d 9.12 d. 8.81 7.76 m 8.16 d{Com[T(MPy)(NPh)PI}C14 9.12 s 99..1064 bbrr 9.11 d 8.79 8.55 br 8.36The plots of the chemical shifts for the 7, 8, 12, 13 pyrrole protons (singlet) provide anopportunity to measure the pKa values of the diaquo complexes, which is discussed inSection 3.3.6. Spectrum of {Com[cis-B(MPy)B(NPh)PDC13 in D20 did not give sharpsignals, therefore was not further studied.Figure 3.2b is a spectrum of 2 mg of {Coli[Tet(MPy)P]}C14 in 0.5 mL degassedD20 under vacuum; no oxidation is evident and the spectrum is that of a paramagneticCo(H) species. The broad signal at 13.7 ppm corresponds to the pyrrole protons which areclosest to the paramagnetic center; the signals of the 2,6-MPy and 3,5-MPy protonsoverlap with each other and result in the signal at 9.88 ppm; and the resonance of themethyl protons is at 5.15 ppm. The proton chemical shifts of this species are dependent onthe concentration and the ionic strength, these findings being discussed later (Section3.3.1.5).The Evan's method, which uses 1H NMR spectroscopy to measure the magneticsusceptibility of a compound in solution, was used in the literature by Pasternack et a!. 12b129Chapter 3 Water-soluble metalloporphyrinato show that the cobalt was in its diamagnetic, +3 oxidation state in a complex ofTet(MPy)P. However, the experiment was performed in D20 and presumably in air, andthus this evidence does not show that the complex in solid state was a cobalt(III) speciesbecause, as shown by Figure 3.2, a cobalt(II) complex in D20 under air is oxidized to acobalt(III) complex.Figure 3.3 shows the spectra of {Co11[Tet(MPy)P]}C14 and {Con[T(MPy)PhP]}C13in DMSO-d6, recorded in air. In spectrum 3.3a for {Coll[Tet(MPy)P]}C14, the pyrroleprotons give a broad signal at 13.9 ppm, and the 2,6-MPy protons a broad signal at 9.8ppm; the 3,5-MPy protons which are farther away from the paramagnetic center give arelatively sharp signal at 10.1 ppm, while the protons of the methyl groups, which arefarthest from the paramagnetic center, give an even sharper signal at 5.30 ppm. Thisspectrum 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 givepeaks similar to those found in spectrum 3.3a for {CoR[T(MPy)PhP]}C14: a broad peak at13.3 ppm corresponds to the pyrrole protons; another broad peak at 9.9 ppm and arelatively 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 and8.45 ppm. The resonance of the methyl protons appears at 5.24 ppm as a singlet. Againthe 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 eachspectrum indicate that two species are present in the solution of each complex, and thedifference between the two species is presumably due to the variation of axial ligands. Theresonances of the 3,5-MPy protons appear at 9.35-9.55 ppm, and the resonances of the2,6-MPy protons appear at 8.75-9.05 ppm. The resonances of the pyrrole protons appear130a II f I I I I f I I I^I I I I^I I 1 1 1 1 I I 111111-1111^1111iIIII14 12 10^ 6PPMFigure 3.3. 1H NMR spectra of Co(II) cationic porphyrinsin 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)Chapter 3 Water-soluble metalloporphyrins9.4^9.2^9.0 8.B PPM1111114.8^ 4.6Figure 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 at9.27 ppm (species 3) and 9.09 ppm (species 4) in the case of the chloride. The possibleaxial ligands for species 1 and 2 giving spectrum a are water and DMSO, ignoring possiblecoordination of C104. The axial ligands for the species 2 and 3 must be the same becausetheir pyn-ole protons give the same signal (9.27 ppm). The 2,6-MPy protonscorresponding to these species give peaks at slightly different positions, presumablycentered at 8.89 ppm for the perchlorate (spectrum a) and at 8.91 ppm for the chloride132Chapter 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 species2 and 3 in spectra a and b. Chloride must be coordinated to cobalt in species 4 simplybecause this species does not exist in the solution of the perchlorate. The actualassignments for the various species in these solutions can not be made with theinformation available.Replacing a neutral axial ligand by a negatively-charged ligand (C1) is likely toshift the pyrrole protons to higher field. This is certainly observed in the 1H NMR - pHstudies, discussed in Section 3.3.6: when H20 is replaced by OH' as a axial ligand, all thesignals shift to higher field. By this observation and a study of the spectra shown in Figure3.4, it is clear that is involved in the coordination shell of cobalt(III) in DMSOsolutions 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 twospecies present in the solution for each system. As discussed above, the actual species insolution are not well defined; the species corresponding to the signals at relatively lowfield is maned #1 (the axial ligand(s) is probably H20 and/or DMSO), and the speciescorresponding to the signals at relatively higher field is named species #2 (at least one ofthe axial ligands is Cl). The chemical shifts of these signals are listed in Table 3.3. Themethyl 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-d6only 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 thoseof 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 in133Chapter 3 Water-soluble metalloporphyrinsTable 3.3; this will be a factor in deciding the relative tendencies to bind the negativelycharged Cl- (vs. water or DMSO).Table 3.3. 1H NMR data of the Co cationic porphyrin complexes in DMSO-d6chloride salts pyrrole 3,5-MPy2,6-MPy2,6-Ph or2,6-NPh3,4,5-Ph^or3,5-NPhColll[T(MPy)PhP] #1 9.23 s, 9.15(AB qt) 9.48 d 8.92 d 8.17 m 7.89 mCol11[T(MPy)PhP1 #2 9.06 s, 8.96(AB qt) 9.40 d 8.82 d 8.11 m 7.85 mCollirT(MPy)(NPh)P] #1 9.26 s, 9.19(AB qt) 9.50 d 9.43 d 8.72 d 8.43 dCox[T(MPy)(NPh)13] #2 9.08 s, 9.00(AB qt) 9.42 d 8.82 d 8.69 d 8.37 dCom[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 dThe 1H NMR spectral data for {Coultret(MPy)PMC104)5 in DMSO-d6 have beenreported before as 9.37 (s, 8H), 9.27 (d, 8H), 8.92 (d, 8H) and 4.80 (s, 12H), but withoutassignments.3 These data do not correspond to those obtained for either of the speciesdiscussed here (Figure 3.4). The authors of this report did not mention any dryingprocedure or elemental analysis; one possibility is that the reported sample contained morewater, 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-porphyrinsFigure 3.5 shows the spectra of Na4{Con[Tet(SPh)P)).4H20 andNa3(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 pyrroleprotons, because these are closest to the paramagnetic center; the sharper signal at 8.8ppm is assigned to the remote 3,5-SPh protons, and the underlying broad signal at 9.2ppm to the 2,6-SPh protons. In the diamagnetic spectrum (b), the sulfonatophenyl protonsgive an AB quartet (8.0 to 8.2 PIN11, Jim = 8.0 Hz), while the resonance of the pyrroleprotons appears at 9.20 ppm as a singlet.The 1H NMR spectra of Na2(Com[PyT(SPh)P]) andNa2{Cou'l(AP)T(SPh)P(OH2)] in DMSO-d6, shown in Figure 3.6, are less defined than the1349.2 9.0 •.11 U.S •.4 9.2 PPliChapter 3 Water-soluble metalloporphirrhoscorresponding spectrum of Conet(SPh)P. The broadening of the peaks may result fromthe coordination of the pyridyl or amine group of one porphyrin molecule as an axialligand at the metal center of a second porphyrin molecule. The peaks due to water in bothspectra 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 freewater, or from the possible formation of hydrogen bonds between water and the pyridyl orthe amine moieties of the porphyrin structures. The Ili NMR spectra of the complexes ina^JIIIIf I1I I^I I14^12^10^ 4^PPMFigure 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).13516^14^12^10^8^4^2^0^-2^-4 PPMFigure 3.6. 1H NMR spectra of cobalt complexes oftris-sulfonatoporphyrins in DMSO-d6.a: Na2{Com[PyT(SPh)P]); b: Na2{ColIVAP)T(SPh)Pli0H2)}•H2O.I.Chapter 3 Water-soluble metalloporphyrinaD20 show two even broader peaks centered at 9.2 and 8.3 ppm. The broadening of thesepeaks in D20 again could result from the coordination of the pyridyl or amine groups. Thespectra of Collcis-(NPh)PyB(SPh)13] in DMSO-d6 and in D20 have the samecharacteristics as described for the tris-sulfonato species.Attempts to synthesize Na2{Coll[PyT(SPh)P]} and Na2{Con[(AP)T(SPh)11) viathe same procedure used for the synthesis of Na4(Co11[Tet(SPh)P]}.4H20 resulted inisolation of mixtures of Co(II) and Co(III) species, as judged by Ili NMR data. This mayresult from the existence of coordinating groups (pyridyl or amine) at the Co center of theporphyrin 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 theCo(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 observedin the spectrum of a mixture of Co(II) and Co(III) complexes of (APh)T(SPh)P inDMSO-d6.3.3.1.3 111 NMR of Cu-porphyrinsThe 11-1 NMR spectra of the Cu-porphyrins give broad signals for the porphyrinprotons. The appearance of these broad signals and the disappearance of the N-pyrroleprotons (at approximately -3.0 ppm, Section 2.2) are evidence for coordination ofcopper(II).The II-I NMR spectra for the complexes of the copper(H) complexes of cationicporphyrins in DMSO-d6 are shown in Figure 3.7. In spectrum a for Cu[Tet(MPy)P], twosignals 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 and2,6-MPy protons, which are closer to the paramagnetic center, are probably too broad to137Chapter 3 Water-soluble metalloporpbyrins111111111i1111111111111111117111111111111111111r1111111111IIIIII111111.11,112^10^8^ 4^ 0^-2 PPMFigure 3.7. 1H NMR spectra of copper(II) complexesof 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.)138chapter 3 Water-soluble metalloporphyrinsbe observed. In spectrum b for Cu[T(MPy)PhP], signals at 9.2, 7.7 and 4.5 ppm areassigned to the 3,5-MPy, 3,4,5-Ph and the methyl protons, respectively. Again the signalsof pyrrole, 2,6- MPy and 2,6-Ph protons are considered too broad to be observed. Inspectrum c for Cu[T(MPy)(NPh)P], the peak at 8.5 ppm is assigned to the 3,5-NPhprotons, and the other signals are assigned similarly to those of spectra a and b, as 9.2ppm to the 3,5-MPy protons and 4.5 ppm to the methyl protons. The assignmentsgenerally are supported by the relative intensities of the signals; for instance, the ratio ofthe 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 apattern for the aromatic protons similar to that of the cationic ones: one broad peak at 7.8ppm for Cu[Tet(SPh)P], two broad peaks at 7.8 and 7.0 ppm for Cu[(AP)T(SPh)P], andtwo broad peaks at 7.8 and 7.3 ppm for Cu[PyT(SPh)P]. In the last two cases, the peak at7.8 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 thespectrum of Cu[(AP)T(SPh)P] is assigned to the 3,5-APh protons (2H), and the signal at7.3 ppm in the spectrum of Cu[PyT(SPh)P] to the 3,5-Py protons (2H). The only signalobserved in the spectrum of Cu[Tet(SPh)P] (at 7.8 ppm) is assigned to the 3,5-SPhprotons. Again, the signals for the pyrrole, 2,6-SPh, 2,6-APh and 2,6-Py protons areprobably too broad to be observed in this spectrum.3.3.1.4 1H NMR of Zn-porphyrinsThe 1H NMR spectra of the Zn complexes have the same characteristics as thoseof 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 pyrrolesignals, shift to the higher field. The signals are assigned similarly to the assignments of thefree bases (Tables 2.11 and 2.14), and are listed in Table 3.4.139Chapter 3 Water-soluble metalloporphyrinaTable 3.4. 111 NMR data for Zn porphyrin complexes in DMSO-d6Zn[Tet(MPy)P] 9.42 d3,5-MPy9.00 spyrrole8.87 d2,6-MPy4.73CfliZn[T(MPy)PhP] 9.42 d3,5-MPy9.03 s; 8.93 AB qt,pyrrole8.93 d2,6-MPy8.18 d2,6-Ph7.86 m3,4,5-Ph4.71CH/Zn[Tet(SPh)P] 8.78 spyrrole8.13 d2,6-SPh8.00 d3,5-SPhZn[(APh)T(SPh)P] 8.88 d, 8.75 mpyrrole8.12 d, 2,6-SPh7.99 d, 3,5-SPh7.83 d2,6-APh6.97 d3,5-APh5.46 sNH,Zn[PyT(SPh)P] 8.78 mpyrrole8.13 d, 2,6-SPh8.01 d, 3.5-SPh9.04 s(br)3,5-Py8.27 s(br)2,6-Py3.3.1.5 1H NMR studies on the aggregation of Con complexesAggregation 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 atroom temperature, while addition of DMF to the solution under air prior to freezingresulted in a spectrum due to the presence of the monomeric Co(11)-porphyrin and itsdioxygen complex (C(III)-02).'9 These findings suggested that the Co(ll) porphyrinaggregates in aqueous solution and, when organic solvent is added, the aggregation breaksdown, thus leading to the ESR spectrum of the monomer and (in the presence of 02) thesuperoxide complex. In this thesis work, the aggregation of {ConfTet(MPy)PDC14 andNa4{Coll[Tet(SPh)P] were studied in a preliminary manner by 11-1 NMR in D20 underanaerobic conditions.Figure 3.8 shows the 1H NMR spectra of {Co11[Tet(MPy)P])C14 in D20 undervacuum at concentrations of 2 x 10-3 M (a), 2 x 10-3 M at g = 0.2 M (NaC1) (b), and 1 x10-2 M (c). The signal of the pyrrole protons (13-14 ppm) shifts to higher field, and thesignal of the MPy protons (9.5-10 ppm) remains essentially at the same position (verysmall shifts to lower field can be observed), when either ionic strength or concentrationincreases. These findings possibly result from aggregation, though no model can beproposed from the data available.140Limh....e........00Chapter 3 Water-soiable metalloporphyrins11111111111FIIIIIIIiiiiimiiiiIIII14^12^10^8I^'511^11111^II4 PPMFigure 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.141Chapter 3 Water-soluble metalloporphyrinsSpectra of Con[Tet(SPh)P] in D20 under N2, and the conditions for eachspectrum, 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 byoxidation of the Co(II) complex by residual oxygen in D20. In spectrum a, the very broadsignal centered at —12.0 ppm can be assigned to the pyrrole protons, the broad signalcentered at about 9.6 ppm (labeled V) can be assigned to the 2,6-SPh protons, and arelatively sharp signal at 9.15 ppm which can be assigned to the 3,5-SPh protons. There issome degree of overlap for the last two signals. When the concentration increases from0.004 to 0.008 M (from spectrum a to b), the signal for the pyrrole protons shiftssignificantly to higher field (-12.0 to —11.3 ppm) and the signal for the 3,5-SPh protonsshifts slightly to lower field. The shift of the signal for the 2,6-SPh protons is notobservable because the signal overlaps with the signal of the 3,5-SPh protons. Spectrum cis obtained at the same concentration as spectrum b, but NaC1 is added to a concentrationof 0.2 M. Again, the pyrrole signal shifts to even higher field, while that for the 2,6-SPhprotons shifts further to lower field (now at 9.32 ppm), and the two signals overlap. Theremay be a broad peak in the region of 7-10 ppm in this spectrum because the base linecannot be phased to give a straight line, and this peak could be the signal for the 2,6-SPhprotons. Spectrum d, obtained at higher concentration (0.03 M) without salt, is almost thesame as spectrum c. When salt is present in a solution at this higher concentration, thespectrum changed dramatically as shown in spectrum e; the peak of the 3,5-SPh protonsshifts back to higher field (from 9.35 ppm in spectrum d to 9.00 ppm in spectrum e), and apeak which might be assigned to the 2,6-SPh protons appears at 8.1 ppm. The signal forthe pyrrole protons is probably buried under the observed peaks. Again, no model can beproposed based on these data; however, more than one process appears to be involved inthe aggregation of this metalloporphyrin over the range of concentrations and ionicstrengths used because of the complexity of the signal shifts for the 3,5-SPh protons.142Ce^PfalooMomay.mmommlima1001010111000061~010Ma111111111111111111111111111111iiiiiiiiiiiilliiiiirilliwiiiiiiIIIIIIIIIIITilifilifillimili15^14^15^1k^11^10^ 7 PPM^6Figure 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).143Chapter 3 Water-soluble metalloporphyrins3.3.2 Syntheses3.3.2.1 Synthesis of Co-cationic porphyrin complexesOne original purpose of this project was to synthesize Com complexes of the water-soluble 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 wasadded to precipitate the metallated porphyrin as a perchlorate salt; this was washed withwater. 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 underair in the present work showed the presence of a Co(III) complex, which agreed with theliterature. 12a' b The 111 NMR spectrum of this product in D20 in air also showed thecompound was a Com complex (spectrum a, Figure 3.2, p.128). Further characterizationby both 111 NMR in degassed D20 under vacuum (spectrum b, Figure 3.2) and 11-1 NMRspectrum in DMSO-d6 (spectrum a, Figure 3.3, p.131) showed the compound made wasin fact paramagnetic, implying a complex of Co(Il); the perchlorate salt was shown by 1HNMR 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+ froma 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 amixture 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 ofCo(III) complexes, as described in Section 3.2, the solutions after purification procedures144Chapter 3 Water-soluble metafloporphyrinswere always acidified, and lyophilization was used in some cases to preserve the acidity ofthe solution (see Section 3.3.2.3 for a discussion about the reaction conditions).3.3.2.2 Synthesis of Co-anionic porphyrin complexesIn an effort to prepare Na3{ComiTet(SPh)Ph, a literature method reported for thesynthesis 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 tocoordinate to the Com-porphyrin, as shown by a 1I-1 NMR spectrum in DM50-d6 of thecrude product which showed a peak at -1.57 ppm; this disappeared in a spectrum of thepurified 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 Concomplexes with the latter dominating. The existence of acetate in the reaction mixture mayfacilitate the oxidation of the Con complex to Com through coordination.26Coordinated and free acetate were removed from the crude product by extractionwith CHC13 under acidic conditions, and the inorganic salts left over were removed bydialysis. The low yield for Com[Tet(SPh)P], compared to the yields for cobalt complexesof the other anionic porphyrins (Section 3.2), mainly resulted from the loss of the complexin the dialysis process.Attempts to synthesize Na{Com[c-BPyB(SPh)11) failed in that a mixture ofcobalt(II) and cobalt(III) complexes was always isolated, as judged by 1H NMR inDMSO-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 10-5M in distilled water could not be oxidized by air to a purely cobalt(III) species, as judgedby 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 thecorresponding Co(III) species, Section 3.3.5).145Chapter 3 Water-soluble metalloporphyrins3.3.2.3 Reaction conditions for the synthesis of cobalt porphyrinsIn order to understand the process of the synthesis, especially regarding theoxidation state of cobalt, the effects of a number of reaction conditions were studied.Reaction timeThe reaction of [Tet(MPy)P]C14.4H20 and CoC12.H20 (1:5 mole ratio) inrefluxing aqueous solution under air was monitored by UV-visible spectroscopy. After 45min reaction time, metallation was essentially completed, as the four visible maximabetween 500 and 700 rim had been replaced by a singe absorbance at —540 rim; and thespectrum was typical of a Co(II) complex (see Table 3.8, p.158). The oxidation state ofCo 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 to431 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 aqueoussolution28 was never observed, even up to 24 h. Prolonged reaction times also resulted in alower yield of product and more impurities. In fact, when the porphyrin and excess CoC12were refluxed in water for 2 days, UV-visible spectroscopy indicated that almost all of theporphyrin had been destroyed.1H NMR spectra of DMSO-d6 solutions of the precipitate , formed by addingsodium perchlorate to the mixture of [Tet(MPy)11C14 and CoC12 at different reactiontimes, were also recorded in order to study the effect of reaction time on the oxidationstate 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 mixturecontaining about the same amount of Co" and Com species was observed.146Chapter 3 Water-soluble metalloporphyrinsBased on the observations above, possible reactions occurring in aqueous solutionare tentatively proposed to be [H2P4+ is used to present the porphyrin free base cationTet(MPy)11:(1) Co2+(aq) + H2P4+(2) CoffP4+ + H+ + 1—402(3) CoInP5+ + Co2+(aq) —(4) Co3+(aco + —21 H20Co1134+ + 2H+ConIP5+ + 1H202Col[134+ + Co3+(acp1CO2+(acp + —02 + H+43.13.23.33.4The metallation (eqn 3.1) first occurs, and then the Co(II) complex is oxidized by air (eqn3.2, see Section 3.3.5). The oxidation equilibrium favors the Co(III) complex as observedby 1H NMR (spectrum b, Figure 3.2, p.128) in aqueous solution. However, a subsequentmetal ion exchange reaction might result in the formation of some Co(10-porphyrinspecies, 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 destroythe porphyrin structure. Aggregation of the Co(II) species at the concentration and ionicstrengths used for the synthesis might also play a role in the observed phenomena; this isdiscussed later in this section (eqns 3.2 and 3.5).AcidityFor the synthesis of Cow[Tet(MPy)P], samples collected from evaporation of thesolutions after Cl- ion exchange were always mainly the Co(II) complex, whileevaporation of acidified solution gave a Co(III) complex, as judged by 1H NMR spectra inDMSO-d6.Use of the tosylate salt of the porphyrin or use of Co(acetate)2 resulted in moreCo(III) in the product mixture of porphyrin complexes precipitated by NaC104 than whenusing chloride salts as the starting materials (for a reaction time of 10 h). This may be147Chapter 3 Water-soluble metalloporphyrinaexplained in that, in the refltudng process, protons (produced from the replacement of theN-pyrrole protons by cobalt) may be lost in chloride media in the form of HC1, while theformation of acetic or tosylic acid may help to preserve the acidity. Possible coordinationof anions may also have some effect on the Co-redox equilibrium, as discussed in the caseof 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 thecationic and anionic porphyrins as described in the synthesis procedures (Section 3.2). Forinstance, when a sample of Na4Coll[Tet(SPh)13] was dissolved in water to a concentrationof approximately 10-5 M, and the solution stirred in air until the UV-visible spectrumshowed that Cou[Tet(SPh)P] (the Soret band at 411.8 nm, Figure 3.14) was oxidized toCom[Tet(SPh)13] (the Soret band at 423.9 nm), evaporation of the solution to dryness on arotovap gave a residue which was a mixture of Co(II) and Co(III) (according to the NMRspectrum). However, when this solution was acidified and evaporated to dryness at lowertemperature (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 becompleted in aqueous solution at neutral conditions (Figure 3.2, p.128), and yet acidity isneeded to ensure a Co(Ill) product in the synthesis procedure. It may be that aggregationof Co(II) complexes is important in the redox process. Aggregation of water-solubleporphyrins and metalloporphyrins in aqueous solutions is well known.30 Although reportson 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 beenreported to aggregate.25,32,38 Some Ili NMR evidence for the aggregation of Co"complexes is presented in Section 3.3.1.5.148Chapter 3 Water-soluble metalloporphyrimCo(111)-porphyrins have been reported not to aggregate in aqueoussolutions, 12a,13,31 and this was confermed with an anionic Co(III)-porphyrin in the presentwork (Section 3.3.5).The reactions involving the Co(ll) and Co(I11) redox process and the evaporationprocess are possibly related to the equilibria shown below using the Co complexes of[Tet(MPy)P]4+ as an example:Co1134+ + H+ + -102 —... Com135+ + —1 H20 3.24 2n [Co11114+ [ConP4+ln 3.5Eqn 3.2 is the redox reaction and eqn 3.5 represents the aggregation process for theCo(l) complexes. If a solid sample of {Co11[Tet(MPy)PDC14 is dissolved in water to forma dilute solution (pH —8) under aerobic conditions, the 02 can oxidize the Co(ll) complexto a Co(III) complex as shown in equation 3.2. When this solution is concentrated byevaporation, Co1134+, although presents in a small amount, will start to aggregate (eqn3.5), and this shifts eqn 3.2 to the left. Consequently, at the point of dryness the complexis in its reduced, aggregated Coll state. If the pH is changed from 8 to 3 (0.001 M acid wasused for the synthesis, Section 3.2), the ratio of Co(II) to Co(III) species governed by eqn3.2 is reduced by 105. The increase in concentration of the total cobalt from a dilute to asaturated solution of the complex (e.g., at pH 3) is probably not sufficient to shift theequilibrium of eqn 3.3 to effectively form the Co(II) complex. This suggested mechanismis 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 spectrumappeared at 423.9 nm (the maximum for the Co(III) complex, Section 3.3.5). In thefollowing 2 h, a sample was taken out every 15 min and a spectrum was recordedimmediately after dilution to approximately 10-5 M; and then another spectrum of the149Chapter 3 Water-soluble metalloporphyrinssame dilute sample was run again 5 min later. The latter spectrum was found to have anabsorbance approximately 10% higher at 423.9 nm than the first spectrum for everysample, which was always essentially the same. This can perhaps be explained by assumingthat in the oxidized concentrated solution (-0.01 M), only a small portion (-10%) of thecomplex was present as Con9jTet(SPh)11 (the majority being in the aggregated forms), andthus 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 weredissociated into monomer and were oxidized to Co(III) (eqn 3.2), and this resulted in theabsorbance increase. Unfortunately, this portion of the Co(II), if it does exist, can not bedefinitely and directly detected by UV-visible spectroscopy because of the poor sensitivityof the method, nor by NMR spectroscopy because a Coo species, especially in theaggregated forms, gives only broad signals in the 1H NMR spectrum.TemperatureNeta5 has demonstrated that when an aqueous solution of Na3Com[Tet(SPh)P] isevaporated to dryness, and the residue then heated to 120 0C, part of the resulting productis reduced to Col[Tet(SPh)P]. This agrees with the finding in the present work that amixture of Co(II) and (III) complexes resulted from evaporation of a neutral solution ofCom[Tet(SPh)P], this is originating from air-oxidation of ColiTet(SPh)P1 (p.149). Theobservation of Neta has been stated by Hambright and Langley14 to result from the heatingof 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 vacuumovernight, and no reduction was found. Also, neutral solutions of {Com[Tet(MPy)P1) C15were evaporated to dryness either by lyophilization or roto-evaporation, and the residuesdried at 100°C or room temperature under vacuum; {Co9ITet(MPy)PDC14 was the onlycobalt-containing product, as indicated by 1H NMR. These findings indicate that reduction150Chapter 3 Water-soluble metalloporphyrinsoccurs during the process of concentration, and not via the heating process as suggested inthe literature." The conversion found by Neta probably resulted from the evaporationprocess at the relatively high pH value (> 6) of the solution (see previous page).Lyophilization has the advantage of maintaining the higher oxidation state ofcobalt probably because it prevents an increase in concentration and retains the acidity ofthe solution, both of these factors having effects on the oxidation state of the isolatedproduct as discussed above.3.3.2.4 Synthesis of Cu-porphyrins and Zn -porphyrinsSynthesis of the copper and zinc complexes was first tried using a reactioninvolving Cu(OH)2 (or CuO) and ZnO, respectively. The advantage of using these metalsources with low solubilities was that the excess starting metallic material could be filteredoff at the end of the reaction. However, reaction of PyT(SPh)P with Cu(OH)2, CuO orZnO gave a very low yield of the required complex, because of the observed association ofthe product with the metal precursor (Cu(OH)2, CuO or Zn0), possibly via coordinationof the pyridyl group. Samples synthesized with Cu(OH)2, CuO or ZnO had to be dialyzedto obtain analytically pure products. It was also difficult to filter the muddy lookingsuspensions of Cu(OH)2, CuO or ZnO. The syntheses starting with CuC12 and ZnC12 weremore convenient. Dialysis of the resulting copper and zinc porphyrin complexes resulted inhigh yield, probably because of the aggregation of these complexes at the dialysisconcentrations used (0.01 -0.005 M).323.3.3 Mass spectra3.3.3.1 Mass spectra of cobalt complexes of the anionic porphyrinsFigure 3.10 shows the anionic FAB mass spectrum (thioglycerol matrix) ofNa3{Com[Tet(SPh)P](OH2)} .3H20 and Figure 3.11 shows the suggested molecular15192695097490956I100071, 111111109710340 —900Figure 3.10. Anionic FAB mass spectrum of Na3{Co11i[Tet(SPh)]P(OH2)}•3H20.152100 — -509021050^ 1100Chapter 3 Water-soluble metalloporphyrinsNa 03SNo 03SFigure 3.11. The suggested molecular fragments for three of the major peaks inthe mass spectrum of Na3(Co111[Tet(SPh)P](OH2)}.3H20.153Chapter 3 Water-soluble metalloporphyrinsTable 3.5. Mass spectra of some anionic Co-porphyrinsNa2{Cox[(APh)T(SPh)1311947NaH{CoRAPh)T(SPh)13])9251H2{CoRAPh)T(SPh)11}Na2{Com[PyT(SPh)11}933NaH{Co[PyT(SPh)Pj}911!H2{Co[PyT(SPh)11)Na{ Coli(cis-NPh)PyB(SPh)11 )8981Na{CoRNPh)PyB(SPh)P11876H{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 porphyrinsThe mass spectra of the cobalt cationic porphyrins were recorded using thecationic FAB technique (3-nitrobenzylalcohol matrix). The major peaks above massnumber 500 are listed in Table 3.6.Table 3.6. Mass spectral data for some cationic cobalt porphyrins{Conl[Tet(MPy)P(OH)}C14 734M-4C1-0H-1720!M-4C1-0H-CH1704M-4C1-0H-2CH3-1{C09IT(MPy)Ph(OH2)}C14 7191 704 689M-4C1-14, 0 M-4C1-1-170-CH/ M-4C1-H70-2CH1(Com[T(MF'y)(NPh)P(OH2)1C14 7641 748 734M-4C1-H70 M-4C1-1170-CH/ M-4C1-H70-2CH1{ConTB(MPy)B(NPh)P(OH2)}C13 792 7781 732M-3 C1-1470-1 M-3 C1-HO-CH M-3 C1-11,70-CH1 -NO7II: The major peaks.A peak at 690 in the spectrum of {Colll[Tet(MPy)P](OH))C14 is also relativelyintense, and may be assigned as M-4C1-0H-3CH3. There is a peak at 735 in the spectrumof {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 coordinatedwater after loss of two protons.154Chapter 3 Water-sotable metalloporphyrias3.3.4 Magnetism and coordination of the cobalt-porphyrin complexesThe magnetic susceptibilities of solid samples of some of the cobalt porphyrinswere measured using a magnetic susceptibility balance at room temperature (21.0°C).xg = 01-(R-R0)/109.mwhere^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 sampleRe = reading for empty tubeM = sample mass in g.The molar susceptibilities (xm) obtained by multiplying xg by the molecular weight of thecompound are listed in Table 3.7.Table 3.7. Magnetic moments of some Co-PorphyrinsXm x 103 Xm' x 103 1-1 (3-M.) unpairedelectronsNa4 {Coll[Tet(SPh)P]} .4H20 7.39 7.87 4.32 3Nal {Com[Tet(SPh)P](OH7)) •1411,0 10.6 11.1 5.04 4Na7{Com[(APh)T(SPh)P(OH,)]).1170 3.94 4.42 3.24 2Nal {Coni[PyT(SPh)P]) 1.39 1.87 2.11 1(Cog[Tet(MPy)P]}C14•21-1?0 3.34 3.82 3.01 2{Com[Tet(MPy)P](OH0)C15-2H70 0.45 0.93 1.48 . 1(?)Diamagnetic susceptibilities of porphyrin free-bases have been reported.32 Thereported molar diamagnetic susceptibility of TetPhP (Xm = 4.81 x 10-4 c.g.s. units) is usedto correct the Xm values. The corrected Xm values (Xm') are also listed in Table 3.4, aswell as magnetic moment I/ which can be calculated as: IA = 2.84 (Xmi•T)1/2 B.M.(T is thetemperature in K).The values of the magnetic moments listed in Table 3.7 are all significantly higherthan the theoretical values calculated from the spin-only expression155Chapter 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-irinteractions between the metal and the porphyrin system. Further investigations on themagnetochemical aspects of these complexes are essential.The magnetic moments of Co-water-soluble porphyrins have not been reported inthe literature. However, some five-coordinated Co(III) complexes (Figure 3.12) have beenfound to be paramagnetic with 2 unpaired electrons.33XX = 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 aplanar, macrocycfic N4 ligand set; further, the four nitrogen have two negative chargeswhich are delocalized within the aromatic systems. When an extra ligand binds to thesefive-coordinate complexes, the resulting six-coordinate species becomes diamagnetic.33Examples of five-coordinate Co(III) porphyrin complexes are known.34 Themagnetic data presented here strongly suggest that cobalt in these Co(1.1.1) complexes ofwater-soluble porphyrins is five-coordinate in the solid state, while it is shown later that inaqueous solutions two water molecules are present as axial ligands (Section 3.3.6). It ispossible that in the process of lyophilization or drying, one of the coordinated watermolecules 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, theparamagnetism is lost (as judged by 1H NMR data, Section 3.3. 1) following coordinationof the solvent in the sixth site. A six-coordinate formulation for the Co(III) porphyrin156Chapter 3 Water-solabk metidloporphyrinecomplexes in the solid state (e.g., {Com[Tet(SPh)19(OH2)2}) would be inconsistent withthe 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, thecomplexes are nearly always five-coordinate.26The paramagnetism of the Co(III) complexes presented in Figure 3.12 has beenrationalized, in simple ligand field terms, as a system with a strong in-plane ligand-field ofthe Schiff base and a weak axial ligand of the halogen, which results in the dxy and dz2orbitals being of similar energy and hence a (dxz)2(dyz)2(dxy)1(dz2)1 configuration for themeta136 (see the energy splitting diagram37 shown below, intermediate position). In theporphyrin systems, the axial ligand (H20) is stronger than a halogen, and the d orbitalsmay split in a manner closer to that of a square pyramid system. In the case ofCom[Tet(SPh)P](OH2), 4 unpaired electrons are detected, consistent with a high spinspecies shown on the right-hand side of the energy splitting diagram. For theCo11[Tet(SPh)13] complex a high spin square planar Co(1)N4 system (shown on the left-hand side of the diagram) would fit the experimental data.—A— dx2..y2 ^ _L_ dx2_y2dx2-y24.-'7-T dz2—I— dxy^dxy^ii,,,:,.- --ir dx z dyz.--la .--- ^ dxy.^......_.4 dz2 d72 .- .-.^...d^d d- d -----"-'"xz, yz^xz, yz inewav=d orbital splitting forsquare planar COI)^square planar^ square pyramid Co(M)--> stronger axial ligandThe same schemes do not fit the data for the Co(ITI and II) complexes ofTet(MPy)P. One possible explanation for this is that there may be some metal-metal157Chapter 3 Water-soluble metalloporphyrinsinteractions 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 unpairedelectrons for each molecule. Such interactions, if they do exist, are less defined forCoilr[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 ofthe pyridyl or amine substituent coordinating, which would change the energy splittingdiagrams compared to that for Com[Tet(SPh)P]; the possibilities for a Co-Co interactionpromoted by aggregation of the porphyrin structures are higher than in the case ofCom[Tet(SPh)P] because there is less static electronic repulsion when one of the chargedgroups is replaced by a neutral group. The rationale used for the 2e magnetism of thecomplexes shown in Figure 3.12 could also be applied to Com[(APh)T(SPh)P(OH2)], withthe aminophenyl possibly acting as the weak axial ligand. More detailed investigation isrequired 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 unpairedelectron, although a difference in geometry between the coordination of the pyridyl andthe coordination of the aminophenyl can be imagined (Section 3.3.7).3.3.5 UV-visible spectraThe UV-visible spectral data for the Co-porphyrin complexes dissolved in water at1.0 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 boratebuffer), 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 differentexperimental conditions, such as the pH value, ionic strength and concentration.158Chapter 3 Water-soluble ntetalloporphyrinsTable 3.8. UV-visible spectral data for Co-porphyrins (a)[Ex10-3]___Xmax Xmax [Ex10-3]{Co11[Tet(MPY)Pi}C14•2F170 428.7^[119] 540.0^[12.7]{Com[Tet(MPy)P](H0)}04-21-170 434.4 [190] 548.0 [16.2]{Com[T(MPy)PhP}(1-170))C14.2H,0 432.0 [162] 545.0 [14.8]{ Coll T(MPy)(NPh)P] (1-170)) C14-H70 431.9 [158] 545.0 [14.3]{ Com[B(MPy)B (NPh)P] (11, 0) } Cla .21170 431.9 [117] 545.0 [10.5]Na4 ( Con[Tet(SPh)P]1•9H20 411.8 [198] 529.5 [13.4]Nal {Colll[Tet(SPh)P](H70)} • 141170 423.9 [219] 539.5 [14.4]Na, {Com[(APh)T(SPh)P](H70) } 430.8 [145] 539.5 [13.1]Nal {Com[PyT(SPh)P] } 429.0 [150] 545.0 [11.4]Na { C om[cis-(NPh)Py(SPh)P](1-1? 0)) • 2H,0 431.0 [125] 547.0 [11.6]A: wavelength (Xmax) in tun, [extinction coefficient (E) in cm-1M-1].The Soret band of CoIf Tet(SPh)P] is obviously sharper and more intense than thecorresponding Soret band for the other cobalt anionic porphyrins, and the latter (the lastthree entries of Table 3.8) appear at longer wavelengths. The shifting of this X and thebroadness of the bands might result from some coordination of the pyridyl or atninophenylmoiety at the cobalt center. The coordination of stronger ligands than water at axial sitesof such metalloporphyrins generally results in the shifting of the Soret X to longerwavelengths.23The Com[PyT(SPh)P] species obeyed Beer's law in distilled water from 1 x 10-6 to1 x 10-4 M. This agrees with an earlier finding that Co(111) complexes of water-solubleporphyrins do not aggregate in aqueous solutions. 12a,13, 31The ColliTet(MPy)P] and Cog[Tet(SPh)P] species are stable in the solid statetoward air for at least days, but are air-sensitive in aqueous solution; this is demonstratedby the II-I NMR spectra in Figure 3.2 (p.128), and by the UV-visible spectra shown inFigures 3.13 and 3.14. The data in Figure 3.13 show the process of oxidation ofColl[Tet(MPy)P] by 02, with isosbestic points appearing at 428, 475 and 540 nm.159Chapter 3 Water-soluble metalloporphyrins350^450^550^65011MFigure 3.13. UV-visible spectra of Con[Tet(MPy)11 at 8.7 x 10-5M, under variousconditions 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 10min 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).160Chapter 3 Water-soluble metalloporphyrins350^450^550^650nFigure 3.14. UV-visible spectra of Coll[Tet(SPh)P] in H20 at 6.9 x 10-5M, at variousconditions at room temperature.Spectra were taken: under argon; 1 min later after 02 was bubbled through the solution for 20 s; another10 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 timeafter the solution was exposed to air.161Chapter 3 Water-soluble metalloporphyrinsA similar set of spectra was reported for the electrochemical reduction of a solution ofCoaget(MPy)I1,28 under anaerobic conditions, where different isosbestic points wereseen at 425 and 450 nm in the Soret region. The differences are probably due to theinvolvement of a superoxo intermediate which has been observed previously by ESR whenCon[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 couldbe 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 . 8In 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 3.9Spectrum e in Figure 3.13 is likely to correspond to the peroxy dimer according to thefindings of Evan and Wood.8 The solution corresponding to spectrum e (e = 1.34 x 105cm-'M-1) was then exposed to air, and the spectrum of the solution changed slowly in thenext 10 h, in that the Soret band shifted to slightly longer wavelength and increased inintensity by –35%, finally reaching the spectrum of the Co(III) monomer.28 A solution ofCon[Tet(MPy)11 was acidified with HC1 solution to pH = 3 and exposed to air, when aCo(III) monomer spectrum was achieved in 1 h, and there was no more subsequentchange; this reaction is probably the combination of those of eqns 3.6, 3.7 and 3.9. Thedata suggest that the Co(Il') complex synthesized is highly likely to be a monomer and nota 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' spectrawere reported when Coni[Tet(SPh)P] was reduced by methanol in a photochemicalreaction under vacuum, but more isosbestic points were observed in this methanol system162Chapter 3 Water-soluble metalloporphyrins(at 360, 419, 454, 534 and 615 nm).21 The similarity of the final spectrum in Figure 3.14to the initial spectrum in the methanol system suggests that the final product in theautomddation process is same as the starting compound in the methanol system, which isconsidered21 to be a Co(III) monomer. The four negatively charged sulfonatophenylgroups will make the metal center more electron rich compared with that inColl[Tet(MPy)P] which has four electron-withdrawing methylpyridinium groups; theoxidation reaction should be more favorable for the sulfonatophenylporphyrin system.The copper metallation of the porphyrins can be readily monitored by UV-visiblespectroscopy. The replacement of the four visible bands (500-650 nm) of the freeporphyrins by one major band (530-550 nm) with a weak shoulder band (575-585 nm) inthis region is very characteristic for the metallation. Because of the known aggregationproperties of copper complexes of water-soluble porphyrins in aqueous solutions,32,38 theUV-visible spectra are almost certainly dependent on concentration and ionic strength, butthe details of these dependences are beyond the scope of this thesis work. Table 3.9 showsthe 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] Xmax [Exl 0-3] Xmax [EX 1 0-3](CUITet(MPY)P1)C14: 101170 422 [200] 545 [16.1] 585 [3.8]{Cu[T(MPy)Ph13] } C11 -101170 421 [95] 546 [8.3] 583 [2.4]{Cu[T(MPy)(NPh)PDC11.6H10 421 [158] 546 [12.9] 583 [2.8]Na4{Cu[Tet(SPh)13]) -101170 410 [466] 538 [19.2] 575 [3.1]Nal { Cu [(APh)T(SPh)P] ) .91170 409 [376] 538 [17.5] 575 [2.6]Nal {Cu[PyT(SPh)P] } .91170 411 [314] 540 [16.7] 578 [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 complexesThe 11-1 NMR spectra of the Com porphyrin complexes in D20 show a pHdependence which is attributed to the equilibria presented in equations 3.10 and 3.11:Co lliP (1120)2n^ComP(H20)(OH)11-1 + H+^Kai^3.10163Chapter 3 Water-soluble utetsdloporphyrinsConiP(H20)(OH)n-1^ComP(OH)2n-2 Fi+^"2^3.11P = Tet(MPy)P, n = 5+;P = T(MPy)PhP/P , T(MPy)(NPh)P, n = 4+P = T(SPh)P, n = 3-The^N1VIR spectra in D20 at various pD values for Com[Tet(MPy)P] andCom[Tet(SPh)P] are shown in Figures 3.15 and 3.16, respectively. In these spectra, all thesignals shift to higher field when the pD increases, and this is attributed to conversion ofcoordinated D20 to OD-; presumably enhanced electron donation to the metal centerresults in increased shielding of the porphyrin protons, especially the pyrrole protonswhich are closest to the metal. At higher pD, the resolution of the signals for the MPyprotons in the spectrum of Cos[Tet(MPy)11 becomes somewhat poorer, presumably dueto OD- associating with the MPy groups. On the other hand, in the case ofColiTet(SPh)13], at lower pD possible association of protons with the sulfonate groupsdecreases the resolution of the sulfonatophenyl signals.The 1H NMR spectra of Con[T(MPy)PhP] and Coffi[T(MPy)(NPh)P] at differentpD values have the same characteristics as that of Com[Tet(MPy)13]. Spectra of the otherCo"-porphyrins in D20 solution do not give sharp signals (Section 3.1), and thereforewere not studied at different pD values.The dependence of the chemical shift of the pyrrole protons on the pD valuesmeans that the pKa values for eqns 3.10 and 3.11 can be measured by NMR. Asolution of the Cog-porphyrin (1.0 x 10-3 M) in D20 was prepared; then to half of thesolution was added solid NaOH to make CNaoH = 0.1 M, and to the other half was added6.0 M HC1 to make Clio = 0.1 M. Aliquots were drawn into NMR tubes during thetitration of the acidic solution with the basic solution, and the pD values were monitoredby a 'pH' meter. NMR samples prepared in this way had essentially identicalconcentrations of the porphyrin and ionic strengths (C 1.0 x 10-3 M, 1.1 = 0.1 M) withknown pD values. 1H NMR spectra were recorded at 23°C. The pKa values were164I^4e 0ala; cy,^eI^ ,,f'//pD = 2.05,,Chapter 3 Water-soluble metaDoPorPhYrhupD = 6.00S11611%millail° .4141"P444".....b...„,„,,,,,,.....001a•qr.qr.••••a;1ildei1pD = 12.00 milinquillimulipuquillimpupliquumpupulinupuquumnimil9.8 9.6 9.4 9.2 9.0 8.8 8.6 8.4 8.2 PONOFigure 3.15. 1H NMR spectra of 10-3 M Colli[T(MPy)Pj in D20 at 23°C atdifferent pD values.Assignments are discussed in Section 3.3.1.165$,sgx‘9 tysitt`,9s1c°^kto°^0%.°°1P13pD = 3.00p.1601011WMI^\1111"114~14114111141#A1dkimminerwpoithoo,pD = 7.02Chapter 3 Water-soluble metalloporphyriaspD = 10.10a11111^[111111111111^111111id.°^9.5 9.0^8.5^8.0 PPMFigure 3.16. 1H NMR spectra of 10'3 M Com[Tet(SPh)PJ in D20 at 23°C atdifferent pD values.Assignments are discussed in Section 3.3.1.166Chapter 3 Water-soluble metalloporphyrinsobtained 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 Figures3.17 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 conventionalmethod" was used to determine the equivalent points, and the pKa values obtainedtogether with available literature values are shown in Table 3.10.Table 3.10. pKa values for some cobalt(M) diaquo porphyrin complexespKal pKa2 conditions (ref.)Conget(MPy)11(H70), 6.0 10.0 C=2 x 10-5 M (12a)^a5.9 ±0.1 9.9±0.1 C=4.34 x 10-6 M (3)^a5.46 ± 0.09 10.7 ± 0.8 1.t =1.0 M [NaC104]25°C (13)ComfTet(MPY)PND20)2 5.5 ± 0.1 10.7 ± 0.1 C=1.0 x 10-3M23°C (present work)Con11[T(Wy)PhP](D20)2 6.2 ± 0.1 10.7 ± 0.1 C=1.0 x 10-3M23°C (present work)Co111[T(MPy)(NPh)P](D20)2 5.9 ± 0.1 10.8 ± 0.1 C=1.0 x 10-3M23°C (present work)CollTet(SPh)P](-120)2 5.72± 0.01 11 =1.0 M (NaC104)25°C (23) .7.02 9.76 IA =1.0 M (NaC104)25°C (40)ConitTet(SPWP20)2 7.6 ± 0.1 11.8 ± 0.1 C=1.0 x 10-3M23°C (present work)a: temperature is not reported.The pKa values obtained in this work for Com[Tet(SPh)P](D20)2 are higher thanthose obtained for Com[Tet(MPy)P](D20)2, and this can be understood because the latterhas four electron-withdrawing cationic groups on the porphyrin ring resulting in a1679.3 -9.25 -9.2 -9MMINIMMIChapter 3 Water-sohthie metalloporphyrim.9.358.95 -8.93^5^7^9^11^13pDFigure 3.17. pKa measurements for Co0i[Tet(MPy)F1(D20)2 from II-I NMR data at23°C.1689.35 —9.15ONIM,9.1 —9.05Chapter 3 Water-salable metalloporphyrias9.459.4I^2^4^6^8pDI10^12 14Figure 3.18. pKa measurements for Co1itTet(SPh)Pp20)2from 1H NMR data at23°C.169Chapter 3 Water-soluble metalloporphyrinsrelatively electron deficient cobalt center, which makes the coordinated water more acidic.In the case of Com[Tet(SPh)ITD20)2, the negatively charged sulfonatophenyl substituentsresult in a relatively electron rich cobalt center which makes the coordinated water lessacidic. Similarly, when a MPy group is replaced by the less electron-withdrawing phenylgroup, i.e., in going from Conget(MPy)11(1320)2 to Com[T(MPy)PhI](D20)2, the pKalvalue increases; and when this phenyl group is replaced by the more electron-withdrawingnitrophenyl group (Com[T(MPy)P1111(D20)2--->Com[T(MPy)(NPh)P](D20)2), the pKaivalue decreases. The pKa2 values of the three cationic cobalt porphyrins studied wereessentially the same.All of the literature pKa values listed in Table 3.10 were measured by UV-visiblespectrophotometric titrations in H20. There is some disagreement between some of thesepublished 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 andH20 should be negligible in these measurements.3.3.7 Hydration and elemental analysisThe hydration of water-soluble metalloporphyrins has been observed by severalauthors in the literature. 14,15,19,20 Hydration is also a general observation for water-solubleporphyrin free-bases (see Section 2.3.1). The degree of hydration is determined byelemental analysis, and is observed also by Ill NMR measurements in DMSO-d6. Ofinterest, no hydration is observed in the sample of Na2{Co111[PyT(SPh)P]), althoughhydration (including possibe coordination ) is observed in all the other metalloporphyrinssynthesized 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). TheCom[(APh)T(SPh)P] species would not be able to form such a tetramer because of the170Chapter 3 Water-soluble metalloporphyrinsdifferent coordination direction of the arninophenyl group and, in the solid state, the axialligand for this metalloporphyrin is most probably a water molecule, as shown by thecorrect elemental analysis for Na2{ComRAPh)T(SPh)PK0H2)).r171Chapter 3 Water-soluble metalloporphyrinaReferences-Chapter 31^J•W• Buchler, in "The Porphyrins and Metalloporphyrins", K.M. Smith, ed., ElesvierScientific 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).172Chapter 3 Water-soluble metalloporphyrias25^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 ScientificPublish 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).173Chapter 4 Aggregation of porphyrinsChapter 4 Aggregation of porphyrins4.1 IntroductionThe importance of porphyrin chemistry is briefly reviewed in Chapter 1. In thestudies of porphyrin chemistry, especially in aqueous solutions, aggregation is oftenencountered.' An understanding of the extent, the kinetics, and the models of theaggregation of porphyrins and metalloporphyrins is crucial to interpretation of kinetics andthermodynamic data obtained in studies on these compounds concerning their substitutionand oxidation-reduction reactions,2 DNA-porphyrin associations,3 and in vivo behaviors.4Because of the practical and intrinsic importance of aggregation in the chemistry andcharacterization of porphyrin compounds, this chapter is devoted to the aggregation of thewater-soluble, porphyrin free-bases synthesized in this thesis work (Chapter 2).Resulting from the hydrophobic properties of the porphyrin ring, aggregation is ageneral property of porphyrin compounds in aqueous solutions. Three types ofinteractions have been proposed in connection with the aggregation of porphyrincompounds:5 (1) n-rc interactions for porphyrin free-bases, (2) metal-n interactions formetalloporphyrins, and (3) metal-side-chain interactions (coordination) formetalloporphyrins. The last two types of interactions are often found in non-aqueoussolutions 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 toface" interaction is proposed. With the synthesis of water-soluble porphyrins, whichstarted in the early seventies, the focus of studies on aggregation of porphyrins shifted tothis new class of compounds.' Many methods have been employed in these studies174Chapter 4 Aggregation of porphyrinsincluding: UV-visible spectrometry,3,8 fluorescence spectrometry,9,0 temperature-jumpkinetic studies," and NMR spectroscopy.12,13 However, limited by the availability of thenumber of synthetic, water-soluble porphyrins, most of the studies have been on just a fewsymmetric porphyrins. The synthesis of a large variety of water-soluble porphyrins in thepresent work provided an opportunity for further studies on the relationship between theaggregation 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 ExperimentalAll 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 porphyrinswere studied.A buffer solution was made by dissolving NaH2PO4 (0.69 g, 0.0050 mol) andNa2HPO4 (0.71 g, 0.0050 mol) in distilled water (1.00 L). The pH value of this buffersolution, measured by a pH meter (Radiometer Copenhagen PHM25) with a glasscombination electrode (Cole-Parmer Chemical Co.), was 7.03 ± 0.01, and the ionicstrength was 0.013 M. Stock solutions of the porphyrins (1.0 x 10-4 M) were made bydissolving a known amount of the solid porphyrin (— 2 mg) in the required amounts of thebuffer solution (-15-25 mL). The solutions at other concentrations were made by dilutionof the stock solutions with buffer or buffer and methanol using volumetric analysisapparatus. Solutions of the porphyrins in pure water were made in the same way but usingdistilled water instead of the buffer solution.Absorbance spectra were measured on a Perkin Elmer 552A spectrometer usingquartz cells (0.100, 0.500, 1.00, 2.00 cm) at 20.0°C. The ordinate scale of the absorbance175Chapter 4 Aggregation of porphyrinsplots, controlled electronically in the spectrometer, was set proportional to the product ofconcentration (C) and light-path length (b) for each of the solutions of a porphyrin ofabsorbance A; thus the vertical axis was proportional to A/bC (equal to e, the molarabsorptivity or extinction coefficient), and if Beer's law was obeyed, the spectra of thesesolutions would be identical. For instance, a solution at concentration C was measuredusing a cell of path length b at a scale S; another solution at concentration of 0.500 C wasmeasured 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 measuredusing a 10 b cell path length at scale 0.3 S. This method was checked by recording spectraof 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, while2-D 1H NMR data were recorded on a 400 MHz spectrometer; spectra were measured atroom temperature (23-25°C) and with external reference (DDS = 0 ppm).4.3 Results and discussion4.3.1. UV-visible spectra in aqueous solutionsThe 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'slaw.1 Dimerization has been proposed as a structural model for most of theaggregations. 8,11,14,15 The most important spectral evidence for dimerization is theobservation of an isosbestic point in the normalized spectra in dilution studies where theproduct of concentration and light-path length is kept constant.16 Dimerization ofmonomers is the simplest model to rationalize such evidence, but higher aggregation (forexample tetramerization from initially present dimers) can also explain the data. Little176Chapter 4 Aggregation of porphyrinsevidence supporting the monomer-dimer model could be found in the literature forsynthetic porphyrins.In this work, the appearance of the spectra should be of the same type as thoserecorded in the literature method where the product of concentration and light-path lengthwas kept constant at one absorbance scale,16 but the experiments could be controlled moreconveniently using the variation of absorbance scale and the variation of cell path length.4.3.1.1 Tet(SPh)PThe spectra of this porphyrin in distilled water at different concentrations areshown in Figure 4.1. The molar absorptivity of the Soret band at 410.0 nm remainsessentially the same (e = 5.0 x 105 M-1 cm-1) when the concentration increases from 0.50x 10-6 to 4.0 x 10-6 M (see inset in Figure 4.2), and increases when the concentrationincreases from 6.0 x 10-6 to 4.0 x 10-5 M; the molar absorptivity at 392 nm increasesthroughout with increase of concentration.The Beer's law plots for the spectra in distilled water are shown in Figure 4.2 (410nm and 392 nm data; some spectra are not shown in Figure 4.1 for the sake of clarity). InFigure 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 higherconcentrations) deviate positively from this straight line. [e increases with increasingconcentration: the molar absorptivities calculated for the spectra at 1.0 x 10-5 M (e = 5.3 x105), 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 thanthe e value at lower concentrations]. Fleisher et a/.14 had interpreted the data for thisporphyrin 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 10-6 M, phosphate buffer) at 412 nm with e = 5.10 x 105 M4 cm-1; these values essentially177Chapter 4 Aggregation of porphyrbuagree with the average of the e values obtained here. The deviation from Beer's law ismore obvious in Figure 4.2B (X = 392 nm).6.0Figure 4.1. The normalized spectra of Tet(SPh)P at various concentrations indistilled water.The concentrations are 5.0 x 10-7, 1.0 x 10-6, 2.0 x 10-6, 4.0 x 10'66.0 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 atdifferent concentrations. As the concentration increases, A.max remains the same (411.3nm), 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 lawdeviation is shown in Figure 4.4.178Chapter 4 Aggregation of porphyrinaA^data at 410 nm1 612010.4144s - AO°..dirr4 4PFP-AAO°01 02 03r.,0Pe .0.5^1^1.5^2^2.5^3^3.5^4Concentration x 105 (M)data at 392 nm543210 0^0.5^1^1.5^2^2.5^3^3.5^4Concentration x 105 (M)Figure 4.2. Beer's law diagrams for Tet(SPh)P in distilled water.2420161284179Chapter 4 Aggregation of porphyrinsFigure 4.3. The normalized spectra of Tet(SPh)P at various concentrations in aphosphate 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.252015105 i^2^3^4^5^6Concentration a 105 (M)Figure 4.4. Deviation from Beer's law for Tet(SPh)P in a phosphate buffersolution.180Chapter 4 Aggregation of porphyrhisThese findings are contrary to the early report, as noted above, that Tet(SPh)Pobeys Beer's law at concentrations between 1 x 10-4 to 1 x 10-9M, at pH = 7.0 [p P4 0 andp = 0.10 M (NaC104)] or pH = 13 (0.1 M Na0H). 14 However, the data agree with laterreports in that Tet(SPh)P aggregates at 20°C at ionic strength p. = 0.05 M (NaNO3) asobserved 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 useda dimerization model to rationalize the data, and the relevant equilibrium constants (Kvalues) for the monomer/dimer equilibrium are summarized in Section 4.3.5).The aggregation observed here in distilled water and in the buffer solution mostprobably is a dimerization process as rationalized in the literature.8,12 Alternatively, theremight be two stages of the aggregation process; e.g. the aggregation in the buffer solutioncould be an oligmerization process. However, a monomer-dimer model (discussed Section4.3.4) is supported by studies of the behavior of this porphyrin in mixtures of methanoland buffer solutions (Section 4.3.2). The equilibrium constant for this presumeddimerization in the buffer solution is presented in Section 4.3.5.4.3.1.2 PyT(SPh)P and (APh)T(SPh)PThe patterns of the normalized spectra at different concentrations. for the buffersolutions of PyT(SPh)P and (APh)T(SPh)P are similar. The former has an isosbestic pointat 402.8 nm (Xmax = 410.7 nm at C = 1.00 x 10-6 M), and the latter has two isosbesticpoints at 404.8 and 428 nm (Amax = 412.4 nm at C = 1.00 x 10-6 M). The Xmax valuesshift to shorter wavelengths for both porphyrins with increasing concentration. Again, theisosbestic points indicate two species in equilibrium in both systems. The normalizedspectra of (APh)T(SPh)P are shown in Figure 4.5 as an example. Negative deviationsfrom Beer' law are obviously observed in the spectral data for both porphyrins. The Beer'slaw plots (A/b vs. C) for these porphyrins in the buffer solution are shown in Figure 4.61813{30^410 40 4K)-500nm3.03Chapter 4 Aggreption of porphyrins(not all the spectral data collected for the Beer's law plot are shown in Figure 4.5 forreasons of clarity in this figure). Negative deviations from Beer's law can be also observedfor both porphyrins from the Beer's law plots, although the plot in Figure 4.6B might wellbe taken to be a straight line; the corresponding spectral data of Figure 4.5 more clearlyshow the deviation.Figure 4.5. The normalized spectra of (APh)T(SPh)P at various concentrationsin 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.182A^ PyT(SPh)P (410.7 nm)2520101505Chapter 4 Aggregation of porphyrins0^1^2^3^4^5^6^7^8^9Concentration x 105 (M)(APh)T(SPh)P (412.4 run)2015500^1^2^3^4^5^6^7^8^9^10Concentration x 105 (M)Figure 4.6. Beer's law deviation for PyT(SPh)P and (APh)T(SPh)P.183Chapter 4 Aggregation of porphyrinsThe aggregation of PyT(SPh)P porphyrin in distilled water has also beenexamined. The maximum absorbance (410 nm) essentially obeys Beer's law (e = 4.3 x 105M-1 cm-', at concentrations from 0.05 x 10-5 to 1.0 x 10-5 M), but the molar absorptivitiesin the 350-390 nm region increase with increasing concentration. No isosbestic point wasobserved.Again, the observed aggregation in the buffer for these two porphyrins might be amonomer-dimer processes, and the equilibrium constants for presumed dimerization arepresented in Section 4.3.5.4.3.1.3 trans-BPyB(SPh)PThe normalized spectra in distilled water for this porphyrin (Figure 4.7) are muchmore complicated than those for Tet(SPh)P. There are two bands in the Soret region, oneat 400-410 nm and the other at 442 nm. It also can been seen that there are probably twopeaks 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 theconcentration increases by 5 times to 5.0 x 10-6 M, a shoulder band at approximately 400nm appears with this peak, while the molar absorptivity in this region decreases and that ofthe band at 442 nm increases; on increasing the concentration above 5.0.x 10-5 M, themolar absorptivities of the broad bands at 400 - 410 nm increase as the the shoulder peakbecomes more intense, and that of the band at 442 nm now decreases. This behavior withvarying concentration was quite reproducible. All these observations indicate that thisporphyrin shows complicated aggregation processes under these conditions, although anisosbestic point is observed at 423 nmIn buffer solutions, the aggregation behavior is completely different. Only one bandis observed in the region of 400-410 nm in the normalized spectra(Figure 4.8). At low184Chapter 4 Aggregation of porphyrinsconcentrations (<1 x 10-5 M) the major absorbance is at 410.0 mn, which is close to theXmax values of the tetralds- and tris-sulfonatoporphyrins, while at higher concentrationsthe absorbance at 443.0 nm becomes the major one. There is a clean isosbestic point at421.3 nm.Figure 4.7. The normalized spectra of trans-BPyB(SPh)P at variousconcentrations 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.185Chapter 4 Aggregation of porphyrinsFigure 4.8. The normalized spectra of trans-BPyB(SPh)P at variousconcentrations 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 x3.0 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 aggregationprocess observed here in the buffer solution is a monomer-dimer equilibrium. The processobserved 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.186Chapter 4 Aggregation of porphyrins4.3.1.4 cis-BPyB(SPh)P, cis-B(APh)B(SPh)P and trans-B(APh)B(SPh)PThe normalized spectra at various concentrations in a buffer solution of these threeporphyrins show that they do not conform to a simple aggregation model in the range ofconcentrations studied.Figure 4.9 shows the normalized spectra of cis-BPyB(SPh)P; there would be twoisosbestic points at 398 and 419 nm if the spectrum at 1.0 x 10-6 M were ignored. Thisprobably means that above this concentration, one aggregation equilibrium dominates and,below this concentration, another equilibrium becomes significant. There appears to betwo bands overlapping in the Soret region, the A, values being approximately 409 and402 rim. At lower concentration (0.10-3.0 x 10-5 M), the 409 rim band dominates, while athigher 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 aremore complicated than for cis-BPyB(SPh)P. From 1.0 x 10-6 to 1.0 x 10-5 M, the Xmaxshifts from 410 to 418 rim, but when the concentration increases from 1.0 x 10-5, to 8.0 x10-5 M, Xmax shifts back to shorter wavelengths from 418 to 412 rim. There would be twoisosbestic 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 twoabsorbance maxima in the Soret region as shown in Figure 4.11. The extinction coefficientat —415 rim decreases with increasing concentration from 0.50 x 10-5 to 1.0 x 10-5 M andthen increases with increasing concentration from 1.0 x 10-5 to 4.0 x 10-5 M, beforedecreasing 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 atthese concentrations. This figure again shows that the aggregation is a multiple-step187Chapter 4 Aggregation of porphyrinsprocess (see also Section 4.3.2). It should be noted that the molar absorptivities of thisporphyrin at either of the absorbance maxima are only about 10% of that for the 410 nmmaxium of Tet(SPh)P.Figure 4.9. The normalized spectra of cis-BPyB(SPh)P at variousconcentrations 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.1880 350^380^410^440^ 500nmFigure 4.10. The normalized spectra of cis-B(APh)B(SPh)P at variousconcentrations 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 x6.0 x 10'5,7.0 x 10-5, 8.0 x 10 M in sequence as shown by the arrows.1890 350^390 430^470 510Q50Chapter 4 Aggregation of porphyrhnFigure 4.11. The normalized spectra of trans-B(APh)B(SPh)P at variousconcentrations 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).190Chapter 4 Aggregation of porphyrins4.3.1.5 Tet(MPy)PThe tosylate salt of this porphyrin was reported by Pastemack et al. in 19728 toobey Beer's law and was considered to remain as a monomer in aqueous solution atconcentrations of 1 x 10-6 to 6 x 10-5 M at 1 M ionic strength. Later on, it becamegenerally accepted that this porphyrin did not aggregate in this concentration range.' In1983, Kano et al. reported that aggregation of the tosylate salt of this porphyrin can bedetected at low concentrations (1 x 10-8 - 2 x 10-7 M) by fluorescence spectroscopy.9 Theexistence of aggregated forms at 1 x 10-6 M was also shown in this report by usingfluorescence spectra as a function of temperature and methanol content. Beer's lawbehavior was also reported in this paper for both the chloride and tosylate salts atconcentrations between 10-5 - 10 "3 M. However, these findings were strongly disputedsubsequently by Pasternack et al. in 1985.17 Also in 1985, a report on the aggregation ofthe chloride salt of this porphyrin was published by Brookfield et a!. 18, in which deviationfrom Beer's law was observed at lower concentrations where A/b vs. C gave a curve; atconcentrations of 5 x 10-6 to 7 x 10-5 M, A/b vs. C gave a straight line, but this straightline 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, moreaggregation data were published by Kano et al.1° to support their earlier findings9 that theporphyrin tosylate aggregates at low concentrations. The dispute about the aggregationstill continued in a paper published again by Pastemack's group in 1988.3 Recently, 1HNMR data have provided evidence for some interaction between the porphyrin and thetosylate counterion.19 1H NMR spectroscopy has been also employed in studies on theaggregation of the porphyrin chloride salt in D20, 10,17,20 and there is general agreementamong these authors that aggregation is only observed by 1H NMR at concentrationsabove —0.01 M, although the data reported by different authors were quite different. Thecopper(II) complex of this porphyrin has also been used as a probe in order to comment191Chapter 4 Aggregation of porphyrinson the aggregation of the free base, and the complex was found to aggregate at 0.5 Mconcentration and 0.2 M ionic strength by ESR.21As shown in Figure 4.12A, marked changes are found in the spectra of theporphyrin chloride in distilled water. The Amax shifts to shorter wavelengths and the molarabsorptivity increases with increasing concentration. These results simply indicate that thechloride salt of this porphyrin does not obey Beer's law in distilled water over the wholerange of concentrations from 2.5 x 104 to 1.0 x 10-4 M, and the deviation from Beer's lawis positive.3.0,-...Ec..)..,cb■••••■C.)siZ•••■ 1.41t0 ^i^350 400 450Figure 4.12A. The normalized spectra of Tet(MPy)P chloride at variousconcentrations 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.1921 2130 OAOA0.30201 0.8070.5120.90.60.3o1284o0^1^2^3^4^5 7^8^9^1028242016Chapter 4 Aggregation of porphyrinsThe plot of A/b vs. C at 422.0 nm is shown in Figure 4.12B, and the inset of Figure 4.12 Bis an enlargement of the lower concentration region.Concentration x 105 (M)Figure 4.12B. Plot of A/b vs. concentration for Tet(MPy)P chloride in distilledwater.The results shown in Figure 4.12B agree with the report by Brookfield et al. onthe Beer's law plot in similar conditions," but do not agree with the reports that thisporphyrin obeys Beer's law by Pasternack et al. (tosylate salt, p = 1 M, C> 10-6 M)8 andby Kano et al. (chloride and tosylate salts C> 10-5 M).9 In conclusion, this porphyrin, atleast as the chloride salt, aggregates in distilled water over the whole range of193Chapter 4 Aggregation of porphyrhisconcentrations examined; the presumed monomer-dimer equilibrium constant is presentedin Section 4.3.5.Aggregation is also observed in a buffer solution (IA = 0.013 M). The buffersolution spectra of the Soret band region are shown in Figure 4.13. As in the distilledwater system, the extinction coefficient of the Soret band increases with increasingconcentration from 1.3 x 10-6 to 8.0 x 10-5 M. The Xmax first shifts to slightly longerwavelength, and then shifts to shorter wavelengths when the concentration increases from2.5 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 variousconcentrations.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.194ampter 4 Aggregadon of porphyrins4.3.1.6 T(MPy)PhPFigure 4.14 shows the normalized spectra of the chloride salt of this porphyrin atvarious concentrations in the buffer solution. There is a positive deviation from Beer's lawfor the Soret band of this porphyrin, from C = 1.0 x 10-6 to 4.0 x 10-5 M, while no morespectral changes are observed at C = 4.0, 6.0, 8.0 and 10 x 10-5 M. These data indicatethat the aggregated form in the observed equilibrium dominates exclusively aboveconcentrations of 4.0 x 10-5 M, and this aggregated form might just be the dimer.350^400^ 450nFigure 4.14. The normalized spectra of T(MPy)PhP at different concentrationsin 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 asshown 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 linemight be drawn approximately, although deviation from Beer's law is observed at thelowest concentrations (0.10 to 4.0 x 10-5 M) as evident in Figure 4.14. This indicates wellthat the plot of A/b vs. concentration sometimes is not as sensitive as the normalized195Chapter 4 Aggregation of porphyrinsspectra for detecting aggregation phenomena. Deviation from Beer's law is also observedmore clearly in the normalized spectra (Figure 4.5) than in the plot of Ab vs. C (Figure4.6B) in the case of (APh)T(SPh)P in the buffer solution.242016-› 128400^1^2^3^4^5^6^7^8^9^10Concentration x 105 (M)Figure 4.15. A plot of A/b vs. concentration for T(MPy)PhP in a phosphatebuffer.4.3.1.7 T(MPy)(NPh)P and cis-B(MPy)DPhPThese two porphyrins were also studied in the same range of concentrations in abuffer solution. The C values at Xmax for both increase with increase of concentration from1.0 x 10-6 to 4.0 x 10-5 M, and then decrease with increase of concentration from 4.0 x 10-5 to 10.0 X 10-5 M. The itinwc at 1.0 x 10-5 M is at 419 nm for the mononitro compoundand at 420 nm for the cis- compound. The Xmax shifts "back and forth" at the lowerconcentrations (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 multi-stage process. The normalized spectra of cis-B(MPy)DPhP are shown in Figure 4.16 inthe two concentration regions, as an example of these two porphyrins.196Chapter 4 Aggregation of porphyrinsFigure 4.16. The normalized spectra of cis-B(MPy)DPhP at variousconcentrations 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 10-510.0 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.1970^!350 45 SOOnm4.04I4I4Figure 4.17. Absorbance spectra of (APh)T(SPh)P in aqueous buffer/methanolmixtures.Chapter 4 Aggregation of porphyrins4.3.2. Affects of methanol on aggregation of porphyrins.As noted by others, some organic solvents can break down aggregation ofporphyrins, and methanol, glycerol and acetone have been found to have a dramaticeffect.9,21,22 The effects of adding methanol to the buffer solution of some porphyrins werestudied in this thesis work.In Figure 4.17, the absorbance spectra of 1.0 x 10-5 M (APh)T(SPh)P in mixturesof the buffer solution and methanol are shown.The content of methanol: 0, 10, 20, 30, 50% (v/v) in sequence as indicated by the arrows.198Chapter 4 Aggregation of porphyrinsThe Soret band increases significantly in intensity (by 44%) and shifts slightlyto longer wavelengths (410.9 to 412.7 nm) when the methanol content increases from 0to 50%. Isosbestic points are observed at 395, 401, 431 and 455 nm in Figure 4.17 (onexpansion), and these isosbestic points may imply that the data result from a shiftingequilibrium of two species.The spectra of 1.0 x 10-5 M Tet(SPh)P in the mixtures of buffer solution andmethanol (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 Soretband increases in intensity by 8% and A.„.x shifts to longer wavelengths (411.3 to412.0 nm) when the content of methanol increases from 0 to 30%. The spectrum in40% methanol is the same as in 30% methanol.Similar characteristic spectral changes also occur for PyT(SPh)P (at 1.0 x 10-5M) 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 inintensity by 32% and Xmax shifts to longer wavelength (410.1 to 411.3 nm) when thecontent of methanol increases from 0 to 30%.More marked spectral changes occur in the case of 1.0 x .10-5 M trans-BPyB(SPh)P when the content of methanol changes (Figure 4.18). In pure buffersolution, there are two peaks (at 410 and 443 nm) at about the same intensity (Figure4.8, p.186). In a 10% methanol solution, the band at 410 nm becomes 2.2 times moreintense than in pure buffer, while the intensity of the band at 455 nm decreases by27%; when 30% methanol is present, the intensity of the band at 410 nm is 7 times thatin the buffer, and the band at 455 nm disappears completely. There is an isosbesticpoint at 424 nm.199Chapter 4 Aggregation of porphyrnn2.54^350 390 430 470 500nmFigure 4.18. Absorbance spectra of trans-BPyB(SPh)P in aqueousbuffer/methanol mixtures.The content of methanol. 0 10, 20, 30% (VAT) in sequence as indicated by the arrows.200o.^,550nm40A2.0-0 350 390 430 470 510Chapter 4 Aggregation of porphyrinaSpectral changes for trans-B(APh)B(SPh)P (at 1.0 x 10-5 M) under the sameconditions are shown in Figure 4.19A. In the spectrum at 0% methanol, the majorabsorbance 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) increasesdramatically and, in a mixture of 1:1 methanol and buffer solution, the intensity of thisband is about 12 times higher than in neat buffer solution, while the other band (at 455nm) disappears completely. There is no isosbestic point.Figure 4.19A. Absorbance spectra of trans-B(APh)B(SPh)P in aqueousbuffer/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 cis-B(APh)B(SPh)P in the mixtures of buffer and methanol (0-50%, v/v). The spectra of cis-201Chapter 4 Aggregation of porphytinsB(APh)B(SPh)P in the mixtures are shown in Figure 4.19B as an example. In thespectrum at 1.0 x 10-5 M at 0% methanol, the Soret band is unsynunetric which mayindicate that there are two overlapping bands. When 10% methanol is present, the Xmaxshifts to shorter wavelength (from 419 to 407 rim) to give a more symmetric band with a25% increase in intensity. When the content of methanol increases from 10 to 50%, the Xmax shifts back to longer wavelengths (from 407 to 417 rim), and the intensity of the Soretband increases by additional 71%. 350Figure 4.19B. Absorbance spectra of cis-B(APh)B(SPh)P in aqueousbuffer/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 atabout 409 nm is slightly more intense than the band at about 402 rim (Section 4.3.1.4 and202Chapter 4 Aggregation of porphyrinsFigure 4.9, p.188). When the methanol content is increased, the band at 409 nm increasesin intensity and shifts to longer wavelengths. In the spectrum in 50% methanol, theintensity of this band has increased by —100% and shifted to 411 tun, while the band at402 nm cannot be observed.4.3.3 1H NMR studies on aggregation of porphyrins4.3.3.1 1H NMR studies on the aggregation of T(MPy)PhPThe Ill NMR spectra of T(MPy)PhP in D20 at various concentrations are shownin Figure 4.20 (a-d), and the 1H NMR spectrum of this porphyrin in DMSO-d6, which wasfound to be concentration independent (0.002-0.02 M), is also shown in this figure (z). Aslisted in Table 2.14 (p.65) and discussed in Section 2.3.2, the signals in spectrum z areassigned as follows: the doublet at 9.50 ppm to the 5,10,15-(3,5-MPy) protons; thedoublet at 9.03 ppm to the 5,10,15-(2,6-MPy) protons; the singlet at 9.2 ppm and the ABquartet 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; andthe multplet at 7.9 ppm to the 20-(3,4,5-Ph) protons. The methyl protons appear as asinglet at 4.71 ppm and the N-pyrrole protons appear as singlet at -3.04 ppm, and thesepeaks are not shown in this figure. In a spectrum in D20 (spectrum d for instance), eachof the signals of the 5,10,15-(3,5-MPy), 5,10,15-(2,6-MPy) and 20-(3,4,5-Ph) protons issplit into two signals; the pyrrole protons give rise to a broad signal at 8.9 ppm, and thesignal of the methyl protons overlaps with the solvent peak. The assignments of the signalsin spectrum a and d are listed in Table 4.1, and labeled in Figure 4.20. Results from twodimension NMR spectrum of this porphyrin at — 0.01 M in D20 show that all the signalsassigned to the same substituent couple to each other, thus supporting the assignments.203Chapter 4 Aggregation of porphyriesCH I1^''OH^N-4" H3L H6 ^H2/y 0 \ X 0--NH^N--------^H^H3N HN i,^ 0 Ni- CH35\^26-^H/ 6 H 50 H^H2+ H5^H3111111/111^1111114111111111111111119.5^9.0 8.5^8.0^7.5^7.0 PPM^6.5Figure 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-d6.204Chapter 4 Aggregation of porphyrinaWhen the concentration increases, all the signals shift to higher fields, but todifferent 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 tospectrum 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 M0 A 0 a * 0 +10-(3,5-MPy) 10-(2,6-MPy) 5,15-(3,5-MPy)5,15-(2,6-MPy)2044-Ph) 20-(2,6-Ph) 2043,5-Ph)d 9.14 8.72 9.07 8.57 7.76 7.83 7.64a 9.07 8.35 8.73 7.71 7.53 6.78 7.15pattern d d d s(broad) t d tArs 0.07 0.37 0.34 0.86 0.23 1.05 0.49The 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 broadnessof the 1H NMR signal of the pyrrole protons, in the case of Tet(SPh)P in D20, has beenattributed to slow tautomeric exchange of the central deuterium between the pyrrolenitrogen atoms.12 However, the observed concentration dependence of the pyrrole signalin Figure 4.20 may be some evidence that this broadness is, at least partially, related toaggregation of this porphyrin.From Figures 4.20 and 4.21, it can be seen that the splitting of the signals of the10-(3,5-MPy) and 5,15-(3,5-MPy) protons becomes larger when the concentrationincreases; the extension of the two lines in Figure 4.21 corresponding to these two signalsmay reach the same chemical shift (-9.15 ppm) at a concentration of zero (whenaggregation should be eliminated); this agrees with the fact that these two signals appearidentical when aggregation is eliminated, as observed in the spectrum in DMSO-d6. Thesame observation can be made for the signals of the 5,10,15-(2,6-MPy) protons whichmay give a common resonance at -8.80 ppm at C = 0, and the 20-(3,4,5-Ph) protonswhich may give a common resonance at -7.80 ppm at C = 0. The chemical shifts at2059.56.5Chapter 4 Aggregation of porphyrinsconcentration of 0 obtained by extending the lines in Figure 4.21 are not the same as thechemical shifts observed in the spectrum in DMSO-d6; this difference presumably resultsfrom differences in the interactions in the porphyrin monomer with the solvents.o^0.004^0.008^0.012^0.016^0.02^0.024concentration (M)—A— 1042,6- —0-- 5,1543,5- —X— 5,15-(2,6-1043,5-MPy) 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 concentrationof T(MPy)PhP.Aggregation can be observed by comparing spectrum d (0.002 M in D20) andspectrum z (in DMSO-d6) in Figure 4.20. This observation agrees with the deviation fromBeer's law at much lower concentrations (10-6 - 8 x 10-4 M in buffer solution) observed byUV-visible spectra (Section 4.3.1.5).A structural model for this porphyrin can be suggested based on the changes of thechemical 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 porphyrinsits on top of the other with a 180° rotation, and with one of the porphyrin planes206Chapter 4 Aggregation of porphyrinsdisplaced along the axis connecting carbons 10 and 20 (Figure4.22A). Higher aggregation occurs in a similar fashion as shown in Figure 4.22B, in whicha trimer is given as an example. Because aggregation (possible dimerization) is observed at10-6 to 10-4 M, at least in a buffer solution (Section 4.3.1.5), the aggregation observedhere 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 NMRspectra where only one signal is observed for the 5,15-(2,6-MPy) protons (this should be adoublet, 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 ringsin 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 phenylring, are closest to the centers of the adjacent porphyrin rings, and this makes their signalshift 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 shiftaccordingly to their distances from the adjacent porphyrin rings; for example, the signal of10-(3,5-MPy) protons is little shifted at the various concentrations, as the protons arequite 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 ofTet(MPy)P, in which one porphyrin sits on top of the other with a 45 degree rotation, andall the methylpyridinium groups are identical. This model is not applicable to T(MPy)PhPat this concentration because of the difference between the 10-MPy and 5,15-M-Py groupsobserved in the 1H NMR spectra. The aggregation model for Tet(MPy)P is furtherdiscussed in Section 4.3.4.207Chapter 4 Aggregation of porphyrinaAB41■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 situatedbetween the charged pyridinium centers.)208Chapter 4 Aggregation of porphyrins4.3.3.2 1H NMR studies on the aggregation of PyT(SPh)PFigure 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 concentrationindependent. 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. Thesignals 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 anda 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 the2,6-SPh protons resonate at lower field than the 3,5-SPh protons, the signals at the higherfield (at 7.22 and 7.43 ppm in spectrum b) are assigned to the 2,6-SPh protons. Thisassignment has been proven by partial deuteration of the 2,6-SPh protons withinTet(SPh)P for spectra measured at similar concentrations.12 The pyrrole protons give amultiplet at 8.87 ppm in spectrum y; these protons give a very broad signal at 8.6 ppm inspectrum a, and this shifts to higher field and broadens as the concentration increases. Thesignal of the pyrrole protons becomes too broad to be seen in spectra e and f. In spectrumf, most of the signals are broad.Table 4.2. Chemical shifts in spectra of PyT(SPh)P.0 A 0 0 a +5-(3,5-Py) 5-(2,6-Py) 10,20-(3,5-SPh) 10,20-(2,6-SP1i) 15-(3,5- 15-(2,6-SPh) SPh)Y 9.03 8.27 8.04 8.28 8.04 8.28b 8.72 7.50 7.85 7.22 7.96 7.43f 7.92 6.55 7.33 6.55 7.75 7.2pattern 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 0.23209S03-H5o°xChapter 4 Aggregation of porphyrios1^r1^1^1^1^r^i^II^III^IIIIII^11,11111!IIIIII9•0 8.5 8.0 7.5 7.0^6.5^6.0 PPMFigure 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 10-3e: C = 1.0 x 10-2, f: C = 2.0 x 10-2 M in D20; y: in DMSO-d6.210Chapter 4 Aggregation of porphyrinsThe differences of the chemical shifts of the signals (Acs) in spectra b and f arealso listed in Table 4.2. The chemical shifts of the signals are plotted vs. concentration inFigure 4.24.96o^0.004^0.008^0.012^0.016^0.02^0.024P5-(3,5-Py)concentration (M)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 ofT(SPh)PyP.Similar to the spectral changes in Figure 4.20, all the signals shift to higher fieldswhen the concentration increases, and a similar aggregation model (Figure 4.25) can besuggested for this porphyrin in these conditions.2115^ 55-—/^/-^/\----} Oil^t i^ 12(3^-_/^ \\--_ r- " ^i^12 (3- -\05Chapter 4 Aggregation of porphyrinsAFigure 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)212Chapter 4 Aggregation of porphyrinaSomewhat different from the cases for T(MPy)PhP, the extensions of the lines (ofFigure 4.24) for the 10,20-(3,5-SPh) protons and for the 15-(3,5-SPh) protons do notconverge at zero concentration, and neither do the lines for the 2,6-SPh protons. Thisperhaps indicates that the aggregation of this porphyrin at the noted concentrations isbeyond the monomer-dimer process, and the trends for the changes in the chemical shiftswith 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 Mto 0.02 M) for the protons on the same substituent show more significant differences thanthe related values of A" (from 0.002 M to 0.02 M) in Table 4.2. For example, the valuefor the 10-(2,6-MPy) protons (0.37 ppm) and the 10-(3,5-MPy) protons (0.07 ppm) inTable 4.1 are significantly different compared to the corresponding values in Table 4.2 forthe 15-(2,6-SPh) protons (0.23 ppm) and the 15-(3,5-SPh) protons (0.21 ppm). This mayindicate that the side displacement in the model for this sulfonatoporphyrin is smaller thanin the model for the pyridiniumporphyrin (Figure 4.22), and that the 15-(2,6-SPh) protonsare also significantly shielded by the ring current of the pyridyl ring of the adjacentporphyrins. Thus, the distance between two adjacent SPh groups might be close enough toshift upfield all the SPh group signals, which also means that the 10,20-SPh groups mightbe unable to rotate freely. This suggestion is supported by the broadness of the signals forthe 10,20-SPh protons (Figure 4.23), and is also plausible because the SPh rings wouldhave more 1C-7C interaction than the MPy rings which are positively charged because of thedelocalization of the charge from the nitrogen atoms.Figure 4.25A shows a dimer model; Figure 4.25B shows the plausible interactionof the pyridyls with the porphyrin rings and the ability of 15-SPh to rotate; and Figure4.25C shows the potential interactions between the 10 and 20-SPh groups.213Chapter 4 Aggregation of porphyrins4.3.4 Aggregation modelsSeveral aggregation models have been suggested for metalloporphyrins in non-aqueous solution,2,6,24 and for natural porphyrins in aqueous solution,' but discussion onmodels for synthetic, water-soluble porphyrins is rare in the literature, though theaggregation of symmetric, synthetic porphyrin free-bases including Tet(SPh)P,11.12Tet(mpy)p,2,10,9,1 8 Tet(CPh)P (5,10,15,20-tetracarboxyphenylporphyrin),8 andTet(tAPh)P [ 5, 10,15,20-tetra(4-trimethylammoniumphenyl)porphyrin] 15 has beenintensively studied.4.3.4.1 Monomer dimerization or oligomerizationThe monomer-dimer equilibrium model was first proposed for the aggregation fora natural porphyrin because isosbestic points were observed in the normalized spectra indilution experiments. However, as noted by White' in 1978 in a review about aggregationof porphyrins and metalloporphyrins: "it is conceivable that the isosbestic points may befortuitous, 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-dimerequilibrium." 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 beenreported, and thus this monomer-dimer equilibrium has been used as a general aggregationmodel for all porphyrin free-bases.In this work, there is evidence against the monomer-dimer process as a generalmodel because of the complexity of the normalized spectra in the dilution experiments forsome 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-214Chapter 4 Aggregation of porphyrinaB(MPy)DPhP (Section 4.3.1.7, p.196; Figure 4.16, p.197). The complexity of thesespectra just cannot be explained using a monomer-dimer model.Evidence has also been found to support the monomer-dimer model for certainporphyrins: Tet(SPh)P, (APh)T(SPh)P, PyT(SPh)P and trans-BPyB(SPh)P. For theseporphyrins, isosbestic points have been observed in the normalized spectra in both dilutionexperiments in a buffer solution and in the methanol-effect experiments. The existence ofthe isosbestic points in the spectra in the methanol experiments for Tet(SPh)P, forexample, 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, ispresent because organic solvents are known to break down aggregation quite efficiently.9This assumption is also supported by the fact that there is no more spectral change whenthe methanol content increases from 30% to 40%. Thus, the two species in equilibrium at0% methanol should be monomer and dimer. The spectrum at 0% methanol alsocorresponds to one in the figure of the normalized spectra in the buffer solution (Figure4.3, p.190), in which isosbestic points are also present, and the two species in equilibriumat different concentrations should again be monomer and dimer. This discussion can beextended 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 foundto either support or oppose the monomer-dimer model in the buffer solution, although theprocesses observed by UV-visible are likely to be a monomer-dimer process; this isbecause the aggregation is 'limited' in both cases, i.e., the points in the Beer's law diagramat higher concentrations (>2 x 10-5 M) tend to give a straight line plot (Figures 4.12B and4.15).It has been clearly shown in this work that model and degrees of aggregation areaffected strongly by the nature of the substituents, the number of charged substituents and215Chapter 4 Aggregation of porphyrinsthe position of the substituents. Thus, the aggregation model can be quite different fromone porphyrin to another.4.3.4.2 Structural models4.3.4.2.1 A model for the tetra-ionic porphyrinsA "face-to-face" dimer with an angle of 45° between corresponding porphyrin axeshas been suggested by Kano et al.10 according to NMR and fluorescence spectral data. Afigure of this model is presented in Figure 4.26. A model for higher aggregation at higherconcentrations (> 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 protonNMR signals of the 2,6-SPh protons and the 3,5-SPh protons of shift to higher field whenthe concentration increases, and the former shifts more than the latter; (2) there is only onedoublet for the 2,6-SPh protons and one doublet for the 3,5-SPh protons, which impliesthat the planes of the sulfonatophenyl groups are either parallel to the porphyrin ring orare freely rotating, and in the "face-to-face" model the sulfonatophenyl groups should beable to rotate freely. This model is optimized for other factors, such as charge distributionand steric hindrance.4.3.4.2.2 Models for the tri-ionic porphyrinsFor T(MPy)PhP and PyT(SPh)P, a "slide-over" model (Figures 4.22 and 4.25) hasbeen suggested in this thesis work according to 1H NMR data (Section 4.3.3). Differentfrom the the "face-to-face" model, this "slide-over" model takes advantage of theexistence of a hydrophobic phenyl group, which can form a relatively strong interaction216Chapter 4 Aggregation of porphyrins, + _/:)-----\Figure 4.26. Aggregation models for Tet(MPy)P.A: a model for a dimer; B: a model for a trimer.A217Chapter 4 Aggregation of porphyrinawith an adjacent porphyrin ring(s), and this interaction could facilitate the rotation of thisring to make it parallel to the adjacent porphyrin ring(s). There are no obvious factors thatprevent the dimer from further aggregation.Shifts of the 1H NMR signals of porphyrins with variation of concentration havebeen reported for porphyrins in organic solvents, and similar "slide-over" models have alsobeen suggested.2,5This "slide-over" model may be appropriate for the aggregation of other tri-ionicporphyrins, including T('MPy)(NPh)P and (APh)T(SPh)P (this work), and PhT(SPh)P.8There is similarity among these porphyrins in that they both possess three charged meso-substituents and one lipophylic meso-substituent.4.3.4.2.3 Models for bis-ionic porphyrinsUV-visible studies on the aggregation of the bis-ionic porphyrins show that thecompounds [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 themonomer-dimer equilibrium in the range of concentrations studied. Although in the buffersolution, the behavior of trans-BPyB(SPh)P may conform to a simple monomer-dimeraggregation, in distilled water this porphyrin may also participate in higher aggregation. Itcan be concluded generally that these porphyrins tend to aggregate more than the tetra- ortri-ionic porphyrins because they have more lipophilic substituents. Unfortunately, 1HNMR spectra in D20 at room temperature of these porphyrins are uninformative becauseonly less defined broad peaks can be observed, which again probably result fromoligomerization processes. Several plausible aggregation models can be suggested forthese porphyrins, and in solution one porphyrin could exist as a mixture of some of theseforms. Among these possibilities, a model for trans-B(MPy)DPhP, based on the "slide-218Chapter 4 Aggregation of porphyrhoover" model for T(MPy)PhP (Figure 4.22), is presented in Figure 4.27; as shown, theformation of a trimer, tetramer or higher aggregated forms can be achieved without sterichindrance. The UV-visible and NMR spectra suggest that a mixture of the forms may bepresent in an aqueous solution, and this makes further characterization of this porphyrin inaqueous solution very difficult.4.3.5 The equilibrium constants for dimerizationIf dimerization of monomers is the only process observed in the concentrationranges 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 buffersolution, and Tet(MPy)P in distilled water (Figure 4.12A), the following equations can bewritten for analysis of the UV-visible spectral data:CT = 2 CD + Cm^ 4.12M^D K = CD / Cm2^4.2A = emCm + eDCD^ 4.3Where 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:8Am -A = [(2em - eD)(4KCT +1 -1/1+ 8KC )]/8K,where Am= Cm X CT. Rearrangement gives:219Chapter 4 Aggregation of porphyrinaMI■111.--MINMEON■1■1C}■•■■•■■-■■•■411■1•1111M--M■■•■•■■---(11)a4■■■■■■F-III■MONIN11■0111111■1■110---•■••■••■!---■■1•11M-.■111■11■INIMMEN■-■00■01■1■Ml■MMENIMMINHINIIMINI■•■••1=11.00--11.1•■■-•■1■---•■•■11Figure 4.27. Aggregation models for trans-B(MPy)DPhP.a: a model for a dimer; b: possibilities for a tetramer.a220Chapter 4 Aggregation of porphyrhuA -A(4CT - 8  m^)K + 1 = 1.11 +8KC T2& _ 6M DA -APutting (4C -8 m^) — B, this equation becomes B2K + 2B - 8CT =0,T 2e ^eM D= 8CT- 2Band K B24.4Thus, once £m and ED values are chosen, then the K value, and the A values at eachconcentration can be calculated using eqn. 4.4; and the calculated curve can be comparedto the experimental curve for a best-fit.For Tet(SPh)P, the molar absorptivity in distilled water at low concentrations4 x 10-6 M) at 410 nm (inset in Figure 4.2A) was used as Em for the calculation of K inthe buffer solution and ED was varied a best-fit curve (data from Figure 4.3, p.180). Theresultant curve with the experimental data and the Beer's law straight line for the assumedmonomer are shown in Figure 4.28A. The K and ED values obtained in this way, togetherwith 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 distilledwater at 410 nm at concentrations from 5 x 10-7 to 1.0 X 10-5 M, where Beer's law wasfound 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 (dataof Figure 4.8, p.186), the em values were first estimated according to the molarabsorptivity of these porphyrins in mixtures of the buffer solution and methanol, and thenoptimized for best-fit curves. For Tet(MPy)P in water, both Em and eD were varied, and asatisfactory 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 orpossibly from further aggregation beyond dimerization at this concentration. The best-fitcurves are shown in Figures 4.28A and 2.28B. The K values, the Em and ED values arelisted in Table 4.3.221Chapter 4 Aggregation of porphyrinsTable 4.3. Equilibrium K values for dimerization of porphyrins.K (M-1) Conditions &ma, epaX (nm)Ref.Tet(SPh)P 1.5 x 104 pH= 7.04 5.0, 1.8 present work0.010 M phosphate buffer 411Tet(SPh)P 9.6 x 104 pH = 7, 0.05 M NaNO3 temp.-jump"Tet(SPh)P 2.2 x 104 pH = 6.4 4.91, 4.2 UV-visible120.100 M KNO1 412Tet(SPh)P 1.5 x 104 pH = 7.0 5.1, 3.9 UV-visible120.028 M phosphate buffer 412+ 0.050 M KNO3Tet(SPh)P 1.6 x 104 pH = 9.0 4.7, 4.0 UV-visible120.100 Na(0Ac) buffer 412PyT(SPh)P 8.9 x 104 pH = 7.04 4.2, 1.8 present work0.010 M phosphate buffer 411(APh)T(SPh)P 1.0 x 105 pH = 7.04 3.0, 2.9 present work0.010 M phosphate buffer 412PhT(SPh)P 4.28 x 104 pH = 7.5, 0.1 M KNO3 4.00, 2.46 UV-visible8413trans- 4.9 x 107 pH = 7.04 2.6, 0.49 present workBPyB(SPh)P 0.010 M phosphate buffer 410Tet(MPy)P 7.3 x 105 distilled water 0.50, 5.0 present work422a 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 similarconditions (pH = 7.0, 11 = 0.1 M) using the same technique.12 However, this value is about6 times lower than the value obtained by a temperature-jump technique." This differencemay result from a difference in experimental conditions or most likely from the lowaccuracy of the temperature-jump teclmique.8 The em value for this porphyrin obtainedhere is basically the same as the reported ones,12 but the ED value is only about half of thereported value.12 Again this difference might result from the slightly different experimentalconditions or from the errors in the treatments. From a comparison of the values obtainedunder the same conditions for different porphyrins in the present work, the K values are inthe order of Tet(SPh)P < PyT(SPh)P 5. (APh)T(SPh)P << trans-BPyB(SPh)P. This order222Tet(SPh)P at 411.32420 —16 —fi. 12 —8 _0 Experimental^ Calculated—I^ Bee rs law for themonomer4 -0^1^2^3^4^5^6^7PyT(SPh)P at 412.4 nm2520 —15 —110 —0 Experimental^ Calculated—I— Beer's law for the monomerI I^I^I^I1^2^3^4 5^60I^I^I7^8^9Chapter 4 Aggregation of porphyrinaConcentration x 105 (M)Concentration x 105 (M)Figure 4.28A. Best-fit curves for the UV-visible spectral data of Tet(SPh)P andPyT(SPh)P in an aqueous buffer.223••••••0^ExperimentalCalculated•,!a-- -1- — Beer's law tor themonomerI^i2015--tZ 1050augger 4 Aggregation of porphyrins(APh)T(SPh)P in a phosphate buffer (412.4 nm)2^3^4^5^6^7^8 9^10Concentration x 105 (M)Tet(MPy)P in distilled water at 422.0 nm0^1^2^3^4^5^6^7^8^9^10Concentration x 105 (M)Figure 4.28B. Best-fit curves for the UV-visible spectral data of (APh)T(SPh)P inan aqueous buffer and Tet(MPy)P in distilled water.224Chapter 4 Aggregadon of porphyrinscorresponds inversely with the number of charges, i.e., the less charged structure gives thehigher 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 valuesare also similar to that reported for another tri-anionic porphyrin, PhT(SPh)P.8The K values obtained here are most probably the equilibrium constants for themonomer-dimer process, because evidence has been found in this work to support themonomer-dimer model under the conditions used (Section 4.3.4.1, p.214) for theporphyrins listed above, except Tet(MPy)P; a monomer-dimer equilibrium also seemslikely, however, for Tet(MPy)P, as judged by the literature.9.10,184.3.6 SummaryAggregation is a general phenomenon for porphyrin free-bases in aqueoussolutions, and is found for all the water-soluble porphyrins studied in this thesis work. Thedegree of aggregation is probably related to many structural factors, including the ioniccharge (a tetra-ionic porphyrin probably aggregates in a fashion different from that a tri-ionic 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)Pappear to aggregate differently under the same conditions--see Figures 4.8 and 4.9); andthe nature of the substituents (a difference is observed between T(MPy)(NPh)P andT(MPy)PhP--see Sections 4.3.1.6 and 4.3.1.7). Although some structural models relatedto the porphyrin structures have been suggested in this thesis work, the findings present apreliminary 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 ofoligomerization may exist simultaneously in solution making such studies difficult.225Chapter 4 Aggregation of porphyrinsReferences-Chapter 41^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, 1983,186710^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).226Chapter 4 Aggregation of porphyrina14^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).227Chapter 5 In vitro activitiesChapter 5 In vitro studies of selected syntheticporphyrins and metalloporphyrins5.1 IntroductionSome in vitro studies on the radiosensitizer activities of synthetic porphyrins andmetalloporphyrins have appeared in the literature' (Chapter 1). Preliminary investigationson the in vitro activities of some of the porphyrins and metalloporphyrins synthesized inthis 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 toxicityof the drugs in oxic vs. hypoxic conditions. The toxicities of most of the synthesizedwater-soluble compounds in both oxic and hypoxic conditions are presented in thischapter.As discussed in Chapter 1, one of the goals of this project was to designporphyrins as radiosensitizers. With the assay routinely used to assess radiosensitizationabilities of drugs, low radiosensitization efficiencies were found in this thesis work inmammalian cells for porphyrins and metalloporphyrins, including those metalloporphyrinswhich have been reported earlier in the literature to have high efficiencies."The degree of lipophilicity of the porphyrins synthesized in this thesis work wasvaried by changing the number of ionic groups and probably by the symmetry (cis-isomervs. the trans-isomer). The partition coefficients between 1-octanol and water of someporphyrins were determined and are presented in Appendix B. The effects of charges onthe porphyrin free-bases on cell accumulation were also tested.228Chapter 5 In vitro activitiesA few of the porphyrin free-bases were also tested for their photosensitizationproperties.Table 5.1 shows the assays and the compounds tested. The selections of thecompounds for each assay are discussed in the later sections of this chapter. Partitioncoefficients of the porphyrin free-bases are presented in Appendix B. Due to timelimitation, 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 water-solubilities, such as trcms-B(MPy)DPhP, trans-B(MPy)B(NPh)P, and cis- and trans-B(APh)B(SPh)P. Some of the results were consistent with the original ideas of designingthe chemical structures, some were not. Further investigations are essential in order toestablish possible correlations between chemical structures and the biological activities.5.2 Materials and methods5.2.1 Cell growth, maintenance and treatmentThe cells used in the toxicity and radiosensitization assays were obtained from aCHO (Chinese hamster ovary) cell line. The cells were routinely grown in a spinner cultureflask 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 ofabout 1 x 105 cells/mL to maintain an exponential growth (doubling time wasapproximately 13 h).Drug accumulation and photosensitization assays were performed using cells froman HT-29 (human-cancer tumor) cell line which was maintained as monolayers in229Chapter 5 In vitro activitiesTable 5.1. In vitro tests and the tested compoundscode compound^a toxici ty radiosen-.^.^.sttizationcelluptakephotosen-sitization1 Tet(MPy)P x x x x2 T(MPy)PhP x x x x3 T(MPy)(NPh)P x x x x4 cis-B(MPy)DPhP x x5 cis-B(MPy)B(NPh)P x x x6 Tet(SPh)P x x x7 PyT(SPh)P x x x x8 (APh)T(SPh)P x x x9 cis-BPyB(SPh)P x x x10 cis-(NPh)PyB(SPh)P x x x11 trans-BPyB(SPh)P x x x12 Co[Tet(MPy)P] x x13 Co[T(MPy)PhP] x x14 Co[T(MPy)(NPh)P] x x15 Co [cis-B(MPy)B(NPh)P] x16 Co[Tet(SPh)P] x x17 Co[PyT(SPh)P] x x18 Co[(APh)T(SPh)P] x x19 Co[cis-(NPh)PyB(SPh)P] x20 Cu[Tet(MPy)P] x x21 Cu[T(MPy)PhP] x22 Cu[T(MPy)(NPh)P] x23 Cu[Tet(SPh)P] x24 Cu[PyT(SPh)P] x25 Cu[(APh)T(SPh)P] xa^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 theaxial ligands in aqueous solutions.The oxidation state of copper in complexes 20-25 is "+2".Assays carried out.230Chapter 5 In vitro activitiesRPMI+/+ 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 wereplated 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-rayirradiation, or light irradiation) were vortexed, and then centrifuged (7 min); the cells werethen 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 solutionswere plated into 5 cm Petri dishes prepared previously (filled with 5 mL a+/+ medium forCHO cells or RPM1+/+ for HT-29 cells, and kept in a tray incubator for 24 h). Thenumber of cells plated into the Petri dishes was such as to ensure 100-200 colonies perdish. 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-29cells), and the colonies were then stained using a methylene blue solution and counted. Atleast two or three parallel experiments were performed. The plating efficiency (PE) andsurviving fraction (SF) were calculated as:number of coloniesPE —^ , for all the treatments;number of cells platedPE (at time t) SF —^, for toxicity experiments; andPE (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 cellsfrom solutions (usually 7-9 min). The cell concentration (cells/mL) was determined using a"Coulter Cell Counter" (Coulter Electronic Inc.). Tray incubators (National Inc.) operatedat 37°C with a 95% air and 5% CO2 gas flow. Nitrogen used to produce hypoxic231chapter 5 In vitro activitiesconditions was oxygen-free grade (Linde Specialty Gas, Union Carbide). Plastic Petridishes and tissue culture flasks (Falcon, Becton Dickinson and Co.) were used for all theexperiments. The preparations of the solutions, including PBS, media (a+/-, a-/-, a+/+,RPMI-/+, and RPMI+/+), trypsin, and methylene blue, are described in Appendix A. A10% (v/v) ScintiGest solution was made from ScintiGest (a tissue solubilizer, FisherScientific Co.) and doubly distilled water.5.2.2 Drugs and drug solutionsThe tested compounds (see Table 5.1) were synthesized and purified as describedin Chapters 2 and 3. Stock solutions of the tested compounds at 1.0 mIvl were made bydissolving the compounds in double distilled water (warming was occasionally required tomake these solutions).Solutions used for testing of the compounds (conventionally called drug solutionsin biological laboratories) were made from dilution of the stock solutions using theappropriate medium, and the solutions were sterilized by filtering using 10 mL disposablesyringes and Nalgene 0.22 p.m filter units.5.2.3 Toxicity in oxic and hypoxic conditionsThe toxicities of the compounds were measured by incubating CHO cells with thedrug solutions for various time intervals under 1 atmosphere of either air or nitrogen, asreported in the literature.2 Approximately 2 x 106 cells were harvested from a spinnerculture for each of several vessels (Figure 5.1) using a centrifuge. The cells wereresuspended 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, thenfiltered. An aliquot of 9 tnL of each drug solution was added into each of two vessels (foroxic and hypoxic conditions, respectively). Medium (a+/-, 9 mL) was added to each of the232Gas Inlet^Gas OutletGlass Tubefor SamplingCell SuspensionChapter 51n vitro activitiescontrol vessels (for oxic and hypoxic conditions respectively). The vessels weremaintained at 37.4°C in a Labline Instruments "Orbit Shaker Bath", which was in a warmroom at 37°C. The hypoxic and oxic conditions were created by flowing humidifiednitrogen and air, respectively, through the vessels for 1 h. Then the resuspended cellsolution (1 rnL per vessel) was added, the concentration of the drugs becoming 100 JAM atthis time by the dilution of the cell solution. A small amount of the solution was left in thepipette to avoid introducing air into hypoxic vessels. Samples of 1 mL were taken fromeach vessel at incubation times of 0, 1, 2, and 3 h. The samples then were treated asdescribed in Section 5.2.1.Figure 5.1. The vessel for toxicity assay.233Chapter 5 In vitro activitiesToxicity experiments for HT-29 cells were performed with subconfluentmonolayers of cells. Cells (0.5 x 106) were seeded in 5 cm Petri dishes two days beforeexperiments, 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 usingRPM14+ medium. The cell subconfluent monolayers in the Petri dishes were rinsed withPBS; and then each of the drug solutions was added to two Petri dishes (5 mL drugsolution to each Petri dish). The Petri dishes were then immediately covered withaluminum foil to avoid receiving light. One of the two Petri dishes with the same drugsolution 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 wererinsed with PBS, trypsinized and added into dilution tubes which contained 10 mL PBSsolution; approximately 2 x 105 cells were taken from each dilution tube and added toanother dilution tube containing 10 inL RPM14+ medium and; the cells in the mediumsolutions were treated as described in Section 5.2.1.5.2.4 Radiosensitization in hypoxic conditionsA published procedure was followed for the radiosensitization assays.2,3 Irradiationwas performed using an X-ray source (Philips, 250 kv, 0.5 mm Cu), using the set-upshown in Figure 5.2. Glass "duck vessels"3 with magnetic stir bars were used. Radiationdoses of the set-up were measured using a Precision Electrometer (Victoreen, Model500).Drug solutions of about 17 niL for each drug were made by dissolving thecompounds (drugs) in a medium solution (a+/- or a-/-, depending on the design of theexperiment), at a concentration of C'. These solutions were filtered for sterilization, and14.5 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. The234Chapter 5 In vitro activitiesvessels were then immersed into a water bath at 37.4°C with magnetic stir bars. Nitrogengas 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 andresuspended 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 avoid14.5introducing air into the vessel). The concentration of the drugs became C (i.e., 150 C') atthis time resulting from the dilution of the cell solution. The cells were incubated in thewater-bath (37.4°C) with the drug solutions (or medium only for the control) for 1 h, thenchilled with ice-water for at least 5 min before irradiation. The vessel to be irradiated wasimmersed in 1 L ice-water in a plastic container which was placed on top of the head ofthe 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-outletarm of the duck vessel at doses of 0, 5.0, 10.0, 15.0, 20.0, and 25.0 Gy into dilution tubescontaining 9 mL a+/- medium; the dose rate was typically —5 Gy/min. These samples werethen treated as described in Section 5.2.1.Gicitlet Stir motorGas inletCell suspension /\\\' \\Ice waterMagneticstir barX—ray beamFigure 5.2. Set up for the radiosensitization assay.2351CHO CELLS- \^4c1)\ N.A.Y+._T:02Chapter 5 In vitro activitiesThe survival curves were obtained by plotting log SF vs. dose.4 The effect ofoxygen in CHO cells is shown in Figure 5.3 as a example of radiosensitization (oxygenbeing a natural sensitizer, see Chapter 1). The sensitization enhancement ratio (SER) wascalculated at 1% survival (SF = 0.01), which is commonly chosen for comparison.4Dose without drugSER —^(at 1% survival)Dose with drug0^5^10 15 20 25 30DOSE (Gray)Figure 5.3. A representative example of survival curves (02 effect, SER - 3).(adapted from ref. 4)236Chapter 5 In vitro activfties5.2.5 Cell accumulationCell accumulation was determined using a fluorometric assay.5 Cells (1 x 106 HT-29) were seeded into 10 cm Petri dishes with 15 mL RPMI+/+ medium two days beforethe experiments, and the dishes were incubated in a tray incubator. The drug solutions (17mL) at 100 plvl were made from each of the drug stock solutions using RPM14+ mediumon the day of the experiment. The Petri dishes with subconfluent monolayers were rinsedwith PBS solution three times to remove traces of serum in the medium for cell growth toavoid interactions between the drugs and serum; then 5 inL of a drug solution were addedinto each of three Petri dishes. The dishes were wrapped with aluminum foil immediatelyafter 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 PBSsolution three times. The cells were then harvested using a rubber policeman instead oftrypsinization in order to avoid any possible interactions between trypsin solution and thedrugs, and resuspended into 10 mL of PBS solution by vortexing (for another wash) indilution tubes. Cells in a Petri dish without the drug treatment were harvested into adilution tube containing 10 mL of PBS solution as a control. Cells in two control Petridishes (without the drug treatment) were also harvested in the same way and counted; andthe 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 offas carefully and as completely as possible. ScintiGest solution (10%, 5.00 mL) was addedto each of the dilution tubes, which were then wrapped with aluminum foil to preventpossible photo-destruction of the porphyrin drugs. The solutions were kept at 37°C (in awarm room) for at least 24 h to digest the cells. Standard solutions at 0.10 - 5 tM, madeby diluting 100 11M drug solution using 10% ScintiGest, were also wrapped withaluminum foil and kept at 37°C for the same time as the unknown samples. Some sampleswere found to be too concentrated for the measurements, and were diluted by 10-50237Chapter 5 In vitro activitiestimes, and the diluted samples were again wrapped with aluminum foil and kept at 37°Cfor at least 12 h to assure reproducible results.The concentrations of the drugs in the digested solutions were measured byfluorescence using a Farrent Optical System 3 Scanning Spectrometer at the maxima forexcitation (scanned from 380-500 nm) and emission (scanned from 600 -700 nm); thewavelengths are listed in Table 5.2 for each of the compounds tested. Results werecalibrated by standard solutions of each drug. At least two or three experiments wereperformed for each test.Table 5.2. Fluorescence maxima for excitation and emission (a)Com-poundXexcitation(nm)emission(nm)Corn-poundXexcitation(nm)Xemission(nm)1 445 635 8 417 6472 435 637 9 413 6413 445 645 10 416 6456 415 642 11 413 642•7 413 642a: See Table 5.1 for identification of the compounds; compounds 4 and 5 are not listed in this tablebecause irreproducible data were collected (see Section 5.3.3).5.2.6 PhotosensitizationThese experiments were carried out following an established procedure6 withminor changes. HT-29 cells (1 x 106) were seeded into tissue culture flasks two daysbefore experiments. Drug solutions (20 mL) at 50 pdv1 were prepared by diluting the stocksolutions (1.0 mM in distilled water) with RPM14+ medium on the day of experiments,238Chapter 5 In vitro activitiesand filtering. The flasks with cells were rinsed with sterilized PBS solution three times toremove the serum, and then a drug solution (15 mL) was added to each of two flasks. Theflasks 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 flasksrinsed with 0.1% trypsin solution, and cells were trypsinized (6 min) and counted. 2 x 106cells were taken and washed with 10 mL PBS solution, resuspended in 1 mL cold PBSsolution, and chilled in ice.A tunable light source (Photo Technology International Inc., Model 500) wasadjusted to the wavelength (±10 nm) required for the experiments. A Petri dish was placedon ice which was covered with a black cloth. To this Petri dish, 120 pl. of cell solutionwere delivered and irradiated to a certain dose; 100 ilL solution was recovered and addedto a dilution tube containing 9.9 nth of RPMI-/+ medium. The doses used were typically0, 0.25, 0.50, 0.75, 1.0, 1.5, 2.0 J/cm2 (maximum exposure being --1 minute) and a newPetri dish was used for each sample. The samples in the dilution tubes were treated asdescribed in Section 5.2.1.5.3 Results and discussion5.3.1 Toxicity of porphyrins and metalloporphyrinsToxicities 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 1AMafter 1 to 3 h incubation (37°C) using CHO cells. The toxicities, under oxic conditions, ofcompounds 4, 5, 9, 10 and 11 toward HT-29 cells in the absence of light at incubationtimes of 1 and 2 h were also tested.Generally, under conventional experimental conditions in which laboratory lighthad not been avoided, porphyrin free-bases tested were found to be non-toxic to CHO239Chapter 5 In vitro activitiescells; however, non-reproducible results were observed for compounds 2 and 3. Photo-dynamic toxicity induced by light at certain wavelengths might explain the cell killing inthe experiments where these two compound were found to be toxic, although none of theporphyrin free-bases was found to be photo-dynamically toxic toward HT-29 cells withred light (630 ± 10 tun, Section 5.3.4). The control data in photo-dynamic toxicityexperiments also show that the porphyrin free-bases (including compounds 2 and 3) arenot toxic to HT-29 cells under mdc conditions for 1 h incubation without light. Theporphyrin free-bases (4, 5, 9, 10 and 11) were also found to be non-toxic to HT-29 cellswithout 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 andco& conditions, even after 3 h incubation. However, the cobalt complexes of the cationicporphyrins (12-14) were found to be slightly toxic after incubation periods of 2 and 3 hunder hypoxic conditions and non-toxic under mdc conditions; the toxicities at 100 1.11v1were 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 resultsfor compound 12 is presented in Figure 5.4.Compounds 1, 6 and 16 have been reported as non-toxic to V79N cells, whilecompound 12 has been reported to be slightly toxic to these cells (50% survival after 1 hincubation) under oxic conditions.' The results found in this thesis work essentially agreewith these reported data.That the cobalt(III) complexes of the cationic porphyrins are more toxic underhypoxic than under oxic conditions toward CHO cells may be related to the expected easeof reduction of the complexes compared to the other compounds tested, because of the240Chapter 5 In vitro activillesrelatively high oxidation state of the metal and the positive charges on the porphyrinligands.0 Control Air—X— Control Nitrogen—0-- Comp.12 Air--+— Comp.12 Nitrogen0.10^i^ 2^ 3Time ( h )Figure 5.4. Toxicities of compound 12 (100 pM) under hypoxic and oxicconditions.Although some selective toxicities of cobalt complexes of cationic porphyrinstoward hypoxic cells have been detected, these compounds are unlikely candidates asbioreductive drugs because the toxicities are relatively low at these concentrations. Inspite of this, the results show that modification of the chemical structures has somebiological effect.241Chapter 5 In vitro acdthiesThe toxicities of compounds 15 and 19 were not tested because the materials wereonly synthesized and purified at a late stage of this thesis work. These compounds at100 jz.M were not toxic in CHO cells after 1 h incubation under hypoxic conditions inradiosensitization experiments (Section 5.3.2).5.3.2 Radiosensitization under hypoxic conditionsThe cobalt complexes (12-19), one copper complex (20) and the nitro-porphyrinfree-bases (3, 5, 10) were tested as potential radiosensitizers, because the introduction ofreducible 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-basesare either weak radiation protectors or show no effect at all; and metalloporphyrins areweak sensitizers (see Table 5.2). All of the metalloporphyrins are slightly more effective ina medium without serum (a-/-) than in a medium with serum (a+/-). Serum has beenreported to reduce accumulation of Photofrine (hematoporphyrin derivatives) in cellssignificantly,7 and thus may have similar effects on the cell accumulation of the porphyrinsconcerned 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 slightlyradio-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 mightprovide relatively high lipophilic and electron affinic properties (Chapter 1). Compound20, a copper complex, showed a radiosensitization efficiency similar to that of the cobaltcomplexes (12 - 19).242Chapter 5 In vitro acdvitleaTable 5.3. Radiation enhancement ratio for selected porphyrins and metalloporphyrinsPorphyrin free-bases (100 mM) Metalloporphyrins (100 InM), —Code SER (a+/-) SER (a-/-)! Code SER (a+/-) SER (a-/-)1 0.98 12 1.15 1.192 1.00 13 1.0511 1.113 0.92 14 1.08 1.095 0.92 15 1.226 1.04 16 1.087 1.00 17 1.08 1.088 0.96 18 1.07 1.1310 0.94 19 1.1320 1.08 1.14a: 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.' Thiscurrent 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 literaturereport, a different cell line was used; cell monolayers instead of cell suspensions wereirradiated; and Hanks' balanced salt solution (MSS) (Gibco) instead of medium solutionswas used for the drug solutions. It is possible that comparable results would be obtainedusing the same experimental conditions as reported. Of note, the characterization ofcomplexes 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 aweak radiosensitizer, while compound 3 is a weak radioprotector.2430.0010.10.01—0— Control—0— Compound 18IChapter 51n vitro activitieso^5^10^15^20^25Dose (Gr)Figure 5.5. Surviving curves for radiosensitization by compound 18,a weak radiosensitizer.(hypoxic conditions in a-/- medium, SER = 1.13)244— C>— Control—0-- Compound 35^10^15^20^25Chapter 5 In vitro activities0.10.010.0010.0001 ^0Dose (Cr)Figure 5.6. Surviving curves for effect of compound 3 with radiation,a weak radioprotector.(hypoxic conditions in a-/- medium, SER = 0.92)2452.50Chapter 5 In vitro act/vides5.3.3 Accumulation of porphyrin free-bases in HT-29 cellsThe accumulation of the porphyrin free-bases in HT-29 cells was measured, andthe results are shown in Figure 5.7.1^2^3^6^7^8^9^10^11CompoundsFigure 5.7. Results of drug accumulation in HT-29 cells.Compounds 1 - 3 are cationic porphyrins, while the rest are anionic porphyrins. Itcan be seen by comparing the accumulation values of compound 1 to those of compounds2 and 3, that the accumulation values change dramatically when the total number ofcharges 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 experimentsand were in the range of 12-25 mmol/million-cells (i.e., 5 - 10 times the values for 2 or 3).246Chapter 5 In vitro activitiesThe variations in the uptake values for 4 and 5 probably result from the aggregationproperties of these porphyrins (Chapter 4). Aggregation of porphyrins affects the size ofthe species in the solution and therefore may well affect the accumulation in cells, andpossibly the spectral measurements for the concentration. The aggregation could dependon experimental conditions, particularly on the concentrations of the cells and the particlesfrom cell digestion which could vary in the experiments.For the anionic porphyrins, the accumulation value also increases when the numberof 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) (3and 10), the amine group (8), and the pyridyl group(s) (7 and 9-11) seems to have littleeffect on drug accumulation in cells. "Charge geometry" [cis (9) vs. trans (11)], shows alarge 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 cellmembranes. For porphyrins having the same number of charges (1 vs. 6; 2 and 3 vs. 7 and8), the cationic porphyrins have much higher accumulation values than the anionicporphyrins.5.3.4 Photosensitization of porphyrin free-basesSynthetic porphyrins, including Tet(SPh)P and tetrakis(3-hydroxyphenyI)-porphyrin, have been reported8,9 to have some photosensitizer properties superior to thoseof the clinically used drug Photofin II® (see Chapter 1). The quantum yield of singletoxygenl° and the DNA damage" produced by light and some other synthetic porphyrinshave also been studied with respect to PDT.An in vitro assay using human tumor cells6 was used to test the photosensitizationof some porphyrin free-bases synthesized in this thesis work.2470.2II00tc 0.1a*MI.tt0rn0.01 i^Chapter 5 In vitro activitiesAmong the porphyrins tested [1(0.03 and 0.05 mM), 2(0.03 mM), 3(0.03 and 0.05mM), 7(0.05 mM), 9(0.05 mM), and 11(0.03 mM)] using red light (X = 630 ±10 nm), only9 shows some photosensitization (Figure 5.8). Although 1 has been reported to have highefficiency for producing singlet oxygen in a chemical assay," this porphyrin shows noeffect in the cellular assay under the conditions used.o^0.5 1^1.5^2Dose (J/cm2)Figure 5.8. Photosensitization using 630 nm radiation bycompounds 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 lightwavelength, the concentration of drugs, and the cell lines should be carried out forcomplete evaluation of the compounds as photosensitizers. The other porphyrin free bases,besides the ones tested here, are also of interest as possible photosensitizers.248Chapter 5 In vtro ActivitiesReferences-Chapter 51^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^B.A. Moore, B. Palcic and L.D. Skarsgard, Radix. Res., 67, 459 (1976).3^L. Parker, L.D. Skarsgard and P.T. Emmerson, Radiat. Res., 38, 493 (1969)4^B. Palcic, J. Brosing and L.D. Skarsgard, Br. J. Cancer, 46, 980 (1982).5^M. Korbelik and J. Hung, Photochem. PhotobioL, 53, 501 (1991).6^M. Korbelik, G Krosl, H. Adomat and K.A.Skov, Photochem. PhotobioL, 55 Suppl.,54S (1992).7^M. Korbelik, Photochem. Photobiol., 56, 391 (1992).8^M.D. Berenbaum, S.L. Akande, R. Bonnett, H. Kaur, S. Ioannou, R.D. White andU.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).249Chapter 6 Conelink= and future workChapter 6 Conclusions and suggestions for future workThe purpose of this project was to design, synthesize and test new porphyrincompounds as potential radiosensitizers. Other possible applications of the new porphyrinsto cancer treatment were also of interest. In conclusion:(1) modification of porphyrins as a methodology in porphyrin synthesis has beendeveloped,(2) aggregation of porphyrin free-bases has been studied and the understanding ofthis aspect of porphyrins has been advanced,(3) some chemistry of metalloporphyrins, especially of cobalt porphyrin, has beenstudied in depth,(4) and some in vitro investigations have been initiated.6.1 Synthesis of porphyrinsAlthough examples of modifications of synthetic porphyrins can be found in theliterature,' it is the first time that a wide range of porphyrins has been synthesized usingthe method of pyrrole condensation and subsequent modification. In this thesis work,more than 40 porphyrins have been synthesized, and most of them have been fullycharacterized. Among these porphyrins, 22 are new compounds, and 15. are soluble inwater. A novel class of porphyrins with three different meso substituents has beensynthesized. The 1H NMR spectra of the pyrrole protons of the porphyrin with lowersymmetry are particularly of interest, and some work has been devoted to the assignmentsof the signals in these spectra.Aggregation of the water-soluble porphyrins was subsequently encountered in thestudies of both the porphyrin free-bases and metalloporphyrins, and studied. It can begenerally concluded that all of these porphyrin free-bases aggregate to a certain degree in250Chapter 6 Coneintions and future workaqueous solutions. Aggregation models are suggested, and a novel "slide-over" model issuggested for tris-ionic porphyrins; some equilibrium constants for themonomer.-..- - dimer process were evaluated.6.2 Metallation of porphyrinsThe synthesis and characterization of two known cobalt pozphyrins, two knowncopper porphyrins, seven new cobalt potphyrins and four new copper porphyrins werecarried 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 andcharacterization, because of the complexity of the systems resulting from air oxidation andaggregation. A reproducible synthesis for Co(fl) or Co(ll) porphyrin complexes has beendeveloped here, and the mechanisms involved in this synthesis reaction have been exploredto some degree. Some properties of the cobalt complexes in aqueous solutions, forexample the pKa values of the diaquocobalt(1.11) porphyrins, have been studied.6.3 In vitro studiesThe porphyrin free-bases and the metalloporphyrins are non-toxic to CHO cells atconcentrations of 100 IIM or 25 gM.The accumulation of the porphyrins in HT-29 cells is related to the porphyrinstructure. Generally, the fewer charges on molecules, the greater the accumulation. Forexample, the accumulation of the T(MPy)PhP porphyrins with three plus charges is about10 times higher than that of a porphyrin [Tet(M13y)P] with four similar charges. Forporphyrins with the same number of charges, the positively charged porphyrinsaccumulate to a greater extent than the negatively charged porphyrins. For example, theuptake of T(MPy)PhP (2, +3 charge) is about 23 times higher than the uptake ofPyT(SPh)P (7, -3 charge).251M = 2H or Co(III), etc.Chapter 6 Conch:dons and future workAlthough some of the porphyrins showed moderate effects as radiosensitizersand/or photosensitizers, none of these encouraged further assessment in vivo. Thesefindings are contrary to a literature report2 in which different experimental conditions wereused. The introduction of cobalt(11) and nitro groups does improve the radiosensitizingabilities of the porphyrins, but this effect is smaller than anticipated. The results of the invitro activities of the porphyrin compounds are very preliminary, and further assessmentrequired to permit any conclusions regarding the potential of these porphyrin compoundsas anticancer drugs.6.4 Suggestions for Future Work6.4.1 Synthesis of porphyrinsUsing the method developed in this work, many porphyrins of interest can besynthesized. For example, porphyrins with nitro-imidazole moieties may have higherradiosensitizing 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 (and252\ CIPt-H2NH 2NNNS03-Chapter 6 Conclutions and future workmetallation), or a condensation of 4-pyridinecarboxaldehyde, 2-imidazolecarboxaldehydeand pyrrole with subsequent nitration, methylation (and metallation).6.4.2 Other metallationsAnalogues of cisplatin have been of interest as second or third generation drugs formany years.3 Taking advantage of the reported tumor-accumulation ability of theporphyrins, tumor selectivity of the drug might be realized by a synthesized porphyrinanalogue of cisplatin. A structural example of the suggested compounds is drawn below(A). The peripheral coordination of the porphyrins to other metal complex moieties (forexample Ru(NH3)5) is also of interest for formation of potential anti-cancer drugs; anexample is shown below (B). The lipophilicities of these compounds can be varied bychaffing 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 thanone "active" center may be introduced using porphyrins with more peripheral coordinatinggroups (e.g., cis- or trans-BPyB(SPh)P).SO 3-^SO 3"SO 3-^SO 3"M = 2H or Co(III), etc.^ M = 2H or Co(III), etc.A253Chapter 6 Conc.Inflow and future workOther 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 studiesFurther biological studies should be carried on the newly synthesized, porphyrincompounds. Of immediate interest would be to study the tumor accumulation propertiesof the compounds. If these porphyrin compounds have very good tumor selectivities, thenthey have several potential applications in oncology: (a) as targeting agents, (b) as drugs inchemotherapy (for example, like the one suggested for the cisplatin analogue), (c) as drugsin boron neutron capture therapy (BNCT)4 or (d) as contrast agents in magnetic resonanceimaging (MRI).5254Chapter 6 Conelndona and future workReferences - Chapter 61^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 andC.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).255AppendixAppendix A Solutions in cell bioloovA.1 a-mediumAn alpha-modification of Eagle's minimum essential media (MEM, Gibco) wasused in all procedures involving the maintenance or incubation of CHO cells. Threedifferent forms of the media were used, oc-/-, cc+/- and a+/+ depending on therequirements 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 roomtemperature, 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 theabove solution before filtration; the pH of the resultant solution was adjusted to 7.30 with4 M NaOH, and then filtered to produce a+/+ media.The a+/- media were made up by adding 10% (v/v) of bovine serum to thecc-/- media.All media were stored at 4°C.A-2 RPMI mediumOne packet of RPMI powder, NaHCO3 (20 g) and 10,000 units of Penstrepantibiotic (Gibco) were added to 10 L of doubly distilled water and the solution wasstirred for 2 h at room temperature, then sterilized by filtration through a 0.22 micronfilter 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.256AppendixA-3 PBS (phosphate buffer saline) solutionNaC1 (160 g), KC1 (4g), Na2HPO4 (23 g) and KH2PO4 (4 g) were dissolved indistilled 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 whenrequired.A-4 Methylene-blue solutionMethylene-blue (2 g) was dissolved in distilled water (1 L), and the solution wasallowed 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 byfiltration. The solution was stored at - 10 °C.257AppendixAppendix B Partition coefficients of porphyrinsThe porphyrin free-bases were dissolved in octanol-saturated distilled water tomake porphyrin solutions (10 mL each porphyrin) at 5 x 10-6 M. This concentration waschosen for the convenience of spectral measurements. The absorbances of these solutionsat 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 (600RPM, 5 min) in order to separate the layers. The absorbances of the aqueous layer at thesame wavelengths as the measurements of the Ae values were recorded as the Af values.Partition coefficient (P) values were then calculated as: P= A` - Af . The P values obtainedA fare listed in the following table.porphyrin label used inChapter 5charge A.max(nm) PTet(MPy)P 1 +4 421 0.44T(MPy)PhP 2 +3 421 0.88T(MPy)(NPh)P 3 +3 422 1.2cis-B(MPy)DPhP 4 +2 421 17cis-B(MPy)B(NPh)P 5 +2 421 2.8trcms-B(MPy)DPhP +2 418 13trans-B(MPy)B(NPh)P +2 420 3.0Tet(SPh)P 6 -4 411 <0.01PyT(SPh)P 7 -3 411 <0.01(APh)T(SPh)P 8 -3 413 <0.01cis-BPyB(SPh)P 9 -2 410 0.23cis-(NPh)PyB(SPh)P 10 -2 413 0.82trans-BPyB(SPh)P 11 -2 409 0.16cis-B(APh)B(SPh)P -2 413 0.71trans-B(APh)B(SPh)P -2 418 0.29It should be noted that these P values are accurate only if Beer's law is obeyed bythese porphyrins in aqueous solutions up to 5 x 10-6 M; this has been found to be true forthe tricationic, tetraldsanionic and trisanionic porphyrins (Chapter 4 of this thesis), and nottrue for the other porphyrins. In any case, these measurements provide a good indicationof the trends of the change of the P value as the structure changes, although the error isestimated to be 10-25% for the porphyrins not obeying Beer's law.258Appendix C ill NMR titration curves for Co(III) diaouo porphvrin complexes9.3I I^I^I^I^I2^4^6^8^10^12^14pD(Col11[T(MPy)PIT](0D2)2}C14 ( pKai = 6.2 ± 0.1; pKa2 = 10.8 ± 01).9.2 — -9.9o259ANL9.298.9a0^Appendix9.30^2^4^ 10^12^14pD{ Colli[T(MPy)(NPh)P(OD2)2] ) C14 ( pKa = 5.9 ± 0.1; pKa2 = 10.8 ± 01).260

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0061766/manifest

Comment

Related Items