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Synthesis and photochemistry of AZO-type derivitized porphyrins with crosslinking applications Desjardins, Angèla M. 2000

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SYNTHESIS AND PHOTOCHEMISTRY OF AZO-TYPE DERIVITIZED PORPHYRINS WITH CROSSLINKING APPLICATIONS by A N G E L A M . DESJARDINS B.Sc, University of Western Ontario, 1998  A THESIS SUBMITTED IN P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES D E P A R T M E N T OF CHEMISTRY  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A September 2000 © Angela M . Desjardins, 2000  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g of t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  Department of  Ctt6rA\STflJf  The U n i v e r s i t y of B r i t i s h Columbia Vancouver, Canada  ABSTRACT  The objective of this work was to synthetically modify porphyrins such that they would possess a functionality that would crosslink within its biological environment upon photoactivation, in photodynamic therapy applications.  The reaction of diazomethane and protoporphyrin dimethylester (9) yielded a series of three novel pyrazoline porphyrins (10 - 12) that were isolated and characterized.  These  were investigated for their ability to extrude molecular nitrogen on thermal and photochemical activation with both near-UV and long wavelength light to yield a 1,3biradical intermediate that may provide a site for potential crosslinking. Upon activation three cyclopropane derivatives (15 - 17) were formed in high yield, presumably through the 1,3 biradical intermediate.  These novel cyclopropane porphyrin derivatives were  purified and characterized.  The optimized diazomethane  reaction was then carried out on a chlorin (18), a  photosensitizer currently undergoing clinical trials for P D T , to yield the first example of a pyrazoline derivatized methylpyropheophorbide (MePPP, 19). Activation with heat, U V  ii  light or long wavelength light yielded the cyclopropane product of MePPP  (20),  analogous to the photoreaction described for the model PPDME pyrazoline reactions. These studies represent the first example of long wavelength activation of a photoactive functionality at the porphyrin periphery.  Attempts to trap the biradical intermediates produced on photoactivation of the pyrazolines were carried out in solution.  Unfortunately, no trapped products were  isolated, presumably because the lifetimes of these intermediates proved to be too short due to efficient formation of the cyclopropane.  The second photoactive functionality that was pursued is anticipated to yield a much longer lived carbene intermediate that should be more effective in cross-linking studies. The synthetic target was the diazirine of TPP (25).  Five of the eight reactions planned in  the synthesis of (25) were optimized, and scaled-up. Each of the intermediates (21, 22, 23, 24) were isolated and characterized.  The reactions leading up to the novel  trifluoroacetyl-TPP (23) provide the first example of introducing a trifluoroacetyl group at the (3 position of the porphyrin.  23  in  T A B L E OF CONTENTS Abstract  ii  Table of Contents  iv  List of Figures  vi  List of Schemes  viii  List of Tables  ix  Acknowledgements  x  1  Introduction 1.1  1.2  Tetrapyrrolic Macrocycles  1  1.1.1  Background  1  1.1.2  Nomenclature  3  1.1.3  Structural Characteristics  4  1.1.4  Optical Absorption Spectra  6  1.1.5  Preparation of Porphyrins  9  Photodynamic Therapy  12  1.2.1  Mechanism of Photosensitization  13  1.2.2  Type I Reactions  16  1.2.3  Type II Reactions  17  1.3  Photoaffinity Labeling  18  1.4  Research Objective  24  iv  2  Results and Discussion 2.1  Synthesis and Charaterization of Protoporphyrin Pyrazolines  28  2.2  Thermal and Light Activated Chemistry of Protoporphyrin Pyrazolines..  47  2.2.1  Thermal Activation  48  2.2.2  Photochemical Activation  51  2.3  Synthesis and Characterization of Methylpyropheophorbide Pyrazoline..  58  2.4  Photochemistry of Methylpyropheophorbide Pyrazoline  64  2.5  Progress Towards the Synthesis of Diazirine Tetraphenylporphyrin  81  3  Conclusions and Suggestions for Further Studies  4  Experimental  96  4.1  Instrumentation and Materials  99  4.2  Photochemical Studies-General Procedures  103  4.3  Preparation of Protoporphyrin Pyrazolines 10,11, and 12  104  4.3.1  109  4.4  4.5  Photoproducts of Protoporphyrin Pyrazolines 15,16, and 17  Preparation of Methypyropheophorbide Pyrazoline 19  113  4.4.1  Photoproduct of Methylpyropheophorbide Pyrazoline 20  116  Progress Towards the Synthesis of Diazirine Tetraphenylporphyrin 25...  117  Appendix: Crystallographic Analysis of 17  125  References  131  V  LIST OF FIGURES Figure 1.1:  Important tetrapyrrolic backbones  1  Figure 1.2:  Protoporphyrin IX  3  Figure 1.3:  Porphyrin numbering and nomenclature  3  Figure 1.4:  The 187t-electron pathway  4  Figure 1.5:  Q-band porphyrin spectra for metal-free porphyrins  7  Figure 1.6:  Wavelength dependent penetration of light through tissue  13  Figure 1.7:  Modified Jablonski diagram for a typical photosensitizer  15  Figure 1.8:  Schematic of photoaffinity labeling  19  Figure 1.9:  Photoaffinity taxol analogue  21  Figure 1.10:  Photoactive functionalities  26  Figure 2.1:  BPD-diacid  28  Figure 2.2:  1,3-Dipolar cycloaddition  31  Figure 2.3:  Estimated energy of frontier n orbitals for conjugated vinyl group and diazomethane  32  Figure 2.4:  Orbital coefficients for diazomethane  32  Figure 2.5:  ' H N M R of B-ring pyrazoline of PP-DME, with expansions of pyrazoline signals  34  Expansion of H M Q C spectrum, illustrating within the pyrazoline of 10  36  Figure 2.6:  Figure 2.7  Figure 2.8  l 3  C - ' H associations  Expansion of C O S Y spectrum, highlighting correlations within the pyrazoline  39  Expansion of correlations  42  NOESY  spectrum,  illustrating  Figure 2.9:  1  H N M R spectra of 9 and 12  Figure 2.10:  Course of thermal cycloelimination of nitrogen  meso  proton  44  vi  48  Figure 2.11:  Schematic of T L C plate: Thermolysis of di-adduct pyrazoline  Figure 2.12:  UV-vis spectra photoproduct  Figure 2.13:  of  A-ring  pyrazoline  of  PP-DME  and  50 its 52  Changes in ' H N M R spectra of pyrazoline (11) and photoproduct (16)  53  Figure 2.14:  *H NMR spectrum of 16  54  Figure 2.15:  X-ray crystal structure of 16  56  Figure 2.16:  Transmittance of red light filter  57  Figure 2.17:  UV-vis spectra of 18 and 19 in CH C1  Figure 2.18:  ' H NMR spectra of 19 and 20  66  Figure 2.19:  UV-vis spectra of pyrazoline (19) and its photoproduct (20)  67  Figure 2.20:  UV-vis spectra of chlorin bands, monitoring photoconversion of 19  2  2  60  to 20  69  Figure 2.21:  Emission spectra of 19 and 20  70  Figure 2.22:  Course of photochemical cycloelimination of nitrogen  71  Figure 2.23:  Energy level diagram of porphyrin and pyrazoline  73  Figure 2.24:  Cyclic voltammogram of 19 measured in CH2CI2  75  Figure 2.25:  Energy diagram for the formation and dissociation of pyrazoline....  77  Figure 2.26:  Foscan®  81  Figure 2.27:  Diazirine-TPP  81  Figure 2.28:  Predicted photochemistry of diazirine-TPP and diazirine-DPP  83  Figure 2.29:  ' H NMR spectra of trifluoroacetyl-TPP and intermediates  94  Figure 2.30:  Coupled F spectra of 22 and 23  95  1 9  vii  LIST OF SCHEMES  Scheme 1.1:  Protoporphyrin derived from blood  10  Scheme 1.2:  Chlorins derived from leaves  11  Scheme 1.3:  Synthesis of TPP  11  Scheme 1.4:  Photochemistry of nitrenes  20  Scheme 1.5:  Photochemistry of diazirines  22  Scheme 2.1:  Reaction of PP-DME and diazomethane  28  Scheme 2.2:  Numbered B-ring pyrazoline  35  Scheme 2.3:  Temperature Ranges required for thermolysis compounds  in cyclic azo 47  Scheme 2.4:  Photochemistry of pyrazoline functionality  51  Scheme 2.5:  Numbered pyrazoline of MePPP  59  Scheme 2.6:  Formation of 20 from 19  64  Scheme 2.7:  Preparation of Fluorinated Porphyrins  84  Scheme 2.8:  Synthesis of 3-trifluoromethyl-3-phenyl diazirine  84  Scheme 2.9:  Progress towards the synthesis of diazirine-TPP (25)  85  Scheme 2.10:  Formation of trifluoromethanol-TPP  91  Vlll  LIST OF TABLES  Table 2.1:  H M Q C correlations within 10  37  Table 2.2:  Pyrazoline methylene coupling constants  38  Table 2.3:  N O E S Y correlations for the A and B-ring pyrazolines of PP-DME..  41  Table 2.4:  Thermolysis of pyrazolines  49  Table 2.5:  H M Q C correlations for 19  61  Table 2.6:  Summary of redox potentials measured in CH2CI2 at 22°C vs. Fc....  74  Table 2.7:  Results of crosslinking attempts in various reagent solutions  79  Table 2.8:  TPP metallation conditions  87  Table 2.9:  Formylation of NiTPP  88  IX  ACKNOWLEDGEMENTS  For the opportunity to work in such a dynamic laboratory, I would like to thank Professor David Dolphin. The last two years have been a very exciting time in the Dolphin labs, and I count myself among the most fortunate graduate students to have been able to work on a great project in this unique learning environment.  Every member of the Dolphin research group has contributed to my learning experience at U B C , and I am sincerely grateful. Jill MacAlpine extended her wisdom and friendship to me even before my arrival in Vancouver, and I thank her. Alison Thompson was an invaluable sounding board who offered encouragement both in my laboratory and academic pursuits, not to mention being an exquisite dinner partner! Ethan Sternberg was a mentor, a fountain of knowledge and creative suggestions, and I count him among my true friends.  This page would not be complete without mentioning my classmates.  To Michael  Fenster, Roger Linington, Jeffery Flemming and Jomi Samuel, thank you for the late study nights and the great laughs!  To my family, who has always offered me unconditional love and support, thank you for caring for me half a country away and for being so proud of me.  And to Mark, who has read and re-read every word of this thesis, who was my best critic and is my biggest fan. This is for you L O M L . It represents the end of one stage of my life, and the beginning of our life together.. .1 can't wait!  Introduction  1  Introduction  1.1 Tetrapyrrolic Macrocycles  1.1.1  Background  It is somewhat surprising, but true, that a single family of pigments is responsible for making our blood red and the grass green. These pigments are based on a macrocyclic ring of carbon and nitrogen atoms, some of the best examples of which are the ironcontaining porphyrins found in heme (of hemoglobin) and the magnesium-containing reduced porphyrin (or chlorin) found in chlorophyll. The macrocycles are made up of 1  four pyrrolic moieties, often joined together via methine bridges (Figure 1.1).  H •H  Porphyrin  Chlorin  H H H H-  H "H  Bacteriochlorin  Isobacteriochlorin  Figure 1.1 Important tetrapyrrolic backbones  1  Introduction  The ubiquity and diversity of tetrapyrrolic macrocycles have prompted much research into their structural determination, isolation, total synthesis and function.  Since their  elucidation early in the 20 century , porphyrins have come a long way; particularly in th  2  their diverse applications. Over sixty years ago, the first porphyrins were isolated from oil shale.  3  These 'petroporphyrins' now serve as chemical markers for new oil reserves,  and recent advances continue to fuel this field of investigation, known as organic geochemistry.  Today, research into metalloporphyrins has led to their integration in  4  many fields of study, including medicinal chemistry. These efficient chelators have the ability to harness radioactive metals and can thus be used in diagnostic pharmaceuticals.  5  One need only scan through recent porphyrin abstracts to recognize their wide range applications, including molecular wires in nanotechnology,  catalysis,  light emitting  diodes, enzyme mimetics, industrial paint dyes, and optical recording materials. 8  9  10  The Dolphin lab has been particularly interested in those characteristics of porphyrins that make them well-suited for use in photodynamic therapy (PDT). Both non-metallated (free base) and a variety of metallated porphyrins have shown promise as potential photosensitizers.  11  It is the ability of these extended aromatic molecules to absorb long  wavelength light that enables them to sensitize oxygen, which can then incite the degradation of tumors and other hyperproliferative tissue. Section 1.2 will elaborate on PDT and the use of porphyrins as photosensitizers.  2  Introduction  1.1.2  Nomenclature  The nomenclature of porphyrins and related systems is often difficult to understand, due to the lingering presence of trivial naming schemes. naming scheme was others ' ' 12  of  13  trivial  14  developed  The initial  by Fischer and  in the 1930's and utilizes a large number names  in  conjunction  with  a  basic  Figure 1.2 Protoporphyrin IX numbering system.  For example, protoporphyrin IX  (Figure 1.2), one of the naturally occurring porphyrins, was assigned its numeral designation as it happened to be the ninth isomer discovered in a series. Other important trivially named porphyrins are etioporphyrin, rhodoporphyrin, and phylloporphyrin, on which the classification of their optical spectra is based (see Section 1.1.4).  A revised form of porphyrin nomenclature was proposed in I960 and adopted in 1988 15  16  by an IUPAC-IUB joint commission.  It accounts for all substituents, including the  pyrrolic nitrogen in a systematic way.  The numbering system is as shown, for the  simplest porphyrin, named porphine (Figure 1.3). Positions 1, 4, 6, 9, 11, 14, 16, and 19 are the a-positions, 2, 3, 7, 8, 12, 13, 17, and 18 are the (3-positions and 5, 10, 15, and 20  Figure 1.3 Porphyrin numbering and nomenclature  3  Introduction  are the meso-positions.  The pyrrole rings are lettered clockwise, starting from the top  left, A through D.  Alongside the systematic IUPAC nomenclature, for the sake of brevity and perhaps tradition, the old type nomenclature has been retained in some cases. IUPAC allows a semi-systematic nomenclature based on a dozen trivial names. Any compound that has the basic structure of protoporphyrin keeps protoporphyrin as the root, and modifications are systematically added to the name.  Thus, the compound resulting from methyl  esterification of the propionic acid chains of protoporphyrin IX is called protoporphyrin dimethylester (PP-DME), and not 2, 7, 12,  18-tetramethylporphyrin-3,8-divinyl-13,17-  dimethylpropranoate.  1.1.3 Structural Characteristics  Porphyrins are a unique class of molecules thanks to their structural  and inherent  electronic characteristics. The feature that accounts for most of the porphyrins unique behaviour is their aromaticity. They are planar (for the most part) and possess 22 71electrons,  18 of which participate in a  conjugated pathway (Figure 1.4). these  molecules  are  very  Thus,  stable,  have  reduced heats of combustion, characteristic Figure 1.4 The 18Tt-electron pathway  17  bond lengths, and a large ring current.  4  Introduction  The two remaining exocyclic double bonds can be reduced without compromising the aromaticity of the molecule, but do markedly change some of the molecules' properties (see section 1.1.4). chlorin.  The reduction of one of these bonds leads to the formation of a  When both double bonds are reduced, the result is either a bacteriochlorin -  opposite rings, or an isobacteriochlorin- adjacent rings. Figure 1.1 depicts each of these classes of reduced porphyrins.  The effect of large ring current is best observed in 'H-NMR spectra of porphyrins. Both the raeso-protons and (3-pyrrole protons are deshielded, and are shifted downfield (5=8 to 10 ppm). The inner N-H protons find themselves inside the ring current, are shielded, and appear far upfield (5 = -1 to -5 ppm)  18  relative to tetramethylsilane (TMS).  It is  important to note that the protons on these nitrogens are not localized on any two nitrogen atoms, but exchange rapidly between all four nitrogens.  The resultant signal  visible in N M R spectroscopy is a single peak representative of both central N - H protons.  The central imino nitrogen atoms are basic, and can be protonated to form the dication in strong acid. The central amine groups can be deprotonated, but strong base is required to form the dianion. The pKi and p K 2 values of the N-H protons are both approximately 16, while p K is 5 and pIQ is 2. 3  19  These properties of the central nitrogen atoms, together  with their shape and size make porphyrins excellent chelators. Introduction of divalent metals replaces the central amine hydrogen atoms, and the porphyrin ligand exists formally as its dianion in a metal complex. Such changes in the core of the macrocyle can alter the porphyrin's reactivity at its periphery substantially.  5  Introduction  Porphyrin reactivity can be simplified, perhaps naively, by returning to the fundamental aromaticity of the macrocyle. Porphyrins undergo many of the electrophilic substitution reactions  characteristic  of aromatic  compounds  formylation, acylation and deuteration.  including nitration,  halogenation,  Porphyrins differ from molecules such as  benzene, i n that there are two different sites on the macrocycle where electrophilic substitution can take place with different reactivities; the meso-position and the pyrrole 21  (3-position.  W h i c h of these sites is activated depends on how electronegative is the  porphyrin.  This can be controlled by the choice of metal to coordinate to the central  nitrogen atoms.  1.1.4 Optical Absorption Spectra  Functionalized porphyrins come in a variety of shapes and sizes.. .and colours! The vivid green, purple, and red hues of these molecules stem from their absorption of nearultraviolet and visible light in the electromagnetic spectrum. between 390 and 425 n m ,  22  The most intense band,  is called the Soret band. The Q bands, situated between 480  and 700 nm, vary in number and intensity depending on the substitution pattern of the porphyrin and whether it is metallated or protonated.  Metal-free (free base) porphyrins have four Q bands, and are labeled I V , III, II, and I, in increasing order of wavelength.  There are four classifications for porphyrins of this  nature, etio-type, rhodo-type, oxo-rhodo type and phyllo-type.  Figure 1.5 depicts the  appearance of each type of spectrum, and the nature of the porphyrin's substitution.  6  Introduction  When the relative intensities are such that IV>III>II>I, the spectrum is said to be etiotype after the etioporphyrins in which the (3-substituents are all alkyl groups.  When  electron withdrawing-groups such as carbonyl or vinyl groups are found in the (3positions, a subtle change is observed such that III > IV > II > I. This is called a rhodotype. spectrum because of the slight red shift that is also observed.  When two such  electron-withdrawing groups are situated on opposite pyrrole rings in the macrocyle, the rhodofying effect is intensified to give an oxo-rhodo-lype spectrum in which III > II >IV  IV alkyl substituents in [3-positions  electron withdrawing groups (carbonyl, vinyl) in (3-positions  electron withdrawing groups are on opposite pyrrole units  (3-positions are unsubstituted, or meso positions are occupied  500  600  Figure 1.5 Q-band porphyrin spectra for metal-free porphyrins  7  Introduction  > I.  When some of the pyrrole p-positions are left unsubstituted, or when a meso-  position is occupied, the phyllo-type spectrum is obtained, and the intensities of the Q bands are such that IV > II > III > I.  Addition of strong acid to the porphyrin protonates the central imino nitrogen atoms. The macrocyle is more symmetrical (than the free base) as a result, and the Q band spectrum is simplified by the collapse of four bands to two. By the same token, addition of a metal increases symmetry, thus metalloporphyrins generally have two Q bands.  Chlorin spectra are characterized by a marked increase in intensity in the longest wavelength Q band, as well as a bathochromic shift (>25 nm).  The phenomenon is  somewhat counterintuitive, as one would expect decreased conjugation to result in a higher energy transition, or lower wavelength shift.  However, selection rules and the  allowedness of this transition contribute extensively in the chlorin spectra. Section 2.3 contains the U V - visible spectrum of one such chlorin, methylpyropheophorbide.  Many theoretical chemists have elucidated the origins of these spectra. In the 1960's, Gouterman developed the four-orbital model theory that simplifies the task because it 23  considers only the two highest occupied molecular orbitals (HOMOs), and the two lowest unoccupied molecular orbitals (LUMOs) of a porphyrin. A comprehensive presentation of this theory requires an extensive explanation nonetheless, and is beyond the scope of this work.  8  Introduction  The differences in electronic spectra of tetrapyrroles can serve as an important diagnostic tool. The nature of a compound can thus be identified, and the spectra are invaluable in monitoring the progress of reactions. Finally, it is these optical properties of porphyrins that make them so valuable as phototherapeutic agents.  Their absorption of long  wavelength visible light, in regions where most living tissue does not absorb light, enables them to photosensitize oxygen, and kill surrounding cells in desired regions. This property of tetrapyrrolic macrocyles, as well as an overview of PDT will be further described in Section 1.2.  1.1.5  Preparation of Porphyrins  None of the porphyrins used as starting materials in this thesis were synthesized in house. Each of the porphyrins that has been synthetically modified was chosen because of its inherent potential as a photosensitizer, and its availability. Although many elegant total syntheses of porphyrins have been reported over the years, these are invariably multi18  step, time consuming, and low yielding.  Because the targets herein may have  applications as pharmaceutical agents, the starting materials have to be inexpensive, and the transformations kept to a minimum.  In recognition of these facts, and the diversity of the three classes of porphyrins used, the preparation of PP-DME, methylpyropheophorbide (MePPP), and tetraphenylporphyrin (TPP)  will now be described briefly. In the case of PP-DME and MePPP, nature has  done most of the work for us. Starting from mammalian blood which has been strained  9  Introduction  and heparinized (decoagulated), hemin (Fe(III) protoporphyrin IX chloride) is separated. The blood is poured into hot acetic acid containing sodium chloride, and the hemin separates on cooling.  24  Several isolation techniques exist ' , all which lead to a 25  26  porphyrin that can be converted to various other derivatives by chemical manipulation of its substituents (Scheme 1.1). 27  BLOOD  hematoporphyrin IX  Scheme 1.1 Protoporphyrin derived from blood  Chlorophyll a and b can be extracted from leaves simply by boiling them in methanol or acetone.  The two chlorophylls are separated by column chromatography, with the a-  conformer being in a 3:1 excess. Removal of magnesium metal, and transesterification yields the methylpheophorbide, which is subsequently converted to the MePPP (Scheme 1.2).  10  Introduction  LEAF TISSUE  o u!° i'm „ R = CHO, chlorophyll b a:b = 3:1 =  methylpheophorbide '  phl  methylpyropheophorbide  Scheme 1.2 Chlorins derived from leaves TPP is the simplest porphyrin to make (Scheme 1.3).  The synthesis involves refluxing  pyrrole with benzaldehyde in proprionic acid for a half hour. The purple solid is filtered off after cooling, and then washed to free the porphyrin from soluble poly-pyrrolic impurities. The low yield (20-25% based on pyrrole)  is offset by the ease of synthesis.  The mechanism is based on the formation of a carbonium ion, the result of a pyrrole attack (a-position) on a protonated aldehyde. Another pyrrole then attacks the carbonium ion to give a meso-substituted dipyrrylmethane. tetrapyrrylcarbinols are formed.  Chain building continues until  Ring closure follows to give porphyrinogens, which  oxidize in air to porphyrins.  Scheme 1.3 Synthesis of TPP 11  Introduction  1.2 Photodynamic Therapy  Photodynamic therapy is a minimally invasive, two-step medical procedure that uses light-activated drugs called photosensitizers to treat a range of diseases involving rapid cell growth, such as cancerous tumors or abnormal blood vessels."  First, the  photosensitizer is administered intravenously and, once in the bloodstream, associates with lipoproteins.  Rapidly dividing (neoplastic) cells require more lipoproteins for  membrane construction than normal cells, thus a higher concentration of the drug accumulates in these tissues. The photosensitizer drug is then activated by exposure to a pre-calculated dose of light at a particular wavelength. Once activated, the drug converts oxygen found in the cells into highly energized singlet oxygen. Singlet oxygen can react with subcellular components such as proteins and lipids, which disrupts normal cellular function and results in killing the cells. Lasers and fiber optics are used to deliver light when treating deeper internal organs.  Photodynamic therapy is a relatively safe  treatment, since without exposure to light the drug has no effect. Furthermore, the drug accumulates primarily in diseased cells as described above, thus upon irradiation the effects on surrounding healthy tissue are minimized. Patients treated with photodynamic therapy will experience photosensitivity- skin sensitivity to bright light - for anywhere from 24 hours up to six weeks depending on the photosensitizer used.  12  Introduction  1.2.1  Mechanism of Photosensitization  Photosensitizers  are generally porphyrogenic compounds with strong absorption  coefficients at wavelengths in the (red) region (> 630 nm) of the electromagnetic spectrum. Namely, the Q bands must be used as a means of photosensitization since the presence of endogenous chromophores such as hemoglobin and light scattering result in poor penetration of tissue at wavelengths below 630 nm. Between 630 nm and 750 nm, light penetration through human tissue doubles  (Figure 1.6), and allows efficient  Q. CD  T3  £Z o  "5 <D  cz CD Q. CD  > . O CD CD CD >  CD  550  630  700  W a v e l e n g t h (nm;  800  Figure 1.6 Wavelength dependent penetration of light through-tissue  excitation of the photosensitizer drug, causing the most phototoxic effect.  30  Compounds  absorbing energy above 800 nm do not have a large enough gap between the triplet and ground states to be able to generate singlet oxygen.  13  Introduction  These properties of light penetration through skin are superfluous to note to any child who has succumb to the temptation of holding a flashlight in his mouth. Although we might have felt as though the eerie red hue given off by our cheeks was because of our blood, our skin and various biomolecules were in fact acting to filter the light. Thus it is obvious that the red light would penetrate most effectively!  A modified Jablonski diagram, shown in Figure 1.7, best illustrates the principles of photosensitization.  Non-radiative processes are depicted by curly arrows.  Energy  absorption (photons) is denoted by straight upward arrows, while radiative processes such as fluorescence and phosphorescence are shown with straight downward arrows. The porphyrin photosensitizer absorbs radiant energy, and this photon promotes an electron from the porphyrin's outer shell ground state (So), to an excited state (S ). The molecule n  is then likely to lose energy via internal conversion (IC), until it reaches the first excited state (Si).  The lifetime of the singlet excited state is usually not more than a few  nanoseconds, since the electron can readily undergo internal conversion to its ground state (Si—>So + heat), or fluoresce (Si—>So + hv'). However, under certain circumstances it is possible for one of the electrons to undergo the 'forbidden' process of flipping its spin (known as intersystem crossing, ISC). Now both electrons have parallel spin, which results in the triplet excited state of the sensitizer (Si—>Ti + heat).  Before the triplet excited state can lose its energy, the electrons must first pair their spins again.  As these processes have low probability (spin-forbidden), compared with the  singlet excited state, the triplet excited state has a much longer lifetime (microseconds to  14  Introduction  seconds). One of the possible routes the first triplet state may take to the ground state is via phosphorescence ( T | - » S + hv"). Triplet excited states can undergo many types of 0  reactions. In considering those important to PDT, the other possibilities involve nonradiative interactions of the triplet excited state with an easily oxidized or reduced substrate ("Type I" reactions), or spin-exchange with ground state triplet exchange. The latter "Type II" process allows the excited triplet state of the sensitizer to pass its excitation energy (not an electron) to the triplet ground sate of the oxygen. The sensitizer thus returns to its singlet ground state, while oxygen is converted to a singlet excited state. Both "Type I" and "Type II" reactions are responsible for the cytotoxicity of the drug.  1  UJ  PHOTOSENSITIZER  OXYGEN  Figure 1.7 Modified Jablonski diagram for a typical photosensitizer  15  Introduction  The efficiency with which this internal system crossing to the triplet state occurs is quantifiable, and essentially determines the limit of effectiveness of a photosensitizer. The triplet quantum yield is the ratio of the amount of triplet sensitizer formed to the amount of singlet sensitizer initially formed.  1.2.2  Type I Reactions  Photoprocesses of the Type I nature involve reaction of the sensitizer (SENS) in its triplet excited state ( SENS*), with other molecules in its environment. Because of its unpaired 3  electrons (the promoted electron and the residual electron left by excitation), the triplet state is more readily oxidized and reduced. A photosensitizer in its triplet excited state can abstract an electron or a hydrogen atom from a substrate (SUB) as shown:  3  SENS* + SUB  -» (SENS*)" + (SUB*)  3  SENS* + SUB-H -> SENS-H + SUB*  +  The resultant, highly reactive, radicals can behave in a number of ways. Among these, the semi-reduced sensitizer is capable of reacting with molecular oxygen to generate the superoxide radical (02»)~. The free radicals formed upon hydrogen abstraction can also react with molecular oxygen, in this case forming peroxides, also capable of initiating radical chain reactions.  16  Introduction  1.2.3  Type II Reactions  This second series of reactions stems from the triplet excited state of the sensitizer shunting its energy to triplet ground state oxygen. Formation of highly reactive singlet oxygen by this means is very efficient because it is 'spin allowed'. It is worthy to note that in water (the biological medium) the lifetime of singlet oxygen is only 4 p:s , thus 31  reactions attributed to it must be localized near the site of its generation. Singlet oxygen reacts with biological substrates in a variety of ways . 32  1) Ene Reaction. Ethenes that have allylic hydrogens readily yield hydroperoxides in photo-oxygenation processes by reaction with '0 . 2  The 0 l  2  ene reaction involves  abstraction of the allylic hydrogen, and a shift of the ethene bond. Tryptophan and derivatives of this amino acid are susceptible to singlet oxygen attack:  2) [2+2] Cycloaddition. Electron rich ethenes, such as enol ethers, vinyl sulfides, and enamines which do not have allylic hydrogens, undergo photo-oxygenation to give dioxetanes.  These unstable species undergo decomposition to give two carbonyl  compounds:  o  A*X  o  Introduction  3) [2+4] Cycloaddition to 1,3-dienes. Singlet oxygen behaves as a powerful dienophile in the Diels-Alder reaction and cyclic endoperoxides can be produced in this way, as the following reaction in the formation of ergosterol peroxide illustrates:  4) The photooxidation of sulfides to sulfoxides was originally reported by Schenck and Krauch in 1962 . 33  It is now generally accepted that the reaction proceeds via the  lowest excited state of molecular oxygen, singlet oxygen. The major product is the sulfoxide, with varying amounts of sulfone, depending on the substrate and reaction conditions.  34  The following example depicts the oxidation to methionine sulfoxide:  1.3 Photoaffinity Labeling  Historically, photoaffinity labeling has been used to locate active sites and binding sites in enzyme-substrate complexes (Figure 1.8).  As a result of covalent bonding between  substrates and a variety of cellular components, enzymes, membranes , protein 35  structures,"  neural receptors , and R N A or D N A structures' have been successfully  18  Introduction  labeled. Each of these successes yields information about its respective target. Vigorous application of the technique can reveal the sequence of amino acids involved in binding, and effectively paint a picture of the active site in an enzyme.  Enzyme  Substrate  Enzyme-Substrate Complex  Photolabeled Enzyme  Labeled Fragment  Figure 1.8 Schematic of photoaffinity labeling  Photoaffinity labeling typically requires chemical modification of the substrate so that irradiation of the resultant complex will produce a reactive intermediate that will bond covalently to the enzyme.  39  A critical element of photoaffinity labeling is synthesis of  the modified substrate, and often the substrates have radioactivity incorporated to allow tracking of the tagged binding site. Synthetic methods for introduction of radiolabels and choice of radiolabeling reagents are also important aspects of this technique , but do not 40  relate to the present research topic.  Photolysis of the modified substrate generates an intermediate whose behaviour will dictate the success of its labeling, thus the intermediate precursor must be chosen wisely. Several classes of reactive intermediates have been exploited in photoaffinity labeling; the predominant ones being nitrenes, carbenes and radicals. Each of these will be briefly introduced in this section.  19  Introduction  3r  recombine  ISC  H atom abstraction radical c o u p l i n g  1  2 Scheme 1.4 Photochemistry of nitrenes  Nitrenes stemming from aryl azides have been studied extensively and appear throughout the literature. In spite of the frequent use of azides, it has been pointed out recently that this moiety "rarely gives stoichiometric labeling." are generally less that 30%.  42  41  In fact, yields of azide photolabeling  Low cross-linking yields in aryl azide applications can  result for several reasons. The most probable complication arises from the chemistry that follows irradiation of the aryl azide (1) (Scheme 1.4), which shows that irradiation produces a singlet nitrene that intersystem crosses to the ground state triplet. (At low temperatures, some chemistry from the singlet is observed to form 2.) The triplet nitrene is expected to behave like a diradical, and is hoped to effect useful binding by hydrogen abstraction then radical coupling.  43  In addition to the low reactivity of the ground state  triplet, simple phenyl azides suffer from complications of instability (they are easily reduced) and high energy irradiation wavelengths. Most of these chromophores have to be excited at 254nm, a wavelength that can substantially damage biological systems . 39  A representative example of the use of azides in photoaffinity labeling is found in the work of Swindell et. al.  44  Taxol , a promising anti-cancer drug, is known to interfere 45  20  Introduction  with the normal function of microtubules in rapidly dividing cells.  46  To understand the  interaction between taxol and microtubules, the researchers efforts were directed toward determining the structure of the binding site(s) of the drug on microtubules. This was accomplished by the synthesis of a photoaffinity taxol analogue that bears an azide photoreactive moeity in the A-ring side chain of taxol (Figure 1.9).  ACC;  ,O OH  The tritiated  [~H] version of this taxol analogue (3), was also prepared, carrying tritium on the  P h C  TAXOL  aromatic rings of the N-acyl subtituents for  3  °  OAc  2  X = PhCONH X = 4-(N )-C H CONH 3  6  4  ease of detection. The results of this work determined that 3 labels the TV-terminal domain of (3-tubulin  36  Figure 1.9 Photoaffinity taxol analogue  with specificity, and that this specificity coupled with the  efficiency of labeling make it an attractive candidate for further characterization of the taxol binding site(s) on microtubules.  In general, radical intermediates are ideally suited for photoaffinity labeling.  39  They are  known to abstract hydrogen atoms from virtually any site, are more reactive with C - H bonds than are nitrenes and have less propensity for intramolecular rearrangements than carbenes. Unfortunately, many free radical reactions including intramolecular hydrogen abstraction and bond formation between the initial radical pair can minimize their ability to crosslink. Single electron transfer to generate long -lived charged species, can either minimize effectiveness or be conducive to covalent bonding between nucleophilic and electrophilic sites. Examples of radical generating photoprobes include: benzophenone  21  47  Introduction  (whose triplet diradical has a lifetime of 300ns in the absence of easily abstractable AQ  hydrogen atoms), enones  (which have a higher propensity for 2+2 cycloaddition with  alkenes), and various diazo/diazonium compounds whereby loss of N  2  reactive intermediate .  photosensitizers  49  The first series of potential crosslinking  results in the  appearing in this thesis are pyrazolines, which are expected to behave as biradicals upon photoexcitation.  Diazirines were first proposed as photochemical reagents by Smith and Knowles in 1973 , but most of the recent work with diazirines exploits the photochemical reactivity 50  of the trifluoroethyldiazirinephenyl group . 51  Irradiation of a substituted diazirine (4)  (Scheme 1.5) has been shown to give carbene (5) and the corresponding diazo compound (6).  Carbenes have the ability to insert into carbon-hydrogen bonds (Sol-H) to yield  products like 7.  The diazirine unit is small, non-bulky, and lipophilic.  chromophore that extends significantly into the 300 nm range.  It has a  Many applications of  photo cross-linking have been reported for this functionality. Some of the disadvantages that have been reported include the triplet carbene reacting with molecular oxygen to give the corresponding ketone,  and the photoactive diazo intermediate having a different  22  Introduction  excitation wavelength than the diazirine. efficient  Despite these potential drawbacks, its  53  insertion capabilities coupled with its stability in mild acidic or basic  environments make it a most attractive photocrosslinking functionality. 54  Researchers  at Columbia University  55  reported the synthesis,  radioisotopic labeling, and resolution of a phenylalanine analog, 3[p-[3-(trifluoromethyl)-3H-dizirin-3-yl]phenylalanine (8), containing the 3-(trifluoromethyl)-3H-diazirinyl group. Like all diazirines, 8 absorbs in the near U V (^ x 350; e 265), at wavelengths which  COO-  ma  do little damage to most biological samples.  In this case, they  hoped the analog would be useful in preparing peptide and polypeptide  photoaffinity  polypepetides,  reagents.  Understandably, receptors  including various hormones,  neurotransmitters,  for peptides and and toxins,  are of  considerable interest.  In summary, the literature abounds with examples of successfully labeled binding sites in a wide range of supramolecular systems. ' ' 56  57  58  That the field of photoaffinity labeling is  immense and growing rapidly testifies to the utility of this analytical procedure.  39  It is  clear from the information in this section that the advantages and disadvantages of any given photoactive moiety must be weighed carefully in each application, since many variables play a role in the efficiency and selectivity of photolabeling.  Although the  objective of the work in this thesis does not involve labeling of a specific binding site, the intent is to capitalize on the mechanism of photoaffinity labeling. Futhermore, the design  23  Introduction  of the crosslinking photosensitizers that are envisioned herein is based on literature precedence for the crosslinking ability of the functionalities.  1.4 Research Objective  As the search for more effective and less costly photosensitizers continues, so does the investigation of their characteristics and capabilities. There is a great need to understand the critical structural and electronic factors that control these molecules' reactivities and bioactivities.  In addition, the mechanisms by which phototoxins biodistribute are just  beginning to be understood, and it would be very desirable to increase their selectivity for diseased tissue.  Photosensitizers  are generally porphyrogenic compounds with strong absorption  coefficients at wavelengths in the 650 nm (red) region of the electromagnetic spectrum. At this wavelength, human tissue is the most transparent to light, and allows efficient excitation  of  the  photosensitizer  drug,  causing  the  most  phototoxic  effect.  59  Unfortunately, this strength of light is present in ambient daylight and patients of PDT have experienced varying degrees of skin photosensitivity resulting from residual photosensitizer in healthy tissue.  Although photosensitizers tend to accumulate in  cancerous tissue, their transport by lipoproteins results in their presence throughout systemic circulation.  This 'passive selectivity' is a result of the pharmacokinetic  behaviour of the drug. Results have shown ' 60  61  that photosensitizers are taken up and  released rapidly by all cell types. A l l cells retain a proportion of the drug taken up, and  24  Introduction  cancerous cells have demonstrated an ability to take up increased amount of photosensitizer.  This equilibrium requires that enough drug be administered to ensure  effective photosensitization of the target cells, but is also to blame for residual skin photo-sensitivity.  This side-effect of PDT has prompted an exploration of possible  structural modifications of the photosensitizers, and new ways of using visible light for their photoactivation. One approach is to develop a method of binding the drug once it has reached the diseased tissue, creating an 'active selectivity'. If the drug could be fixed in the desired cells, the possibility of multiple irradiations and new approaches to PDT are envisioned. Such a process might allow for the use of much lower doses and also be more sparing to healthy tissue.  Photoreactive crosslinkers offer an efficient means of conjugation with proteins and various other cellular components. Many azo-type (N=N) functionalities are known to form biradical , carbene , or nitrene 62  63  64  intermediates upon irradiation.  Photoaffinity  labeling techniques have long capitalized on these highly reactive intermediates that can crosslink carbon-carbon multiple bonds, or insert into carbon-hydrogen (C-H) bonds within the targeted cells. ' ' 65  66  67  Figure 1.10 depicts a generalized scheme for some of the  reactivity of the aforementioned reactive intermediates.  The objective of this work is to synthetically modify porphyrins such that they possess a functionality that could crosslink within their chemical environment. Thus, the synthetic targets will be designed according to precedence for the crosslinking moiety's aptitude, as well as the known photosensitizing ability of the porphyrin starting material. With the  25  Introduction  hv -N, Pyrazoline-modified Drug  N=N  Reaction of Biradical  hv  —)  Drug bound to cellular component  hb-^CH  -N, Reaction of Carbene  Diazirine-modified Drug  cm  hv -N,  Azide-modified Drug  Drug bound to cellular component  Reaction of Nitrene  Drug bound to cellular component  Figure 1.10 Photoactive functionalities  potential for these molecules to become PDT agents of the future, it is imperative that transformations be kept to a minimum and that scale-up procedures be considered. Each of the novel synthetic targets will be isolated and fully characterized.  More fundamentally, this work presents an opportunity to examine the photochemistry involved in activating such a crosslinking moiety through absorption of light by the porphyrin chromophore. Each of the  successfully  26  modified porphyrins will be  Introduction  photoactivated,  and their photochemistry  observed  both  at long  and near-UV  wavelengths. The photochemical products will be isolated and fully characterized. An attempt will be made to understand the photochemical phenomena in question, and experiments carried out to further explain the results. In addition, a series of crosslinking experiments will be performed, to assess the modified porphyrins abilities to crosslink within their chemical environment.  27  Results and Discussion  2  2.1  Results and Discussion  Synthesis and Characterization of Protoporphyrin Pyrazolines  A serendipitous discovery in the Dolphin lab prompted much of the research described in this chapter.  An esterification  reaction involving diazomethane and benzoporphyrin diacid (Figure 2.1) was left overnight erroneously, and resulted in a myriad of products in addition to the expected esters of the proprionic acids. The identity of the products was not known,  0 H  H 0  Figure 2.1 BPD-diacid  but it was rationalized that some of the products could be due to diazomethane reacting with the activated olefins of the benzoporphyrin. In order to explore this possibility further, a simpler porphyrin was investigated.  It was discovered that the reaction of  diazomethane and protoporphyrin dimethylester, 9 (PP-DME, the dehydrated version of the naturally occurring starting material for many photosensitizers,) generated three addition products. The three pyrazoline porphyrins (10-12) shown in Scheme 2.1 depict one set of the many structural identities that were initially hypothesized for the products.  N  Scheme 2.1 Reaction of PP-DME and diazomethane  28-  Results and Discussion  Initial research in this project involved the optimization and full characterization of the aforementioned reaction and products.  The yields obtained are respectable  (77%  recovery for reaction Scheme 2.1), but in order to isolate sufficient amounts of the monoadduct pyrazolines, the reaction must be stopped before completion.  Thus the overall  conversion of starting material to products must be high, but if a huge excess of diazomethane is present and left to react, the mono adducts will go on to form the diadduct.  The reaction was not as simple as first anticipated. Gaseous diazomethane is formed on base catalyzed decomposition of N-methyl-N-nitroso amines , and is carried by an 68  ethereal distillate. The low solubility of porphyrins in most solvents was further reduced by addition of ether. In fact, the ether resulted in the precipitation of the porphyrins from solution throughout the course of the diazomethane addition. Methylene chloride was found to be the best solvent for PP-DME. Under dilute porphyrin solution conditions, the conversion to products was very poor. The most successful reactions resulted from a compromise of the conditions: dissolving the nitroso compound in a minimum amount of ether, and adding more methylene chloride to the stirring reaction mixture upon complete addition of diazomethane.  Most references for diazomethane reactions on small molecules cite usage of an excess 69  of approximately 10:1 diazomethane: substrate. With high molecular weight porphyrins and their inherent low solubility it was found through successive increases that a molar ratio of 50:1, diazomethane:porphyrin was required for successful conversion.  29  In  Results and Discussion  addition, because the reaction is sluggish and must be left for 12 hours, a method of containing the gaseous diazomethane in the reaction mixture was required. On several occasions, built-up pressure resulted in septa that were either blown off the reaction flask, or torn open. An alternate approach was found in sealing a balloon over the reaction flask, thus containing the reactant, but allowing for expansion.  Once these problems were overcome and successful conversion was achieved, the task of isolation and characterization began. The three products were observable as red bands by T L C (5% T H F in CH2CI2 eluent). material.  All three bands were more polar than the starting  The slowest moving band was established as the di-adduct, not only for its  increased polarity (Rf u = 0.09), but also because the other two bands disappeared on completion of the reaction. The mono-adduct structural isomers differed only slightly by chromatography (Rfn = 0.43 and Rf 10 = 0.33), rendering separation extremely difficult. The aforementioned R values would probably not present much of a separation challenge f  in small molecule chemistry. High molecular weight porphyrins however, are notorious for tailing and unexpected diffusion behaviour. Large scale separation was only achieved using the Chromatotron®.  Identifying the product pyrazolines was challenging for several reasons.  To our  knowledge, this presents the first example of pyrazoline-modified protoporphyrins. There is precedence for the regioselectivity of diazomethane reactions with many familiar substrates, but none could be found involving a conjugated vinyl group of a porphyrin.  30  Results and Discussion  Before embarking on the task of deciphering extensive NMR spectra, some thought was given to the reaction mechanism.  The pyrazolines are formed as a result of a concerted [47U+2TX] cycloaddition.  The 1,3  70  dipolar molecule is diazomethane, and the vinyl group is the dipolarophile.  Most  examples in the literature cite diazomethane reactions with electron-poor species , or 68  with alkenes conjugated to strong electron-withdrawing substituents.  It was thus quite  surprising to find that these vinyl groups, conjugated to the electron-rich porphyrin system,  reacted to form the products.  The stereochemistry  of the  1,3-dipolar  cycloaddition reaction is known to be stereospecific syn addition , but in the present case 71  of addition to a vinyl group, need not be addressed.  While the products in Scheme N  2.1 show only one arrangement  QN:  II  OR  II II  ©ftO rCH  N  Ar \\. .  NI-^ ONO  J 3 C Hl 0  (  2  N  ( E J ^  H  ^  2  of the pyrazolines, there were  Vs.  two to consider. When both the  ..^~~^OCH  [  2  \\  © N ^ OR  N  CH,  ©Nj  -  — ^  I  N  (U)  1,3 dipole and the dipolarophile are unsymmetrical, as they are in  F  i  g  u  r  e  2  .  2  1,3-dipolar cycloaddition, where 'S' =  symmetrical conformation and 'IT = unsymmetrical the  formation  of  the  protoporphyrin pyrazolines, there are two possible orientations for addition (Figure 2.2). Both steric and electronic factors play a role in determining the regioselectivity of the addition, and can be predicted considering orbital coefficients and by calculation or estimation of the relative energies of the interacting orbitals.  72  The most favorable  Results and Discussion  1 .8 LUMO  1 .0  -9.0  -9.1  H O M O  CH N =N 2  R = c o n j u g a t e d g r o u p with m o d e s t e l e c t r o n - r e l e a s i n g c a p a c i t y  Figure 2.3 Estimated energy of frontier n orbitals for conjugated vinyl group and diazomethane (Relative energies are given in eV) orientation is that which involves complimentary interaction (smallest energy gap) between the frontier orbitals of the dipole and dipolarophile. Figure 2.3 depicts the estimated energy of the frontier TZ orbitals, examined by experimental means in the early work of K. H . Houk,  72  for the interacting molecules.  According to these relative  energies, we are lead to conclude that the L U M O of the conjugated alkene is more likely to react with the H O M O of the diazomethane.  The last piece of the puzzle to answer the question of regioselectivity involves the orbital coefficients of the reactants. Diazomethane has several contributing resonance structures, the two most prominent ones shown  HOMO  LUMO  reacting in Figure 2.2, but which of the orbitals will react with which is not  0 78 ' f") 0.13p,0.61  0  7  4  f~) f%0-50  obvious. The H O M O and L U M O % MOs of the most common 1,3-dipoles were calculated by Houk et. al. , and those of  Figure 2.4 Orbital coefficients for diazomethane  32  Results and Discussion  diazomethane are shown in Figure 2.4. If the vinyl group of the porphyrin is likened to one on styrene, the literature cites the larger orbital coefficient to be on the terminal 72  carbon of the double bond. Thus the atom with the larger terminal coefficient, in this case the carbon atom of the H O M O , will become bonded preferentially in the transition state to the terminal carbon of the vinyl group of the porphyrin to yield the unsymmetrical pyrazoline ('U', in Figure 2.2).  Of course the best way to reinforce this conclusion based on theoretical calculations, is to prove it experimentally.  The characterization of each of the isolated protoporphyrin  pyrazolines was a challenging and rewarding process. pyrazolines was  questioned,  Initially, the stability of the  because *H N M R spectra could only be obtained  immediately upon preparation of the sample. It has since been determined that the small amount of acid present in CDC1 (and reagent grade C H 2 O 2 ) is enough to cause the 3  degradation of these porphyrins, which are otherwise stable at room temperature.  The first series of experiments will address the structural assignment of the pyrazoline (ie; 'S' vs. ' U ' , Figure 2.2). To simplify matters, the spectroscopic investigation of only one of the mono-adducts will be presented. Following the assignment of the pyrazoline, the A and B-ring adducts will be differentiated by spectroscopic means. characterization of the di-adduct will be discussed briefly.  33  Finally, the  Results and Discussion  Results and Discussion  The spectra obtained, albeit first order, are complex because of the number of signals, and particularly due  to  the  heterocycle.  long-range  coupling  within  the  A spectrum of 10 (Scheme 2.2), and  expansions of the pyrazoline heterocycle signals are shown in Figure 2.5. There is ample precedence for the assignment of those signals that are not labeled,  Scheme 2.2 Numbered B-ring pyrazoline  thus only those particular to the novel pyrazoline moeity will be discussed here. The distinctive multiplet at 6.80 ppm that integrates for a single proton can be assigned to the 'ipso' position (HL-8 ) of the pyrazoline. The coupling pattern was difficult to decipher 1  and was initially thought to be a triplet of triplets, indicating a symmetrical pyrazoline ('S', in Figure 2.2).  This being the case, the splitting patterns and shifts of the four  methylene proton signals were even more puzzling, and hindered rotation of the azo moiety was considered.  The orientation of the pyrazoline was further investigated using H M Q C (Heteronuclear Multiple Quantum Coherence) spectra, to determine the assignment of the pairs of methylene signals to their respective carbon atoms. Thus, if one of the multiplets around 5 ppm and another around 2 ppm correlated to a single carbon, the difference in shift within a pair of methylene signals could be attributed to some shielding effect, resultant from hindered rotation. The results however, explicitly countered this theory and point to the unsymmetrical arrangement of the pyrazoline ('U', in Figure 2.2).  The methylene  protons that are deshielded and downfield (H -8 : 5.45 ppm and Hp-8 : 4.74 ppm) 3  0  35  3  Results and Discussion  Results and Discussion  correlate to a single carbon atom, presumably because they are adjacent to the nitrogen. The pair of signals further upfield (H -8 : 2.74 ppm and H -8 : 2.11 ppm) similarly 2  2  M  N  correlate to their own carbon signal (Figure 2.6). HMQC C Correlations (125.8 Mhz) I J  'H NMR (500 Mhz) 5 ppm (mult., J (Hz) )  Assignment H-x H-10 H-15 H-20 H-5 H-3 H -8' H-3 H-3 H -8 Hp-8 H-13 H-17' H-13 H-17 H-7 H-2 H-12 H-18 H-13 H-17 HM-8 1  L  2  2  9.95 9.79 9.69 9.57  (s, (s, (s, (s,  1H) 1H) 1H) 1H)  8.05 (m, 1H, J  TRANS  = 17.8, J =  11.5)  CIS  6.81 (m, 1H, J _M, L-N= 9.8, J . , L-P= 2.6) L  6.23 (m, 1H, J  L  TRANS  = 17.8, J  6.10 (m, 1H, J = 11.5, J CIS  GEM  GEM  G  =  =  1.2) 1.2)  3  5.46 (m, 1H, J -P= 17.9, J - = 9.8, J -N.O-L= 2.6)  3  4.75 4.28 4.24 3.66 3.65 3.49 3.46 3.43 3.42 3.21 3.18 2.72 2.10  0  1  3  3  1  1  1  1  2  2  HN-8  a  0  0  M  0  (m, 1H, J _o= 17.9, J _ . . = 9.3, J . =2.8) (t, 2H, J = 7.8) (t, 2H, J = 7.8) (s, 3H) (s, 3H) (s, 3H) (s, 3H) (s, 3H) (s, 3H) (t, 2H, J = 7.8) (t, 2H, J = 7.8) (m, 1H, J - =13.0) (m, 1H, J - =13.0) P  P  M  N  M  N  M  P  N  P L  97.62 96.86 96.66 95.89 129.99 85.89 120.57 120.57 77.81 77.81 21.65 or 21.62 21.65 or 21.62 51.69 or 51.67 51.69 or 51.67 11.42 12.41 11.83 11.51 36.79 (2xC) 36.79 (2xC) 27.72 27.72  MH  -4.20 (s, 2H) a- Only those J values that could be unambiguously assigned are recorded Table 2.1 H M Q C correlations within 10.  By pairing the proton and carbon signals of 10, each of the primary, secondary and tertiary carbon atoms were assigned (Table 2.1). This exercise would prove invaluable to further characterization.  The multitude of overlapping quaternary signals were not  assigned.  37  Results and Discussion  In order to understand the complex and long-range coupling within the pyrazoline, C O S Y (1H, 1H Correlation Spectroscopy) spectra were acquired. Figure 2.7 is an expansion of the most informative region of the spectrum.  It is clear that protons 'O' and 'P' are  strongly coupled, which reinforces the H M Q C data indicating their attachment to the same carbon. The same holds true for ' M ' and 'N'. Also noteworthy is the strong correlation between protons ' M \ 'N' and ' L ' . This supports the earlier conclusion that the remaining methylene signals ('O' and 'P') are downfield as a result of their proximity to the electron withdrawing nitrogen atom. The obvious question that follows is: 'Why then, is proton ' L ' so far downfield?'.  Proton ' L ' finds itself directly outside the ring  current of the porphyrin. Like the  protons, it is deshielded by the current and gets  meso  shifted to the aromatic region of the spectrum.  With a clear picture of the coupling partners, the J values could thus be deduced, and the spatial orientation of the protons assigned (Table 2.2). would later be confirmed by N O E spectroscopy).  (These preliminary assignments The Karplus relationship and a  molecular model kit provided a way to estimate the stereochemistry along the methylenemethylene bond of the pyrazoline.  JO-P  17.9 Hz  JM-N  13.0 Hz  JOM  9.8 Hz  Jp_M, Jp-N  9.3 Hz  Jo-N  2.6 Hz  Table 2.2. Pyrazoline methylene coupling constants  38  Results and Discussion  39  Results and Discussion  With the knowledge acquired from the COSY, an explanation for the long range (4bond) coupling remained.  Thinking the pyrazoline might be puckered, one might  propose a 'W' arrangement of these protons to explain the large J values of 2.6 Hz (L-O, transoid) and 2.8 Hz (L-P, cisoid) respectively.  Brief experimentation with molecular  modeling (MM) negated this possibility. As it were, there is abundant precedence in the literature for the coupling phenomena occurring within the pyrazoline, entitled homoallylic coupling.  74  Nuclear spin-spin coupling constants over four single bonds and  one double bond are said to be of the homoallylic  type between protons H and HB in A  the system H A — C ] — C 2 ^ = C 3 — C 4 — H B with carbon atoms C] and C4 having tetrahedral hybridization. Many examples  76  can be found in the literature for which C2 and/or C 3  have been replaced by sp -hybridized nitrogen atoms. Furthermore, it has been shown 2  77 that in cyclic systems  such as the 5-membered pyrazoline ring, the interaction of the  protons in question is enhanced because of a double homoallylic path ; namely, across 78  the methylene bridge and across the azo bond. Thus the mislabeled 'triplet of triplets' at 6.81 ppm (Figure 2.5), is actually a doublet of doublet of doublet of doublets, and reaffirm the asymmetric orientation of the pyrazoline.  The consistency among the spectra of each of the mono-adduct pyrazolines required that this extensive spectroscopy to be carried out on one of the structural isomers only. Outside of  small  indistinguishable.  shift  differences,  the  spectra of  the  respective  isomers  are  An additional task however, presented itself in assigning the  respective 'A' and 'B' ring adducts. NOESY (Nuclear Overhauser Effect Spectroscopy) correlations were used in these assignments, and were carried out on each of the isomers  40  Results and Discussion  to positively identify them.  The exercise was fundamental, but interesting enough to  briefly discuss the strategy involved.  A-ring  B-ring  N O E S Y Correlations" (500 MHz)  H-x H-10 H-15 H-20 H-5 H-8  Med. H-8 , str. H-8 , str. H-12 Str. H-13 , str. H-17 Str. H-18 , str. H-2 Str. HL-3 , str. H-7 Str. H-10, str. H-8 Str. H-2 , str. H-5, str. H - 3 , med. Hp-3, w. H - 3 w. H-7 , med. H-10, str. H-8 Str. Hp-3 , str. H - 3 , med. H - 3 Str. H - 3 , str. H - 3 , med. H -3' Med. H-12 , str. H-13 , str. H-15 Str. H-15, str. H-17 , med. H-18 Str. H-5, w. H-8 Str. HL-3', str. H-20 2  1  1  1  1  1  1  1  2  1  HL-3  H-5 H-20 H-15 H-10 H-3'  1  1  1  1  1  Ho-3 Hp-3 H-13 H-17 H-7' H-2 H-12 H-18 H-13 H-17  3  M  1  3  2  3  1  3  M  3  0  2  0  1  H-3 H -8 Hp-8 H-13' H-17 H-12 H-18 H-7 H-2 H-13 H-17 2  2  N  M  3  L  1  2  2  1  1  2  1  1  1  2  2  1  1  Str. H-10.med.H-13 med. H-17 , med H-17 , str. H-20 str. H-13 str. H-17  1  1  1  2  1  1  2  1  2  str. H - 3 , str. H -3', str. H -3 , med. Ho-3 str. H - 3 , str. H - 3 , w. H -3' HN-3 b- str. = strong, med. = medium, w. = weak 2  HM-3  2  3  N  L  P  3  2  2  3  M  0  2  N  1  3  1  2  HL-8  2  2  1  1  N  H-8  1  1  2  M  1  N O E S Y Correlations (500 MHz) Str. H-7 , w. H-3 Str. H-18 , str. H-2 Str. H-17', str. H-13 Str. H L - 8 , str. H-12 w. H-5 Str. H-7', str. H-10, str. H - 8 , w. Hp-8 , med. H - 8 Str. H-2' b  H-x  L  Str. Hp-8 Str. H - 8 , med. H - 8 , w. H - 8 ' Med. H-12', str. H-13 , str. H-15 Str. H-15, str. H-17 , med. H-18 Str. H-10, med. H-13' Str. H-20, med. H-17' Str. H-5, str. H -8' Str.H-3 , str. H-20 Str. H-13' Str. H-17' 3  3  2  0  M  L  2  2  1  L  2  Str. H - 8 , str. H - 8 \ med. H - 8 2  HM-8  2  HN-8  2  N  L  3  P  str. H - 8 , med. H -8' 2  M  L  Table 2.3 N O E S Y correlations for the A and B-ring pyrazolines of PP-DME  In the case of the B-ring isomer for example (Figure 28 . -NOESY), signals from the vinyl group, pyrazoline, and proprionate side chains are readily identified. The meso protons (H-5, H-10, H-15, H-20) resonate downfield, between 9.57 and 9.95 ppm. The methyl groups  (CH3-7, CH3-I2, CH3-I8, CH3-2), 1  1  1  1  occur as four sharp singlets between 3.42  and 3.49 ppm. The meso proton H-20 is the only one in contact (through space) with two  41  Results and Discussion  Results and Discussion  porphyrin methyl groups (CH -2' and CH3-I8 ) and is assigned at 9.69 ppm. The C H 1  3  3  18 is distinguished from the C H - 2 ' by its N O E to the methylene groups of one of the 1  3  proprionate side chains (CH.2-17 ). 1  From the H-20 meso proton, the following  connectivities are therefore traced around the ring: CH3-I8 <— H-20 meso —> CH3-2 —> 1  vinyl -3  1  1  -> H-5 meso - » CH -7' - » pyrazoline-8 - » H-10 meso -> CH -12' - » 1  3  proprionate-13 . 1  3  The H-15 meso proton, between the two proprionate side chains,  resonates at 9.79 ppm and exhibits the expected N O E correlations to each.  Thus the two structural isomers, indistinguishable by mass spectrometry, UV-vis,  l 3  C  NMR, and ' H N M R spectroscopies, and barely separable by chromatography were assigned their respective conformations using 2D N M R spectroscopy . These findings can be related back to the initial chromatographic results; the faster moving regioisomer (Rf = 0.43) is 11, and the slower moving one (Rf = 0.33) is 10. This conclusion is supported by the crystal structure of the photoproduct 11, which is discussed in the next section.  The di-adduct pyrazoline of protoporphyrin-DME (12) did not present the isolation challenges of the A and B-ring adducts. It was much more polar than either of the monoadducts, thus distinguishable by T L C , as well as by MS and UV-vis spectroscopy. The extensive 2 D N M R experiments to assign the regiochemistry of the pyrazoline rings were carried out on the single adducts, and could be extrapolated to similarly assign those of the di-adduct pyrazolines. There are however, some interesting features of the di-adduct that should be noted. Figure 2.9 displays the proton NMR spectrum of the starting  43  Results and Discussion  Results and Discussion  material, PP-DME, and 12. On first observation, one of the most startling features of the di-adduct spectrum was the presence of two central amine peaks at -3.75 ppm (unfortunately, only a large expansion would illustrate this effectively).  Although it is  known that there are two central amine protons, normally their rapid exchange on the NMR time scale does not allow them to be resolved separately.  The peaks were  indicative of something far more fundamental; the presence of more than one porphyrin in solution. On reflection, this makes perfect sense because in the case of the di-adduct, two stereocenters are created at each of the pyrazolines, and the result is a pair of diastereomers. The presence of these diastereomers would have made the assignment of the pyrazoline signals far more difficult, because of the signal overlap in each of the multiplets (H : 6.91, H : 5.55 and 5.50, H : 4.82 and 4.78, H : 2.84, H : 2.24 and 2.22). L  0  P  M  N  Some J values could be assigned unambiguously, but only thanks to the fact that they had been determined previously in the mono-adduct pyrazolines.  Unique to the di-adduct, in comparing it with the starting material PP-DME (9), is the complete disappearance of the vinyl signals (a-vinyl: 8.2 ppm, (3-vinyl:6.32 ppm). The last observation made of the di-adduct proton NMR spectrum is perhaps more subtle. The meso protons, in the aromatic region of the spectrum (around 10 ppm) have a different pattern in the di-adduct.  This could be a result of two factors.  First, the  presence of diastereomers creates new signals which may not overlap exactly. It is more likely however, that the change in symmetry brought about in the A and B rings of the porphyrin alters the shift of the meso protons.  45  Results and Discussion  In summary, Scheme 2.1 accurately depicts the products formed in the reaction of diazomethane and protoporhyrin dimethylester. The spectroscopic (and crystallographic) evidence supports the assumptions made in adopting the theoretical calculations from the literature. The mystery of the overnight diazomethane reaction is solved, and three novel pyrazoline porphyrin derivatives have been identified.  46  Results and Discussion  2.2  Thermal and Light Activated Chemistry of Protoporphyrin Pyrazolines  It is well known that cyclic azo compounds including heteroaromatics with a N=N bond of strong double-bond character, such as pyrazolines, can eliminate molecular nitrogen on addition of sufficient thermal energy or on electronic excitation.  79  Some substrates  decompose very quickly, while others are moderately or even extremely resistant to N 2  elimination.  In fact, the range of activation energies for the pyrrolytic extrusion of  molecular nitrogen covers values as low as 15-20 kcal/mol to as high as 40-45 kcal/mol . 60  The observed rates of decomposition and temperatures vary dramatically. For example, 2,3-diazabicyclo[2.2.2]octa-2,5-diene phase pyrolysis of benzotriazole (14)  (13) 81  80  decomposes at -78 °C, whereas the gas-  (Scheme 2.3) occurs at 1100 °C. In order for the  porphyrin pyrazolines described in this work to be applied as crosslinking agents in PDT, they would have to be stable at physiological temperatures. As a result, both the thermal and photochemical activation of the protoporphyrin pyrazolines will be examined in this chapter.  Scheme 2.3 Range in temperatures required for thermolysis in cyclic azo compounds  47  Results and Discussion  2.2.1 Thermal Activation  It was anticipated that the protoporphyrin pyrazolines in this work would also undergo a thermal cycloelimination reaction through the extrusion of nitrogen. The course of the cycloelimination of nitrogen on addition of thermal energy is shown in Figure 2.10 (adapted from literature ). In the rate determining step, substrate (A) cleaves a single bond of the azo function either homolytically or heterolytically, giving rise to a generally energy-rich diradical (C) or zwitterionic species (only the former will be illustrated here for clarity), (A—>B—>C). Nitrogen is eliminated in a second activated step, and the primary fragment, namely a diradical (E), is formed (C—>D—>E). This second diradical intermediate subsequently forms the cyclopropane (F) by ring closure (E—>F).  Reaction coordinate  •  Figure 2.10 Course of thermal cycloelimination of nitrogen  48  Results and Discussion  Each of the product pyrazolines was investigated for its thermal stability towards cycloelimination by looking for decomposition products at a variety of temperatures in various refluxing solvents. The fear that these pyrazolines were not stable at or near room temperature was very real, and was intensified when it seemed as though they were breaking-down before NMR spectra could be obtained (Section 2.1). It was reassuring to find that the pyrazolines could sustain relatively high temperatures for extended periods of time with little or no decomposition. In fact, even in refluxing toluene they undergo conversion to their thermal products relatively slowly (Table 2.4).  In addition, it was  encouraging to observe that both the A and B-ring pyrazoline adducts reacted cleanly and gave a single product each, which were presumed to be their respective cyclopropanes. The thermal products were visible by T L C as faster moving pink spots (Rf 15 = 0.55, and R f i 6 = 0.53, 10% EtAce in CH2CI2 eluent) above their respective pyrazolines (Rf 10= 0.18 and R 11 = 0.21). f  Pyrazoline  Time required for complete thermolysis (reflux in toluene) to cyclopropanes  B-ring Isomer (10) A-ring Isomer (11) Di-Adduct (12)  23 h 21 h 47 h  Table 2.4 Thermolysis of pyrazolines  The di-adduct (12), on the other hand, formed three products in the course of the reaction. For the di-adduct it was thought that the three pink bands visible by T L C (Figure 2.11) represented the two possible products formed by cycloelimination from the pyrazoline moieties on the A and B ring, and a third product from cycloelimination from both (17).  49  Results and Discussion  Thus the two bands directly above the diadduct pyrazoline represented the products whereby one ring-pyrazoline had reacted while the other remained intact, and the fastest  moving  band  represented  the  porphyrin where both pyrazolines reacted to give  the  di-cyclopropane  product  (17).  Disappearance of the two middle spots in the T L C as the reaction proceeded helped to verify this suspicion. Figure 2.11 Schematic of T L C plate: Thermolysis of di-adduct pyrazoline (8% EtAce/CH Cl eluent) 2  The intermediates  were not isolated.  2  The thermal reactions described above were performed on relatively small scales, and the products were not fully characterized. The identities of the thermal products (15, 16, 17) were confirmed by MS, and by comparing the Rf values and UV-vis spectra of the products in question to their photochemical counterparts, which were characterized extensively and are discussed next. In summary, these simple thermal studies verified that the pyrazolines are stable to relatively high temperatures and have an extremely long half-life at physiological temperature, which made them suitable for the photochemical studies described next.  50  Results and Discussion  2.2.2  The  Photochemical Activation  protoporphyrin pyrazolines were then investigated  for their susceptibility to  eliminate nitrogen on electronic excitation, in accordance with the thrust of the project, to create a reactive intermediate that will crosslink cellular components. The spectral characteristics  82  of the cw-azoalkanes  (pyrazolines) reveal that the photochemical  excitation process is n-7t* derived. The n-it* absorption of a pyrazoline lies in the 300400 nm range. Although the n-7t* transition is much weaker (ca. 100-fold) than the K-K* transition (e = 10 -10 ), the lower energy light of the former is convenient for the 4  5  photoextrusion of nitrogen from azoalkanes. "  Scheme 2.4 Photochemistry of the pyrazoline functionality  Irradiation of degassed, dilute solutions [3x10 M] of the protoporphyrin pyrazolines 10, _  11 and 12 in benzene with 350 nm light produced a single photoproduct in each case. The  photochemistry is thought to proceed initially through a diazenyl biradical  intermediate (11a), followed by extrusion of nitrogen, yielding a biradical (lib), which 84  reacts intramolecularly to form the cyclopropane (16). Scheme 2.4 illustrates the proposed reaction scheme for the A-ring pyrazoline (11).  51  Analogous reactions would  Results and Discussion  lead to the formation of the respective cyclopropanes (15, 17) of the other mono-adduct (10) and the di-pyrazoline (12) as well.  350  400  450  500  550  600  650  700  W a v e l e n g t h (nm) Figure 2.12 UV-vis spectra of A-ring pyrazoline of PP-DME and its photoproduct  Unfortunately, the absorption spectra of the photo-products did not differ markedly from that of the pyrazolines, and the reaction could not be monitored conveniently by UV-vis spectroscopy (Figure 2.12). The photoproducts were visible by T L C , and appeared as a faster moving pink bands above the pyrazoline (identical R values as the thermal f  products, Section 2.2.1). Additional characterizations of the product cyclopropanes (15, 16, 17) were carried out by mass spectrometry, elemental analysis and ' H N M R spectroscopy, and are outlined in detail in the experimental section.  Figure 2.13  compares the proton N M R spectra of the pyrazoline (11), and its photoproduct (16) to illustrate the changes that are observed. What is readily apparent is the disappearance of the pyrazoline signals, and the appearance of distinctive signals in the upfield region of  52  Results and Discussion  7.0  6.5  6.0  5.5  5.0  4.5  4.0  3.5  3.0  2.5  2.0  1.5ppm  Figure 2.13 Changes in H N M R spectra of pyrazoline (11) and photoproduct (16) [  the spectrum (H-3 : 3.06 ppm, H-3 : 1.66 and 1.45 ppm) that can be attributed to the 1  2  cyclopropane. Figure 2.14 shows expansions of these cyclopropane signals. Regrettably, the signal at 1.45 ppm is partially obscured by residual water in the sample. Normally, one would expect to find these signals even higher upfield (between 0 and 0.5 ppm) , but 85  what cannot be dismissed is the large deshielding effect of the porphyrin ring. Similar features were found for the cyclopropane photoproduct 15.  A notable spectroscopic distinction between the di-adduct pyrazoline (12) and its photoproduct di-cyclopropane (17) is the absence of diastereomers  in the latter.  Formation of the cyclopropanes removes the stereocenters in the pyrazolines and the  53  Results and Discussion  Results and Discussion  result is a single product visible in the NMR spectrum (complete signal assignment can be found in the experimental section of this work).  With all these pieces of the puzzle in place, there was not much doubt with respect to the identity of the photoproducts. Throughout the course of the characterization however, an attempt was made to grow single crystals of each of the products. Single crystals of freebase porphyrins, as it happens, are very difficult to grow and present a diffraction challenge to the crystallographer. Their planar nature yields very thin crystals and they tend to stack and twin. The twinning in particular makes it difficult to isolate single crystals, thus it was a fortuitous event that the structure of the photoproduct of the A-ring pyrazoline was obtained (Figure 2.15). As mentioned in Section 2.1, this X-ray structure also helped to confirm the N O E assignment  of the A versus B-ring adducts.  Unfortunately, suitable crystals for X-ray analysis of the other cyclopropane products could not be isolated.  In Section 1.2.1, the phenomena of long wavelength light penetration through tissue was explained. Out of curiousity, and as a result of the findings detailed in the next section of this work, the possibility of activating the protoporphyrin pyrazolines with long wavelength light was investigated.  With the prospect that these compounds may  someday play a role as PDT photosensitizers, red light activation would be very beneficial. The first challenge in this experiment was finding a suitable filter to allow for selective long wavelength irradiation and restrict short wavelength irradiation.  The  protoporphyrin family of tetrapyrroles have but a weak absorption at long wavelengths,  55  Results and Discussion  Figure 2.15 X-ray crystal structure of 16  56  Results and Discussion  thus the window for activation was not very big; a suitable filter was found and its transmission spectrum is shown in Figure 2.16.  The  results  of  the  long 70  wavelength activation Each  of  photochemical were the  A  encouraging. and  B-ring  pyrazolines (10, 11), as well as  Corion 600  60  0 o  1 I  to  \  50  40 30 20 10 0  the  di-adduct pyrazoline  formed  their  (12)  respective  10  11  350  I  400  1! 11  I ' I 1  450  11  500  1111  I  550  1111  i  1  l_rn  600  I  1111  650  I ' ! 11  700  1  750  Wavelength (nm)  cyclopropanes (15, 16, 17) on  Figure 2.16 Transmittance of red light filter  irradiation with filtered long-wavelength red light. Degassed dilute benzene solutions of the pyrazolines with similar concentrations to those irradiated with 350 nm light were exposed to the red light, and complete conversion to corresponding cyclopropane(s) (identical to those obtained with 350 nm irradiation) was achieved in approximately 48 hours.  The fact that this low energy light was sufficient to bring about the extrusion of molecular nitrogen was surprising since the pyrazoline chromophore is known to absorb between 300-350 nm. Thus, the efficient nitrogen extrusion to form the cyclopropanes using long wavelength irradiation is not expected and these results raise many interesting photophysical questions.  These, in addition to a series of experiments exploring the  crosslinking abilities of this series of pyrazolines will be discussed in Section 2.4.  57  Results and Discussion  2.3  Synthesis and Characterization of Methylpyropheophorbide Pyrazoline  As mentioned in Section 1.2.1, the efficacy of a porphyrin-based PDT drug depends on its ability to absorb light in the red or near infrared region of the absorption spectrum. The nature of the optical spectrum is determined by the level of saturation of the tetrapyrrolic macrocycle.  Consequently, research towards new photosensitizers  (so-  called second-generation photosensitizers) in recent years has focused on chlorins and bacteriochlorins.  IQ  Methylpyropheophorbide  (MePPP, 18)  and its derivatives  meet many of the important criteria for a suitable PDT agent. MePPP is an effective producer of singlet oxygen.  A chlorin,  this chromophore has an appreciable absorption in the visible region at wavelengths greater than 630 nm (Figure 2.17). Derived from a natural product, the drug consists of a single isomer.  OCH  3  Several derivatives have also demonstrated an ability to localize in desired  tissue, and clear from the body in a relatively short time post-irradiation.  88  In addition to  these chemical, photochemical and biological features, practical considerations such as ease, yield and cost of production (it comes from spinach!) also contribute to its strengths.  These attributes, and the presence of a single vinyl substituent on the A-ring of the macrocycle prompted the present investigation of a cycloaddition reaction similar to that  58  Results and Discussion  performed on PP-DME described in the previous section.  Initially, it was feared that  multiple products might result from the reaction of diazomethane and 18. The reaction of diazomethane with ketones is known and has been studied for some time.  68  In the case of  cyclic ketones, such as the one in the exocyclic ring fused with the C-ring of 18, the dominant reaction pathway is expected to be ring expansion. This can be complicated by the conflicting migratory aptitude of the involved carbon atoms , and reaction of the 89  product with excess diazomethane to produce undesired higher homologues.  90  Fortunately, the reaction of diazomethane and 18 formed a single product.  On one  occasion, when the reaction was left for an extended period of time, a second product was visible by T L C (R = 0.2 vs. R 19 = 0.44 and R =0.67, 10% EtAce/Ch Cl eluent), but f  f  r 18  2  2  not enough was present for isolation or characterization. From the beginning, the main product of the reaction was expected to be the pyrazoline of MePPP (19, Scheme 2.5) based on the experience with diazomethane and PP-DME. The increased solubility of the starting material in C F L C L (compared with PP-DME) and the experience gained from the previous reactions facilitated this procedure greatly. A  19 Scheme 2.5 Numbered pyrazoline of MePPP  large excess of diazomethane: porphyrin (50:1) was still required, but the fact that only one product was formed and  that the reaction could go to completion, simplified the isolation. purified by column chromatography to give a final yield of 78% yield.  59  The product was  Results and Discussion  350  400  450  500  550  600  650  700  750  W a v e l e n g t h (nm) Figure 2.17 UV-vis spectra of 18 and 19 in CH2CI2. The spectrum of 19 is scaled by a factor of three for ease of comparison.  As mentioned on several occasions,  the absorption characteristics of the chlorin  distinguish it from other types of tetrapyrrolic macrocycles. The first indication that 19 had in fact been formed was given by the UV-vis spectrum of the product (Figure 2.17). The expected cycloaddition of diazomethane  would bring about a reduction in  conjugation. As a result, we might expect to see a hypsochromic (higher energy) shift in spectrum of the product pyrazoline. It is not an extremely large shift, but the difference in the first Q-band is noteworthy; Q  {  (MePPP, 18): 669 nm vs. Qj (19): 663 nm. The  spectra are very similar, indicating the overall electronics of the chlorin have not been altered drastically by the pyrazoline functionality. Slight variations in the Q-bands can be quite informative nonetheless. Overall, it was a relief to find that the intense Q-band of 19 had not shifted to much lower wavelengths, as it is hoped that this chlorin will be photoactive at long wavelengths.  60  Results and Discussion  Characterization of 19 was also facilitated by the extensive N M R spectroscopy experiments carried out previously on the pyrazolines of PP-DME.  Figure 2.18 (p.66)  displays the NMR spectrum of 19, and the familiar pyrazoline signals are present: H -3': L  6.67 ppm, H - 3 : 5.47 ppm, H -3 : 4.76 ppm, H - 3 : 2.75 ppm and H - 3 : 2.11 ppm. 3  3  0  Assignment H-x H-10 H-20 H-5 H -3' L  H -3 H-15 H-15 Hp-3  3  0  1  1  3  2  P  9.51 8.96 8.57 6.67 5.47 5.26 5.11  (s, 1H) (s, 1H) (s, 1H) (m, 1H, J . , L-N= 9.7, J (m, 1H, J -P= 17.6, J . (d, 1H, J = 19.6) (d, 1H, J = 19.6) L  M  0  0  1H, J . = 18.2,  J .  (m,  1H, J  J  4.29  (m,  1H, Jdoub = 8.1,  3.68 3.65 3.59 3.25 3.17  (t, 2H, J = 7.4) (s, 3H) (s, 3H) (s, 3H) (s, 3H)  HM-3 H-17 H-8 H-8 H-17 HN-3  2.75 2.68 2.55 2.28 2.28  (m, (m, (m, (m, (m,  1  1  1  1  1  P  d  0  o  u  b  = 7.4,  P  L  M  L-P= 2.2) 10.1, J -N,O-L= 2.5)  . . 0  =  M  (m,  1  1 3  a  4.48  5  HMQC C Correlations (125.8 MHz)  8 ppm (mult., J (Hz) )  4.76  1  N  H NMR (500 MHz)  !  H-18 H-17 H-17 H-17 H-7 H-2 H-12  2  2  M  0  48.01 , . =  q u a r t  P  9.1,  N  =  J d b = 2.2, OU  J - = P  L  3.1)  7.1) Jd b= 0 U  2.2)  78.01 51.01 51.75 19.38 51.61 11.94 11.45  1H, J -N=19.7, J -p= 7.6, J -o, M-L= 2.4) 1H) 1H) 1H) 1H) M  104.08 96.69 93.01 85.32 78.01  M  M  11.11 27.32 29.85 30.93 29.85 27.32  2.11 (m, 1H, J -M=19.6, J -L,N-O=9.7 H-18 1.80 (d, 3H, J = 7.4) 23.10 H-8 1.67 (t, 3 H , J = 7.7) 17.33 NR -1.77 (s, 2H) a- Only those J values that could be unambiguously assigned are recorded 2  N  N  1  2  Table 2.5 H M Q C correlations for 19.  61  Results and Discussion  The consistency within the signals of the pyrazoline functionality for PP-DME and MePPP are quite remarkable in fact.  Comparing the pyrazoline signals of the mono-  adduct 11 with 19 for example, the difference in shift varies only ±0.14 ppm in the most extreme case, and the coupling constants differ by less than 0.4 Hz in all cases. The complications that arose in characterizing 19 were a result of two factors. In forming the pyrazoline, a new stereocenter is created at C ' (Scheme 2.5). 3  Relative to the fixed  natural stereocenters at Q 7 and Ci8, a diastereomeric mixture results. Expansion of the NR signal at -1.77 ppm reveals a broad peak (broader than that which results from the rapidly exchanging amine protons), indicative of two species in solution. Other features such as fine splitting in the meso protons (H-5: 8.57 ppm, H-10: 9.51 ppm, and H-20: 8.96 ppm) also point to the presence of diastereomers.  The other difficulty in completing the assignment of signals of 19 was a result of the large number of signals found in the region of the H and H N  M  pyrazoline signals (2.11 -  2.75 ppm). Also found in this region, and overlapping with the pyrazoline signals, are the methylene signals from the ethane moiety (H-8 ) and from the methyl propyl ester 1  (H-17 ). H M Q C spectroscopy was used to resolve the signals in question, by assigning 1  each of the proton signals to their respective carbon signals (Table 2.5). Thus the pairs of proton signals were resolved, J values were extracted, and each of the 38 protons signals of 19 was assigned.  It was reassuring to observe the proof of concept in forming the resultant pyrazoline of MePPP. The reaction of diazomethane with the vinyl group of a chlorin, known for its  62  Results and Discussion  PDT potential, was successfully carried out.  Furthermore, the predictions based on  theoretical calculations in Section 2.1 were once again supported experimentally in forming the unsymmetrical pyrazoline functionality. With the characterization complete, the more interesting task of exploring the photochemistry of this novel chlorin can now be explored in the next section.  63  Results and Discussion  2.4  Photochemistry of Methylpyropheophorbide Pyrazoline  The pyrazoline of methylpyropheophorbide (19) makes for an interesting photochemical study. A chlorin, this molecule has a strong Q-band absorption at 665 nm (e = 52, 700, in benzene) whose intensity is almost half that of its Soret band (411 nm, e = 107, 100) (Figure 2.19).  As mentioned previously in Section 1.2.1, this region of the visible  spectrum is particularly useful for light penetration through tissue.  In Section 2.2.2, the photochemistry of the PP-DME pyrazolines was investigated.  In  this section, the photochemistry of 19 will be discussed. In particular, an attempt will be made to rationalize the photochemistry that takes place both from irradiation in the nearU V region (350 nm), and at long wavelengths (672 nm). To the best of our knowledge, this is the first report of photochemical activation of functionalities at the periphery a chlorin derivative.  Since chlorins are known to be efficient singlet oxygen sensitizers, irradiations were performed in Ar or  N  saturated  2  solutions.  Irradiation of a dilute [3 x 10' M], 4  oxygen purged solution of 19 in Scheme 2.6 Formation of 20 from 19  b  e  n  z  e  n  e  w  i  t  h  3  5  0  n  m  H  §  h t  produced a single photoproduct. It was expected that similar chemistry to the pyrazolines of PP-DME (Scheme 2.6) would be observed. Thus, via the proposed diazenyl biradical  64  Results and Discussion  intermediate, extrusion of nitrogen, and intramolecular closure of the resulting biradical, a cyclopropane (20) would be formed.  The photoproduct was visible by T L C as a faster moving (R = 0.61, 10% EtAce in f  CH C1 eluent), single light green spot above the pyrazoline (Rf = 0.48). This decrease in 2  2  polarity in the photoproduct was the first good indication that the cyclopropane had been formed.  N M R spectroscopy was relied upon again in the identification of 20.  Figure 2.19  compares the spectra of the pyrazoline (19) and photoproduct (20). Particularly notable is the disappearance of the pyrazoline signals (H -3', Ho-3 , H -3 , H - 3 , H -3 ). The 3  3  L  P  2  M  2  N  methylene cyclopropyl signals (H-3 : 1.57 ppm, and H-3 : 1.30 ppm) are somewhat 2  2  difficult to observe due to some residual ethyl acetate (q: 3.65, s: 2.00, t: 1.25 ppm) and water (s: 1.5 ppm) in the sample.  Their J values (1.9 and 1.6 Hz respectively) were  extracted from expansions however, and correlate with those expected for cyclopropyl protons.  The distinctive multiplet observed at 2.80 ppm, seems to couple with the  methylene cyclopropane signals at 1.57, and 1.30 ppm. (J ~ 7 Hz); although the ambiguity of the splitting made it difficult to assign a definitive value. Other differences in the spectrum, such as the shifting meso protons between 8 and 10 ppm all indicate that the macrocyle has been altered, but it is difficult to rationalize each of these subtle changes. A comprehensive assignment of each of the signals can be found in the  65  Results and Discussion  Results and Discussion  experimental section. Coupled with MS, and UV-vis spectroscopy data, the proton N M R is nonetheless very telling and confirms the formation of the cyclopropyl photoproduct  (20).  MePPP Pyrazoline MePPP Cyclopropane  350  400  450  500  550  600  650  700  750  Wavelength (nm)  Figure 2.19 UV-vis spectra of pyrazoline (19) and its photoproduct (20)  PDT agents are activated at long wavelengths.  It is obvious from the absorption  characteristics of 19, that it too absorbs low energy light.  What was not known was  whether the same photochemistry, if any, could be observed by irradiation in this low energy absorption. Thanks to a light emitting diode (LED) panel that emits at a single wavelength (672 nm) in the red region of the optical spectrum, this aspect of the photochemistry was explored.  Irradiation of an oxygen purged, dilute [3 x 10' M] solution of 19 in benzene with 672 4  nm light produced a single photoproduct. The T L C and the spectral characteristics of this  67  Results and Discussion  product were identical to that obtained on 350 nm excitation; suffice to say that isolation and all characterization led to the identification of 20 again. Fundamentally, we did not expect this chemistry to be possible at such a long wavelength activation. This is based on the fact that only 43 kcal/mol of energy are produced by 672 nm light and may not be sufficient to initiate a reaction that involves the cleavage of a C - N covalent bond in the pyrazoline.  In order to ensure that the product observed was due to photochemistry and not a thermal reaction that was taking place as a result of the heat given off by the L E D , some simple control experiments were carried out.  The temperature of the photoreaction was  monitored by placing a thermometer in the solution (inserted through the rubber septum, which was then sealed). At no time did the temperature of the solution rise above 30 °C. By way of comparison, after heating 19 in various solvents it was found that efficient conversion to 20 required over 42 hours of refluxing in toluene. In addition, concurrent with every photoreaction, a 'dark' reaction was carried out. A foiled vial containing the same solution as that which was irradiated was placed in front of the light source, and its products monitored. No products were ever observed from any of the dark solutions. The evidence herein suggests that photochemistry at both long (672 nm) and short (350 nm) wavelengths leads to efficient extrusion of nitrogen from the pyrazoline moiety to yield the cyclopropane derivative 20.  As mentioned in Section 2.2.2, from the spectral characteristics of cz's-azoalkanes  82  (pyrazolines) it is clear that the excitation process in light activation of simple systems  68  Results and Discussion  involves the n-71* state which lies in the 300-400 nm range. Since pyrazolines are known to absorb in the near U V they would not be expected to be directly excited at such low energy wavelengths. This poses an interesting mechanistic and photophysical question.  The UV-vis spectra of 19 and 20 are very much alike (Figure 2.19). With the intensity of the chlorin bands (Qi 19: 665 nm, 52,700 vs. Qi 20: 658 nm, 46,400) and the observed subtle blue shift (~7 nm) between reactants and products, the reaction progress was monitored.  By watching the spectrum change as the photoconversion proceeded, we  gained insight into the mechanism of this reaction and got an idea of the rate of conversion.  The following spectra (Figure 2.20) were taken at timed intervals (-5-10  minutes), and show the distinct disappearance of the pyrazoline, and formation of the  1.2-1  Isosbestic Point  1 9  o c  0.8  .Q 0  CO .a  <  0.6 0.4 0.20  640  1 , 1 1 j , , 1 r—, 1 1 1 1 650  660  1  111  670  !  1  680  1  1  1  1  1  r  690  1  1  I  1  700  W a v e l e n g t h (nm) Figure 2.20 UV-vis spectra of chlorin bands, monitoring photoconversion of 19 to 20  69  Results and Discussion  cyclopropyl MePPP.  The isosbestic point was identified at 662 nm. Identifying an  isosbestic point was informative, because it meant that one product (19) was being converted to another (20), without an intervening product. Slight disparity between U V vis measurements is apparent in Figure 2.20. This is the result of the fact that the L E D could not be installed inside the UV-vis chamber, so there was a temperature discrepancy between the lamp and the recording instrument which might affect peak position. While the reaction proceeded quite efficiently with long wavelength excitation, the rate of conversion decreased as the reaction proceeded and complete conversion of solutions of 19 in benzene (monitored by TLC) took several hours. This is most likely due to an inner filter effect of the product competing for light absorption as the reaction proceeds. However, it is noteworthy that this photoreaction could indeed be taken to completion cleanly, with no secondary photolysis products evident.  The emission spectra of the pyrazoline of MePPP (19) and its cyclopropane (20) were also obtained. Irradiation at both the Soret band at 350 nm and at longer wavelength of 666 669 Cyclopropane Pyrazoline  C D O c  C D 8  s  -I /  i  o  i  i  i: 600  620  640  660  680  700  Wavelength (nm)  Figure 2.21 Emission spectra of 19 and 20.  70  720  740  Results and Discussion  665 nm yielded identical fluorescence spectra (Figure 2.21). It is therefore assumed that fluorescence decay competes with reaction from the pyrazoline. The emissions appear in the region where they would be expected; each show only a minor Stake's shift (19: 658—>666, A = 8 nm , 20 665—>669, A = 4 nm respectively.) Relative quantum yields of emission of 19 and 20 were not estimated.  Extensive research of a wide variety cyclic azo compounds by Herbert Meier , has 79  contributed to a more thorough understanding of their photochemical behaviour. Figure 2.22 illustrates graphically what is known about the photochemical cycloelimination of nitrogen and reviews the proposed mechanism.  Cyclic azo compounds like the  pyrazoline can be activated by direct irradiation, usually in the longest wavelength band  s J=^= 0  Figure 2.22 Course of the photochemical cycloelimination of nitrogen. Adapted from Ref. #79  71  Results and Discussion  (350 nm), and often also by triplet-sensitization (Sens.)- It is rarely possible to reach the T| level by intersystem crossing (ISC) without a sensitizer, so most reactivity ensues from the singlet state, ^-elimination can occur directly from Si or T | , by crossing of a repulsion term (predissociation), or by internal conversion (IC) to a highly vibrationally excited electronic ground state S . V  0  According to many theoretical studies,  91  the  conversion from the biradical resultant from nitrogen extrusion to cyclopropane involves a very small energy barrier. Therefore, it is not a kinetically relevant intermediate and the rate-determining step involves the initial homolysis of the C-N bond in the pyrazoline. The question that remains is why does the long wavelength excitation of 19, which is presumably excitation of the porphyrin, lead to efficient elimination of nitrogen from the pyrazoline unit?  Of the possible explanations, intramolecular energy transfer from the porphyrin to the pyrazoline is tempting because of the porphyrins known sensitizing abilities. Thus the porphyrin moiety (M*) would absorb the incident light and transfer its excitation energy to the pyrazoline moiety (Q), affecting the photochemistry without the latter having to absorb the incident light.  M * + Q -> M + Q*  Energy transfer of this type could be ruled out if we consider the intermolecular case of an excited state chlorin reacting with pyrazoline. This is based on the fundamental requirement for energy transfer that the energy level of Q be lower than (or at the very  72  Results and Discussion  92  most equal to) that of M * .  Thus if we approximate the energy levels available to the  porphyrin, and consider that the singlet states of the n-Tt* transition of pyrazolines have excitation energies Es of ca. 70-90 kcal/mol and for the triplet states E T is ca. 50-60 kcal/mol  (illustrated in Figure 2.23), energy transfer is an extremely endothermic  process. 80 kcal/mol  A  34.4 kcal/mol  672 nm  Figure 2.23 Energy level diagram of porphyrin and pyrazoline.  Another possible mode of action for extrusion of nitrogen, in particular in vivo, might be a photoinduced electron transfer, although there is no precedence for this is reaction with pyrazolines. Again, if we consider the porphyrin and pyrazoline as separate molecules one can estimate the feasibility of an electron transfer process.  Generally, a  photochemical electron transfer occurs only when it is exergonic or <5 kcal mof endergonic.  93  1  The free energy change for a photoinduced electron transfer can be  determined from the oxidation potential of the electron donor (E°D), the reduction potential of the electron acceptor ( E ° ) and the excited state energy of the sensitizer A  73  Results and Discussion  (E ,o), using the relationship A G 0  = 23.06 kcal mol [ ( E ° - E ° ) - E , ] - C ; where C is a -1  E T  D  A  0  0  term that accounts for solvation. In order to estimate the feasibility of electron transfer using this relationship, the redox properties of the molecules must be known. Each of the starting material MePPP (18), the pyrazoline (19), and the photoproduct cyclopropane (20) were examined by electrochemical methods.  Table 2.6 lists both the reduction and oxidation potentials of the methylpyropheophorbide series, while Figure 2.24 depicts the cyclic voltammogram of the pyrazoline. The C V reveals the reversible nature of the reduction waves of 19 (18 and 20 are also reversible). The radical anions and dianions are therefore stable on the time scale of these experiments, with no chemistry occurring on addition of electrons to these molecules. If any chemical reactions were occurring from the radical anion or dianion, the CV's would be irreversible. The reduction potentials do not vary a great deal, which tells us that these particular functionalizations of MePPP did not change the overall electronics of the molecule to a great extent. On reduction, two consecutive electrons are accepted by the porphyrin macrocycle in each of 18, 19 and 20. The oxidations are also reversible; the only notable feature of the oxidation potentials is that 19 and 20 are somewhat easier to oxidize than MePPP.  Compound  Reduction 1  Reduction 2  Oxidation 1  Methylpyropheophorbide (18)  -1.47  -1.73  0.61  Pyrazoline of MePPP (19)  -1.48  -1.76  0.45  Cyclopropane of MePPP (20)  -1.52  -1.83  0.43  Table 2.6 Summary of redox potentials measured in dichloromethane at 22°C versus Fc. Details in the experimental section.  74  Results and Discussion  R2 Rl  Current  J  02  N  R4  01  1.5  1.0  R3  0.5  0.0  - 0 . 5 -1.0  -1.5  -2.0  E vs. S C E Figure 2.24 Cyclic voltammogram of 19 measured in methylene chloride containing 0.2M B u N P F on a Pt electrode at 0.2V s". 1  4  6  With the estimates for the reduction potential of the porphyrin from the electrochemistry and the E o = 1.8 eV (or 43 kcal mol" ) available from the absorption and fluorescence 1  0i  data, the feasibility for an electron transfer can be estimated.  In order for electron  transfer from the pyrazoline to the porphyrin to be exergonic the oxidation potential of the pyrazoline would have to be less than 0.3 V . Even accounting for the solvation term, the oxidation potentials of pyrazolines are too high to make electron transfer feasible in this case.  Nonetheless, the electrochemical data acquired for these compounds may be useful in future applications. In biological systems, donors and acceptors are ubiquitous. When  75  Results and Discussion  the question of whether or not electron transfer will occur with this drug and a specified substrate, this redox information will enable the investigator to predict the outcome.  All these investigative experiments, UV-vis product study, emission spectra, and the electrochemistry, provide valuable information about the pyrazoline of MePPP (19) and its photoproduct cyclopropane (20). The one message that is clear, is that it is wrong to think of the pyrazoline as a distinct entity from the porphyrin.  Initially, the lack of  conjugation between the pyrazoline and the porphryin led to this assumption, but many examples exist of molecules where two chromophores are separated by far more than a single carbon, and interaction between the groups is still observed. ' 94  9 5  All the evidence  suggests that this functional group is an integral part of the chromophore, and is intact with the overall electronic structure of the macrocyle.  Throughout this quandary, a statement made early in our amazement that long wavelength light was able to effect the observed chemistry kept resurfacing: "About 75 kcal/mol are required to break the C-N covalent bond, and only 43 kcal/mol of energy are produced by 672 nm light." Although this is still true, what may have been neglected is the overall free energy of the reaction. In a theoretical study by Branchadell, the picture 91  becomes more clear. They investigated the mechanism of addition of diazomethane to ethylene, as well as the nitrogen elimination reaction from the cycloadduct. considering entropy and enthalpy values  96  the exergonicity of these reactions is  highlighted. Figure 2.25 is a free energy diagram for the reactions of the pyrazoline.  76  In  Results and Discussion  N; N  ••N  I 1  N  23.5  +CH N 2  2  35.2  f 23.0 1.  N W  N 43.4  ^7  Figure 2.25. Energy diagram for the formation and dissociation of pyrazoline (kcal/mol)  In summary, upon absorption of a photon of long wavelength light, 19 forms an excited state with sufficient energy (672 nm = 43 kcal/mol) to extrude nitrogen from its pyrazoline moeity (E  a  = 35.2 kcal/mol). While the mechanism involved in long  wavelength irradiation is not known, it is be feasible to imagine a mechanism similar to that invoked by the short wavelength irradiation The fact that this low energy light is enough to bring about the observed photochemistry at the periphery of a porphyrin is very encouraging for the design of future potential crosslinking photosensitizers.  77  Results and Discussion  2.4.1  Crosslinking Attempts in Solution  Until now, the photochemistry that has been discussed was carried out in benzene; an 'inert' solvent in terms of its potential to react with radicals in solution. The title of this thesis reminds us of the impetus for synthesizing these pyrazoline porphyrins: "Synthesis and  Photochemistry  Applications."  of  Azo-Type  Derivitized  Porphyrins  with  Crosslinking  It is hoped that these derivitized PDT agents will have the ability to  crosslink within their biological environment.  As mentioned in Section 1.3, these  functionalities were chosen based on their ability to form reactive intermediates on photoactivation. Before attempting in vitro assays on venerable cell lines with this series of compounds, several experiments were carried out in solution where we attempted to trap the 1,3 biradical intermediate. Literature precedence was found that stated that 1,3diradical species like the intermediate created from photolysis of the pyrazolines can be trapped by alkenes , isocyanates ' , and enamines , which led to some of the first 97  98  attempts in Table 2.7.  99  100  The results were disappointing to say the least.  Only the  cyclopropane was observed, except for those cases where degradation of the starting material or polymerization of the solution made it impossible to recover any products.  A paper by Jerome Berson gave hope to these crosslinking efforts citing their experience with various substrates:  "The efficiency of the cycloaddition, as measured by the  competition between adduct formation and cyclization, is greater with conjugated olefins than with  simple  ones.  1,3  -Cyclohexadiene,  2,4-hexadiene,  methyl acrylate,  acrylonitrile, fumaronitrile and crotononitrile all form adducts in high yield, whereas  78  Results and Discussion  cyclohexene, dihydropyran, and 1,2-diethoxyene do not...conjugated acetylenes do not seem to be good trapping agents...decomposition of cyclic azo X in the presence of dimethyl acetylenedicarboxylate gives no adduct and a substantial amount of a polymeric product."  97 ( c )  Even though a lesson was learned from these previous attempts -not to try  to crosslink with D M A D  or unconjugated  olefins- efforts  to react the porphyrin  pyrazolines with conjugated olefins were unsuccessful.  Substrate  Concentration  Result  Styrene (solution in benzene) Styrene (neat) Dimethylacetylene Dicarboxylate (neat) Tributyltinhydride (neat)  0.28M 8.70M  Diethylamine (neat) Ethyl Acrylate Cyclohexadiene  9.70M 9.23M 10.57M  N o reaction N o reaction N o reaction/ Polymerization N o reaction/ Polymerization Degradation of S M N o Reaction  8.10M 3.72M  -  Table 2.7 Results of crosslinking attempts in various reagent solutions (no reaction is synonymous with cyclopropane formation and recovery)  The conclusion, it seemed, was that the only hope for trapping this incredibly short-lived reactive intermediate would be in a very concentrated environment. However, even upon irradiation in neat solutions of the solvents listed in Table 2.7 only the cyclopropane was formed.  The lifetimes for the biradicals mentioned in the Berson papers were on the  order of x = 5-20 ns. A quick calculation comparing the resultant rate of intramolecualr cyclisation with the bimolecular crosslinking reaction leads us to believe that the lifetime of the biradical created from the porphyrin pyrazolines is much shorter than originally anticipated.  Other factors, including sterics, may also be inhibiting the bimolecular  reaction.  79  Results and Discussion  Solution studies alone may not reveal the modified photosensitizers' abilities.  crosslinking  In vivo reactions of this nature would likely occur more readily due to the  proximity of the crosslinking agent and closely associated cellular components.  A  variety of in vitro testing regimes are available to assess these crosslinking systems in a biological environment, and will be discussed in Chapter 3. 61  Since the lifetimes of biradical intermediates are very short, and because these can undergo hydrogen atom abstraction and other competitive reactions, their efficacy as crosslinking agents may be limited.  For this reason, other (longer lived) reactive  intermediates will also be investigated, and the progress towards one such functionality is discussed in the next section.  On a positive note, were nothing to come of the pyrazoline as a crosslinking agent, it is nonetheless a fortuitous event to have discovered a means of functionalizing porphyrins with cyclopropyl moieties.  The cyclopropane ring, due to its unusual bonding and  inherent ring strain (27.5 kcal/mol) is unique among carbocycles in both its properties and reactions. Thus cyclopropane derivatives provide building blocks of unprecedented synthetic potential.  Moreover, natural and synthetic cyclopropanes bearing simple  functionalities are endowed with a large spectrum of biological properties ranging from enzyme inhibition to antibiotic, antiviral, antitumor and neurochemical properties.  101  These novel cyclopropyl derivitized photosensitizers could therefore hold promise in areas not yet considered.  80  Results and Discussion  2.5  Progress Towards the Synthesis of Diazirine Tetraphenylporphyrin  The second series of compounds of interest is not dependent  upon a naturally occurring starting  material.  This  becomes  an  important  consideration for potential PDT agents. With the fear of contaminants causing illness such as 'mad cow disease' threatening natural blood sources, it OH  seems judicious to explore alternative porphyrin  Figure 2.26 Foscan® families.  As  mentioned  in  Section  1.1.5,  tetraphenylporphyrin (TPP) is the simplest and least expensive porphyrin to make. These attributes combined make such a parent molecule very attractive to the pharmaceutical industry.  In addition, the phenyl groups of TPP are easily modified, and many  derivatives are available. An example of one such TPP-derived photosensitizer exists in 'Foscan' ' 102  103  (Figure 2.26), a promising drug that is currently undergoing clinical trials  in Europe.  The  modification to TPP envisioned for a model  crosslinker  herein  is  inspired  by  the  efficient  crosslinking abilities of the diazirine functionality (Figure  2.27).  As  mentioned  in  Section  1.3,  25  photochemically induced extrusion of nitrogen from  Figure 2.27 Diazirine-TPP  diazirine yields a relatively long-lived and reactive  81  Results and Discussion  carbene species that is capable of insertion into C - H bonds or addition to C-C multiple bonds. From the wide variety of functionalities able to produce reactive intermediates including phenyl azides, diazo groups, and benzophenone derivatives, the diazirine was chosen because of its particular attributes. Recent literature accounts also emphasize the functionality's ability in photolabeling of biological substrates. ' ' 63  is small, non-bulky, and lipophillic.  104  105  The diazirine unit  The carbene intermediate derived from photolysis  of the diazirine has a lifetime in the milliseconds , unlike the biradical intermediate 50  derived from the pyrazolines, increasing its propensity to react with its surroundings. Its chromophore extends significantly into the 300 nm range, which means that its activation is non-destructive to biological tissue and also increases the chances of its activation by the porphyrin's absorption. The discoveries in the previous sections, which indicate the porphyrin is capable of long wavelength absorption and consequent activation of functionalities at its periphery, are encouraging for the predicted photochemistry of this target molecule.  At this time, it should be noted that there exists a parent molecule that might be more appropriate for this model photocrosslinking compound; one based on diphenylporphyrin (DPP). Figure 2.28 illustrates the predicted photochemistry for a diazirine-modified DPP (either in the (3 or meso position) versus that expected from diazirine-TPP. Thus in the case of diazirine-TPP, there is a great likelihood that the reactive intermediate carbene will react intramolecularly with an adjacent phenyl ring to form a stable exocyclic sixmembered ring. Functionalized DPP, on the other hand, could only react with a carbonhydrogen bond four bonds away, resulting in a highly strained four-membered ring.  82  Results and Discussion  Additionally, the envisioned functionalizations of DPP would likely be easier because the (3 and meso positions are less sterically hindered than in TPP. Ph  Ph  Ph  Figure 2.28 Predicted photochemistry of diazirine-TPP and diazirine-DPP This work however, describes the progress towards diazirine-TPP and not diazirine-DPP. Quite simply, we have chosen TPP over DPP because TPP can be purchased, whereas the synthesis of DPP is lengthy, arduous, and has low yields.  106  Synthesis and subsequent  derivatization of DPP may still prove to be worth the time investment in the future, however it was felt that proof of concept could be illustrated with the TPP derivative, and the photochemistry could nonetheless be explored upon successful synthesis of the diazirine functionality.  83  Results and Discussion  An additional point of interest regarding the trifluoromethyl diazirine functionality is the presence of fluorine atoms. In porphyrin and chlorin systems, it has been shown that overall lipophilicity of the molecules plays an important role in PDT efficacy. Coupled with the interesting solubility properties brought about by the incorporation of fluorine functionalities, there exists another intriguing aspect.  I 9  F NMR can be used to provide  the pharmacokinetic profiles of these photosensitizers, thus greatly increasing the ability to study mechanistic aspects of in vivo photodynamic therapy.  107  The synthesis of 25 is based on a combination of two published synthetic strategies. The transformations that lead to the trifluoroacetyl-TPP (23) are based on a modified procedure by Ravindra Pandey , who worked on octaethylporphyrin and functionalized 108  it at he meso position (Scheme 2.7). From the trifluoroacetyl towards the diazirine, the reactions are planned according to a series of reactions that have previously been carried out on aryl systems (Scheme 2.8) 51  Et  Et  Et  Et  Et  Et  Et  Et  Et  Et  Et  Et  Scheme 2.7 "Preparation of Fluorinated Porphyrins" ,OTos O  HN—NH  CF R'  3  1)NH OH  Ag O  2  2) TosCI, pyr  N=N z  R'  R'  "tosyloxime'  R'  'diaziridine"  Scheme 2.8 Synthesis of 3-trifluoromethyl-3-phenyl diazirine  84  "diazirine'  Results and Discussion  The methodology discussed below illustrates the first example of the utility of the trimethylsilyltrifluoromethyl (TMSCF3) reagent for introducing a trifluoromethyl group at the [3-position of the porphyrin macrocycle. The electron withdrawing groups most commonly employed at the meso positions of the porphyrins are the perhalophenyls and perfluoalkyl groups obtained by total synthesis.  109  In addition, the reaction of porphyrin  (22) (obtained by treating the related formyl analog with T M S C F 3 ) , with tetrapropyl ammonium perruthenate (TPAP)-N-methyl-morpholine N-oxide (NMO) provides the first example of introducing the trifluoroacetyl group at the P-position of the porphyrin system 23 (Scheme 2.9).  Scheme 2.9 Progress towards the synthesis of diazirine-TPP (25)  85  Results and Discussion  The synthesis towards 25 got off to a slow start because of the difficulties encountered in finding a suitable metal to coordinate the central nitrogen atoms. attempts on the free base of TPP were unsuccessful. metals is known to activate porphyrin reactivity.  110  Direct formylation  Introduction of divalent central  This is thought to occur as a result of  the increased electronegativity and the 'ruffling'"', or deviation from planarity, known to occur in the porphyrin macrocycle. Certain trends have been observed; for example, the following  metalloporphyrins'  MgP>ZnP>CuP>NiP>PdP.  112  degree  of  electronegativity  follows  the  order,  The problem that often follows however, is the harsh  conditions required to demetallate the porphyrin after subsequent modifications. To this end, a certain amount of trial and error must be carried out before finding the conditions appropriate for the required reactivity. The first attempts to formylate TPP in the series of reactions in this work were carried out using nickel as the central metal.  Table 2.8 illustrates the progress made in finding suitable metallation conditions from the vast number found in the literature." '" ' 3  4  115  Most of the problems encountered centered  around the major difficulty of getting both the free base porphyrin and the metallic reagent simultaneously into the same solution under reactive conditions. This is due to the fact that good solvents for the porphyrins in their unionized forms are generally poor solvents for the simple metallic ions and vice versa. Hence the yields were low, the rates of reaction slow, and it was very inconvenient to prepare a large amount of material at one time.  In addition, work-up and product purification were both troublesome and  tedious. The use of D M F as a reaction solvent seemed to represent a compromise to the  86  Results and Discussion  problem. While the solubility is low at room temperature, at the reflux point all of the porphyrinic material was soluble.  Nickel Salt Ni(COCH ) 3  Ni(acac)  2  2  NiCl »6H 0 2  2  Reaction Time  Solvent  Temperature  Yield  1 week  CHC1  100°C  3%  24 h  o-xylene  150°C  10-55%  6-24 h  DMF  180°C  95%  3  Table 2.8 TPP metallation conditions Subsequent to metallation, the task of formylating nickel TPP remained an arduous one. The Vilsmeier-Haack procedure is well known for the formylation of aromatic rings. In the formylation of nickel TPP however, the HC1 biproduct of the reaction resulted in the demetallation of the porphyrin. This competing reaction resulted in decreased yields of the desired product, and an increase of TPP in the product mixture which proved impossible to remove. Table 2.9 lists the various modifications that were attempted to increase product yield. Initially, it was thought that reactant concentrations might be the cause for poor reactivity and these were adjusted accordingly. When the suspicion of acid formation arose, two strategies were attempted to resolve the problem. First, the reaction mixture was evacuated upon formation of the Vilsmeier complex in an attempt to remove the HC1 formed.  The second approach involved the addition of a proton  sponge, namely l,8-bis(dimethyl amino) naphthalene . 107  It was hoped that this strong  base would neutralize the acid formed, without interfering with the reaction thanks to the base's weak nucleophilic character. The results varied, and it seems the benefit of the proton sponge was outweighed by the amount of sponge required and the difficulty in isolating the product from the intractable mixture.  87  Results and Discussion  NiTPP  POCl  0.149  DMF  Proton Sponge  Reaction Time  Yield  0.013  0.020  -  48 h  33 %  0.223  13.17  19.84  -  8h  28 %  3.100  31.00  46.50  -  24 h  30%  0.797  95.64  143.46  23.33  14 h  40%  3.172  380.68  571.02  95.19  12 h  20%  0.760  107.28  129.14  50.74  24 h  30 %  3  Table 2.9. Formylation of NiTPP (all concentrations shown are in millimoles)  Amidst the frustration of these early formylation reactions, a colleague in the Dolphin group facing similar challenges discovered an alternative published method. The answer was found in a translated article from a Russian publication, by Ponomarev and Maravin.  116  It so happens that the cobalt complex  considerably more reactive to formylation.  of tetraphenylporphyrin is  Metallation is comparatively facile, and  involves the addition of cobalt acetate in methanol to a refluxing solution of the porphyrin. The reaction is monitored by UV/vis spectroscopy and takes less than an hour to complete. In addition to the obvious advantages in synthesis, it was a relief to forgo the handling of the nickel salts, whose toxicity has been documented extensively."  7  The method of formylation proposed shortened the reaction time to 30 minutes, decreased the amount of reagents consumed, and lowered the temperature of the process. At the end of the reaction (determined by chromatography) the product is not hydrolyzed, rather the intermediate, a metallated phosphorus complex is isolated. Treatment of the latter with sulfuric acid not only results in the desired aldehyde, but also serves to remove the metal simultaneously.  Inspection by T L C did reveal a secondary product, however  88  Results and Discussion  not enough of this product was isolated for extensive characterization. The suspicion is that this side product is the result of attack on the formyl group, or perhaps sulfonation of the porphyrin ring. Nonetheless, yields increased to 65% and product isolation was much easier than with the nickel-TPP.  It should be noted, however, that even this method of formylation leaves much to be desired. The characteristically low solubility of porphyrins, TPP in particular, requires large volumes of solvent.  For example, for 0.744 mmol of CoTPP, 300 mL of  dichloroethane, followed by 500 mL of water, and 300 mL of benzene in the sulfuric acid work-up were used. Many would consider this a large scale reaction, and yet less than 400 mgs of product are obtained. In addition, attempts at scaling-up the reaction were unsuccessful.  The products listed in the experimental section of this work are all free base porphyrins. Although progress in this series was also made on the metallated NiTPP, namely as far as the oxime, these products have not been included on the basis of redundancy. The superior yield and ease of preparation of the free base formyl-TPP via CoTPP will undoubtedly be the one to explore further.  The conversion of formyl-TPP (21) to trifluoromethanol-TPP (22) (Scheme 2.9) proved as elusive as the formylation did initially.  Although Pandey  1 0 7  reported a similar  reaction on a formyl group in the meso position of octaethylporphyrin in excellent yields (95%), the communication gave no indication of the proprotions of reagents, solvents or  89  Results and Discussion  work-up used.  A paper by Prakash et. al.  118  which reports the nucleophilic  trifluoromethylation reaction of carbonyl compounds by Rupert's reagent's" (TMSCF3) 9  was referred to, but gives no porphyrinic examples.  It recommended the use of an  "equimolar excess" of T M S C F 3 for every mole of aldehyde substrate. The reaction did not work on the porphyrin until the ratio of T M S C F had systematically been increased to 3  15 times that of the formyl-TPP. Throughout the assays, it was thought that an increased amount of tetrabutylammonium fluoride (TBAF) catalyst might also improve the yield of the reaction.  This error in logic resulted in many unsuccessful reactions, as well as  expensive reagents squandered.  In fact, one mechanistic explanation points to a  competition for the T M S C F 3 reagent between the fluorine generated from T B A F and the reaction site on the porphyrin. What was far more successful in increasing the reaction yield, was maintaining dry reaction conditions to assure that TMSCF3 would not be hydrolyzed. Once this was realized, and all extra precautions were taken to assure dry reaction conditions (including distillation of the solvent-THF) a much better conversion (89%) was achieved.  The final hurdle in this reaction was overcome by the discovery that trifluoroacetic acid (TFA) was not an appropriate acid for cleaving the TMS from the intermediate 21a to afford the alcohol (Scheme 2.10).  This milder acid was suggested for use with the  porphyrin, but it turns out that [0.5M] HC1 is required to cleave the strong oxygen-silicon bond. The HC1 protonated the porphyrin as well, turning the solution a vibrant green colour, but was easily neutralized by washing with saturated sodium acetate solution.  90  Results and Discussion  21  22  21a  Scheme 2.10 Formation of trifluoromethanol-TPP  An oxidation similar to that of trifluoromethanol-TPP (22) to trifluoroacetyl-TPP (23) was also referred to by Schiau et. al.  120  Ley and co-workers  121  first described the use of  tetrapropylammonium perruthenate (PfyN) (R.UO4) and N-methylmorpholine N-oxide for the oxidation of alcohols to aldehydes and ketones.  Similar to the aforementioned  formation of trifluoromethanol, several reaction conditions had to be worked out to apply the chemistry to the porphyrin molecules in question.  Again, the amount of reagents  needed to be increased (6.4: 1, NMO:alcohol instead of the suggested 3.2:1). It became particularly important to assure the driest possible conditions in this case as well. It is suspected that water in the N M O and solvent (CH2CI2) was responsible for repeatedly poisoning the ruthenium catalyst.  Once precautions for a dry reaction were taken, the  yield for the reaction increased from 20 to 74%. It was also found that addition of one or two extra equivalents of catalyst, towards the end of the reaction, helped to push it to completion. The remaining 26% can be attributed to a substantial impurity, visible by T L C as a purple band directly under the desired product. Despite efforts to characterize this side-product, MS, N M R and UV-vis spectroscopies only revealed it to be a porphyrin of lower molecular weight than the desired product.  91  Results and Discussion  In Scheme 2.7, the conversion of trifiuoroacetophenone to the tosyloxime is shown in one step. The oxime is not purified or characterized in the literature procedure, presumably 51  due in part to the ease with which the tosylation is carried out on that intermediate. Unfortunately, the tosylation of the trifluoromethyl oxime-TPP (24) was never achieved in the present work. The oxime (24) is in fact, the last intermediate in the synthesis to be isolated and characterized. One of the challenges in the formation of the oxime was finding a suitable solvent for the reaction, and making the necessary changes in reaction to accommodate this solvent.  The literature precedence cited a reaction carried out in  ethanol, in which the pH would be continually adjusted to 6 with addition of 4M NaOH. Not only would the porphyrin be insoluble in ethanol, but also any suitable organic solvent would not allow the pH to be easily monitored. Addition of molar equivalents of NaOH was not successful, nor was the use of excess hydroxylamine»HCl. After some thought about the equilibrium involved in the reaction, it was decided to attempt the reaction in a high boiling, dry solvent with molecular sieves. The hope was that the water could be driven off, pushing the reaction to the products side of the equilibrium. After only one hour of reflux in dry benzene with molecular sieves, a light green spot (Rf 4 2  =0.42, 100% CH C1 eluent) appeared below that of the trifluoroacetyl-TPP (R 2  2  f 2 3  =  0.61). Many hours later when T L C revealed near-completion of the reaction, the product was isolated at 60% yield. It is anticipated that upon scale-up of future reactions, the use of a Dean-Stark trap will further improve the yield of this reaction.  92  Results and Discussion  It is difficult to postulate about the reasons for the seemingly facile tosylation reaction not working.  By this point in the synthesis, despite efforts to scale-up, there was only a  minute amount of 24 left and it was time to 'pass the torch' to the graduate student who would assume ownership of this project in the future.  Trifluoroacetyl-TPP, the first example of a trifluoroacetyl group in the (3 position, and its intermediates were fully characterized by standard methods. The 'H NMR spectra shown in Figure 2.29 highlights the subtle changes in the aromatic region of the spectra, that support the signal assignments; these are outlined in detail in the experimental section. The aldehyde signal and [3-proton signals at 9.46 and 9.24 ppm respectively, compare favourably with the literature." The introduction of the trifluoromethyl group is far 6  enough removed from the P-proton that its shift is hardly affected (9.24 - 9.12 ppm). The aldehyde signal disappears, and the proton signal from the alcohol carbon appears further upfield, at 8.84 ppm. Oxidation to the trifluoroacetyl results in the disappearance of the 8 proton altogether, and the p-proton is shifted back to 9.25 ppm. 1  The  1 9  F spectra shown in Figure 2.30,  illustrate how simple fluorine coupling  experiments confirmed the oxidation of trifluoromethanol-TPP to trifluoroacetyl-TPP. Thus the fluorine nuclei in the trifluoromethyl moiety are coupled to the single proton on the carbon also bearing the alcohol, producing a doublet (-76.87 ppm).  The same  trifluoromethyl moiety in the trifluoroacetyl functionality, on the other hand, does not have any neighbouring protons and appears as a singlet (-73.88 ppm). These fluorine nuclei also  93  Results and Discussion  Results and Discussion  appear to sense the deshielding effect of the carbonyl, which results in their shifting downfield by 3 ppm.  -76.40  -73.80  -73.90  -74.00  -74.10  -74.20  -74.30  Figure 2.30 Coupled F spectra of 22 and 23. l 9  Despite literature precedence for each of the reactions applied towards the synthesis of 25, many difficulties arose in adapting the procedures to porphyrin chemistry. As a result of optimizing five of the eight reactions in the formation of the diazirine, scale up is now possible and large amounts of the trifluoroacetyl have been formed. It is anticipated that the last three steps in the reaction are feasible, and that upon completion of the synthesis the interesting photochemical properties of 25 will be explored.  95  Conclusion  3  Conclusions and Suggestions for Further Studies  In the first part of this thesis, the synthesis, isolation and characterization of a series of novel pyrazoline porphyrins (10, 11, 12) were described. These azo-type derivatized PPDME  porphyrins were found to extrude molecular nitrogen on thermal and light  activation. Irradiation with U V light and long wavelength light were found to initiate the observed photochemistry,  and the mechanism presumably involved the stepwise  formation of a 1,3-biradical. A single product for each respective pyrazoline porphyrin was formed on completion of both the thermal and photochemical reactions (15, 16, 17). These novel cyclopropane derivatives were also purified and characterized.  The optimized diazomethane reaction was also carried out on a chlorin to yield a novel pyrazoline derivatized MePPP (19).  Like the PP-DME pyrazolines, activation with heat,  U V light and long wavelength light yielded the cyclopropane product of MePPP (20). The  optical characteristics  of the  pyrazoline MePPP  made  for an  interesting  photochemical study. The photoconversion was monitored by UV-vis spectroscopy to reveal that this photochemical reaction can be taken to complete conversion to the respective cyclopropane, without any intervening products.  The phenomenon of long wavelength activation in these systems is very fortuitous considering the potential application of crosslinking photosensitizers in PDT.  The  mechanism of activation was addressed, in particular the ability of the low energy photoactivation to lead to nitrogen extrusion. We believe that the mechanisms of the  96  Conclusion  short and long wavelength photochemistry are the same and that this highly exothermic cycloelimination reaction of pyrazolines is possible with the wavelength of light used. That the porphyrin macrocycle has the ability to absorb light at low wavelengths and activate photoactive moieties at its periphery is very encouraging for the design of future potential crosslinking photosensitizers, and is a key discovery of this work.  Several attempts to trap the biradical intermediates produced on photoactivation of the pyrazolines were carried out in solution, to investigate the potential efficacy for crosslinking. Unfortunately, the lifetime of these biradical intermediates proved to be much shorter than expected.  No adducts were formed with the crosslinking substrate  solutions, even when the pyrazoline porphyrins were dissolved in neat substrate solutions.  Although these results were disappointing, solution studies alone may not  reveal the modified photosensitizers crosslinking abilities in vivo.  A variety of in vitro and in vivo testing regimes are available to assess these crosslinking systems. Initial experiments were scheduled to take place at Q L T Inc., but could not be carried out this past year due to the company's many commitments. The assays that are foreseen will focus on establishing whether crosslinking improves the cellular retention of the photosensitizer upon light activation. L1210, a tumor cell line, will be incubated in the dark with the MePPP pyrazoline 19 and the unfunctionalized MePPP 18. Incubation will be followed by irradiation with the appropriate long wavelength of light. The cells will then be washed after 1 hr of incubation, followed by the extraction of drug from the cells.  61  Comparisons between crosslinked systems, (light activated and dark control), and  97  Conclusion  cells incubated with unfunctionalized photosensitizers will provide evidence for selective retention in a biological environment.  The last section in this work described the progress made towards the synthesis of a diazirine derivatized porphyrin (25).  It is anticipated that this photoactive functionality  will yield a longer-lived carbene intermediate upon photolysis, endowing it with greater crosslinking abilities. Each of the reactions carried-out on TPP had to be modified for porphyrin chemistry from the so-called small molecule literature precedents to yield the desired products. The reactions leading up to trifluoroacetyl-TPP (23) provide the first example of introducing a trifluoroacetyl group at the (3 position of the porphyrin. Each of the intermediates (21, 22, 23, 24) were isolated and characterized. Five of the eight reactions planned in the synthesis of (25) were optimized and scaled-up. This will afford the new investigator with a sufficient amount of the trifluoroacetyl intermediate (23) to pursue the remaining reactions effectively.  Early in the designs of this project, an azide derivatized porphyrin was also envisioned. The nitrene intermediate has been applied extensively in photoaffinity labeling studies, making the azide functionality a worthy synthetic target.  The success in this project  elicited the interest of another graduate student who is currently working towards the synthesis of the diazirine, and has also made progress towards a nitrene derivatized porphyrin.  98  Experimental  4  Experimental  4.1  Instrumentation and Materials  Elemental Analyses (EA) Microanalyses were carried out in the microanalytical laboratory in the Department of Chemistry, University of British Columbia by Mr. Peter Borda using a Carlo Erba Elemental Analyzer 1106.  Analysis was attempted on all pyrazoline and cyclopropyl  derivatives of protoporphyrin and methylpyropheophorbide, as well as trifluoroacetylTPP.  In many cases, the results are unacceptable due to the likely presence of water that  could not be removed by heating, because of the thermal lability of the compounds in question.  Mass Spectra (MS) Mass spectrometric analyses were carried out by the B. C. Regional Mass Spectrometry Center at the University of British Columbia, Department of Chemistry. Low and high resolution mass spectra were obtained by liquid secondary ion mass spectrometry (LSIMS), and were determined on a KRATOS Concept IIHQ hybrid mass spectrometer. Molecular ions are designated as M . +  UV-vis Spectra UV-vis spectra were taken on a Cary 50. Wavelengths for each absorption maximum (^max)  are reported in nanometers (nm), and extinction coefficients (e (M cm"')) are _l  given in parentheses.  99  Experimental  Fluorescence Instrumentation The fluorescence measurements were performed on a SLM-AMINCO  AMINCO-  Bowman Series 2 Luminescence Spectrometer using a pulsed xenon lamp as the excitation source.  Excitation was at 350 nm and 660nm with a 2 nm bandwidth and  emission was at 430 nm with 16 nm bandwidth. Fluorescence emissions were corrected for lampfluctuationsusing the reference signal from the excitation source.  Electrochemical Studies Cyclic Voltammetry was carried out at the University of Western Ontario, with the assistance of Prof. Mark Workentin using an E.G. & G. PAR 283 potentiostat interfaced to a personal computer using PAR 270 electrochemistry software.  The electrochemical  cell was maintained at 25° C and contained 0.1 mol L - l T E A P in 25mL of methylene chloride purged by argon. At the beginning of every experiment the working electrode, a glassy carbon 3 mm disk, was freshly polished with 1 mm diamond paste and ultrasonically cleaned in ethanol for fifteen minutes.  The counter electrode was a  platinum plate and a silver wire immersed in a glass tube containing 0.1 M T E A P in the desired solvent with a fine sintered bottom was used as a quasi-reference. Ferrocene was used as an internal redox reference; the potential was calibrated against the saturated calomel electrode (SCE).  To compensate for the cells internal resistance the iR  compensation was adjusted to at least 95% of the oscillation value. experiment, 1 m M sample of the appropriate porphyrin was used.  100  In a typical  Experimental  Nuclear Magnetic Resonance Spectrometry (NMR) N M R spectra were recorded either by the author, by Dr. Nick Burlinson, by Marietta Austria or Liane Darge of the University of British Columbia Chemistry Department N M R Service Laboratory.  *H NMR Proton nuclear magnetic resonance spectra ('HNMR) were recorded on the following spectrometers:  Bruker WH-400 (400 MHz), Bruker AV-400 (400 MHz) and Buker  AMX-500 (500 MHz). The positions of the signals are given as chemical shifts (8) in parts per million (ppm) with respect to tetramethylsilane (TMS) at 8 0 ppm; however, the internal reference standard used in each case was the residual proton signal present in the deuterated solvent. Reported chemical shifts are followed in parentheses by the number of protons, the multiplicity of the peak, the coupling constant (J) in Hz, and the atomic assignment. The following abbreviations are used in reference to the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets, m = multiplet, br = broad. In some cases, in order to ascertain structures, H M Q C (Heteronuclear Multiple Quantum Coherence, ' H - ' C correlation) and N O E S Y (Nuclear 3  Overhauser Effect Spectroscopy) experiments on the Bruker AMX-500 spectrometer, C O S Y (Correlated SpectroscopY), NOESY and homonuclear decoupling experiments on the Bruker WH-400 spectrometer were carried out.  101  Experimental  1 3  C NMR  The following spectrometers were used to record the carbon nuclear magnetic resonance spectra ( C NMR): Bruker AM-400 at 100.6 MHz, Bruker AV-300 at 74.6 MHz, and l3  Bruker AMX-500 at 128.5 MHz. All spectra were determined with broad band proton decoupling.  The position of the signals are given as chemical shifts (8) in parts per  million (ppm) with respect to tetramethylsilane at 8 0 ppm; however, the internal reference standard used in each case was the central transition of the solvent carbon atom. Reported chemical shifts are followed in parentheses by the carbon assignments, which were often made possible by an APT (attached proton test).  Crystallographic Analysis (X-RAY) The X-ray crystal structure was determined using single-crystal X-ray analysis, on a Rigaku / A D S C C C D area detector with graphite monochromated M o - K a radiation, and was drawn with a locally modified version of the ORTEP program at the 50% probability level. Structures were determined by Dr. Brian Patrick in the Chemistry Department of the University of British Columbia.  Chromatography Chromatographic purifications of compounds were carried out using silica gel 60, 70-230 mesh, supplied by E . Merck Co. Thin layer chromatography (TLC) was carried out on pre-coated silica gel plates (Merck 60, 230-400 mesh, with aluminum backing and fluorescent indicator  (F254).  Preparative thin layer chromatography was prepared on pre-  coated 10 x 10 cm 0.5 or 1 mm thick Whatman or Merck silica gel plates.  102  Experimental  Preparative thin layer chromatography was also carried out using the Chromatotron®, when necessary, whereby circular plates of Merck 60, 230-400 mesh with fluorescent indicator  (F254)  were prepared on site.  Reaction Conditions Due to the inherent light sensitivity of these compounds, all reactions were performed in a blacked-out fume hood or surrounded by aluminum foil.  Reagents and Solvents Unless otherwise specified, reagents were used as supplied by the Aldrich Chemical Company. Solvents were reagent or HPLC grade and purified using standard literature methods when necessary.  Deuterated solvents were supplied by Cambridge Isotope  Laboratory.  4.2  Photochemical Studies - General Procedures  Irradiation Sources Photochemical irradiations were carried out with either a 250W Osram H L X 64655 arc lamp in an Oriel lamp housing, a Rayonet Photochemical Chamber Reactor (Model RPR100) or a 672 nm Light Emitting Diode. The light output from the Oriel lamp passed through a glass filter: P70-600-S-533G-Corion  103  Experimental  Solution State Irradiation Spectral-grade solvents were used for irradiations. For preparative-scale irradiations, the solution of the substrate in the appropriate solvent was placed in a 50 mL septum-sealed tube and deoxygenated with nitrogen or argon for 30 minutes, with stirring, prior to reaction.  Efficient stirring was maintained throughout the irradiation period when  possible.  For irradiations carried out with the Osram lamp and the L E D , the substrate  was placed 6-10 cm from the source, and supported on a retort stand. In the case of the Rayonet Reactor, the tubes were suspended by copper wire in the chamber and were not stirred during irradiation.  Analysis of Photochemical Reactions The photochemical reactions were monitored by thin layer chromatography. To insure exclusion of oxygen, these were purged with nitrogen or argon, a large needle was inserted through the septum and aliquots were removed with a capillary. In some cases UV-vis spectrometry was also performed on these aliquots.  4.3  The  Preparation of Protoporphyrin Pyrazolines 10,11 and 12  pyrazolines of protoporphyrin-dimethylester were synthesized  according to a  modified procedure for the reactions of diazomethane reported by Black.  68  A  diazomethane apparatus appropriate for production of (1-50 mmol) of diazomethane was used. First, the cold finger was filled with acetone and dry ice. A solution of potassium hydroxide (13.36 g, 0.238 mol) in water (20 mis) was then added to the reaction vessel,  104  Experimental  followed by ethanol (10 mis).  A 60 ml separation funnel was then placed over the  reaction vessel, sealed and supported by a rubber septum that had a whole bored through it. The 100 ml receiver flask containing PP-DME (9) (0.5 g, 0.846 mol) in a minimum amount of methylene chloride and a stir bar was placed under the condensor, and cooled in an ice bath. An ether trap was not used. The separation funnel was then charged with diazald (12.74 g, 0.59 mol) in a minimum amount of diethylether (~ 40 mis). (Often, solutions were sonicated to facilitate dissolution). The reaction vessel was heated to 60° in a water bath, and the apparatus was kept behind a blast shield from this point on. Diazald in ether was slowly added to the reaction vessel via the separation funnel, at a rate attempting to match that of the formation of yellow diazomethane gas apparent on the condensor. Once the addition was complete, the separation funnel was flushed with a minimum amount of ether, to dissolve residual amounts of diazald.  The 'diazo-balloon' was then attached to the top of the receiver flask, and placed behind the blast shield or in a canister, left to stir in the dark, overnight.  The reaction was  followed by thin layer chromatography using only make-shift plastic capillaries to remove aliquots (any sharp edges solicit the risk of detonation of diazomethane). In the event the reaction had not proceeded to completion, the solvent (and diazomethane) were evaporated and the reaction mixture re-exposed to the aforementioned diazomethane procedure. (The advent of the 'diazo-balloon' greatly reduced the need for re-exposure.)  After satisfactory conversion, the reaction was quenched by removal of solvent and diazomethane-carrying ether (usually in fume hood, under a gentle stream of nitrogen.)  105  Experimental  Separation of the three pyrazoline products was only effective using the chromatotron, starting 100% methylene chloride to introduce the product mixture to the circular silica plate, and increasing the polarity thereafter (0.5% -> 5 % THF in CH C1 eluent). The 2  2  recovered yield of the three products was 78%, in a ratio of 1(10): 1(11):2(12). Recovered starting material accounted for a remaining 20%.  EA : Calculated for C 7H4oN 0 : C, 70.23; H , 6.37; N , 13.28. Found C, 69.85; H , 6.39; 3  6  4  N, 12.57  MS LR +LSIMS (matrix: thioglycerol) : Exact mass calculated for C37H oN 0 (M+l): 4  6  4  633.75. Found 633.  MS HR +LSIMS (matrix: thioglycerol) : Found C, 37; H , 41; O, 4; N , 6 (M+l) 633.31948, (dev 0.87). X  H NMR (400 MHz, CDC1 ): 5 9.94 (1H, s, H-5), 9.91 (1H, s, H-20), 9.83 (1H, s, H-15), 3  9.61 (1H, s, H-10), 8.14 (1H, m, J  JL-O, L-P= 2.5, H -8'), 6.29 (1H, m, J L  = 17.8, J = 11.5, H-3 ), 6.80 (1H, m, J 1  TRANS  TRANS  CIS  = 17.9, J  106  U M  , L-N= 9.7,  = 1.3, H-3 ), 6.13 (1H, m, J = 11.5, 2  GEM  CIS  Experimental  J  = 1.3, H-3 ), 5.45 (1H, m, J - = 17.8, J -M= 9.8, J . , O-L= 2.7, H -8 ), 4.74 (1H, m, 2  GEM  3  0  P  0  0  N  0  J _o= 18.4, Jp. , . = 9.4, J . =2.8, Hp-8 ), 4.28 (2H, t, J = 7.5, H-13 ), 4.27 (2H, t, J = 3  P  M  P  N  1  P L  7.5, H-17 ), 3.65 (3H, s, H-13 ), 3.64 (3H, s, H-17 ), 3.54 (3H, s, H-12 ), 3.51 (3H, s, H 1  5  5  1  18'), 3.48 (3H, s, H-7 ), 3.47 (3H, s, H-2 ), 3.21 (2H, t, J = 7.7, H-13 ), 3.20 (2H, t, J = 1  1  2  7.7, H-17 ), 2.74 (1H, m, J -N=22.4, J -P=9.7, J -L=3.1, H -8 ), 2.11 (1H, m, J - =22.7, 2  2  M  M  M  M  N  M  JN-L, N-P=9.7, J -o=3.2, H -8 ), -3.72 (2H, s, 7VH) 2  N  N  UV-VIS (CH C1 ) X 2  2  (rel. intensity): 406 (1), 502 (0.08), 536 (0.06), 572 (0.05), 626  max  (0.02)  OCH  3  H3C0  11  E A : Calculated for C 7H oN 04 : C, 70.23; H , 6.37; N , 13.28. Found C, 69.88; H , 6.34; 3  4  6  N, 12.58 MS L R +LSIMS (matrix: thioglycerol) : Exact mass calculated for C H4oN 04 (M+l): 37  6  633.75. Found 633. MS HR +LSIMS (matrix: thioglycerol) : Found C, 37; H , 41; O, 4; N , 6 633.31937, (dev 0.69).  107  (M+l)  Experimental  *H NMR (400 MHz, CDCI3): 5 9.95 (1H, s, H-10), 9.79 (1H, s, H-15), 9.69 (1H, s, H 20) , 9.57 (1H, s, H-5), 8.05 (1H, m, J  = 17.8, J = 11.5, H-8 ), 6.81 (1H, m, J . , L-N= 1  trans  9.8, J . , L-P= 2.6, H -3'), 6.23 (1H, m, J L  11.5, J  0  L  cis  trans  = 17.8, J  L  = 1.2, H-8 ), 6.10 (1H, m, J = 2  gem  cis  = 1-2, H-8 ), 5.46 (1H, m, J -P= 17.9, J -M= 9.8, J -N,O-L= 2.6, H -3 ), 4.75 (1H, 2  gem  M  3  0  0  0  0  m, J -o= 17.9, Jp. , P-N= 9.3, J . =2.8, H -3 ), 4.28 (2H, t, J = 7.8, H-13 ), 4.24 (2H, t, J = 3  P  M  P L  1  P  7.8, H-17 ), 3.66 (3H, s, H-13 ), 3.65 (3H, s, H-17 ), 3.49 (3H, s, H-7 ), 3.46 (3H, s, H 1  5  5  1  2 ) , 3.43 (3H, s, H-12 ), 3.42 (3H, s, H-18 ), 3.21 (2H, t, J = 7.8, H-13 ), 3.18 (2H, t, J = 1  1  1  2  7.8, H-17 ), 2.72 (1H, m, J -N=13.0, H -3 ), 2.10 (1H, m, J -N=13.0, H -3 ), -4.20 (2H, 2  2  M  M  2  M  N  s,/VH) 1 3  C NMR (125.8 MHz, CDC1 ) (quaternary carbons are unresolved, C = 19): 5 97.62, 3  96.86, 96.66, 95.89, 129.99, 85.89, 120.57, 77.81, 21.65, 21.62,51.69, 51.67, 11.42, 12.41, 11.83, 11.51, 36.79 (2xC), 27.72. UV-VIS (CH C1 ) 2  2  X  max  (rel. intensity): 404 (1), 502 (0.08), 536 (0.06), 572 (0.05), 626  (0.02)  108  Experimental  E A : Calculated for CsgH^NgC^ : C, 67.64; H , 6.27; N, 16.61. Found C, 67.50; H , 6.26; N, 15.60 MS L R +LSIMS (matrix: thioglycerol) : Exact mass calculated for CjgH^NgC^ (M+l): 675.79. Found 675. MS  HR +LSIMS (matrix: thioglycerol) : Found C, 38; H , 43; O, 4; N, 8 (M+l)  675.33975, (dev-1.45). *H NMR (400 MHz, CDC1 ): 8 10.08 (1H, s, H-20), 10.03 (1H, s, H-15), 9.81, 9.79 (1H, 3  s, H-5), 9.73, 9.71 (1H, s, H-10), 6.91 (2H, m, H - 3 \ 8 ), 5.55, 5.50 (2H, m, J - = 17.9, 1  L  0  P  J -M= 10.0, J -N,O-L= 2.5, H - 3 , 8 ), 4.82, 4.78 (2H, m, H -3 , 8 ), 4.37 (2H, t, J = 7.4, H 3  0  0  3  3  0  3  P  13'), 4.36 (2H, t, J = 7.4, H-17 ), 3.64 (3H, s, H-13 ), 3.64 (3H, s, H-17 ), 3.60 (3H, s, H 1  5  5  18'), 3.56, 3.55 (3H, s, H-7 ), 3.54, 3.53 (3H, s, H-12 ), 3.50, 3.49 (3H, s, H-2 ), 3.26 1  1  1  (2H, t, J = 7.7, H-13 ), 3.25 (2H, t, J = 7.7, H-17 ), 2.84 (2H, m, H - 3 , 8 ), 2.24, 2.22 2  2  2  2  M  (2H, m, H - 3 , 8 ), -3.75 (2H, s, NH) 2  2  N  UV-VIS (CH C1 ) ? w (rel. intensity): 400 (1), 500 (0.10), 534 (0.07), 570 (0.05), 622 2  2  (0.03)  4.3.1  Stirred,  Photoproducts of Protoporphyrin Pyrazolines (15), (16), and (17)  deoxygenated  solutions of  each  of  the  pyrazolines  of protoporphyrin-  dimethylester in benzene were irradiated in the Rayonet reactor (35 A bulbs) and with red-filtered light (Corion 600). The following table outlines the solution concentrations, irradiation times, and isolated product yield:  109  Experimental  Rayonet Photochemical Chamber Reactor Cone. (g/mL), mmol  Time (h)  Product yield (%)  A-ring pyrazoline  3.06 x 10' , 0.017  22.5  82.4  B-ring pyrazoline  5.00 x 10" , 0.016  21  52.4  Di-adduct pyrazoline  2.29 x 10 , 0.014  5  50.0  Time (h)  Product yield (%)  4  J  -4  650 nm filtered light Cone. (g/mL), mmol A-ring pyrazoline  8.00 x 10"\ 0.006  14  35.3  B-ring pyrazoline  2.40 x 10" , 0.019  7  52.3  Di-adduct pyrazoline  2.60 x 10" , 0.019  38  78.5  4  4  Benzene was removed in vacuo, and the residue dissolved in dichloromethane.  The  crude compounds were chromatographed on silica gel 60, 70-230 mesh, 3%-10% EtAce/CH2Cl2 gradient eluent). The appropriate fractions were pooled and evaporated to give the respective yields of the desired cyclopropyl derivatives.  A large proportion of  the starting material was recovered in each case, which accounts for the incomplete conversion to products.  110  Experimental  MS L R +LSIMS (matrix: thioglycerol) : Exact mass calculated for C37H40N4O4 (M+l): 605.747. Found 605. MS  HR +LSIMS (matrix: thioglycerol) : Found C, 37; H, 40; O, 4; N , 4 (M+l)  605.31293, (dev 0.24). *H NMR (400 MHz, CDCI3): 8 10.44 (1H, s, H-5), 10.16 (1H, s, H-20), 10.01 (1H, s, H 15), 10.00 (1H, s, H-10), 8.28 (1H, m, J 17.7, J  = 17.8, J = 11.5, H-3 ), 6.35 (1H, m, J 1  trans  cis  = 1.4, H-3 ), 6.15 (1H, m, J = 11.5, J 2  gem  cis  trans  =  = 1.4, H-3 ), 4.40 (2H, t, J = 7.6, H 2  gem  13 ) , 4.36 (2H, t, J = 7.6, H-17 ), 3.71 (3H, s, H-12 ), 3.68 (3H, s, H-18 ), 3.65 (3H, s, H 1  1  1  1  13 ), 3.65 (3H, s, H-17 ), 3.62 (3H, s, H-7 ), 3.59 (3H, s, H-2 ), 3.27 (2H, t, J = 7.9, H 5  5  1  13 ) , 3.26 (2H, t, J = 7.8, H-17 ), 3.05 (1H, m, 2  2  1  J ub= d0  8.2, J  = 8.2, H-8 ), 1.67 (2H, m, 1  doub  H-8 ), 1.46 (2H, m, H-8 ), -3.76 (2H, s, NH) 2  2  UV-VIS (CH C1 ) l 2  2  (rel. intensity): 402 (1), 502 (0.08), 538 (0.07), 572 (0.04), 626  max  (0.02)  E A : Calculated for C H oN 04 : C, 73.49, H , 6.67; N, 9.26. Found C, 72.02; H , 6.88; 37  N, 7.93  4  4  Experimental  MS L R +LSIMS (matrix: thioglycerol) : Exact mass calculated for C37H40N4O4 (M+l): 605.747. Found 605. MS HR +LSIMS (matrix: thioglycerol) : Found C, 37; H, 41; O, 4; N, 4 (M+l) 605.31293, (dev 0.24). *H NMR (400 MHz, CDCI3): 5 10.38 (1H, s, H-10), 10.14 (1H, s, H-15), 10.07 (1H, s, H-20), 10.02 (1H, s, H-5), 8.28 (1H, m, J 17.7, J  = 17.6, J = 11.4, H-8 ), 6.33 (1H, m, J 1  trans  cis  = 1.4, H-8 ), 6.14 (1H, m, J = 11.5, J 2  gem  cis  trans  =  = 1.4, H-8 ), 4.40 (2H, t, J = 7.7, H 2  gem  13 ) , 4.37 (2H, t, J = 7.8, H-17 ), 3.68 (3H, s, H-12 ), 3.67 (3H, s, H-18 ), 3.64 (3H, s, H 1  1  1  1  17 ), 3.63 (3H, s, H-13 ), 3.63 (3H, s, H-17 ), 3.61 (3H, s, H-2 ), 3.26 (2H, t, J = 7.7, H 1  5  5  13 ) , 3.26 (2H, t, J = 7.6, H-17 ), 3.06 (1H, m, J 2  1  2  doub  = 7.6, J  = 7.6, H-3 ), 1.66 (2H, m, 1  doub  J =2.0, H-3 ), 1.45 (2H, m,J =1.7, H-3 ), -3.75 (2H, s, NH) 2  2  (?)  (?)  UV-VIS (CH C1 ) X 2  2  m a x  (rel. intensity): 404 (1), 502 (0.07), 538 (0.06), 572 (0.05), 626  (0.03) X-RAY : Please refer to Appendix 1  112  Experimental  E A : Calculated for C38H42N4O4 : C, 73.76, H , 6.84; N, 9.05.  Found C, 73.66; H , 7.54;  N, 7.11  MS LR +LSIMS (matrix: thioglycerol) : Exact mass calculated for C38H42N4O4 (M+l): 619.747. Found 619. MS HR +LSIMS (matrix: thioglycerol) : Found C, 38; H, 4 3 ; O, 4 ; N, 4 (M+l) 619.32852, (dev 0.15). *H NMR (400 MHz, CDC1 ): 5 10.41 (1H, s, H-10), 10.39 (1H, s, H-15), 10.04 (1H, s, 3  H-20), 10.02 (1H, s, H-5), 4.41 (2H, t, J = 7.7, H-13 ), 4.39 (2H, t, J = 7.8, H-17 ), 3.68 1  1  ( 3 H , s, H-12 ), 3.67 ( 3 H , s, H-18 ), 3.64 ( 3 H , s, H-17 ), 3.63 ( 3 H , s, H-13 ), 3.63 ( 3 H , s, 1  1  1  5  H-17 ), 3.61 ( 3 H , s, H-2 ), 3.26 (2H, t, J = 7.7, H-13 ), 3.27 (2H, t, J = 7.6, H-17 ), 3.05 5  1  2  (1H, m, H - 3 and H-8 ), 1.66 ( 4 H , m, J 1  1  =1.9, H - 3 and H-8 ), 1.45 ( 4 H , m, J =1.5, H 2  (vic)  2  2  (?)  3 and H-8 ), -3.74 (2H, s, NH) 2  2  UV-VIS (CH2CI2) X  m a x  (rel. intensity): 400 (1), 500 (0.09), 5 3 4 (0.07), 568 (0.05), 622  (0.04)  4.4  Preparation of Methylpyropheophorbide Pyrazoline (19)  The pyrazoline of methylpyropheophorbide was synthesized according to a modified procedure for the reactions of diazomethane reported by Black  In most cases, this  reaction was carried out immediately following the addition of diazomethane protoporphyrin-dimethylester.  to  Thus, enough potassium hydroxide was added to the  reaction vessel to accommodate the formation of diazomathane for both reactions. The portion of the aqueous solution required for this reaction was (2.88 g, 0.051 mol) of  113  Experimental  potassium hydroxide, followed by addition of ethanol (10 mis). The separation funnel was then charged with diazald (2.75 g, 0.013 mol) in a minimum amount of diethylether. Upon formation, the diazomethane was delivered to a 50 ml receiver flask containing methylpyropheo-phorbide(0.5 g, 0.911  mol) in a minimum amount of methylene  chloride. The reaction was then left to stir overnight.  The crude compound was chromatographed on silica gel 60, 70-230 mesh, 2%-10% EtAce/CH Cl2 gradient eluent). The appropriate fractions were pooled and evaporated to 2  yield 0.419 g (78% yield) of the desired pyrazoline. 15% of the starting material did not react, and was recovered.  E A : Calculated for C 5H38N 0 : C, 71.16, H , 6.48; N , 14.23. Found C, 70.72; H , 6.72; 3  6  3  N, 13.63  114  Experimental  M S L R + L S I M S (matrix: thioglycerol) : Exact mass calculated for C H 8 N 0 (M+l): 3 5  3  6  3  591.73. Found 591. M S H R + L S I M S (matrix: thioglycerol) : Found C, 35; H , 39; 0, 3; N, 6 (M+l) 591.30823, (dev -0.23). * H N M R (400 MHz, CDC1 ): 5 9.51 (1H, s, H-10), 8.96 (1H, s, H-20), 8.57 (1H, s, H-5), 3  6.67 (1H, m, J -M,L-N= 9.7, J -O,L-P= 2.2, H -3'), 5.47 (1H, m, J - = 17.6, J - = 10.1, J . , L  L  L  0  P  0  M  0  N  o-i = 2.5, H -3 ), 5.26 (1H, d, J = 19.6, H-15 ), 5.11 (1H, d, J = 19.6, H-15 ), 4.76 (1H, m, 3  1  1  0  J . = 18.2, Jp. , P-N= 9.1, JP-L= 3.1, Hp-3 ), 4.48 (1H, m, J 3  P  0  M  4.29 (1H, m, J  = 8.1, J  D O U B  DOUB  = 2.2, J  D O U B  = 7.4, J  QUART  = 7.1, H-18),  = 2.2, H-17), 3.68 (2H, t, J = 7.4, H-17 ), 3.65 2  DOUB  (3H, s, H-17 ), 3.59 (3H, s, H-7 ), 3.25 (3H, s, H-2 ), 3.17 (3H, s, H-12 ), 2.75 (1H, m, 5  1  1  1  J - =19.7, J -P= 7.6, J -o, M-L= 2.4, H -3 ), 2.68 (1H, m, H-17 ), 2.55 (1H, m, H-8 ), 2  M  N  M  M  1  1  M  2.28 (1H, m, H-8 ), 2.28 (1H, m, H-17 ), 2.11 (1H, m, J . =19.6, J . 1  1  N  M  N  -o=9.7, H -3 ), 2  U N  N  1.80 (3H, d, J = 7.4, H-18 ), 1.67 (3H, t, J = 7.7, H-8 ), -1.77 (2H, s, NH) 1  1 3  2  C N M R (125.8 MHz, CDC1 ) (total C = 35) 210.68, 195.67, 71.28, 160.45, 154.72, 3  150.91, 148.92, 144.99, 141.05, 137.95, 135.96, 135.33, 134.66, 132.81, 130.62, 128.56, 106.28, 104.08, 96.69, 93.01, 85.32, 78.01, 51.75, 51.61, 50.01, 48.01, 30.93, 29.85, 27.32, 23.10, 19.38, 17.33, 11.94, 11.45, 11.11 U V - V I S (CH C1 ) ?i x: 411.0 (107, 100), 506.4 (10, 800), 536.5 (9, 500), 608.0 (8, 070), 2  2  ma  663.5 (46, 061)  115  Experimental  4.4.1 Photoproduct of Methylpyropheophorbide Pyrazoline (20)  A stirred, deoxygenated solution of the pyrazoline of methylpyropheophorbide in benzene (2.75 x 10~ g/mL, 0.023 mmol) was irradiated in front of a 672 nm L E D panel 4  for 14 hours.  Thin layer chromatography revealed completion of the reaction, and  benzene was removed in vacuo.. The residue was dissolved in dichloromethane, and the crude compound  was chromatographed on silica gel 60, 70-230 mesh, 3%-10%  EtAce/CH Cl gradient eluent). The appropriate fractions were pooled and evaporated to 2  give  2  (0.0130  g,  0.023  mmol,  98.6%)  the  cyclopropane  derivative  of  methylpyropheophorbide.  MS L R +LSIMS (matrix: thioglycerol) : Exact mass calculated for C35H N 03 (M+l): 38  4  563.712. Found 563. *H NMR (400 MHz, CDC1 ): 5 9.55 (1H, s, H-10), 9.46 (1H, s, H-20), 8.41 (1H, s, H-5), 3  5.22 (1H, d, J = 19.6, H-15 ), 5.10 (1H, d, J = 19.6, H-15 ), 4.43 (1H, m, J 1  = 7.1, H-18), 4.24 (1H, m, J  1  d o u b  = 8.5, J  doub  = 2.2, J  116  doub  d o u b  = 7.4, J  q u a r t  = 2.2, H-17), 3.68 (2H, t, J = 7.6, H -  Experimental  17 ), 3.64 (3H, s, H-17 ), 3.58 (3H, s, H-7 ), 3.35 (3H, s, H-2 ), 3.24 (3H, s, H-12 ), 2.80 2  5  1  1  1  (1H, t, H-3 ), 2.67 (1H, m, H-17 ), 2.52 (1H, m, H-8 ), 2.30 (1H, m, H-17 ), 2.27 (1H, m, 1  1  1  1  H-8 ), 1.77 (3H, d, J = 7.3, H-18 ), 1.68 (3H, t, J = 7.8, H-8 ), 1.57 (2H, m, J = 1.9, H-3 ), 1  1  2  2  1.30 (2H, m, J = 1.6, H-3 ), -1.65 (2H, s, M i ) 2  UV-VIS (CH C1 ) ^ 2  2  : 410.5 (112,000), 505 (9,800), 536 (9,200), 603 (8,400), 658  (46,400)  4.5  Progress Towards the Synthesis of Diazirine-TPP (25)  Formyl-tetraphenylporphyrin (21)  Formyl-tetraphenylporphyrin (21) was synthesized according to a modification of the procedure reported by Ponomarev . 1  dichloroethane  (300  mL) was  A solution of CoTPP (500 mg, 0.744 mmol) in added  117  to  a  pre-formed  Vilsmeier  complex  Experimental  (dimethylformamide: 1.9 mL, 1.79 g, 24.52 mmol and phosphorusoxychloride: 2.25 mL, 3.70 g, 24.14 mmol) in a 2L RBF and heated at 60°C for 30 minutes.  The solvent was then evaporated in vacuo, and 500 mL of cold water added rapidly to the oily residue. After 10 minutes, the precipitate was removed by water aspirator filtration through a coarse sintered glass filter and left to air dry overnight. The resultant crude salt, a dark green powder, was used in the following steps without purification.  The immonium salt, having been returned to the 2L flask, was then dissolved in 10-15 mL of concentrated sulfuric acid and stirred for an hour. 300 mL of benzene and then a maximum amount saturated sodium acetate solution were added and left to reflux for an hour. Upon cooling, the organic layer was separated and the aqueous layer checked for neutrality. In the event the product was not yet neutralized, the separation funnel was charged with additional sodium acetate solution. The organic layer was passed through cotton and evaporated to dryness.  The solvent was removed by evaporation and the  residue chromatographed on silica gel 60, 70-230 mesh, 50-100% CH2Cl /hexanes as 2  gradient eluent).  Of the four bands apparent, the fastest moving, peach coloured band  accounted for a small proportion of unreacted CoTPP (Rf = 0.8).  The dark green band  (Rf = 0.5) was the desired product, and two lighter green bands (Rf = 0.3, Rf - 0.24) are yet undefined because their presence is so slight. The appropriate fractions were pooled and dried to afford 0.232 g (49%) of the desired purple crystals, formyl-tetraphenyl porphyrin.  118  Experimental  MS L R +LSIMS (matrix: thioglycerol) : Exact mass calculated for C 4 5 H 3 0 N 4 O 3 (M+l): 699.42. Found 699; J  H N M R (300 MHz, CDCI3): 5 9.46 (1H, s, H-8 ), 9.24 (1H, s, H-7), 8.92 (4H, m, H 1  2,3,12,13), 8.82 (2H, s, H-17,18), 8.23 (8H, m, H-5 , 10 , 15 , 20 ), 7.77 (12H, m, H-5 ' , 2  2  2  2  3  4  10 ' ,15 ' , 20 ' ) 3  1 3  4  3  4  3  4  C N M R (75 MHz, CDC1 ) (quaternary carbons are unresolved, C = 28): 5 189.26, 3  142.43, 141.76, 141.57, 134.98, 134.63, 134.58, 134.55, 134.55, 133.34, 133.32, 130.79, 130.05, 130.03, 128.98, 128.14, 127.92, 127.92, 127.36, 127.36, 126.84, 126.84, 126.84, 126.81, 122.60, 120.59, 120.28, 120.00. UV-VIS (CH C1 ) X ax (rel. intensity): 430.9 (1), 466, (0.06), 526.0 (0.07), 567.0 (0.03), 2  2  m  606.0 (0.03), 662.0 (0.03)  Trifluoromethanol-tetraphenylporphyrin (22)  22  Trifluoromethanol-tetraphenylporphyrin  (22)  was  synthesized  modification of the procedure reported by Prakash and Olah."  119  8  according  to  a  All flasks and syringes  Experimental  were flame-dried and cooled under argon.  To a stirring solution of formyl-  tetraphenylporphyrin (21) (25 mgs, 0.039 mmol) in distilled tetrahydrofuran (15 mL) was added TMS-CF3 (1.16 p:L, 0.585 mmol). The mixture was cooled to 0° under argon, and a catalytic amount of T B A F (lOuL , 0.01 mmol) was added. The reaction was monitored by T L C whereby disappearance of the green starting material (R = 0.5) and appearance f  of the reddish, faster moving trifluoromethylated siloxy intermediate (R = 0.78) signaled f  completion of the reaction. Hydrolysis was achieved by addition of 0.5M hydrochloric acid (until the solution takes on a bright green colour). Methylene chloride and a solution of saturated sodium acetate were then added to neutralize the product. The organic layer was separated, the solvent removed in vacuo, and the residue chromatographed on silica gel 60, 70-230 mesh, (50-100% CH Cl /hexanes as gradient eluent) to yield 24.7 mgs 2  2  (89%) of trifuoromethanol-tetraphenylporphyrin. One faint impurity was visible by T L C , a light green spot (Rf = 0.73), but did not impede isolation of the desired product (Rf = 0.34) which moved much slower.  M S L R +LSIMS (matrix: thioglycerol) : Exact mass calculated for C H3|F3N 0 (M+l): 46  4  713.74. Found 713. *H N M R (300 MHz, CDC1 ): 5 9.12 (1H, s, H-7), 8.84 (1H, s, H-8 ), 8.82-8.72 (6H, m, 1  3  H-2,3,12,13, 17, 18), 8.20 (8H, m, H-5 , 10 , 15 , 20 ), 7.76 (12H, m, H-5 ' , 10 ' , 15 ' , 2  2  2  2  3  4  3  4  3  4  20 ' ), 2.99 (1H, br. s., -OH), -2.68 (2H, s, -NH). 3  1 9  4  F N M R (282 MHz, CDC1 ) (coupled) 5 -76.87 (3F, d, 3  UV-VIS (CH C1 ) X 2  2  m a x  -CHOHCF ) 3  (rel. intensity): 420.0 (1), 450 (0.12), 517.1 (0.05), 553.0 (0.02),  596.0 (0.02), 652.0 (0.02)  120  Experimental  Trifluoroacetyl-tetraphenylporphyrin (23)  23  Trifuoroacetyl-tetraphenylporphyrin (23) was synthesized according to a description of the use of tetrapropylammoniumperruthenate (Pr N)(Ru0 ) and N-methylmorpholine 4  4  oxide (NMO) for oxidation of alcohols to ketones by Ley and co-workers. and syringes were flame-dried and cooled under argon. trifluoromethanol-tetraphenylporphyrin  (22)  (250  mgs,  121  All flasks  To a stirring solution of 0.351  mmol)  in distilled  methylene chloride (50 mL) was added N M O (0.46 mL, 2.24 mmol). The mixture was then left to stir at room temperature for 10 minutes, under argon, after which time a catalytic amount of TPAP (25 mgs, 0.07 mmol) was added. The reaction was monitored by T L C whereby disappearance of the dark red starting material (R = 0.4) and f  appearance of the dark green, faster moving trifluoroacetyl-tetraphenylporphyrin (Rf = 0.81) signaled completion of the reaction. The mixture was then washed with water (2 x 50 mL), the organic layer separated and dried through cotton, the solvent removed in vacuo, and the residue chromatographed on silica gel 60, 70-230 mesh, (40-60%  Experimental  CH Cl /hexanes as gradient eluent) to yield 199.3 mgs (74%) of trifluoroacetyl2  2  tetraphenylporphyrin. One substantial impurity was visible by T L C , a purplish band (R  f  = 0.72), directly under the desired product. E A : Calculated for C46H F N40 : C, 77.73, H , 4.11; N, 7.88. Found C, 77.97; H , 4.11; 29  3  N.7.78. M S L R +LSIMS (matrix: thioglycerol) : Exact mass calculated for C H F N 4 0 (M+l): 46  29  3  711.74. Found 711. ]  H N M R (300 MHz, CDC1 ): 5 9.25 (1H, s, H-7), 9.15 (1H, d, H-12), 9.04 (1H, d, H-3), 3  9.00 (2H, d, H-2,13), 8.83 (2H, s, H-17,18), 8.32 (8H, m, H-5 , 10 , 15 , 20 ), 7.81 (12H, 2  2  2  2  m, H - 5 , 10 ' , 15 ' , 20 ' ), -2.43 (2H, s, -NH). 3,4  1 3  3 4  3  4  3  4  C N M R (75 MHz, CDC1 ) (quaternary carbons are unresolved, C = 29): 5 190.06, 3  180.53 (q, J -F = 135 Hz), 142.10, 141.707, 141.51, 141.313, 138.15, 137.70, 136.44, c  136.44, 134.82, 134.73, 134.58, 129.88, 129.39, 128.93, 128.33, 128.33, 127.94, 127.94, 127.00, 126.93, 126.88, 126.85, 122.32, 121.15, 120.61, 120.42, 118.50. 1 9  F N M R (282 MHz, CDC1 ) (coupled) 8 -73.88 (3F, s, -COCF ) 3  UV-VIS (CH C1 ) ? i 2  2  max  3  (rel. intensity) : 429.0 (1), 562.0 (0.07), 562 (0.02), 606.0 (0.02),  665.0 (0.04).  122  Experimental  Trifluoromethyl oxime-tetraphenylporphyrin (24)  s  4  24  Trifuoromethyl  oxime-tetraphenylporphyrin  modified procedure by Brunner et al.  51  (24)  was  synthesized according to a  All flasks and syringes were flame-dried and  cooled under argon. To a stirring solution of trifluoroacetyl-tetraphenylporphyrin (23) (43 mgs, 0.06 mmol) in distilled benzene (15 mL) was added 12 mgs (0.18 mmol) of hydroxylamine hydrochloride (NH OH«HCl). Activated molecular sieves (7A) were also 2  added to the 50 mL RBF. No base was added.  The mixture was then left to reflux  overnight. The reaction was monitored by T L C whereby disappearance of the dark green starting material (23, Rf = 0.72) and appearance of the lighter green, slower moving trifluoromethyl oxime-tetraphenylporphyrin (24, Rf = 0.46) signaled completion of the reaction.  The mixture was then washed with water (2 x 50 mL), the organic layer  extracted with CH2CI2 and dried through cotton, and the solvent removed in vacuo. The crude residue  was  isolated  to yield 26  123  mgs  (60%)  of trifluoromethyl  oxime-  Experimental  tetraphenylporphyrin (24).  One minimal impurity was visible by T L C , a light green band  (R = 0.24), under the desired product, but sufficient amounts could not be isolated for f  characterization. It should also be noted, that on standing the product (24) reverted back to its precursor (23), presumably due to hydrolysis.  M S L R +LSIMS (matrix: thioglycerol) : Exact mass calculated for C H3oF N 0 (M+l): 46  3  5  725.76. Found 725. *H N M R (300 MHz, CDC1 ): 8 9.63 (1H, d, H-12), 8.94 (1H, s, H-7), 8.74 (1H, d, H-3), 3  8.73 (2H, d, H-2,13), 8.64 (2H, s, H-17,18), 8.36 (8H, m, H-5 , 10 , 15 , 20 ), 7.74 (12H, 2  2  2  2  m, H-5 ' , 10 ' , 15 ' , 20 ' ), -1.70 (2H, s, -NH). 3  4  3  4  3  4  UV-VIS (CH C1 ) \ 2  2  3  m a x  4  (rel. intensity) : 435.9 (1), 539.0 (0.04), 582.1 (0.07), 618.0  (0.02), 679.0 (0.04).  124  Appendix: Crystallographic Analysis of 17  EXPERIMENTAL DETAILS  A. Crystal Data  Empirical Formula  C37.50H42.5rjN4O4Cl1.50  Formula Weight  666.45  Crystal Color, Habit  red, block  Crystal Dimensions  0.30 X 0.20 X 0.15 m m  Crystal System  triclinic  Lattice Type  Primitive  Lattice Parameters  a= b = c = a = 0 = 7 =  8.928(2)A 12.015(1) A 17.212(2) A 106.955(7)° 89.471(3)° 106.925(2)°  V = 1683.8(4) A Space G r o u p  Pi (#2)  Z value  2 1.314 g / c m  Fooo  706.00  /z(MoKa)  2.00 c m  3  3  - 1  B . Intensity M e a s u r e m e n t s  Diffractometer  Rigaku/ADSC C C D  Radiation  MOKQ (A = 0.71069 A) graphite monochromated  Detector Aperture  94 m m x 94 m m  D a t a Images  464 exposures @ 58.0 seconds  125  Appendix: Crystallographic Analysis of 17 <f> o s c i l l a t i o n R a n g e  u  oscillation Range  (x=0)  (x=90.0)  0.0 - 190.0° -19.0 - 23.0°  Detector Position  40.32 m m  Detector Swing Angle  -5.57° 50.3°  N o . of Reflections Measured  Total:  12726  U n i q u e : 5576 ( R j Corrections  = 0.054)  n t  Lorentz-polarization Absorption (trans, factors: 0.7284 - 1.0000)  C. Structure Solution and  Refinement  Structure Solution  Direct Methods (SIR97)  Refinement  F u l l - m a t r i x least-squares  Function  Zw(Fo - F c )  Minimized  L e a s t Squares  Weights  p-factor Anomalous  2  w  2  2  = ^ ( k j = l°UFo) +  0.0000 Dispersion  A l l non-hydrogen  N o . O b s e r v a t i o n s (I>0.00<r(I))  5576  No. Variables  504  Reflection/Parameter Ratio  11.06  Residuals: R; R w  0.103 ; 0.154  Goodness of F i t Indicator  1.52  M a x Shift/Error in Final Cycle  0.00  N o . O b s e r v a t i o n s (I>3tr(I))  3026  R e s i d u a l s ( o n F , I>3CT(I)): R ; RW  0.058 ; 0.071  M a x i m u m p e a k i n F i n a l Diff. M a p  0.57  M i n i m u m p e a k i n F i n a l Diff. M a p  -0.62  126  e"/A  3  e"/A  3  atoms  ^Fo ]-i 2  Appendix: Crystallographic Analysis of 17 T a b l e 1. A t o m i c c o o r d i n a t e s a n d B , - , / B 0  atom  x  e ?  occupancy  z  y  B  e 9  occ  Cl(l)  -0.0689(5)  0.5686(3;  0.4369(2)  9.9(1)  1/2  Cl(2)  0.1062(7)  0.4078(6;  0.4550(5)  8.5(2)  1/4  Cl(3)  -0.0555(6)  0.5084(5;  0.5824(2)  12.6(1)  1/2  Cl(4)  0.1948(7)  0.5196(6;  0.4847(3)  7.0(2)  1/4  0(1)  0.4910(5)  0.3519(3;  0.4987(2)  7.37(9)  0(2)  0.2743(4)  0.2218(3;  0.4258(2)  5.54(8)  0(3)  -0.1655(5)  0.0120(3;  0.3821(2)  7.8(1)  0(4)  -0.2405(3)  0.1773(2]  0.4215(1)  4.64(7)  N(21)  0.0958(3)  0.2300(2;  -0.0914(1)  2.11(6)  N(22)  0.4388(3)  0.3288(2;  -0.0601(1)  2.07(6)  N(23)  0.4081(3)  0.2691(2]  0.0913(1)  1.97(6)  N(24)  0.0657(3)  0.1684(2]  0.0604(1)  2.08(6)  C(l)  -0.0629(4)  0.1800(3]  -0.0978(2)  2.23(7)  • C(2)  -0.1324(4)  0.1729(3]  -0.1771(2)  2.27(7)  C(3)  -0.0105(4)  0.2193(3]  -0.2177(2)  2.34(7)  C(4)  0.1318(4)  0.2563(3)  -0.1629(2)  2.07(7)  C(5)  0.2820(4)  0.3100(3]  -0.1819(2)  2.20(7)  C(6)  0.4224(4)  0.3459(3]  -0.1345(2)  2.09(7)  C(7)  0.5746(4)  0.4095(3]  -0.1523(2)  2.01(7)  C(8)  0.6793(4)  0.4314(3)  -0.0878(2)  2.20(7)  C(9)  0.5936(4)  0.3783(3)  -0.0294(2)  2.07(7)  C(10)  0.6507(4)  0.3768(3]  0.0444(2)  2.13(7)  C(ll)  0,5657(4)  0.3274(3]  0.1009(2)  1.94(7)  C(12)  0.6348(4)  0.3348(3]  0.1802(2)  2.07(7)  127  Appendix: Crystallographic Analysis of 17  T a b l e 1. A t o m i c c o o r d i n a t e s a n d B j , / B 0  atom  X  e 9  a n d o c c u p a n c y (continued)  z  y  C(13)  0.5148(4)  0.2797(3)  0.2175(2)  1.98(6)  C(14)  0.3739(4)  0.2378(3)  0.1612(2)  2.06(7)  C(15)  0.2254(4)  0.1776(3)  0.1783(2)  2.03(7)  C(16)  0.0842(4)  0.1446(2)  0.1322(2)  1.98(6)  C(17)  -0.0690(4)  0.0839(3)  0.1503(2)  2.23(7)  C(18)  -0.1762(4)  0.0729(3)  0.0895(2)  2.25(7)  C(19)  -0.0894(4)  0.1265(3)  0.0322(2)  2.10(7)  C(20)  -0.1492(4)  0.1333(3)  -0.0400(2)  2.24(7)  C(21)  -0.3009(4)  0.1255(3)  -0.2025(2)  3.02(8)  C(22)  -0.3638(4)  0.0663(3)  -0.2773(2)  4.11(9)  C(31)  -0.0100(5)  0.2282(4)  -0.3028(2)  3.05(8)  C(71)  0.6046(4)  0.4479(3)  -0.2267(2)  2.49(7)  C(72)  0.5901(5)  0.3554(3)  -0.3083(2)  3.87(9)  C(73)  0.7463(5)  0.4369(4)  -0.2703(2)  4.9(1)  C(81)  0.8507(4)  0.4990(3)  -0.0762(2)  2.73(8)  C(121)  0.8046(4)  0.3936(4)  0.2090(2)  2.99(8)  C(131)  0.5145(4)  0.2658(3)  0.3015(2)  2.43(7)  C(132)  0.4402(4)  0.3522(3)  0.3623(2)  3.02(7)  C(133)  0.4073(6)  0.3130(3)  0.4375(2)  4.2(1)  C(134)  0.2329(7)  0.1723(6)  0.4926(3)  8.3(2)  C(171)  -0.1033(4)  0.0420(3)  0.2249(2)  2.44(7)  C(172)  -0.1337(4)  0.1401(3)  0.2960(2)  3.42(8)  C(173)  -0.1808(4)  0.1007(3)  0.3695(2)  3.17(8)  C(174)  -0.2875(6)  0.1516(4)  0.4967(2)  5.5(1)  128  Appendix: Crystallographic Analysis of 17  Table 1. Atomic coordinates and Bi /B SO  atom  C(181)  X  -0.3493(4)  eq  and occupancy (continued)  y  z  B  eq  0.0148(4)  0.0812(2)  3.08(8)  0.5000  16.1(6)  C(200)  0.0000  0.5000  H(l)  0.356(4)  0.294(3)  -0.036(2)  3.3(8)  H(2)  0.144(4)  0.205(3)  0.035(2)  3.2(7)  H(3)  0.2891  0.3233  -0.2353  2.6226  H(4)  0.7640  0.4140  0.0586  2.4632  H(5)  0.2196  0.1565  0.2295  2.2980  H(6)  -0.2638  0.1031  -0.0514  2.5840  H(7)  -0.3447  0.0697  -0.1710  3.5190  H(8)  -0.3400  0.1960  -0.1858  3.5190  H(9)  -0.4774  0.0445  -0.2779  4.8515  H(10)  -0.3261  0.1184  -0.3124  4.8515  H(H)  -0.3312  -0.0080  -0.2975  4.8515  H(12)  -0.114(5)  0.227(3)  -0.323(2)  5.0(8)  H(13)  0.010(4)  0.152(4)  -0.344(2)  5.1(8)  H(14)  0.060(5)  0.303(4)  -0.311(2)  4.9(9)  H(15)  0.5734  0.5195  -0.2267  2.8432  H(16)  0.5603  0.2684  -0.3124  4.6008  H(17)  0.5442  0.3659  -0.3558  4.6008  H(18)  0.8051  0.5013  -0.2928  5.6488  H(19)  0.8212  0.4038  -0.2494  5.6488  H(20)  0.882(3)  0.544(3)  -0.116(2)  2.4(6)  H(21)  0.918(5)  0.448(4)  -0.086(2)  5.0(9)  H(22)  0.881(4)  0.548(4)  -0.025(2)  4.5(8)  129  Appendix: Crystallographic Analysis of 17  Table 1. Atomic coordinates and B i / B , and occupancy (continued) S 0  X  atom  e  z  y  H(23)  0.868(5)  0.345(4)  0.183(2)  5.4(9)  H(24)  0.846(4)  0.473(3)  0.201(2)  3.8(7)  H(25)  0.823(4)  0.403(3)  0.269(2)  4.4(8)  H(26)  0.4547  0.1819  0.2978  2.8726  H(27)  0.6234  0.2825  0.3217  2.8726  H(28)  0.5155  0.4352  0.3775  3.5526  H(29)  0.3438  0.3519  0.3367  3.5526  H(30)  0.2222  0.2349  0.5407  10.1717  H(31)  0.1341  0.1047  0.4773  10.1717  H(32)  0.3166  0.1390  0.5049  10.1717  H(33)  -0.1969  -0.0297  0.2115  2.9551  H(34)  -0.0136  0.0198  0.2408  2.9551  H(35)  -0.0366  0.2098  0.3117  4.0382  H(36)  -0.2173  0.1673  0.2782  4.0382  H(37)  -0.3295  0.2151  0.5299  6.5600  H(38)  -0.3697  0.0721  0.4839  6.5600  H(39)  -0.1972  0.1485  0.5268  6.5600  H(40)  -0.386(4)  -0.021(3)  0.124(2)  4.6(8)  H(41)  -0.415(5)  0.076(4)  0.081(2)  7(1)  H(42)  -0.387(5)  -0.058(4)  0.031(2)  5.4(9)  H(43)  -0.0465  0.4710  20.1154  B  eq  0.4130  = -7r (t/u(aa') + Un{Wf + U {cc*) + 2V aa'bb' c o s + 2U aa'cc* cos/3 + 2U zbb'cc cosa) 2  2  33  2  l2  130  7  13  2  t  References  References  1.  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