Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Electron nuclear double resonance studies of free radicals trapped in x-irradiated single crystals of… Singh, Baishnab Charan 1980

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

Item Metadata

Download

Media
831-UBC_1980_A1 S55_4.pdf [ 6.7MB ]
Metadata
JSON: 831-1.0047164.json
JSON-LD: 831-1.0047164-ld.json
RDF/XML (Pretty): 831-1.0047164-rdf.xml
RDF/JSON: 831-1.0047164-rdf.json
Turtle: 831-1.0047164-turtle.txt
N-Triples: 831-1.0047164-rdf-ntriples.txt
Original Record: 831-1.0047164-source.json
Full Text
831-1.0047164-fulltext.txt
Citation
831-1.0047164.ris

Full Text

ELECTRON NUCLEAR DOUBLE RESONANCE STUDIES OF FREE RADICALS TRAPPED IN X-IRRADIATED SINGLE CRYSTALS OF PHENAZINE, HIPPURIC ACID AND N-ACETYLGLYCINE by BAISHNAB CHARAN SINGH B.Sc.(Hons.)-1968, M.Sc.-1970, Ph.D.-1977, The U n i v e r s i t y of Utkal, INDIA A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of CHEMISTRY We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA March, 1980 © BAISHNAB CHARAN SINGH, 1980 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i I m e n t o f the r e q u i r e m e n t s f o r an advanced degree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that 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 s t u d y . 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 o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It i s u n d e r s t o o d that c o p y i n g o r p u b l i c a t i o n o f 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 o f a The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date JS" , l j , Supervisor: C. A. McDowell x i ABSTRACT The technique of electron nuclear double resonance (ENDOR) spectroscopy has been used to determine the i d e n t i t y and structure of r a d i c a l s trapped i n room temperature X - i r r a d i a t e d s i n g l e c r y s t a l s of some b i o l o g i c a l l y important compounds, namely, phenazine, hippuric acid and N-acetylglycine. The ESR spectra of phenazine which had not previously been reported, were too complex to be analyzed. However, the high r e s o l u t i o n of ENDOR has made i t possible to study a l l the proton hyperfine i n t e r a c t i o n s i n d e t a i l . The r a d i c a l s are formed as a r e s u l t of the addition of atomic hydrogen to the nitrogen atom of phenazine molecule. The appearance of twice the number of c e r t a i n ENDOR l i n e s than expected from the c r y s t a l symmetry was interpreted by postulating that the r a d i c a l was s t a b i l i z e d i n two d i f f e r e n t conformations. In hippuric acid (N-Benzoylglycine), the stable N-Benzoylaminomethyl r a d i c a l formed by room temperature X - i r r a d i a t i o n of sing l e c r y s t a l s has been i d e n t i f i e d and i t s structure established by de t a i l e d ENDOR studies at 77 K. Besides proton s i g n a l s , ENDOR t r a n s i t i o n s a r i s i n g from the 14 hyperfine and quadrupole int e r a c t i o n s of the N nucleus were detected and a de t a i l e d analysis i s presented. The assignment of exchangeable protons has been accomplished by use of p a r t i a l l y deuterated c r y s t a l s . The r a d i c a l i s most l i k e l y formed from oxidised hippuric acid by depro-tonation and subsequent decarboxylation. E l e c t r o n i c and molecular structure of the stable r a d i c a l produced by room temperature X - i r r a d i a t i o n of si n g l e c r y s t a l s of N-acetylglycine has i i i been reinvestigated by detailed ESR and ENDOR studies. The results indicate that the radical structure and the nature of the wavefunction of the unpaired electron are significantly different from those reported in a previous 'negative' ENDOR study. In agreement with earlier ESR and ENDOR studies, i t is found that the stable radical at room temperature is CH^ CONHCHCOOH. ENDOR lines due to the a-proton, three equivalent methyl protons and six exchangeable protons have been identified and the hyperfine tensors determined. According to the findings, the radical exists in four slightly different conformations. Isotopic substitution with and deuteration of the exchangeable protons were helpful in the analysis of the complex ESR and ENDOR spectra. It has been conclusively shown that the multiplet structure in the ESR spectrum of irradiated N-acetylglycine arises from the interaction of the unpaired electron with 14 protons of the methyl group and the N nucleus. It has been also demonstrated that the radical concentration in the crystal has to be very carefully con-trolled for the success of an ENDOR investigation. In order to confirm the radical identification INDO-MO calculations have been performed on a l l the radicals reported here. Excellent agreement has been obtained between the observed spin densities and those computed by the INDO-MO calculations. i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGMENTS x i CHAPTER ONE: INTRODUCTION 1 CHAPTER TWO: THEORETICAL 11 2.1. Introduction 11 2.2. The Spin Hamiltonian 12 2.2.1. The E l e c t r o n i c Zeeman Interaction 12 2.2.2. The Nuclear Zeeman Interaction 15 2.2.3. The Hyperfine Interaction 16 2.2.3.1. The Dipolar Interaction 16 2.2.3.2. The Isotropic Hyperfine Coupling 18 2.2.4. The Quadrupole Interaction 20 2.3. Theory of ESR 22 2.3.1. Energy Levels for S=%, l=h system 23 2.3.2. ESR Selection Rules 28 2.3.3. Thermal Equilibrium and Spin Relaxation 30 2.3.4. Hyperfine Interaction with More Than One Nucleus 32 2.4. Theory of ENDOR 34 2.4.1. The Isotropic Hyperfine Interaction 34 2.4.2. The Anisotropic Hyperfine Interaction 37 2.4.3. The Quadrupole Interaction 39 2.4.4. ENDOR Spectrum f or an Electron Coupled to More Than One Nucleus 41 2.4.5. Determination of Spin Hamiltonian Parameters 42 CHAPTER THREE: INTERPRETATION OF SPIN HAMILTONIAN PARAMETERS 44 3.1. Introduction 44 3.2. Hyperfine Coupling Tensors for a-Protons 44 3.2.1. The Isotropic Hyperfine Coupling 44 3.2.2. The Anisotropic Hyperfine Tensor 45 3.3. Hyperfine Coupling Tensors f o r B-Protons 47 3.4. Interactions with Distant Protons 49 3.5. I n t e r a c t i ons of Protons Bonded to Atoms Othfir Than Carbon 49 V Page 3.6. Hyperfine Coupling f o r Nitrogen Atoms 50 3.6.1. The Isotropic 1 % Hyperfine Coupling 50 3.6.2. The Anisotropic ^ N Hyperfine Tensor 50 3.7. 1*N Quadrupole Interactions 51 3.8. Spin Density Calculations 52 CHAPTER FOUR: EXPERIMENTAL METHODS 55 4.1. Introduction 55 4.2. The ENDOR Spectrometer 55 4.3. The ENDOR technique 59 4.3.1. C r y s t a l Alignment Through Site S p l i t t i n g 59 4.3.2. Operation of The ENDOR Spectrometer 60 4.3.3. Data Reduction 61 CHAPTER FIVE: ENDOR STUDIES OF X-IRRADIATED PHENAZINE 63 5.1. Introduction 63 5.2. Experimental 65 5.3. Experimental Results 67 5.4. Assignment of the Couplings and Radical I d e n t i f i c a t i o n 73 5.4.1. Radical(I) 73 5.4.2. Radical(II) 78 5.5. Spin Densities 79 5.6. D i r e c t i o n Cosines 80 5.7. Discussion 83 CHAPTER SIX: 1 4 N AND 1H ENDOR STUDIES OF RADIATION DAMAGE IN HIPPURIC ACID 86 6.1. Introduction 86 6.2. Experimental 87 6.3. Experimental Results 91 6.4. Discussion: 96 6.4.1. a-proton Interactions 96 6.4.2. l^N Hyperfine and Quadrupole Interactions 103 6.4.3. Weakly Coupled Protons 107 6.5. Conclusion 113 CHAPTER SEVEN: ENDOR STUDY OF X-IRRADIATED SINGLE CRYSTALS OF N-ACETYLGLYCINE - A REINVESTIGATION 115 7.1. Introduction 115 7.2. Experimental 118 7.3. Results and Discussion 120 7.3.1. Experimental Results 120 7.3.2. Discussion 131 7.4. Concluding remarks 141 v i Page CHAPTER EIGHT: CONCLUSIONS 143 BIBLIOGRAPHY 149 v i i LIST OF TABLES Table Page 1 Proton hyperfine i n t e r a c t i o n tensors for r a d i c a l ( I ) i n X - i r r a d i a t e d s i n g l e c r y s t a l of phenazine. 74 2 Proton hyperfine i n t e r a c t i o n tensors for r a d i c a l ( I I ) i n X - i r r a d i a t e d s i n g l e c r y s t a l of phenazine. 75 3 Spin d e n s i t i e s on the r i n g atoms of N-hydroacridinyl and N-hydrophenazinyl r a d i c a l s . 81 4 Hyperfine coupling tensors for the a-protons of N-BenzoyLaminomethyl r a d i c a l . 100 5 Nitrogen hyperfine and quadrupole coupling tensors i n N-Benzoylaminomethyl r a d i c a l . 104 6 Hyperfine coupling tensors for the weakly coupled protons i n N-Benzoylaminomethyl r a d i c a l . 108 7 Comparison of experimental and calculated (INDO) unpaired electron d e n s i t i e s i n N-Benzoylaminomethyl r a d i c a l . 112 8 Hyperfine coupling tensors for a-proton and methyl protons of hydrogen abstraction r a d i c a l i n N-acetylglycine. 127 9 Hyperfine coupling tensors for the weakly coupled protons of hydrogen abstraction r a d i c a l i n N-acetylglycine. 137 10 Comparison of experimental and INDO unpaired electron d e n s i t i e s i n the hydrogen abstraction r a d i c a l of N-acetylglycine . 140 v i i i LIST OF FIGURES Figure Page 1 Energy l e v e l s of an S=ht I=% system, with f i r s t order ESR t r a n s i t i o n s . 29 2 Energy l e v e l s for ENDOR experiment i n an S=h, l=h system (a) vN>a/2 (b) a/2>vN. 35 3 Energy l e v e l s for an S=h, 1=1 system. 40 4 Block diagram of the ENDOR spectrometer. 56 5 C r y s t a l morphology of phenazine with reference axes. 66 6 Molecular structure and numbering of the ri n g atoms of phenazine. 68 7 (a) F i r s t d e r i v a t i v e X-band ESR spectrum at room temperature and (b) ENDOR spectrum at 77 K of the N-hydro-phenazinyl r a d i c a l i n an X-i r r a d i a t e d s i n g l e c r y s t a l of phenazine for the magnetic f i e l d p a r a l l e l to the b-axis. 69 8 Angular dependence of the ENDOR frequencies of r a d i c a l ( I ) i n an X- i r r a d i a t e d s i n g l e c r y s t a l of phenazine. 71 9 Angular v a r i a t i o n of the ENDOR frequencies of r a d i c a l ( I I ) i n an X-i r r a d i a t e d s i n g l e c r y s t a l of phenazine. 72 10 Spin density d i s t r i b u t i o n i n (a) N-H r a d i c a l s i n molecule A and B of ac r i d i n e and (b) NQQ^-H and N^^-H r a d i c a l s i n phenazine. 77 11 C r y s t a l morphology and axis system of a sin g l e c r y s t a l of hippuric a c i d . 89 12 Molecular structure of hippuric a c i d . 90 13 Typ i c a l ESR spectra of X- i r r a d i a t e d s i n g l e c r y s t a l s of hippuric a c i d . 92 i x Figure Page 14 ENDOR spectra due to the a-protons i n N-Benzoylaminomethyl r a d i c a l i n an X-i r r a d i a t e d s i n g l e c r y s t a l of hippuric acid at 77 K . 93 15 ENDOR spectra due to the weakly coupled protons i n an X - i r r a d i a t e d s i n g l e c r y s t a l of hippuric a c i d . 94 14 16 ESR and ENDOR spectra of N i n X- i r r a d i a t e d s i n g l e c r y s t a l of hippuric acid f o r the magnetic f i e l d p a r a l l e l to a-axis. 95 17 Angular v a r i a t i o n of the high frequency branch of the ENDOR frequencies of the a-protons i n an X - i r r a d i a t e d s i n g l e c r y s t a l of hippuric a c i d . 97 18 Angular v a r i a t i o n of the ENDOR frequencies of the weakly coupled protons i n an X- i r r a d i a t e d s i n g l e c r y s t a l of hippuric a c i d . 98 14 19 Angular v a r i a t i o n of N ENDOR frequencies i n an X - i r r a d i a t e d s i n g l e c r y s t a l of hippuric a c i d . 99 20 Structure of N-Benzoylaminomethyl r a d i c a l 102 21 ENDOR spectrum i n the v i c i n i t y of vp from an X - i r r a d i a t e d s i n g l e c r y s t a l of hippuric a c i d grown from heavy water for the magnetic f i e l d p a r a l l e l to a-axis. 109 22 C r y s t a l l i n e form and cr y s t a l l o g r a p h i c axes of N-acetylglycine. 119 23 Molecular structure and numbering system of N-acetylglycine. 121 24 F i r s t d e r i v a t i v e ESR spectrum of an X- i r r a d i a t e d s i n g l e c r y s t a l of N-acetylglycine for the magnetic f i e l d p a r a l l e l to a*-axis. 123 25 High frequency branch of the ENDOR spectrum due to the a-proton i n t e r a c t i o n i n an X-i r r a d i a t e d s i n g l e c r y s t a l of N-acetylglycine f o r the magnetic f i e l d making 5° with b-axis i n the bc-plane. 124 26 Angular v a r i a t i o n of the high frequency branch of the ENDOR frequencies for the a-proton i n an X-ir r a d i a t e d s i n g l e c r y s t a l of N-acetylglycine. 125 X Figure Page 27 ENDOR spectra i n the v i c i n i t y of vp from an X - i r r a d i a t e d s i n g l e c r y s t a l of N-acetylglycine for the magnetic f i e l d p a r a l l e l to a*-axis: (a) c r y s t a l grown from (b) c r y s t a l grown from D2O. 126 28 Angular v a r i a t i o n of the ENDOR frequencies of the weakly coupled protons i n an X- i r r a d i a t e d si n g l e c r y s t a l of N-acetylglycine. 130 29 Second d e r i v a t i v e ESR spectra of deuterated N-acetylglycine for the magnetic f i e l d p a r a l l e l to a*-axis: (a) -^N-acetylglycine (b) ace t y l g l y c i n e . 134 x i ACKNOWLEDGMENTS I would like to express my deep sense of gratitude to my research director, Professor C. A. McDowell, for his interest and guidance in a l l phases of the present work. I am immensely indebted to my new supervisor, Dr. J. B. Farmer, for meticulously going through the manuscript and making valuable suggestions in the absence of Dr. McDowell because of his serious il l n e s s . I am extremely grateful to Dr. V. P. Chacko for kindly introducing me to the f i e l d of ENDOR spectroscopy and providing his assistance and collaboration throughout the course of this work. Sincere thanks are due to Dr. F. G. Herring for the use of his ESR spectrometer and many valuable suggestions after reading the manuscript of the paper on N-acetylglycine. I wish to thank Dr. S. Rettig of this department for identifying the crystallographic axes in hippuric acid single crystal by X-ray diffraction and Mr. K. Sukal of the electronic shop of this department for providing expert maintenance of the spectrometer. Thanks are also due to Anna for typing the thesis and Monica for tracing the figures. I am grateful to the Canadian Commonwealth Fellowship Committee for the award of a fellowship and the department of chemistry at U.B.C. for providing teaching assistantship. I wish to thank the education department of the Government of Orissa (INDIA) for granting me the leave of absence. Lastly I extend my deep sense of appreciation to my wife, Gita, daughters, Pinky, Chumky and Milky, a l l my relatives and friends for their patience and understanding. x i i DEDICATED TO THE LOVING MEMORY OF MY FATHER CHAPTER ONE INTRODUCTION Radiation chemistry i s the study of the chemical e f f e c t s of high energy i o n i z i n g r a d i a t i o n . High energy r a d i a t i o n includes electromagnetic r a d i a t i o n (x-rays and y - r a y s ) , p a r t i c l e s ( a - p a r t i c l e s , 6 - p a r t i c l e s or electrons, protons and neutrons) and f i s s i o n fragments. Radio chemistry, which i s often confused with r a d i a t i o n chemistry, however, deals with the chemistry of rad i o a c t i v e elements and with the use of radioactive tracers and the measurement of t h e i r r a d i o a c t i v i t y . Nuclear chemistry, on the other hand, i s concerned with nuclear transformations, p a r t i c u l a r l y f i s s i o n products and transuranium elements. The p r i n c i p a l c h a r a c t e r i s t i c of high energy r a d i a t i o n i s that i t causes i o n i z a t i o n and subsequent chemical change during i t s passage through matter. There i s s u f f i c i e n t energy a v a i l a b l e to break any bond, but i n p r a c t i c e c e r t a i n bonds may be broken p r e f e r e n t i a l l y . The i o n i z i n g photon or p a r t i c l e and the displaced electron are often both capable of producing further i o n i z a t i o n . Thus one incident photon may a f f e c t many thousands of molecules. Out of numerous uses of r a d i a t i o n chemistry, b i o l o g i c a l e f f e c t s of r a d i a t i o n have always been of i n t e r e s t . Studies i n t h i s f i e l d range from p h y s i o l o g i c a l and biochemical e f f e c t s i n l i v i n g animals, through those on tissues and other body components to the chemistry of proteins and amino acids. One aim of such work i s the treatment of people inadvertently exposed to i r r a d i a t i o n . Radiation b i o l o g i c a l studies are also helping to reveal the chemistry of many l i f e processes and the formation mechanism of various r a d i a t i o n products. Magnetic resonance spectroscopy has provided a new and powerful approach to the study of r a d i a t i o n e f f e c t s . Most organic molecules contain an even -2-number of electrons and hence are diamagnetic. The net magnetic moment of the nonvalence electrons i n the inner f i l l e d s h e l l s of each atom or of any unshared p a i r of valence electrons i s zero. The net magnetic moment of each pair of valence electrons involved i n bond formation i s also zero, since i t i s axiomatic i n quantum chemistry that covalent bonds are formed by p a i r s of electrons of opposite spin. The e f f e c t of i o n i z i n g r a d i a t i o n i s to disrupt t h i s p a i r i n g . Obviously unpairing occurs during i o n i z a t i o n or whenever a covalent bond i s ruptured. A molecule having an unpaired electron i s c a l l e d a free r a d i c a l . Free r a d i c a l s are necessar i l y para-magnetic. The unpaired electrons i n the damaged species i n t e r a c t magnetically with any nucleus i n the environment which possesses a magnetic moment. This i s e s p e c i a l l y true i n the organic realm where the nearly ubiquitous proton and other magnetic n u c l e i are often the o r i g i n of magnetic i n t e r a c t i o n with the unpaired electron. The f i e l d of magnetic resonance began with the demonstration of Electron Spin Resonance (ESR) spectroscopy by E. Zavoisky"*" i n 1945 who observed the resonance absorption of energy by unpaired electrons i n the presence of a magnetic f i e l d . I n i t i a l l y only paramagnetic t r a n s i t i o n metal complexes were studied because the s e n s i t i v i t y of the instrumentation was very low. 2 By the mid 1950's, however, ESR spectra of organic free r a d i c a l s trapped 3 i n p o l y c r y s t a l l i n e s o l i d s had been observed. In 1956, Uebersfeld and Erb showed that i t was possible, by high energy i r r a d i a t i o n , to form r a d i c a l s i n s i d e a single c r y s t a l and for them to be trapped i n s p e c i f i c o r i e n t a t i o n s . The ESR spectrum of such an oriented r a d i c a l y i e l d s more information about i t s structure than can be obtained from spectra of the r a d i c a l i n solution or i n a p o l y c r y s t a l l i n e s o l i d . The magnetic moment of the unpaired electron i s coupled to the magnetic moments of n u c l e i i n i t s v i c i n i t y . This coupling -3-i s responsible for the hyperfine structure of an ESR spectrum and i s the source of most of the s t r u c t u r a l information obtainable from t h i s spectrum. One component of t h i s coupling i s analogous to the c l a s s i c a l d i p o l e-dipole i n t e r a c t i o n and i s d i r e c t i o n dependent, or a n i s o t r o p i c . The r o t a t i o n a l motion of a r a d i c a l i n s o l u t i o n averages out the anisotropic component so that only the i s o t r o p i c coupling remains. In p o l y c r y s t a l l i n e s o l i d s , the r a d i c a l s are randomly oriented with respect to the magnetic f i e l d , so that the anisotropic contribution to t h i s coupling leads to a b l u r r i n g of the spectrum. ESR technique has been successful i n i d e n t i f y i n g the major r a d i c a l s formed i n s i n g l e c r y s t a l s of large number of organic compounds on the basis of the largest hyperfine i n t e r a c t i o n s . However, d e t a i l e d information on the e l e c t r o n i c structure of the r a d i c a l s could not always be obtained, because important intramolecular hyperfine s p l i t t i n g s were buried within the ESR line-width. Furthermore, more than one r a d i c a l was often formed i n these c r y s t a l s and t h e i r spectra could not always be distinguished. In general, the r a d i c a l s produced by i o n i z i n g r a d i a t i o n i n a l i p h a t i c compounds y i e l d w ell resolved ESR spectra and hence they have been studied i n d e t a i l . In aromatic systems, however, the unpaired electron i s extensively d e l o c a l i z e d and hence coupled to a large number of n u c l e i , a s i t u a t i o n which generally y i e l d s inhomogeneously broadened ESR spectra. For these compounds i n the s o l i d state, the ESR spectra appear very poorly resolved and thus y i e l d l i t t l e information. 4 In 1956 Feher demonstrated the technique of Electron Nuclear DOuble Resonance (ENDOR) i n h i s famous work on defects i n s i l i c o n . The much better r e s o l u t i o n of ENDOR spectroscopy makes i t possible to study the hyperfine i n t e r a c t i o n s of n u c l e i which i n t e r a c t magnetically with unpaired electrons i n both a l i p h a t i c and aromatic r a d i c a l s . One of the main ap p l i c a t i o n s of - 4 -the ENDOR technique i s r e s o l u t i o n enhancement. A c l a s s i c example of t h i s i s Holton, Blum and S l i c h t e r ' s study of the F-centres i n l i t h i u m fluoride"'. Although the ESR spectrum showed one broad stru c t u r e l e s s l i n e , the ENDOR technique allowed them to resolve the hyperfine s p l i t t i n g s out to the seventh coordination sphere. Furthermore, the ENDOR spectrum i s inherently simpler than i t s parent ESR spectrum whose hyperfine structure has approximately 2 n l i n e s for n i n t e r a c t i n g protons. The ENDOR spectrum contains only two l i n e s f o r each group of equivalent protons. SHORT REVIEW ON ENDOR OF STABLE ORGANIC RADICALS; The science of r a d i a t i o n chemistry attempts to e s t a b l i s h the mechanism by which v a r i e t i e s of free r a d i c a l s are formed. By mechanism, we mean the en t i r e sequence of physi c a l and chemical events i n i t i a t e d by the primary event i n which energy i s transferred to matter from a beam of r a d i a t i o n . As mechanisms become c l e a r , the ph y s i c a l , chemical and b i o l o g i c a l consequences of r a d i a t i o n become correspondingly more comprehensible. The ENDOR technique i s being extensively employed for the study of organic and b i o l o g i c a l r a d i c a l s . The subject was f i r s t reviewed by Kwiram^ i n 1971 and subsequently discussed i n two excellent books and some reviews Four types of r a d i c a l s are encountered i n s o l i d organic matter as a r e s u l t of i o n i z i n g r a d i a t i o n . 1. Removal of si n g l e electrons from electron pairs of nonradical molecules (oxidation). M -^S-* [M-] + (Cation) 2. Addition of sing l e electrons to neutral nonradical molecules (Reduction). M [M> ]~ (Anion) -5-3. D i s s o c i a t i o n of covalently bonded atomic hydrogen from nonradical molecules. MH M-4. Association of atomic hydrogen on unsaturated positions of nonradical molecules. +H M > MH-Of these, the i o n i c r a d i c a l s r e s u l t i n g from electron removal or attachment are usually unstable at room temperature and have been found to be stable only at low temperatures. The present thesis deals with the detection and ch a r a c t e r i z a t i o n of stable r a d i c a l s trapped i n organic compounds at room temperature. Hence we s h a l l not discuss the i o n i c r a d i c a l s r e s u l t i n g from low temperature i r r a d i a t i o n . Rather, we s h a l l b r i e f l y discuss here the a p p l i c a t i o n of ENDOR to stable r a d i c a l s mainly from carboxylic acids, amino acids, n u c l e i c acids and aromatics. Carboxylic Acids: The f i r s t observations of ENDOR signals from organic free r a d i c a l s 11 12 were made as early as 1961 by Cole, H e l l e r and Lambe and Kwiram on a series of di c a r b o x y l i c acids. In succinic a c i d , Cole et a l . " ^ have observed the ENDOR l i n e s corresponding to the hyperfine s p l i t t i n g of the r a d i c a l 13 HOOCCHC^COOH, whereas Kwiram and Hyde have suc c e s s f u l l y studied the weak int e r a c t i o n s which caused inhomogeneously broadened ESR spectrum of the 14 r a d i c a l HOOCCQ^gCHCOOH i n a d i p i c a c i d . Kwiram himself has made a deta i l e d study of r a d i c a l s formed on x - i r r a d i a t i o n of g l u t a r i c a c i d . Out of two r a d i c a l s , one was removed by u.v. i r r a d i a t i o n , so that the remaining -6-H0OCCHCH2CO0H could be studied without interference. Wells and Ko''""' have reported temperature dependent sequence of molecular fragmentation processes occurring i n x - i r r a d i a t e d s i n g l e c r y s t a l s of creatine monohydrate, a molecule with carboxylic and guanidine structures. The primary reduction product was found to undergo series of changes leading to deamination followed by hydrogen abstraction and the i n i t i a l oxidized species was found to decarboxylate forming stable r a d i c a l s at ambient temperature. Recently"*"^ free r a d i c a l s produced by i r r a d i a t i n g s i n g l e c r y s t a l s of c i t r i c a c i d , a t r i c a r b o x y l i c a c i d , has been studied by ENDOR. At room temperature, two stable r a d i c a l s were i d e n t i f i e d . One of these was the oxidized species produced by decarboxylation i n a d i f f e r e n t conformation from that at low temperature and the other r a d i c a l showed coupling to only one alpha-hydrogen and thus i t was a hydrogen abstraction r a d i c a l . Amino acids: There has been considerable e f f o r t to understand the mechanism involved i n the production of r a d i a t i o n damage i n amino acids because of t h e i r b i o -l o g i c a l importance i n the synthesis of proteins. Box and coworkers"*"^ have studied the r a d i c a l formation i n glycine hydrochloride x - i r r a d i a t e d at 4.2K. They have shown that upon warming a neutral r a d i c a l -C^ COOn. i s formed. 18 Recently Welter et a l . have reported an ENDOR i n v e s t i g a t i o n of the structure of glycine r a d i c a l i n x - i r r a d i a t e d t r i g l y c i n e sulphate s i n g l e c r y s t a l s at 77 and 300K. ENDOR spectra showed the l i n e s of one a-proton and three nonequivalent 8-protons i n d i c a t i n g strongly hindered r o t a t i o n of the amino group. The structure of the r a d i c a l was also confirmed by INDO ca l c u l a t i o n s . On the other hand, three stable r a d i c a l s have been i d e n t i f i e d 19 i n the study of r a d i a t i o n damage products formed i n 6-alanme * at room -7-temperature. From c o n t r o l l e d warming experiments, the authors have suggested that the primary oxidation and reduction products stable at low temperature decay into the more stable room temperature products. H i s t i d i n e i s one of the more common of the b i o l o g i c a l l y important amino acids characterized by an imidazole r i n g that often contributes importantly to b i o l o g i c a l function. ENDOR spectroscopy was used to characterize more completely the f i n a l hydrogen adduct r a d i c a l obtained on 20 warming the c r y s t a l s i r r a d i a t e d at 4.2K to room temperature . The unpaired electron i s deloc a l i z e d over the imidazole r i n g and experiences a large i n t e r a c t i o n with 8-protons v i a hyperconjugation. Detailed ENDOR i n v e s t i -21 gation on x - i r r a d i a t e d L-asparagine hydrate was undertaken, since ESR data show a great deal of unresolved hyperfine structure. From the agree-ment of experimental r e s u l t s with INDO c a l c u l a t i o n s , i t was concluded that the dominant free r a d i c a l at room temperature i s l^NCOCHCH (NH_)C0„. 22 By x - i r r a d i a t i n g a-amino i s o b u t y r i c acid at 77K, Wells and Box were able to study three d i s t i n c t r a d i c a l s which appeared sequentially on warming. At room temperature, the ENDOR spectrum consists of four l i n e s which are att r i b u t e d to the protons of one r o t a t i n g methyl group and three nonequivalent protons of a nonrotating methyl group. Similar behaviour was noted by Box 23 et a l . i n the study of v a l i n e . They also obtained hyperfine coupling parameters f o r the nitrogen nucleus. 24 Whelan used ENDOR to confirm C-H bond s c i s s i o n product i n glutamic acid and reported the hyperfine i n t e r a c t i o n s f o r the three strongly coupled 25 protons. Castleman and Moulton have examined Serine by x - i r r a d i a t i n g at 77K and i d e n t i f i e d two stable r a d i c a l s by subsequently warming to room 26 temperature. A more d e t a i l e d study on serine s i n g l e c r y s t a l x - i r r a d i a t e d at 300K revealed the ENDOR t r a n s i t i o n of nitrogen i n one of the two stable r a d i c a l s . -8-One of the simplest substituted amino acids with a peptide bond i s N-acetylglycine. Series of attempts through ENDOR have been made within the past decade to investigate the e f f e c t of i o n i z i n g r a d i a t i o n on t h i s linkage that unites amino acids together i n protein. Piazza and 27 Patten measured hyperfine i n t e r a c t i o n for the methyl protons and proposed 28 the r a d i c a l CH^ONHCHCOOH, same as predicted from ESR studies. The 29 negative ENDOR technique has been used to study x - i r r a d i a t e d c r y s t a l of N-acetylglycine at room temperature. Although signals from at l e a s t 13 d i f f e r e n t types of protons were detected, r a d i c a l i d e n t i f i c a t i o n remains uncertain. Nucleic Acids: Knowledge of the r a d i a t i o n chemistry of deoxyribonucleic a c i d (DNA) and i t s constituents i s very important to r a d i o b i o l o g i s t , who seeks to understand the e f f e c t s of r a d i a t i o n on l i v i n g organisms. The technique of ENDOR was not applied to s i n g l e c r y s t a l s of n u c l e i c acid constituents u n t i l 30 1973, when Hampton and Alexander studied one of the r a d i c a l s formed i n x - i r r a d i a t e d s i n g l e c r y s t a l of c y t i d i n e . This was a species with hyperfine coupling to three protons and i s i d e n t i f i e d as a r a d i c a l i n the ribose 31 group, with the spin l o c a l i z e d on C^^ and C^^. Recently Close et a l . have studied the dominant r a d i c a l observed i n room temperature x - i r r a d i a t e d deoxy c y t i d i n e 5'-phosphate monohydrate. With the help of d e t a i l e d SCF-MO cal c u l a t i o n s at the INDO l e v e l , they have shown that 3aH r a d i c a l has the a l l y l i c structure. Perhaps the best known r a d i c a l i n r a d i a t i o n biology i s thymine r a d i c a l . In an attempt to characterize t h i s free r a d i c a l i n i r r a d i a t e d thymidine, 32 Box and coworkers have shown that the r a d i c a l i s formed by the addition -9-of hydrogen to carbon atom. They have also deduced the methyl group r o t a -t i o n a l s p l i t t i n g constant from ENDOR measurements at 4.2K. Recently Huttermann 33 34 and coworkers ' have attempted 5-chloro and 5-bromo analogs of u r a c i l and i d e n t i f i e d a-halo r a d i c a l s formed by hydrogen add i t i o n to the pyrimidine 5,6-unsaturated bond. There have been s u b s t a n t i a l contributions i n the past from t h i s 35 laboratory on nucl e i c acid bases. Herak, K r i l o v and McDowell have studied i n d e t a i l the two types of r a d i c a l s i n x - i r r a d i a t e d deoxy c y t i d i n e - 5 ' -phosphate. One of the r a d i c a l s i s formed by the decomposition of the furanose r i n g , thus giving r i s e to couplings of two protons at C^^ and one at C ^ . In the second r a d i c a l the unpaired electron i s located mostly on C^,. In the study of x - i r r a d i a t e d s i n g l e c r y s t a l s of thymidine, Herak 36 and McDowell have reported a minority r a d i c a l which i s formed by the abstraction of a hydrogen atom from the methyl group. Other studies of stable r a d i c a l s derived from nuc l e i c acid constituents such as 1-methyl 37 38 u r a c i l , cytosine monohydrate etc. have been mainly concerned with long range i n t e r a c t i o n s . Aromatics; I r r a d i a t i o n of aromatic molecules frequently r e s u l t s i n stable r a d i c a l products corresponding to the addition of a hydrogen atom at the double bond. 39 For example Bohme and Jesse studied such a r a d i c a l formed i n anthracene c r y s t a l . ENDOR l i n e s of a l l the C-H protons have been found. . From the agreement of experimental r e s u l t s with McLachlan Theory, i t i s concluded that the r a d i c a l i s formed by hydrogen addition to C^. On the other hand, 40 x - i r r a d i a t i o n of naphthalene s i n g l e c r y s t a l s at room temperature produces two types of a-hydronaphthyl r a d i c a l s which d i f f e r by the p o s i t i o n of hydrogen ad d i t i o n . While these positions i n the molecule are chemically -10-equivalent, i t s c r y s t a l environment i s d i f f e r e n t . This leads to d i f f e r e n t concentrations of the two r a d i c a l s depending on t h e i r d i f f e r e n t forming rates and hence unequal l i n e i n t e n s i t i e s . The complete ENDOR study of r a d i c a l s from y - i r r a d i a t i o n at room 41 42 temperature of sing l e c r y s t a l s of imidazole and 1,2,4—triazole enabled Lamotte and Gloux to deduce the spin density d i s t r i b u t i o n i n the r a d i c a l from the determination of a l l the proton hyperfine tensors. For example, there are three protons i n the parent imidazole molecule. ENDOR spectra show six proton l i n e s f o r a si n g l e c r y s t a l l o g r a p h i c s i t e . Two tensors have nearly zero i s o t r o p i c coupling and have been assigned to two hydrogen bonded protons. This leaves four tensors which s u c c e s s f u l l y account for the hydrogen adduct r a d i c a l . SCOPE OF THE PRESENT WORK: In the work presented here, the technique of ENDOR has been used to study the free r a d i c a l s trapped i n x - i r r a d i a t e d s i n g l e c r y s t a l s of phenazine, hippuric acid (N-benzoylglycine) and N-acetylglycine. A b r i e f introduction to the theory and experimental techniques of ESR and ENDOR w i l l be presented i n order to show how the structure of a r a d i c a l can be determined from spin Hamiltonian parameters. This theory w i l l then be used to in t e r p r e t the ENDOR spectra of r a d i c a l s trapped i n the above mentioned compounds of b i o l o g i c a l importance. The ENDOR study of phenazine and hippuric acid has not previously been attempted. Although a serie s of attempts through ENDOR have been made to study the r a d i c a l i n N-acetylglycine, the nature of r a d i c a l has been a matter of controversy. However, from d e t a i l e d ESR and ENDOR studies, we have been able to e s t a b l i s h the structure of the r a d i c a l . CHAPTER TOO THEORETICAL 2.1 Introduction Several excellent a r t i c l e s ^ ' ^ ^ and books^'^'^ provide comprehensive accounts of both ESR and ENDOR. Therefore t h i s chapter contains a b r i e f discussion on those aspects of the above phenomena which are relevant to the work described i n t h i s t h e s i s . Electron Spin Resonance (ESR) i s a s e n s i t i v e t o o l for the study of free r a d i c a l s . We w i l l r e s t r i c t our discussion to systems having only one unpaired electron, as these are the only systems studied i n the present work. ESR i s sometimes confused with Electron Paramagnetic Resonance (EPR). EPR, s t r i c t l y speaking, r e f e r s to the magnetic resonance of permanent magnetic dipole moments. ESR i s more s p e c i f i c , but i t i s perhaps inaccurate i n i t s implication because o r b i t a l angular momentum as well as spin angular momentum contributes i n general to the e l e c t r o n i c magnetic dipole moment. For a l l p r a c t i c a l purposes, we s h a l l use the term ESR i n the remainder of the t h e s i s . For the general case of a c r y s t a l containing paramagnetic molecules, i t i s necessary to determine the energy l e v e l s by solving the Schrodinger equation for the electrons and n u c l e i i n the e n t i r e c r y s t a l i n the presence of a s t a t i c magnetic f i e l d . However, i t i s not possible to a r r i v e at a s o l u t i o n and one i s forced to si m p l i f y t h i s many body problem. A great s i m p l i f i c a t i o n occurs i f the i n d i v i d u a l free r a d i c a l s are considered to be independent and non-interacting. Another useful approximation i s known as -12-Born-Oppenheimer approximation"'"3 which states that the t o t a l wavefunction of a molecule can be separated into an e l e c t r o n i c and a nuclear part. Electrons move so fast ( i t s mass being very small) that n u c l e i are e f f e c t i v e l y stationary. Thus the e l e c t r o n i c wave function can be obtained with respect to fixed positions of the n u c l e i . The t o t a l Hamiltonian f o r the molecule can be divided into two parts. where J[E describes the e l e c t r o n i c and k i n e t i c terms'^ and ^  ^ describes the magnetic i n t e r a c t i o n s . In order to describe the e f f e c t s of these magnetic i n t e r a c t i o n s , we s h a l l adopt the formalism of a spin Hamiltonian introduced f i r s t by Pryce 55 56 and Abragam ' , whereby ESR and ENDOR spectra are analyzed i n terms of t r a n s i t i o n s between energy l e v e l s which are the eigenfunctions of a Hamiltonian containing only spin operators. The eigenvalues of the spin Hamiltonian are the energies measured r e l a t i v e to the energy of the molecule i n the absence of any magnetic i n t e r a c t i o n s . 2.2 The Spin Hamiltonian: 2.2.1 The E l e c t r o n i c Zeeman Interaction: An unpaired electron i s characterized by a spin vector which gives the value of the spin angular momentum vector fiS. The magnetic moment of the electron possessing both spin and charge i s proportional to tiS_ and i s given by: = -ynS = -gBS (2-2) -13-where y i s a constant c a l l e d the magnetogyric r a t i o of the electron equal to 1.76 x 10^ rad sec ^ G \ g i s a dimensionless constant c a l l e d the electron g-factor which equals 2.0023 for a free electron and i s about 2.0 for most organic free r a d i c a l s and B i s the Bohr magneton represented by n - e T l = 0.927 x 10~ 2° erg G _ 1 (2-3) 2mc For a free electron, the i n t e r a c t i o n of i t s magnetic moment with the f i e l d determines the energy which i s represented by E = -y .H (2-4) —e — In order to obtain the quantum mechanical Hamiltonian, we replace J J ^ by the appropriate operator, eq (2-2) K, = gBS..H (2-5) If the magnetic f i e l d i s assumed to be i n the z - d i r e c t i o n , Hx=H^=0. Thus The energy of t h i s system i s then E = gBHzMg (2-7) where Mg i s the magnetic quantum number representing the value of S z. The -14-spin magnetic moment for S=h system can thus be aligned e i t h e r p a r a l l e l or a n t i p a r a l l e l to the magnetic f i e l d . This gives r i s e to two states of d i f f e r i n g energy with E = ±%gBH (2-8) z The lowest state has the negative sign and corresponds to the magnetic moment aligned p a r a l l e l to the f i e l d and hence the spin a n t i p a r a l l e l to the f i e l d . The d i f f e r e n c e i n energy between the two states can be matched to a quantum of r a d i a t i o n through the Planck-Einstein formula i . e . AE = hv, so that t r a n s i t i o n s can be induced between the l e v e l s by i r r a d i a t i n g at the frequency given by the resonance condition: AE = hv = gBH C2-9) z Our ESR spectrometer operates with v = 9.5 GHz and the f i e l d f o r resonance of a free electron i s about 3,400 gauss. In addition to the contribution of spin angular momentum, there i s usually a cont r i b u t i o n to the magnetic moment from e l e c t r o n i c o r b i t a l motion. The spin o r b i t coupling can only a r i s e for non-spherical o r b i t a l s and introduces an o r i e n t a t i o n dependence into the Hamiltonian. Because g i s a measure of the e f f e c t i v e magnetic moment associated with an angular momentum S_, t h i s o r i e n t a t i o n dependence i s included i n the Hamiltonian as an anisotropy i n the g-factor. A more general expression f o r the e l e c t r o n i c Zeeman i n t e r a c t i o n i n terms of some convenient cartesian axis system i s : -15-&L - ^H.g.S B(H x,H y,H z) ' 8xx 8 x y g x z 8yx g y y g y z \ 8 z x 8 z y 8 z z j (2-10) where g i s a symmetric tensor. A s u i t a b l e reference frame can always be chosen to diagonalize the g-tensor which i s then represented by i t s p r i n c i p a l values: g , g , g xx yy zz In the organic free r a d i c a l s we have studied, the o r b i t a l angular momentum i s strongly quenched and so the g-tensor being r e l a t i v e l y l e s s anisotropic i s close to the free electron value, i . e . , 2.0023. 2.2.2 The Nuclear Zeeman Interaction: I f a nucleus i n the free r a d i c a l also possesses a spin, the magnetic moment of the nucleus can be expressed as: 8 N 6 N ± (2-11) where g^ i s the nuclear g-factor, i s the nuclear magneton and I_ i s the nuclear spin angular momentum operator. Thus the Hamiltonian which r e -presents the e l e c t r o n i c and nuclear Zeeman contributions i s given by = gBH.S - I g ^ . I . (2-12) i = l where the summation i s over a l l n u c l e i with 1^0. -16-2.2.3 The Hyperfine Interaction: If we consider an organic free r a d i c a l containing at l e a s t one magnetic nucleus with rj=0, the unpaired electron w i l l experience a l o c a l magnetic f i e l d due to t h i s nucleus. The magnitude of t h i s l o c a l f i e l d i s determined by the e l e c t r o n i c structure of the r a d i c a l , the magnetic moment of the nucleus and the o r i e n t a t i o n of the nuclear spin i n the t o t a l f i e l d which i t experiences. The electron resonance occurs when the t o t a l f i e l d which i t experiences, made up of contributions from the applied f i e l d of the spectrometer and the l o c a l f i e l d , has the value given by the resonance condition. The value of the applied f i e l d required to cause a resonance i n a p a r t i c u l a r r a d i c a l w i l l therefore depend on the spin states of the magnetic nucleus i n the r a d i c a l . Thus while sweeping the applied f i e l d to record the spectrum, there w i l l be observed as many absorption l i n e s as there are spin states of the magnetic nucleus and the spectrum w i l l contain (21+1) l i n e s . 2.2.3.1 The Dipolar Interaction: The i n t e r a c t i o n energy between two magnetic moments y g and y^ can be derived by considering the energy of one i n the magnetic f i e l d of the other and i s given by He-iifl 3 (vK.r)(y^.r) E = ^ 3 - ^ 5 ^ (2-13) r r where r i s the radius vector from y to y„ and r i s the distance between the — —e —N two moments. The above formula i s v a l i d f o r point d i p o l e s . It gives the energy of i n t e r a c t i o n of two bar magnets i f t h e i r s i z e i s small compared to the distance between them. -17-Th e Hamiltonian for the magnetic interaction of a nuclear and electron spin can be obtained by replacing y g and y^ by their operator equivalents. Thus the dipolar interaction Hamiltonian is represented by The expression (2-14) has to be averaged over the entire probability distribution |iKr)| of the odd electron. If we now try to evaluate a spin Hamiltonian by integrating over the spatial coordinates, we meet a d i f f i c u l t y . In general, the integral w i l l be of the form: ///^*(r,e,4>)itDipiJ;(r,e,(}.)r2sinededrd()) (2-15) and unless the wavefunction goes to zero very rapidly at r=0, the integrand becomes i n f i n i t e at this point. The d i f f i c u l t y is that the point dipole treatment breaks down at very short distances as for classical magnets. To simplify the treatment and obtain useful results, we w i l l proceed by discussing more expli c i t l y the case of an atom with one electron, neglecting the spin orbit coupling. If the unpaired electron i s in a p,d or any other orbital with £>0, the wavefunction goes to zero exponentially as r goes to zero. Expanding the scalar products in Eq. (2-14) in a cartesian coordinate system with the nucleus at the origin, we obtain #-Dip r5 \ ( 3 x 2 - r 2 ) I x S x + ( 3 y 2 - r 2 ) I y S y + ( 3 z 2 - r 2 ) I z S z + 3xy(I xS y +I yS x) + 3 y z ( I y S z + I z S y ) + 3 x z ( I x S z + I z S x ) j (2-16) -18-2 2 2 ^  where r = (x +y +z ) 2 . The int e g r a t i o n over the s p a t i a l part of the electron wavefunction can now be ca r r i e d out term by term and i t i s cl e a r that the spin Hamiltonian f o r the dipo l a r coupling can be written i n the t e n s o r i a l form: H r - = S - A ° ' 1 • Dip = A ° S I + A ° S I + A ° S I (2-17) x x x x y y y y z z z z The coupling tensor A° i s symmetric with i t s diagonal elements given by: " g 3 gN eN tc^T-) •  ±=X' y' Z ( 2 _ 1 8 ) and with i t s off-diagonal elements given by A°. = g B g . T B „ / ^ - \ (2-19) The most important property of the anisotropic hyperfine tensor i s that i t s trace, i . e . , sum of the diagonal elements, i s zero. An immediate consequence of t h i s property i s that for a r a d i c a l undergoing rapid tumbling i n highly f l u i d s o l u t i o n , there i s no contribution to the hyper-f i n e s p l i t t i n g from the dipo l a r c o u p l i n g . ^ A second consequence of the traceless property of the dipo l a r coupling i s that the di p o l a r Hamiltonian averages out to zero whenever the electron cloud i s s p h e r i c a l , as for an electron i n an atomic s o r b i t a l . 2.2.3.2 The Isotropic Hyperfine Coupling: The magnetic moments of the electron and nucleus are also coupled v i a -19-58 the Fermi contact i n t e r a c t i o n which represents the energy of the nuclear moment i n the magnetic f i e l d produced at the nucleus by e l e c t r i c currents associated with the spinning electron. I f we treat the nucleus as a spinning charged s p h e r i c a l s h e l l , the energy of the electron spin magnetic moment i n t e r a c t i n g with the magnetic f i e l d (H) insi d e the sphere i s given by E = - ^ r 3 | ^ ( 0 ) | 2 y ^ . ( 2 y N / r 3 ) (2-20) 2 where r i s the radius of the sphere and |IJJ(0) | i s the p r o b a b i l i t y of findi n g the electron at any point i n the sphere. Substituting the magnetic moments by appropriate spin operators, we obtain the spin Hamiltonian: 8tr „ _ i , i 2, = aS.I = a(I S +1 S +1 S ) (2-21) x x y y z z where a i s the isotropic hyperfine coupling constant which i s proportional to the squared amplitude of the e l e c t r o n i c wavefunction at the nucleus. a = | l g g g N 6 N ^ ( 0 ) | 2 (2-22) The contact i n t e r a c t i o n can only occur when the electron has a f i n i t e p r o b a b i l i t y density at the nucleus. In other words, the electron must have some s o r b i t a l character. -20-The complete Hamiltonian describing the hyperfine i n t e r a c t i o n i s : ft Hyp = tftiso + ft Dip " *JL.I.+S.A°.I = S.A.I = A S I + A S I + A S I (2-23) — = — xx x x y y y y zz z z where A = A° + a xx xx A = A° + a yy yy A = A° + a zz zz Since the dipolar tensor has zero trace i . e . A 0 + A° + A° r xx yy zz a = -kA + A + A ) 3 xx yy zz The evaluation of these parameters provides a valuable method for determining the electron d i s t r i b u t i o n i n a r a d i c a l . 2.2.4 The Quadrupole Interaction: For n u c l e i with l=h, the energy l e v e l s are mainly determined by electron and nuclear Zeeman in t e r a c t i o n s and the hyperfine i n t e r a c t i o n . For n u c l e i with I_l» there i s another contribution to the energies of the hyperfine l e v e l s . This contribution i s due to the nuclear quadrupole moment. Nuclei with I _ l have quadrupole moments because t h e i r charge d i s t r i b u t i o n s can deviate from sph e r i c a l symmetry. The symmetry axis of the charge d i s -t r i b u t i o n s i s also the axis of the spin and magnetic moment. The nuclear quadrupole moment Q, whose magnitude i s a measure of the deviation of the charge d i s t r i b u t i o n from s p h e r i c a l symmetry i s defined as: = o, (2-24) -21-eQ = / p N ( 3 z 2 - r 2 ) d T (2-25) where i s the d i s t r i b u t i o n function of the nuclear charge, z i s the z-coordinate of the charge element at a distance r from the o r i g i n , e i s the proton charge and the i n t e g r a l i s evaluated over the volume of the nucleus. A quadrupole moment has no i n t e r a c t i o n with a homogeneous e l e c t r i c f i e l d . However, i t i n t e r a c t s with an inhomogeneous e l e c t r i c f i e l d , the energy of i n t e r a c t i o n depending on the magnitude of quadrupole moment and the gradient of the e l e c t r i c f i e l d , and i s given by: 1 a,6 EQ " 6 \ Va3 Qa3 ( 2" 2 6> U T 7 where V a3 8a93 ' x » y » z 2 and = f OaS-S^r )P Ndx. The Hamiltonian i n terms of nuclear spin i s given by: 41Q = 61 Zft V l ( I a V W - (2'27) a, ts This can be expressed more sh o r t l y as a tensor coupling of the nuclear spin with i t s e l f . KQ = I-I- 1 <2-28> where P i s c a l l e d the quadrupole coupling tensor. The f i e l d gradient generally obeys Laplace's equation, hence the f i e l d gradient tensor i s trac e l e s s i . e . Z V =0. As a r e s u l t the quadrupole coupling tensor i s also t r a c e l e s s . -22-There i s no f i e l d gradient at the nucleus of a free nitrogen atom because of s p h e r i c a l symmetry, but i n molecules, the non-spherical charge d i s t r i b u t i o n of electrons, p a r t i c u l a r l y the valence electrons, gives r i s e to f i e l d gradients. The quadrupole i n t e r a c t i o n influences the o r i e n t a t i o n of the nuclear spin, whose axis i s colinear with that of the quadrupole moment. Therefore the energies of the hyperfine l e v e l s are determined by the e l e c t r o s t a t i c quadrupole i n t e r a c t i o n i n a d d i t i o n to the magnetic Zeeman and hyperfine i n t e r a c t i o n s . The t o t a l Hamiltonian required to describe a free r a d i c a l i n a s o l i d i s : $C= gBH.S + E (S.A..I. + I 4 . P _ . _ i " S N e NI-Ii) (2-29) i = l The i n t e r a c t i o n does not involve the electron spin e x p l i c i t l y and hence to f i r s t order there i s no strong e f f e c t on the frequency of a AM_-0 ESR t r a n s i t i o n . However, when the energies of the hyperfine l e v e l s are c a lculated to higher order, the quadrupole i n t e r a c t i o n causes a s h i f t i n the t r a n s i t i o n frequencies and i n extreme cases there may be appreciable i n t e n s i t y for c e r t a i n forbidden t r a n s i t i o n s . The quadrupole coupling can be observed more d i r e c t l y i n ENDOR spectra where there can a c t u a l l y be a s p l i t t i n g . Further aspects of the e f f e c t of the quadrupole i n t e r a c t i o n w i l l be described under ENDOR p r i n c i p l e s . 2.3 Theory of E S R : In the following section, i t w i l l be shown how ESR spectra can be described i n terms of the spin Hamiltonian. The f i r s t task i s to f i n d the forms of the stationary state spin wavefunctions and t h e i r energies. -23-The f i n a l step i s to determine which t r a n s i t i o n s may be induced by an o s c i l l a t i n g magnetic f i e l d . 2.3.1 Energy l e v e l s f o r S=h, I=% system: We w i l l discuss i n d e t a i l the case of a free r a d i c a l containing a sin g l e nucleus of spin l=h and generalize the most important r e s u l t s f o r the case of inte r a c t i o n s with several n u c l e i . For s i m p l i c i t y , we w i l l consider r a d i c a l s i n so l u t i o n with only i s o t r o p i c i n t e r a c t i o n . The spin Hamiltonian i s : = g3H. S - gjjSjJH.I + aS. I (2-30) The energy l e v e l s are determined by solving the equation: This equation i s usually solved with the help of perturbation theory, i n which the Hamiltonian i s separated into two d i s t i n c t p a r t s , a n d i £ £ ^ . n i s the main component of the Hamiltonian a n d ^ ^ i s treated as a small perturbation. The spin wave functions are then expressed i n terms of l i n e a r combinations of the basis functions $ which are chosen to be eigenfunctions of The eigenvalues of $ Q are the unperturbed energies e . The per-turbation y i e l d s modified wave functions and energies of the form: <m | n> * = • - Z ^ <f> (2-32) n n I e -e m mf n m n <m|fC. |n><n|# |m> E = E + <n Iff. I n> - Z (2-33) n n 1 1' i e -e m^n m n - 2 4 -The two terms on the r i g h t side of ( 2 - 3 3 ) are the f i r s t order and second order corrections r e s p e c t i v e l y and <n|*f(^ |n> and <m|f(-Jn> are the matrix elements o f ^ ( ^ . The electron has a spin vector S_ equal to y and there are two allowed components of the spin along any chosen d i r e c t i o n , i . e . , z-axis. The two possible spin functions are denoted by symbols |ag> and |Be> with spin quantum numbers Mg = +% and -% r e s p e c t i v e l y . In terms of operator equations'. S a > = M a > z 1 e 1 e h\9> > ( 2 - 3 4 ) z' e ' e For nuclear spins, equations analogous to the above can be written I a > = +h\a> z' N 1 N \ v • ~h V (2"35) The appropriate basis functions are written as the products of the electron and nuclear spin functions: •l = KV' *2 • I'eV' *3 " " B e V ' U = ' W (2"36) These functions are a l l eigenfunctions offCg a n c* t* i e m a t r i x °f H.Q diagonal. For example: -25-K , J a a > = gBHS l a >.|a >- g.T3,,H I a >.I a > • ^•0' e N z e N & N N e z N - %g3H|aeaN> - %g NB NH|a ea N> = (%g3H - %g N6 NH)|a ea N> (2-37) The zero order energy values for the four states are: E i = %ggH - %gNeNH e2 = h * m + ^N 6N H e 3 = -%g8H - %g N6 NH e 4 = -%g3H + %g N3 NH (2-38) The p e r t u r b a t i o n ^ ^ , the hyperfine i n t e r a c t i o n may be expanded as follows: K, . = aS.I = a[S I + S I + S I ] (2-39) , v l z z x x y y The f i r s t term contributes to f i r s t order energies. The e f f e c t of the term a[S I + S I ], however, i s to produce a second order change i n the x x y y ° energies which w i l l be calculated l a t e r . The f i r s t order energy s h i f t s are: -26-<cx a„|aS I la a„> = %a e N 1 z z 1 e N <a B„|aS I la 6 > = -%a e N1 z z 1 e N < B e a N | a S z l j B e a N > - -*a The operator S^I^ gives a diagonal matrix and can be included i n zero order energies. In order to consider the e f f e c t s of a(S I + S I ) on the spin x x y in-states, i t i s convenient to define the ladder operators. S + = S + iS x y S" = S - iS (2-41) x y S +|a > = 0 1 e S +|B > = la > ' e 1 e S~|B > = 0 1 e S"la > = IB > (2-45) 1 e 1 e Similar operators also exist f o r the nuclear spin. The operator ( S I + S I ) can be written i n terms of the ladder x x y y operators a(S I + S I ) = ^{S+I + S " l + ) (2-46) x x y y 2 -27-Th e only nonvanishing matrix elements are: « * B J ( S + r + S I + ) | @ a > = 1 e N e N <B a J ( S + I + S I + ) | a 6 > = 1 e N e N (2-47) The complete matrix of 4t i s thus represented by 0 1 C t X T > 1 e N 0 1 . A T * e N e N e N' e N < B eV e N 1 ^ Z e + Z N - f ) %a -%(ze+zN+f) where Z g = gBH and Z N = g ^ H (2-48) The eigenvalues and eigenfunctions of fa can be determined by di a g o n a l i -zation of the above matrix. The same r e s u l t i s also obtained by using the perturbation formulae (2-32) and (2-33), where the second order terms involve the off-diagonal elements of matrix. The eigenvalues are: 2 E 2 = hgW + %g NB NH - f + A ( g 6 H + g N e N H ) 2 E 3 = -kgw - %g Ne NH - f - A ( g B H4 Ne NH) E 4 = -%g6H + %g N6 NH + f (2-49) -28-For most free r a d i c a l s , the hyperfine coupling energy i s much less than the electron Zeeman energy. Hence the second-order corrections approach zero and can be neglected from the above equations. The energy l e v e l s are shown i n f i g . 1. 2.3.2 ESR s e l e c t i o n r u l e s : I f an o s c i l l a t i n g magnetic f i e l d of strength 2H^cosut produced by microwave r a d i a t i o n i s applied perpendicular to steady f i e l d , H i n x-d i r e c t i o n such that w i s correct f o r resonance, several kinds of spin t r a n s i t i o n s can be induced. Transitions l i k e a a„ 3 a„ where only the e N e N electron spin changes are c a l l e d ESR t r a n s i t i o n s , while those l i k e a a„,-» e N a 6.. which involve the nucleus alone are NMR ones. The t h i r d kind of e N t r a n s i t i o n a 8^->-3 where both spins change has a very low p r o b a b i l i t y and i s known as a "forbidden t r a n s i t i o n " . The r e s u l t i n g time dependent perturbation i s : V(t) = 2(gBH 1S x-g N e NH 1I x)cosu)t = 2V cosa>t (2-50) The t r a n s i t i o n p r o b a b i l i t y for state n to state m i s equal to 2TT 2 P = -j=—I <n| v|m> I <§(to -u>) (2-51) nm -n ' 1 1 1 mn where u> = E - E , expressed i n frequency u n i t s . The deduction of mn m n J s e l e c t i o n r u les i s based on f i n d i n g whether the matrix elements of V between each p a i r of states vanishes or not. As the electron resonance t r a n s i t i o n s are caused by the e f f e c t of H^ on the electron spins, we can omit the nuclear spin operator 1^ from (2-51). Thus -29-H Y P E R F I N E C O U P L I N G N U C L E A R Z E E M A N E L E C T R O N Z E E M A N Z E R O F I E L D + ' / 2 g ^ H v / N - ' / 2 g N / 3 N H + a / 4 + ' / 2 g N / 9 N H / - ' / 2 g N / 9 N H \ - a / 4 E, IOe ,a N > E 2 i a e , £ N > E 4 1&,0N> E 3 l j 8 e a N > F i g . 1. Energy l e v e l s of an S-h, I=% system, with f i r s t order ESR t r a n s i t i o n s . -30-^ - $ . V H ? I < ° I S > I 2 ! < " . . - ) < 2 " 5 2 ) The t y p i c a l matrix element for ESR t r a n s i t i o n i s calculated using the ladder operators. <a a j s | @ a > = «x |Js(S +s")|B > <a |a > e N x e N e e IN IN + IR . = J< (2-53) <a S 3 > = H. Thus the spin quantum numbers i n allowed ESR t r a n s i t i o n s obey the s e l e c t i o n r u l e s : AM = ±1 ; AM = 0 (2-54) S J-These t r a n s i t i o n s marked by the s i n g l e arrows i n F i g . 1, have equal pr o b a b i l i t i e s and hence equal i n t e n s i t i e s . The frequencies of the t r a n s i t i o n s are: hv__ 3 = (%g3H-% N 3 NH+f) - (_% g3H-%g N3 NH-|) = g3H+| H V2 - 4 = (^geH+%g N3 NH-f)-(-%g3H+% g ^ H + f - ) = g 3 H - f (2-55) The f i r s t order ESR spectrum, therefore, consists of two equally intense l i n e s separated by 'a', the hyperfine coupling constant. Since the spectrum i s recorded by varying the magnetic f i e l d , the t r a n s i t i o n corresponding to the largest energy d i f f e r e n c e occurs at the lowest f i e l d . 2.3.3 Thermal equilibrium and spin r e l a x a t i o n : Resonance absorption can be detected only i f there i s a population -31-d i f f e r e n c e between the upper and lower spin l e v e l s . When a macroscopic specimen containing N spins i s placed i n a steady magnetic f i e l d , the degeneracy i s l i f t e d and there are N^ spins i n the lower state and i n the upper. In thermal equilibrium, there i s s l i g h t excess of spins i n the 3-state which gives r i s e to a small temperature dependent para-magnetism. The r a t i o of 3 to a spins i s determined by the Boltzmann fa c t o r : ND/N «= e g 3 H / k T (2-56) At ordinary temperature g3H<<kT and the Boltzmann factor i n (2-56) i s approximately [l+g3H/kT]. On the average (N/2)[l+g3H/kT] electrons have spin 3 and (N/2)fl-g3H/kT] have spin a. The a p p l i c a t i o n of microwave energy i n the proper o r i e n t a t i o n (microwave magnetic f i e l d perpendicular to the s t a t i c magnetic f i e l d ) causes t r a n s i t i o n s between the magnetic l e v e l s . The microwave induced t r a n s i t i o n s have equal p r o b a b i l i t i e s i n either d i r e c t i o n . As a r e s u l t , i t can be shown that a p p l i c a t i o n of the resonant microwave f i e l d r e s u l t s i n exponential decay of the population diff e r e n c e and eventually the l e v e l s w i l l be equally populated. This i s known as saturation. There would then be no net absorption of microwave energy and the resonance absorption l i n e w i l l u l t i m a t e l y disappear. For ESR spectrum to be observed there must then be some mechanism through which the spin system returns to thermal equilibrium. The establishment of thermal equilibrium between the a and 3 states a f t e r a p p l i c a t i o n of steady magnetic f i e l d , H must i n e v i t a b l y require that there are i n t e r a c t i o n s between the electrons and the thermal motion of l a t t i c e or surroundings which cause the spin orientations to change, while the excess energy i s transferred to other degrees of freedom. This process of nonradiative t r a n s i t i o n s between the two states i s c a l l e d spin l a t t i c e -32-r e l a x a t i o n . Because the l a t t i c e i s at thermal equilibrium, the p r o b a b i l i t i e s of spontaneous spin t r a n s i t i o n s up and down are not s t r i c t l y equal. Con-s i d e r a t i o n of these points leads to an expression for the rate of absorption of microwave energy dE dt (2-57) where n Q i s the population d i f f e r e n c e at thermal equilibrium, P i s the t r a n s i t i o n p r o b a b i l i t y and T.. i s c a l l e d spin l a t t i c e r e l a x a t i o n time i . e . , f or the spin system to approach thermal equilibrium). Equation (2-57) indicates that as long as 2PT^<<1, i t i s r e l a t i v e l y easy to avoid saturation. In an ESR experiment, one normally operates with low microwave power to avoid saturation. In addition to s p i n - l a t t i c e r e l a x a t i o n , i n which energy i s transferred from the spin system to the l a t t i c e , there e x i s t spin-spin r e l a x a t i o n mechanisms i n which energy i s r e d i s t r i b u t e d within the spin system. The c h a r a c t e r i s t i c time for spin-spin r e l a x a t i o n or transverse r e l a x a t i o n i s symbolized by T^- Spin relaxations play an important r o l e i n determining the width and shape of resonance absorption l i n e . 2.3.4 Hyperfine Interaction With More Than One Nucleus: The above discussions can be e a s i l y extended for the case where there i s hyperfine i n t e r a c t i o n with several n u c l e i . To f i r s t order, the nuclear Zeeman i n t e r a c t i o n does not a f f e c t the appearance of ESR l i n e positions. Hence the nuclear Zeeman term can be omitted. The spin Hamiltonian for an (time taken for energy to be transferred to other degrees of freedom, -33-l e c t r o n coupled to more than one nucleus can be written as: fc = gSH.S + | a . I ( ^ S (2-58) with eigenvalues: E = g8HM + Za.M O - T l X ( i ) (2-59) S The r e s u l t i n g number of AMg=l, AM^ .=0 t r a n s i t i o n s i s given by: n = TT(2I ( i ) +1) (2-60) i Thus there w i l l be 2 X t r a n s i t i o n s i n the ESR spectrum of an unpaired electron i n t e r a c t i n g with x protons. It often happens that several protons have i d e n t i c a l coupling constants. Such protons are termed equivalent and usually occupy symmetrically equivalent positions i n the molecule. The number of l i n e s i n the spectrum i s reduced but the i n t e n s i t i e s vary. In general, number of l i n e s i n the spectrum of n equivalent n u c l e i i s (2nl+l). Thus the i n t e r a c t i o n of odd electron with n equivalent protons (I=h) r e s u l t s i n (n+1) l i n e s whose r e l a t i v e i n t e n s i t i e s are proportional to the c o e f f i c i e n t s of the binomial expansion of (a+b) n The ESR spectrum of a r a d i c a l i n which the unpaired electron i s coupled to many n u c l e i often contains so many l i n e s that many of them overlap and the r e s u l t i n g broadened spectrum cannot be analyzed. In s o l i d s tate, there i s further complication as a r e s u l t of the ani s o t r o p i c hyperfine i n t e r a c t i o n . The l i n e positions w i l l vary with the magnetic -34-f i e l d o r i e n t a t i o n and unless the l i n e s are w e l l separated, t h e i r angular v a r i a t i o n s cannot be followed. These are two of the major reasons why the ENDOR technique i s so us e f u l . 2.4 Theory of ENDOR: 2.4.1 The Isotropic Hyperfine Interaction: In order to understand the basic p r i n c i p l e s of the ENDOR experiment, i t w i l l be us e f u l to s t a r t with the simplest system, that of an unpaired electron i n t e r a c t i n g with a proton through an i s o t r o p i c hyperfine i n t e r -action. The Hamiltonian i s : ' f t = SBH.S - gjjBjjH.I + a'S.I (2-61) To f i r s t order, the energy l e v e l s f o r t h i s system are determined by: ECMg.Mj) = gBHMg - gNSNHM]. + a ' M ^ (2-62) It i s convenient to work i n frequency units and define g6H/h= v e ; % e N H / h = V a ' / h = a E(Ms,M].)/h = V eM g - + aMgMj. (2-63) F i g . 2(a)(b) shows the energy l e v e l s f o r two cases i . e . v N>a/2 and a/2 > v N r e s p e c t i v e l y . Because the nuclear Zeeman energy i s much smaller than that of the electron, the population differences between the l e v e l s l 0 ^ 0 ^ 5 , a n d l 0 1 ^ ^ can be ignored. The thermal equilibrium populations can be calculated on -35-F i g . 2. Energy l e v e l s f o r ENDOR experiment i n an S=%, I=% system, (a) vN>a/2 (b) a/2>vN. -36-the basis of Boltzmann d i s t r i b u t i o n and can be shown to be 1+e for 6 e state and 1-e for a g state where e=gBH/kT. I f s u f f i c i e n t microwave power i s applied to one of the ESR t r a n s i t i o n s , i.e.,|6 a ><-»|a a >, the populations of the two l e v e l s w i l l be equalized. In the ENDOR experiment, a radiofrequency f i e l d i s applied to the spin system, while continuing to saturate the ESR t r a n s i t i o n . As the radiofrequency i s varied, i t w i l l match the separation of |a g a^> and lot , B > l e v e l s and induce t r a n s i t i o n between them when: 1 e' N v ,. = v —Ha r f N Removal of spins from |a g a^> by t h i s absorption, restores a population d i f f e r e n c e between |8 a > and |a a > l e v e l s and there i s an increase i n ' e, N 1 e, N the ESR absorption. Thus the ENDOR spectrum i s a display of the enhance-ment of a p a r t i a l l y saturated ESR t r a n s i t i o n as a function of the radio-frequency. The response has a linewidth more nearly that of the narrower nuclear resonance absorption than the electron resonance absorption. As the radiofrequency i s further v a r i e d , the separation between |8 a > and |g 6 > l e v e l s w i l l also be matched when: 1 e, N 1 e, N For vN>a/2, the two ENDOR t r a n s i t i o n s are given by v N±a/2 separated by 'a' and centered at v^. For a/2>v^, the two ENDOR t r a n s i t i o n s are given by a/2±v separated by 2v N and centered at a/2. If a/2=v N > then only one ENDOR l i n e w i l l be observed at v..+a/2. -37-I t may be noted that the same two ENDOR t r a n s i t i o n s are observed when either ESR l i n e i s monitored. However, since depends on H, there w i l l be a s h i f t i n the center of spectrum as d i f f e r e n t ESR l i n e s are monitored by sweeping the magnetic f i e l d . 2.A.2 The Anisotropic Hyperfine Interaction: An anisotropic or dipolar hyperfine i n t e r a c t i o n always e x i s t s between two magnetic dipoles , i . e . , electron dipole-nuclear dipole. This i n t e r a c t i o n i s o r i e n t a t i o n dependent. In the l i q u i d phase where r a d i c a l r o t a t i o n i s f a s t , the di p o l a r hyperfine i n t e r a c t i o n averages out on the ESR or ENDOR time scale, so i t does not enter i n the spectra. I f the motion i s arrested, except f o r v i b r a t i o n or some i n t e r n a l motion, as i n s o l i d s , p a r t i c u l a r l y , s i n g l e c r y s t a l s , we encounter anisotropic hyperfine i n t e r a c t i o n to analyze the ENDOR spectra. We consider the t y p i c a l case f o r organic r a d i c a l s i n s i n g l e c r y s t a l s where the hyperfine i n t e r a c t i o n i s g e n e r a l l y a n i s t r o p i c . The spin Hamil-tonian i s : In the preceding section we have seen that, to f i r s t order, the ENDOR t r a n s i t i o n frequencies are independent of the electron Zeeman energy. Thus the ENDOR frequencies can be written as: (2-64) v 2 = K2£.A2.h - 2M sv Nh.A.h + g 2 6 2 H 2 (2-65) where h i s a unit vector along the external f i e l d d i r e c t i o n (H=Hh) The nuclear t r a n s i t i o n s are induced by an o s c i l l a t i n g r f f i e l d H 2 -38-i n the x - d i r e c t i o n i f the s t a t i c f i e l d i s along z. The f i r s t order ENDOR s e l e c t i o n rules can be determined i n a manner analogous to that shown for ESR i n Sec. 2.3.2 using the time dependent perturbation: #C<t) = (geH 2S x-g N B NH 2I x)cosa)t (2-66) The ENDOR s e l e c t i o n rules can thus be found as: AM = 0 , AM = ±1 Thus i f both the ENDOR t r a n s i t i o n s (for I=%) are observed, the hyperfine tensor can be determined using the equation: v 2 - v 2 = 2v h.A.h (2-67) + " p- = -If for example, a c r y s t a l i s mounted i n the spectrometer such that the magnetic f i e l d i s rotated i n the yz plane, the equation (2-67) becomes: 2 2 2 2 v, - v = 2v s i n 6(A )+4v sin6cos6(A ) + 2v cos 6(A ) + P yy P yz p zz (2-68) where 0 i s the angle between the z-axis and the f i e l d d i r e c t i o n . These 2 2 three elements of A can be determined by measuring v + - v for a minimum of three values of 9 i n t h i s plane. The remaining elements can be obtained by remounting the c r y s t a l and ro t a t i n g the magnetic f i e l d i n zx and xy planes. When the hyperfine s p l i t t i n g i s approximately 50 MHz or lar g e r , the second order corrections to the energy l e v e l s become s i g n i f i c a n t . The best way to analyze the ENDOR spectrum i s to use a least squares procedure to -39-match the observed frequencies to those calculated by diagonalizing exactly by computer the Hamiltonian (2-64) for t r i a l values of para-met ers. 2.4.3 The Quadrupole Interaction: In general, information about nuclear quadrupole couplings i s not re a d i l y derived from the normal ESR spectrum unless the couplings are f a i r l y large. However, quadrupole couplings can be measured d i r e c t l y 14 from an ENDOR spectrum. Let us consider a simple case of N nucleus (1=1) with an i s o t r o p i c hyperfine coupling a/2>v^ and a x i a l l y symmetric quadrupole coupling. The spin Hamiltonian i s : The frequencies of ESR t r a n s i t i o n s (AM g=l, AM^O) are independent of P, but those of the ENDOR t r a n s i t i o n s are not. F i g . 3 shows the e f f e c t of quadrupole i n t e r a c t i o n on the energy l e v e l s and the r e s u l t i n g four ENDOR frequencies for 1=1. In the absence of a quadrupole i n t e r a c t i o n , the hyperfine l e v e l s i n each M manifold are equally spaced. Thus there i s only one ENDOR t r a n s i t i o n frequency i n each manifold and the complete spectrum consists of only two l i n e s f or each ESR l i n e . However, with an observable quadrupole i n t e r a c t i o n , the four d i f f e r e n t ENDOR t r a n s i t i o n frequencies are: U = gBH .S - g^AiH-1 + aS .1 + P{I 2 - - k d + l ) } <JV_ e —z —z &N N—z — z —z —z z 3 (2-69) The eigenvalues are: (2-70) -40-+ Z / e / 2 - Z / e / 2 + Q/2 N / \ \ \ - a / 2 + a /2 N / + P/3 -2P/3 + P/3 +P/3 -2P/3 - a / 2 + P/3 r ifc.'> A4 , l fc,0> J A3 114,-1> |-/ 2,-1> A2 F i g . 3. Energy l e v e l s f o r an S=%, 1=1 system. -41-A l = %a + VN - P A2 = ha + VN + P A3 = ha - VN - P A4 = ha - VN + P (2-71) The appearance of ENDOR l i n e s w i l l depend on ESR t r a n s i t i o n being saturated. I f \-h,0> —*\h>0> ESR l i n e i s saturated, a l l the four ENDOR l i n e s can be observed. Saturation of eit h e r of the other two ESR t r a n s i t i o n s r e s u l t s i n only two l i n e s i n the ENDOR spectrum. In addition to a d i r e c t measure of the quadrupole coupling, we can determine the r e l a t i v e signs of the hyperfine and quadrupole couplings from the ENDOR spectrum. I f the signs of A and P are the same, saturation of the low f i e l d (M^=+l) ESR l i n e gives the highest and lowest frequency ENDOR l i n e s . But i f the signs of A and P are opposite, saturation of the low f i e l d ESR l i n e gives the intermediate frequency ENDOR l i n e s . 2.4.4 ENDOR spectrum for an Electron Coupled to More Than One Nucleus: Very often r a d i c a l s of i n t e r e s t contain groups of equivalent and non-equivalent n u c l e i and i f there are several such groups, the ESR spectrum can contain an enormous number of l i n e s . However, the ENDOR spectrum remains comparatively simple. I f the internuclear i n t e r a c t i o n s are neglected, the f i r s t order energies are: E = M gv e - v N(EM^ x )) + I a 1M sM^ l ) (2-72) Applying the ENDOR s e l e c t i o n r u l e s : ^ 5 = 0 , A M ( ± ) = ±1 -42-where the spin of only one nucleus changes, the ENDOR t r a n s i t i o n s are described by Thus, ENDOR spectrum consists of a pair of l i n e s for each i n t e r a c t i n g , nonequivalent nucleus and can be analyzed i n terms of an electron i n t e r a c t i n g with each nucleus separately. Equivalent n u c l e i only contribute a pair of l i n e s to the ENDOR spectrum, although t h e i r i n t e n s i t y depends d i r e c t l y on the number of n u c l e i i f the rel a x a t i o n rates are equal. The ENDOR spectrum i s , therefore, often much simpler than i t s parent ESR spectrum, which for x protons may contain up to 2 l i n e s while the ENDOR spectrum consists of no more than 2x l i n e s . When the r a d i c a l contains several d i f f e r e n t couplings, i t may be that even the ENDOR l i n e s overlap and then f o r the highest p r e c i s i o n , i t may be necessary to study p a r t i a l l y deuterated samples. Replacement of a proton by a deuteron completely removes i t s co n t r i b u t i o n to the spectrum because of the change i n free nuclear frequency. As a r e s u l t , one obtains an unequivocal assignment of the couplings to the various protons i n the r a d i c a l . 2.4.5 Determination of Spin Hamiltonian Parameters: In p r a c t i c e , the spin Hamiltonian parameters are best obtained by comparing the observed frequencies to those calculated by exact, computer dia g o n a l i z a t i o n of the matrix for the Hamiltonian: v r f N + (2-73) '$C = 8SH._S. - g j A j H - I + S.A.I + I.P.I_. (2-74) -43-with appropriate spin functions and t r i a l values of the parameters. The parameters are then adjusted by the method of least squares u n t i l the calculated frequencies match the observed ones. In t h i s work, we have used a computer programme written by Dickinson et a l . ~ ^ ' ^ and Hebden^ based on t h i s approach. This program ca l c u l a t e s the ESR and ENDOR t r a n s i t i o n frequencies for any s p e c i f i e d spin Hamiltonian and performs the le a s t squares refinement of the Hamiltonian parameters. For t h i s program, the values of S and I are read i n , together with the nuclear magnetic moment and the other elements of the spin Hamiltonian (2-74) where these are nonzero. For our study, the e l e c t r o n i c g-tensor was taken to be i s o t r o p i c . The experimental data are read i n as a serie s of observed t r a n s i t i o n frequencies, with the corresponding f i e l d strengths and d i r e c t i o n . For each value and o r i e n t a t i o n of H, the t o t a l spin Hamiltonian i s diagonalized numerically; the t r a n s i t i o n frequencies are calculated and compared to the experimental values and the r e s u l t i n g set of r e s i d u a l s i s used to make a f i r s t order c o r r e c t i o n to the parameters to be refined according to the le a s t squares c r i t e r i o n . The whole process i s cycled u n t i l the errors and parameters remain constant. In general, the i n i t i a l guess at the required parameters need be accurate only to within an order of magnitude and convergence i s usually achieved within 5 i t e r a t i o n s . CHAPTER THREE INTERPRETATION OF SPIN HAMILTONIAN PARAMETERS 3.1 Introduction: Hyperfine tensors are interpreted f i r s t i n terms of the signs and magnitudes of t h e i r components; next they are interpreted i n terms of the d i r e c t i o n cosines for the p r i n c i p a l axes of the hyperfine tensor r e l a t i v e to the c r y s t a l axes. In order to obtain the most d e t a i l e d p i c t u r e of the e l e c t r o n i c structure of a r a d i c a l i n a s i n g l e c r y s t a l , one needs to know the c r y s t a l structure. It i s usually found that organic r a d i c a l s produced i n s i n g l e c r y s t a l s by i o n i z i n g r a d i a t i o n are oriented nearly the same as the undamaged molecules. In t h i s chapter, we w i l l discuss the methods used to deduce the r a d i c a l ' s e l e c t r o n i c structure from the observed hyperfine tensors. F i r s t of a l l we w i l l consider proton hyperfine tensors which are roughly c l a s s i f i e d into four groups based on the magnitude of the anisotropy and on the r a t i o of dipolar hyperfine components to i s o t r o p i c components. These groups correspond to a-protons, 8-protons, more distant protons, including matrix protons and protons bonded to atoms other than carbon. F i n a l l y we w i l l discuss the hyperfine and quadrupole couplings for nitrogen nucleus. 3.2 Hyperfine Coupling Tensors for a-Protons: A proton adjacent to an atom possessing appreciable spin density i n a p-type o r b i t a l and l y i n g i n the node of that o r b i t a l i s conveniently c a l l e d an a-proton. 3.2.1 The Isotropic Hyperfine Coupling: Because the a-proton i s i n the node of the unpaired electron o r b i t a l , i t should have zero i s o t r o p i c coupling. In f a c t , the proton i s experimen-t a l l y observed to have a nonzero coupling which i s d i r e c t l y proportional to the 2p spin population on the adjacent carbon atom. The problem was f i r s t discussed, independently and simultaneously 62 63 65 by J a r r e t t , Weissman , Bersohn and McConnell i n terms of c o r r e l a t i o n of the spins of the electrons i n the C-H bond and the ir electrons i n aromatic compounds. The treatment r e f e r s p a r t i c u l a r l y to the ^C-H fragment and i s therefore applicable with considerable success to both aromatic and a l i p h a t i c systems.^ This c o r r e l a t i o n which can be explained i n terms of e i t h e r valence bond or molecular o r b i t a l theory, r e s u l t s i n a s l i g h t p o l a r i z a t i o n of the electron spins i n the C-H a bond. The extent to which the C-H a electrons are polarized i s d i r e c t l y proportional to the i r-electron spin density, p^ , representing the f r a c t i o n a l p r o b a b i l i t y of f i n d i n g the unpaired electron i n the carbon 2p z o r b i t a l . The p r o p o r t i o n a l i t y i s usually expressed i n terms of McConnell's r e l a t i o n ^ aH = QCH P, where Q i s the p r o p o r t i o n a l i t y constant for a proton i n a C-H fragment and has a value of about -63 MHz. The determination of the i s o t r o p i c hyperfine coupling constant for an a-proton i s thus a very useful method for deducing the e l e c t r o n i c structure of a i r - r a d i c a l . McConnell's r e l a t i o n i s v a l i d f o r the N-H fragments as w e l l , with an appropriate change i n Q. 3.2.2 The Anisotropic Hyperfine Tensor: Experimental determination of a-proton anisotropic hyperfine tensors i n several r a d i c a l s ^ has shown that the p r i n c i p a l values of the tensors -46-have approximately the form: t,0,-t with the p r i n c i p a l value ' t' directed along the X-H bond d i r e c t i o n , p r i n c i p a l value '0' directed p a r a l l e l to the axis of the p - o r b i t a l and the p r i n c i p a l value of - ' t ' directed perpendicular to the X-H bond i n the molecular plane. These observations were f i r s t q u a l i t a t i v e l y explained i n terms of the e l e c t r o n i c structure of a i T - r a d i c a l by Ghosh and Whiffen.^ 7 They explained the case of an i s o l a t e d C-H fragment, with the unpaired electron i n a carbon 2p o r b i t a l . The l o c a l symmetry of the problem required the three p r i n c i p a l axes to l i e along the d i r e c t i o n s experimentally observed for an a-proton. Using the dip o l a r Hamiltonian, they determined the r e l a t i v e sizes and signs of the three p r i n c i p a l values given by: -g3g N B N < ( l - 3 c o s 2 6 ) r - 3 > a v e where r i s the distance from the electron to the nucleus, 6 i s the angle between the radius vector and a p r i n c i p a l axis of the coupling tensor and < > a v e s i g n i f i e s an average over the s p a t i a l d i s t r i b u t i o n of the unpaired 2 electron. The angular function, (l-3cos 0) has a node at 0^=53° d i v i d i n g 2 the space into two parts: i n one part (l-3cos 9) i s p o s i t i v e and i n the other negative. When the magnetic f i e l d being i n Z-direction l i e s along C-H bond, electron w i l l most probably be found inside the cone of h a l f angle, 9^ thus giving a p o s i t i v e hyperfine coupling. When the f i e l d i s along Y-axis i n the plane of the r a d i c a l and i s perpendicular to the C-H bond, the electron would be found outside t h i s cone, hence the hyperfine coupling i s negative. When t h i s f i e l d i s p a r a l l e l to the axis of the p-o r b i t a l i . e . X - d i r e c t i o n , both p o s i t i v e and negative contributions w i l l be appreciable, hence we expect a value much smaller i n magnitude than either of the others and perhaps not far from zero. Comparing these predictions -47-w i t h e x p e r i m e n t a l p o s s i b i l i t i e s , we see t h a t t h e y a r e c o n s i s t e n t w i t h t h e c h o i c e o f n e g a t i v e s i g n f o r t h e i s o t r o p i c c o u p l i n g . a - p r o t o n c o u p l i n g t e n s o r s d i f f e r v e r y l i t t l e f r o m one s a t u r a t e d h y d r o c a r b o n r a d i c a l t o a n o t h e r . I f , h o w e v e r , t h e odd e l e c t r o n i s d e l o c a l i z e d such as i n c o n j u g a t e d and a r o m a t i c compounds, t h e p r i n c i p a l v a l u e s a r e r e d u c e d p r o p o r t i o n a t e l y . Sometimes t h e t o t a l a n i s o t r o p i c h y p e r f i n e t e n s o r o f a p r o t o n does n o t b e a r a s i m p l e r e l a t i o n s h i p t o t h e c r y s t a l s t r u c t u r e . I n such c a s e s , t h e a s s i g n m e n t o f t h e t e n s o r t o a p a r t i c u l a r p r o t o n i s made by c o m p a r i s o n o f i t s d i r e c t i o n s and t e n s o r s w i t h t h e t h e o r e t i c a l ones c a l c u l a t e d a n a l y t i c a l l y by t h e method o f M c C o n n e l l and S t r a t h d e e ^ . M c C o n n e l l and S t r a t h d e e ' s c a l c u l a t i o n i s n o t r e s t r i c t e d t o t h e a n i s o t r o p i c t e n s o r o f an a - p r o t o n , b u t can be a p p l i e d t o t h e i n t e r a c t i o n o f t h e u n p a i r e d e l e c t r o n w i t h any p r o t o n . However , t h e f o r m o f t h e t e n s o r depends on t h e r e l a t i v e o r i e n t a t i o n s o f t h e u n p a i r e d e l e c t r o n ' s o r b i t a l and t h e p r o t o n and t h e d i s t a n c e be tween them. A t s u f f i c i e n t l y l a r g e d i s t a n c e s , t h i s t r e a t m e n t r e d u c e s t o t h e e x p e c t e d p o i n t - d i p o l e f o r m u l a and t h e p r i n c i p a l v a l u e s o f t h e t e n s o r have a x i a l symmet ry . 3 . 3 H y p e r f i n e c o u p l i n g Tenso rs f o r 6 - P r o t o n s : The m e t h y l p r o t o n s i n t h e e t h y l r a d i c a l ( C H ^ ^ Q ^ O a r e two bonds d i s t a n t f r o m t h e u n p a i r e d e l e c t r o n and hence t h e y a r e c a l l e d 3 - p r o t o n s . The B - p r o t o n d i p o l a r t e n s o r s e x h i b i t r e l a t i v e l y s m a l l a n i s o t r o p y a m o u n t i n g t o <10 MHz f o r u n i t s p i n d e n s i t y on t h e a - c a r b o n . The maximum a n i s o t r o p y i s o f t e n VL0% o f t h e i s o t r o p i c c o u p l i n g f o r u n i t s p i n d e n s i t y on an a l i p h a t i c o r a r o m a t i c a - c a r b o n . T h i s s m a l l a n i s o t r o p y i s d u e , o f c o u r s e , - 3 t o t h e g r e a t l y r e d u c e d v a l u e o f < r > compared t o an a - p r o t o n . The c o u p l i n g - 4 8 -t e n s o r s o f t e n show n e a r a x i a l symmetry and c o n s e q u e n t l y t h e o r i e n t a t i o n s o f t h e p r i n c i p a l v a l u e s t e n d t o be r e l a t i v e l y i m p r e c i s e l y d e t e r m i n e d . However, B - p r o t o n s have a l a r g e c o n t a c t h y p e r f i n e i n t e r a c t i o n , a , w h i c h v a r i e s w i t h t h e a n g l e 6 b e t w e e n t h e p l a n e o f t h e C-C and C-H bonds and t h e a x i s o f p o r b i t a l . z a = B 1 + B 2 c o s2 6 ( 3 - 2 ) 2 where B^ and B^ a r e c o n s t a n t s . The cos 8 dependence can be e x p l a i n e d by 69 h y p e r c o n j u g a t i o n . T h i s i s an e x t r e m e a p p r o a c h t o t h e e l e c t r o n i c s t r u c t u r e o f t h e r a d i c a l , b u t i t p r o v i d e s a mechanism whereby t h e e l e c t r o n can p e n e t r a t e i n t o t h e h y d r o g e n I s o r b i t a l s . Such a p r o c e s s assumes t h a t t h e u n p a i r e d e l e c t r o n i n t h e c a r b o n 2p^ o r b i t a l c o u p l e s s l i g h t l y w i t h an e l e c t r o n i n t h e C-H a - b o n d , l e a v i n g t h e o t h e r w i t h a s m a l l p o s i t i v e s p i n d e n s i t y ( t h e s i g n o f i s o t r o p i c c o u p l i n g c o n s t a n t f o r B - p r o t o n s i s i n f a c t f o u n d t o be p o s i t i v e ) . There have been a number o f a t t e m p t s t o i n t e r p r e t t h e w h o l e B - p r o t o n i n t e r a c t i o n i n t e r m s o f s p i n p o l a r i z a t i o n . For e x a m p l e , c a l c u l a t i o n s by M c L a c h l a n ^ and Derbysh i re ' ' " ' " based on t h i s c o n c e p t a l s o y i e l d an a n g u l a r dependence such as t h a t g i v e n i n ( 3 - 2 ) . I n t h i s mechan ism, p o l a r i z a t i o n o c c u r s by t h e a d m i x t u r e o f e l e c t r o n i c c o n f i g u r a t i o n s w h i c h l e a v e u n p a i r e d s p i n i n one o f t h e a t o m i c o r b i t a l s c o n s t i t u t i n g t h e C-H b o n d . There i s no a c t u a l t r a n s f e r o f s p i n as t h e r e i s i n h y p e r c o n j u g a t i o n ; t h e t o t a l s p i n d e n s i t y i n t h e C-H bond r e m a i n s t h e same and t h e change i n s p i n d e n s i t y o f one o f t h e a t o m i c o r b i t a l s i s matched by an o p p o s i t e change i n t h e o t h e r . I n some ways t h i s k i n d o f mechanism a p p e a r s more a c c e p t a b l e , p a r t i c u l a r l y when a p p l i e d t o a r o m a t i c a n i o n s . I n t h e s e , h y p e r c o n j u g a t i o n s u g g e s t s t h a t -49-there i s t r a n s f e r of charge from B-protons to the 2p^ antibonding o r b i t a l s of the r i n g carbons, but these have already accepted one electron i n the formation of the anion from the hydrocarbon and are l i k e l y to r e s i s t further additions. 72 However, recent c a l c u l a t i o n s are tending away from the spin p o l a r i z a t i o n mechanism. Thus hyperconjugation remains a very p l a u s i b l e mechanism based on a widely accepted concept. 3.4 Interactions with Distant Protons: Interactions with more distant protons are r a r e l y observed i n ESR. For instance, where i n t e r a c t i o n with Y -P r°tons has been observed, the 73 s p l i t t i n g i s normally le s s than 1 gauss. There are even one or two 74 cases of i n t e r a c t i o n with s t i l l more distant protons i n aromatic ions. For r a d i c a l s studied i n s o l i d matrices, the linewidths are such that only a- and 3-proton s p l i t t i n g s are observed. It i s through ENDOR that i n t e r a c t i o n s f or such weakly coupled protons have been measured. More distant protons, including protons on adjacent molecules to the r a d i c a l (matrix protons) and hydrogen bonded protons generally exhibit a nearly pure d i p o l a r coupling i n which the maximum anisotropy i s small (<10 MHz), but i s greater than the i s o t r o p i c coupling. A point dipole model for the dipolar i n t e r a c t i o n i s a x i a l l y symmetric along the d i r e c t i o n between the two point dipoles, with the largest p r i n c i p a l component of the dipolar tensor l y i n g along the bond d i r e c t i o n . 3.5 Interaction of Protons Bonded to Atoms Other Than Carbon: In general, the hyperfine tensors of protons bonded to other atoms such as nitrogen and oxygen are somewhat analogous to protons bonded to -50-carbon. A large anisotropy f o r a-protons bonded to nitrogen i s expected. 42 This i s observed i n the 1,2,4-triazole H-adduct r a d i c a l . Protons bonded to oxygen generally show small anisotropy because the protons are often 8 to the s i t e of primary spin d e n s i t y . 7 ^ 3.6 Hyperfine Coupling f o r Nitrogen Atoms: 14 3.6.1 The Isotropic N Hyperfine Coupling: 14 The theory of i s o t r o p i c N coupling i s more complicated than for protons, although no new p r i n c i p l e s are involved. The spin p o l a r i z a t i o n mechanism introduced e a r l i e r which produces spin density at an a-proton also produces spin density at the nucleus of the atom to which that proton i s attached. The expression for t h i s spin density produced at the nitrogen atom y i e l d s a McConnell r e l a t i o n completely analogous to equation (3-1). This simple r e l a t i o n may not hold i f there i s appreciable spin density on adjacent atoms. The unpaired ir-electron density on the adjacent atoms po l a r i z e s the adjoining a-bonds and induces negative spin density 14 i n the nitrogen 2p o r b i t a l . However, the nuclear moment of N i s r e l a t i v e l y small and the p o l a r i z a t i o n produced by spin density on adjacent atoms can 14 often be neglected. Thus the N s p l i t t i n g i s roughly proportional to the odd electron density i n the nitrogen 2p^ o r b i t a l with a Q value of about 75 MHz. 7 6 14 3.6.2 The Anisotropic N Hyperfine Tensor: 14 The anisotropic N-hyperfine tensor possesses a x i a l symmetry. The diagonal hyperfine tensor has the form: (~BQ, -BQ, 2BQ) where: B 0 = 3* B 8N BN < r~ 3 > ( 3 _ 3 ) The d i r e c t i o n of the p r i n c i p a l value 2BQ i s p a r a l l e l to the nitrogen 2p z - 5 1 -o r b i t a l and perpendicular to the molecular plane. The t h e o r e t i c a l value of BQ for a spin density of unity i n the p - o r b i t a l i s estimated to be 47.9 MHz.^ Using the value of BQ from experiment one can estimate the spin density of p^ o r b i t a l of the nitrogen atom. The agreement with the spin density calculated from the i s o t r o p i c coupling of proton attached to nitrogen nucleus gives a clue to assign the tensor to nitrogen nucleus. 14 3.7 N Quadrupole Interactions: P h y s i c a l l y the quadrupole coupling i s s e n s i t i v e to the t o t a l electron density i n contrast to the hyperfine coupling which i s s e n s i t i v e only to the unpaired spin density. Thus quadrupole couplings are more d i r e c t l y r e l a t e d to chemical bond strengths. Because of the prevalence of nitrogen among organic and biochemicals, 14 the N quadrupole coupling i s p o t e n t i a l l y a valuable source of information. The i n t e r p r e t a t i o n of t h i s coupling i s made somewhat uncertain because of complicated bonding on N which makes d i f f i c u l t the estimation of the un-balanced p-electrons and because of some uncertainties i n the f i e l d gradient at the nitrogen nucleus. However, successful r e s u l t s have been obtained 78—80 using approximate methods developed by Townes and Dailey and l a t e r 81—83 modified and extended by Gordy. In i t s simplest forms the theory a t t r i b u t e s the f i e l d gradient to an unequal f i l l i n g of the p - o r b i t a l s i n the valence s h e l l of the atom containing the coupling nucleus, a l l other charges being ignored. If we define an axis system (x,y,z) such that x i s p a r a l l e l to N-C bond, z i s perpendicular to the molecular plane and y i s perpendicular to both x and z, the p r i n c i p a l elements of the t h e o r e t i c a l coupling tensor are given by: -52-X x = [ n x - H i n y + n z ) ] e Q q 2 1 ( ) X y = [ n y - % ( n x + n z ) ] e Q q 2 1 0 *z " I n z - « n x + n y ) ] e Q q 2 1 0 ( 3 _ 4 ) where e i s the proton charge, Q i s the nuclear quadrupole moment, n^, n y , n z are the numbers of unbalanced p-electrons and q2^Q represents the f i e l d gradient due to an electron i n an atomic 2 p - o r b i t a l . e^210 X S a s s u m e c * 14 84 to be -10 MHz for N. Comparison of t h e o r e t i c a l tensors with those from experimental observations does not always y i e l d good agreement. The disagreement between the tensors r e s u l t s from many si m p l i f y i n g assumptions made i n the f i e l d gradient c a l c u l a t i o n s . 3.8 Spin Density C a l c u l a t i o n s : Recently there have been various sophisticated molecular o r b i t a l (MO) methods a v a i l a b l e for the c a l c u l a t i o n of unpaired electron spin density d i s t r i b u t i o n i n ir - r a d i c a l s . Such MO c a l c u l a t i o n s have been advanced to the l e v e l that they can be used to support the assignment of hyperfine couplings to the various r a d i c a l s . The calculated spin d e n s i t i e s should of course be compared with the experimental ones. The a p p l i c a t i o n of experimental techniques, i . e . , ESR and ENDOR to r a d i c a l s produces proton hyperfine coupling constants which can be converted into the experimental spin d e n s i t i e s v i a McConnell r e l a t i o n . -53-In a serie s of papers, Pople and coworkers developed an approximate s e l f - c o n s i s t e n t - f i e l d molecular o r b i t a l s (SCF-MO) theory based on an 85 86 intermediate neglect of d i f f e r e n t i a l overlap (INDO). ' Their aim was to design an approximate a l l valence electron MO method for estimating hyperfine coupling constants i n various r a d i c a l s . In an e a r l i e r approxi-mate MO procedure known as complete neglect of d i f f e r e n t i a l overlap (CNDO) 87—89 devised by Pople and coworkers the one-center exchange i n t e g r a l s were neglected and the r e s t r i c t e d Hartree-Fock wavefunction was used, so that o-TT c o r r e l a t i o n observed i n i s o t r o p i c hyperfine couplings i s not properly treated at t h i s l e v e l of approximation. The INDO approximation i s l e s s severe i n that a l l one-center exchange i n t e g r a l s are retained and the un r e s t r i c t e d Hartree-Fock wavefunction i s used. As a r e s u l t , INDO method i s a s u b s t a n t i a l improvement over CNDO i n any problem where electron spin i s important. The unpaired electron population i s equal to the di f f e r e n c e between a and B-electron d e n s i t i e s : spin a g / r > P P - P (3-5) rs rs rs Form t h i s expression i t i s c l e a r l y seen how a negative spin density can a r i s e at a given point. The simple Hiickel method, on the other hand, i s applicable only to planar conjugated free r a d i c a l and predicts only p o s i t i v e spin d e n s i t i e s . The matrix of elements P_.g 1 U i s usually c a l l e d the spin density matrix. Thus with t h i s matrix, one can obtain the spin density at any point i n the system. Unlike the simple Hiickel and McLachlan methods, which are applicable only to planar molecules, the INDO method i s applicable to molecules of any geometry. I t provides a good means of i n v e s t i g a t i n g the v a l i d i t y of the -54-M c C o n n e l l r e l a t i o n because i t i m p l i e s t h e d i r e c t p r o p o r t i o n a l i t y b e t w e e n t h e u n p a i r e d e l e c t r o n p o p u l a t i o n ( P ^ ) o f t h e a d j a c e n t c a r b o n (Zp^ ) o r b i t a l and t h e u n p a i r e d e l e c t r o n p o p u l a t i o n ( p g ) o f t h e h y d r o g e n I s o r b i t a l . We h a v e , t h e r e f o r e , used t h e INDO a p p r o x i m a t i o n t o c a l c u l a t e t h e s p i n d e n s i t i e s f o r v a r i o u s r a d i c a l s i n o r d e r t o s u p p o r t o u r r a d i c a l a s s i g n m e n t s , t h e compute r p r o g r a m b e i n g p r o v i d e d by P o p l e and c o l l a b o r a t CHAPTER FOUR EXPERIMENTAL METHODS: 4.1 Introduction: This chapter outlines the operation of the ENDOR spectrometer as well as general treatment of ENDOR data. D e t a i l s on c r y s t a l preparation and handling w i l l be discussed i n the chapters dealing with the i n d i v i d u a l samples. 4.2 The ENDOR Spectrometer: The ENDOR spectrometer used i n these experiments has been described 91 i n d e t a i l by a previous worker from t h i s laboratory. The main features of the instrument together with a few modifications are given below and a modified block diagram of the spectrometer i s given i n Figure 4. The spectrometer i s b u i l t around an X-band ESR spectrometer and i s capable of operating i n either the homodyne or superheterodyne mode. Invariably the experiments at the usual temperatures of 77 or 4.2 K require the use of such low microwave powers that the r e s u l t i n g AFC i n s t a b i l i t y and noise l e v e l s make homodyne operation d i f f i c u l t . Hence our experiments were performed e x c l u s i v e l y i n the superheterodyne mode, using an intermediate frequency of 30 MHz. The source of microwave power was a kly s t r o n with a Hewlett-Packard 716B power supply. The microwave frequency was s t a b i l i z e d by phase-lockin to a microwave systems model MOS 1 frequency s t a b i l i z e r . Some of the micro wave power was coupled out of the main waveguide with a 3dB d i r e c t i o n a l coupler and fed into a balanced modulator for the generation of the side bands. The balanced modulator was tuned such that the out-put power at the c a r r i e r frequency, v n was minimum while the power i n the two side band KLYSTRON T T T V ISOLATOR KLYSTRON POWER SUPPLY T F R E Q U E N C Y ! / C O U N T E R PL T T T j SYNCHRONIZER |-20 dB DC. T T T 3 dB D.C. nzr 3=1 ISOLATOR 20 dB DIRECTIONAL COUPLER 20 dB ATTENUATOR 20 dB ATTENUATOR BALANCED 30 MHz MIXER •* AMPLIFIER —^ -FILTER CAVITY PHASE SHIFTER 1 THREE 20 dB ATTENUATORS MATCHED LOAD H 3 __J MAGIC TEE | _ _ f l S O L A T O R [ MICROWAVE SWITCH t I MIXER 1 j ISOLATOR | SAMPLE CAVITY | DETECTOR "j-^ CRO S MODULATION 30 W POWER v COILS AMPLIFIER 30 MHz AMPLIFIER I | RF DETECTOR] RF AMPLIFIER MAGNET IPOWER SUPPLY FREQUENCY SYNTHESIZER AUDIO AMPLIFIER • i y | RAMP GENERATOR FREQUENCY COUNTER DIGITAL EVENT MARKER RECORDER FIELDIAL UNIT ESR -57-was maximum. The main branch of microwave power at the c a r r i e r frequency was led through the three 20dB attenuators to the cavity through a magic Tee bridge. A TE-^Q-L rectangular cavity was modified and designed by Dr. V . P. 92 Chacko for ENDOR work. Two v e r t i c a l columns of f i v e s t r a i g h t gold plated brass posts of 0.5 mm diameter traverse ( i n the x-direction) the cavity p i e r c i n g the two end walls. The posts are insulated from the end walls by a r a l d i t e . The posts are connected by thi n copper wires outside the ca v i t y to form a s i n g l e loop. One end of the loop was connected to the output of an ENI3100L r . f . a m p l i f i e r , the other end was soldered to the body of the cavity to complete the r . f . c i r c u i t . In th i s design, both the microwave magnetic f i e l d , and r . f . f i e l d , are v e r t i c a l , allowing the magnet to be rotated i n the ho r i z o n t a l plane for studies of the anisotropy of the ENDOR spectra. The posts are everywhere perpendicular to the microwave e l e c t r i c f i e l d , which i s i n the y - d i r e c t i o n and have only a minor e f f e c t on the cavity Q. No net f l u x due to the microwave threads the loop; therefore, the loop does not harm the qua l i t y f a c t o r . The spacing between the two columns (5 mm i n our case) i s determined by the sample s i z e . The width of the cavity can be increased to accommodate larger spacing between the two columns. The cav i t y frequency for the T E ^ Q ^ mode i s unaffected by the increased width. The space outside the rectangular cross section solenoid of posts, but inside the cavity provides the return path for r . f . f i e l d l i n e s . A s i m i l a r construction of an ENDOR cavity operating i n the TE201 m ° d e n a s been reported by Castner 93 and Doyle. The c r y s t a l i s mounted on a Perspex wedge and fi x e d onto the side wall of the c a v i t y using s i l i c o n e grease, between the two columns of posts. -58-The r e f l e c t e d microwave power from the cav i t y i s led through the t h i r d arm of the magic Tee to a balanced detector; i t i s detected at 30 MHz, amplified and passed to a PAR model 122 l o c k - i n a m p l i f i e r . The output of the l o c k - i n a m p l i f i e r i s used to provide the Y-drive of an X-Y recorder. The X-drive i s provided by eit h e r the f i e l d sweep from the F i e l d i a l f o r ESR spectra or by a s i g n a l generated by the frequency sweep for ENDOR. The magnetic f i e l d i s provided by a Varian rotatable electromagnet with 9" pole faces c o n t r o l l e d by a F i e l d i a l Mark II power supply u n i t . For ESR work, v a r i a b l e audio-frequency magnetic f i e l d modulation was provided by modulation c o i l s wound on the magnet pole-pieces. The c o i l s were driven by the output of the l o c k - i n a m p l i f i e r . For ENDOR work radio frequency was introduced at the sample s i t e by connecting the r . f . c o i l to the output of an ENI3100L r . f . a m p l i f i e r . The r . f . source was PRD7808 si g n a l generator which can be frequency modulated to a depth of 5 to 50 kHz. The r . f . frequency was varied by sweeping the sig n a l generator by means of a v a r i a b l e voltage ramp generator constructed by the el e c t r o n i c shop of t h i s department. A p a r a l l e l output from the ramp generator was used to drive the X-axis of the recorder. The r . f . frequency was monitored by a Hewlett-Packard 5246L counter which triggered a home-built d i g i t a l event marker producing c a l i b r a t i o n pips i n the spectrum at 1 MHz i n t e r v a l s . The spectrometer was equipped with two concentric pyrex dewars designed to cool the cavity to 4.2 K with l i q u i d helium i n the inner dewar and l i q u i d nitrogen i n the outer dewar. Our experiments were performed with l i q u i d nitrogen i n both dewars. As long as the outer dewar was kept f u l l , there was l i t t l e evaporation from the inner dewar. The outer dewar was r e f i l l e d -59-from time to time without d i s t u r b i n g the spectrometer. The c a v i t y was enclosed by a copper tube and immersed i n the l i q u i d nitrogen. The copper tube cooled the c a v i t y through conduction and prevented the l i q u i d nitrogen from entering the c a v i t y . A.3 ENDOR Technique: 4.3.1 C r y s t a l Alignment Through S i t e S p l i t t i n g : The commonly encountered c r y s t a l s i n which spectra show s i t e s p l i t t i n g are of monoclinic or orthorhombic symmetry and we s h a l l discuss these cases since the c r y s t a l s under i n v e s t i g a t i o n were either monoclinic or orthorhombic. In some cases of s i n g l e c r y s t a l studies, the spectra are symmetrical and can be interpreted straightforwardly i n terms of hyperfine s p l i t t i n g s f or a number of orientations but become more complicated along other o r i e n t a t i o n s . This indicates that there are magnetically inequivalent r a d i c a l s i n the c r y s t a l . These are chemically i d e n t i c a l species but having d i f f e r e n t o r i e n t a t i o n s , so that i n general they w i l l have d i f f e r e n t spectra for a given o r i e n t a t i o n of the magnetic f i e l d . The d i f f e r e n c e between the l i n e positions of the two r a d i c a l s i n the spectrum i s termed s i t e s p l i t t i n g . A monoclinic c r y s t a l has a^b^c, and a=Y=90°^8. The b-axis i s a two f o l d axis. In order to understand how the s i t e s p l i t t i n g v a r i e s with the o r i e n t a t i o n of the magnetic f i e l d , i t i s u s e f u l to think of the orientations of a pair of r a d i c a l s as represented by two vectors. The two r a d i c a l s w i l l have d i f f e r e n t spectra i f the magnetic f i e l d makes unequal angles with the two vectors. I f the b-axis i s i n the plane and the ac-plane i s perpendi-cular to the plane of the paper, the vectors d e f i n i n g the orientations of the two r a d i c a l s l i e i n the plane of the paper symmetrically with respect to b-axis. A magnetic f i e l d p a r a l l e l to b or anywhere i n the ac-plane makes -60-equal angles with the two vectors and so for those o r i e n t a t i o n s , the spectra would show no s i t e s p l i t t i n g . For other o r i e n t a t i o n s , there would be s i t e s p l i t t i n g . In orthorhombic symmetry, there would be four magnetically d i s t i n c t molecules. This sort of c r y s t a l has a^b^c, but a = 8 = Y = 9 0 ° . The b-axis remains fi x e d , but now we f i x a i n the plane of the paper and c perpendi-cular out of the plane of the paper. Out of four vectors, two are below the plane and other two above the plane re l a t e d to the f i r s t two by re-f l e c t i o n through the ab-plane. For a general o r i e n t a t i o n of the magnetic f i e l d , there w i l l be s i t e s p l i t t i n g into four spectra, but i n the ab-, ac-, or bc-planes, the r a d i c a l s become magnetically equivalent i n p a i r s . The only orientations for which there i s no s i t e s p l i t t i n g are when the f i e l d i s along one of the c r y s t a l l o g r a p h i c axes. Radicals trapped i n si n g l e c r y s t a l s are often very p r e c i s e l y oriented and i f there i s appreciable anisotropy, the spectra can be very s e n s i t i v e to s i t e s p l i t t i n g . The most serious uncertainty i n the ENDOR data i s the absolute o r i e n t a t i o n of the c r y s t a l i n the magnetic f i e l d . However, the r e p r o d u c i b i l i t y of the data with d i f f e r e n t c r y s t a l s and the double check provided by the s i t e s p l i t t i n g present i n d i f f e r e n t c r y s t a l l o g r a p h i c systems indicate that ±1° i s a reasonable estimate of t h i s error. Because the c r y s t a l could not be re-aligned without disassembling the cavi t y , i t was very d i f f i c u l t to reduce t h i s error. 4.3.2 Operation of ENDOR Spectrometer: The spectrometer was allowed to s t a b i l i z e for at le a s t an hour with the c a v i t y maintained at 77 K. The ESR spectrum was obtained and a p a r t i c u l a r point on the ESR lineshape was selected for ENDOR study. The -61-microwave power l e v e l for maximum ESR s i g n a l was determined and the ESR t r a n s i t i o n was p a r t i a l l y saturated by increasing the microwave power above t h i s l e v e l . The magnetic f i e l d modulation was then switched o ff and the spectrometer was adjusted f o r higher s e n s i t i v i t y by increasing the l o c k - i n a m p l i f i e r and recorder gains over two hundred-fold. The radio frequency was scanned u n t i l an ENDOR s i g n a l was obtained. The frequency modulation used f or detection was between one and four kHz with a depth depending on the ENDOR linewidth. Once the en t i r e ENDOR spectrum was observed and i t s i n t e n s i t y optimized with the l o c k - i n phase, modulation frequency, f i e l d strength and microwave power l e v e l , the magnet was rotated so that the angular v a r i a t i o n of the spectrum could be studied. The optimum conditions were found to be quite reproducible from day to day. 4.3.3 Data Reduction: The c a l i b r a t i o n of spe c t r a l l i n e positions was achieved by f i t t i n g the positions of frequency markers to a quadratic expression and using t h i s function to inte r p o l a t e the l i n e p o s i t i o n s . A le a s t squares f i t t i n g routine written by Dr. J. A. Hebden for a Monroe 1656 programmable desk c a l c u l a t o r was used f or t h i s . At least 3 c a l i b r a t i o n points were f i t t e d each time and the f i t reproduced the frequencies of the c a l i b r a t i o n markers to within 5 kHz. In most cases, the l i n e p o s i t i o n could be taken as the center of the l i n e , where i t crossed the base l i n e . For overlapping l i n e s which were asymmetric or d i d not cross the base l i n e , the l i n e p o s i t i o n was taken as the point of maximum slope. The ENDOR data were taken i n three orthogonal planes by ro t a t i n g the magnet i n i n t e r v a l s of 2 or 5° depending on the density of l i n e s . The l i n e -62-positions were then plotted as a function of magnetic f i e l d o r i e n t a t i o n f o r each of the three planes. The curves belonging to the same hyperfine coupling i n each of the three planes we re r e a d i l y i d e n t i f i e d by comparing the curves i n d i f f e r e n t planes at coincident axes. Experimentally observed points belonging to the same coupling were then f i t t e d to the spin Hamiltonian with the least-squares-adjustment programme LSF, mentioned e a r l i e r , using an Amdahl 470 V/6 model II computer. CHAPTER FIVE ENDOR STUDIES OF X-IRRADIATED PHENAZINE 5.1 Introduction: A thorough knowledge of the spin density d i s t r i b u t i o n and the geometrical structure of the organic r a d i c a l s can be achieved only through the complete determination of the hyperfine i n t e r a c t i o n tensors ( i . e . , of t h e i r p r i n c i p a l values and di r e c t i o n s ) for the d i f f e r e n t n u c l e i i n the r a d i c a l s under study. This i s possible only by working with the oriented systems such as those having the r a d i c a l species trapped i n a si n g l e c r y s t a l . In general, the r a d i c a l s produced by i o n i z i n g r a d i a t i o n i n a l i p h a t i c compounds y i e l d well-resolved ESR spectra and hence they have been studied i n d e t a i l . In aromatic systems, however, the un-paired electron i s extensively d e l o c a l i z e d and hence coupled to a r e l a t i v e l y great number of nuclear spins. As a r e s u l t , the ESR linewidth becomes e s s e n t i a l l y inhomogeneous and broad. For these compounds i n s o l i d state, the ESR spectra appear very poorly resolved or completely unresolved and 94-96 thus generally y i e l d l i t t l e information. The much better r e s o l u t i o n of ENDOR spectroscopy makes i t possible to study the hyperfine in t e r a c t i o n s of n u c l e i i n such aromatic r a d i c a l s 39 i n s i n g l e c r y s t a l s . Using t h i s technique BShme and Jesse have determined the structure of a stable dibenzo-cyclohexadienyl r a d i c a l produced by room temperature X - i r r a d i a t i o n of anthracene. While ENDOR l i n e s of a l l the C-H protons have been detected, no ENDOR l i n e s are found that could be assigned to methylene protons possibly due to unfavourable r e l a x a t i o n behaviour of ^CH 9 protons. However, from a number of evidences, i t was -64-unequivocally concluded that the r a d i c a l was formed by hydrogen addition. 40 Later, Bohme and Wolf c a r r i e d out de t a i l e d ENDOR studies of X-ir r a d i a t e d naphthalene sin g l e c r y s t a l . From the determination of spin density d i s t r i b u t i o n and hyperfine p r i n c i p a l values of a l l CH and CH 2 protons, they have concluded that two types of r a d i c a l s are formed by addit i o n of a hydrogen atom to the 1 and 4 positions of the naphthalene molecule. The d i f f e r e n t i n t e n s i t i e s for the two types have been ascribed to d i f f e r e n t 41 42 concentrations of the r a d i c a l s . Gloux and Lamotte ' have reported ENDOR investigations of r a d i c a l s i n aromatic h e t e r o c y c l i c compounds. In both imidazole and 1,2,4-triazole systems, t h e i r r e s u l t s correspond to a ir-radi c a l formed by the addit i o n of one hydrogen atom to a carbon of the r i n g . We decided to study phenazine for a number of reasons. The molecule i s very s i m i l a r to anthracene except that i t has two nitrogen atoms. In a l l aromatic compounds, including h e t e r o c y c l i c , so far studied, the ra d i c a l s were shown to be formed by the addition of hydrogen to carbon of the r i n g . P r i o r to t h i s study, an ENDOR in v e s t i g a t i o n of an X-i r r a d i a t e d s i n g l e c r y s t a l of a c r i d i n e (with one nitrogen) was taken up i n t h i s 92 laboratory. I t was found that the r a d i c a l i s formed as a r e s u l t of hydrogen addition to the nitrogen atom of the acri d i n e molecule. I f the hydrogen add i t i o n r a d i c a l detected i n a c r i d i n e was also produced i n phenazine, i t could only a r i s e from hydrogen addition to eit h e r of the two nitrogen atoms of phenazine r i n g . Moreover, phenazine resembles the f l a v i n moiety of r i b o f l a v i n which i s a constituent of several enzyme systems c a l l e d flavoproteins involved 97 i n intermediary metabolism. R i b o f l a v i n i s r e l a t i v e l y heat stable, but -65-s e n s i t i v e to l i g h t and i r r a d i a t i o n . Thus phenazine served as an excellent model system for determining the e l e c t r o n i c structure of the r a d i c a l s i n r i b o f l a v i n . There has not been any report of ESR or ENDOR on sing l e c r y s t a l s of phenazine. Our work i s the f i r s t ENDOR analysis of r a d i c a l formed by hydrogen addition to nitrogen atom of aromatic h e t e r o c y c l i c compounds. We have also observed two types of r a d i c a l s produced by hydrogen addition to either of the nitrogen atoms of phenazine. In t h i s chapter, the r e s u l t s of an ENDOR study of the prominent r a d i c a l s produced by room temperature X - i r r a d i a t i o n of sing l e c r y s t a l of phenazine are reported. No us e f u l r e s u l t s could be obtained from the ESR spectrum because of very poor r e s o l u t i o n as a r e s u l t of the extensive d e r e a l i z a t i o n of the unpaired electron. The hyperfine i n t e r a c t i o n tensors of a l l the protons i n t h i s r a d i c a l could be determined by the ENDOR technique. 14 No ENDOR l i n e s f o r the N n u c l e i were observed. This did not, however, cause any d i f f i c u l t y i n i d e n t i f y i n g the nature of the paramagnetic species. 5.2 Experimental: Single c r y s t a l s of phenazine were grown by slow evaporation of i t s saturated s o l u t i o n i n propanone at room temperature. Well developed c r y s t a l s of approximate dimensions 8x4x3 mm were e a s i l y obtained. Owing to differences i n the r e l a t i v e development of d i f f e r e n t faces, there appeared to be two v a r i e t i e s of c r y s t a l s , but they were found to be 98 c r y s t a l l o g r a p h i c a l l y i d e n t i c a l . The c r y s t a l morphology i s shown i n F i g . 5, along with the cr y s t a l l o g r a p h i c and reference axes. The c r y s t a l structure of phenazine was reported by Herbstein and 99 100 Schmidt and two years l a t e r , by H i r s h f e l d and Schmidt near 80 K. The atomic p o s i t i o n s are e s s e n t i a l l y the same i n both of these works. -66-Fig. 5. Crystal morphology of phenazine with reference axes. -67-Comparison of bond lengths shows that rms di f f e r e n c e between the two o sets of data i s t y p i c a l l y 0.010A. The c r y s t a l i s monoclinic with u n i t c e l l dimensions a=12.967, o b=4.981, c=7.056A, 6=109.0° and space group P2 1/a. There are two molecules per unit c e l l , both of which are magnetically equivalent along the b-axis and i n the ac-plane; i n a l l other orientations they are d i s t i n c t . The cr y s t a l l o g r a p h i c axes were located by external morphology and by i d e n t i -fying the c r y s t a l planes with the help of a two-circle o p t i c a l goniometer. ENDOR measurements were c a r r i e d out i n the reference coordinate system a, b, c* (c*=axb). The molecular structure with the numbering of r i n g atoms i s depicted i n F i g . 6. The c r y s t a l s were X - i r r a d i a t e d at room temperature using a Machlett type OEG-60 X-ray tube operating with a voltage of 40 kV and a beam current of 20 mA. The optimum concentration of the r a d i c a l s was obtained a f t e r the c r y s t a l s were i r r a d i a t e d for about 80 hours. The i r r a d i a t e d c r y s t a l s could be stored i n d e f i n i t e l y i n closed v i a l s at room temperature without any s i g n i f i c a n t decrease i n the r a d i c a l concentration. ENDOR measurements were made at 77 K with the spectrometer described i n chapter four. Since the ENDOR l i n e s were found to be well separated, the spectra were recorded at i n t e r v a l s of 5°, as the magnet was rotated about each of the reference axes. 5.3 Experimental Results: The ESR and ENDOR spectra of an X- i r r a d i a t e d phenazine s i n g l e c r y s t a l for the magnetic f i e l d p a r a l l e l to the b - a x i s - a r e shown i n Figure 7. Although the ESR spectrum i n t h i s o r i e n t a t i o n appears quite well-resolved, the r e s o l u t i o n was very much poorer i n most other o r i e n t a t i o n s . No useful data could, therefore, be obtained from the analysis of the ESR spectra. F i g . 6. Molecular structure and numbering of the ring atoms of phenazine. -69-I I ! I I 1 1 1 16 18 20 22 ENDOR FREQUENCY, MHz F i g . 7. (a) F i r s t d e r i v a t i v e X-band ESR spectrum at room temperature (b) ENDOR spectrum at 77 K of the N-hydrophenazinyl r a d i c a l i n an X-ir r a d i a t e d s i n g l e c r y s t a l of phenazine for the magnetic f i e l d p a r a l l e l to the b-axis. Assignment of the ENDOR l i n e s i s indicated. -70-As can be seen from Figure 7, the ENDOR spectra were, however, quite well-resolved. Not shown i n the f i g u r e are the ENDOR l i n e s i n the immediate v i c i n i t y of which might belong to the weakly coupled intermolecular protons. No ENDOR l i n e s due to the hyperfine coupling of the unpaired electron with nitrogen n u c l e i were observed i n our study. A l l the observed l i n e s a r i s e from i n t e r a c t i o n s with protons. Only those l i n e s appearing at the higher frequency side of were used i n the ana l y s i s . A maximum of 20 ENDOR l i n e s could be observed i n an a r b i t r a r y o r i e n t a t i o n , coalescing into ten when the magnetic f i e l d was oriented along the b-axis or i n the ac-plane. By s e l e c t i v e l y saturating various positions throughout the ESR lineshape, we established that some of the weaker l i n e s belong to a d i f f e r e n t r a d i c a l . The nature and o r i g i n of these weaker l i n e s w i l l be discussed l a t e r . For c a l c u l a t i o n and graphical presentation, was set equal to 13.820 MHz and the observed t r a n s i t i o n frequencies were corrected accordingly. The angular v a r i a t i o n of the proton ENDOR frequencies i s shown i n Figures 8 and 9 i n the three orthogonal planes. Figure 8 presents the high frequency branch of ENDOR l i n e s belonging to r a d i c a l ( I ) , whereas Figure 9 presents those from r a d i c a l ( I I ) . The curves belonging to the same proton coupling i n each of the three planes were r e a d i l y i d e n t i f i e d by comparing curves i n d i f f e r e n t planes at coincident axes (the reference axes). The ESR spectrum i n most orientations consists of an i l l - r e s o l v e d broad l i n e . Therefore the g-tensor could not be determined accurately. For convenience, we used the i s o t r o p i c g-value of 2.0023 i n place of the g-tensor. Approximately 100 data points were used to f i t each tensor and -71-FREQUENCY, MHz 24-T- - — 1 , 1 I 1 1 L- 1 r ' Q 30° 60° b 30° 60° C* 30° 60° a CRYSTAL ORIENTATION F i g . 8 . Angular dependence of the ENDOR frequencies of r a d i c a l (I) in an X-i r r a d i a t e d phenazine. The assignment of various ENDOR l i n e s to s p e c i f i c protons i s indicated on the r i g h t . F i g . 9. A n g u l a r v a r i a t i o n o f t h e ENDOR f r e q u e n c i e s o f r a d i c a l ( I I ) i n X - i r r a d i a t e d p h e n a z i n e . -73-the rms error was t y p i c a l l y l e s s than 10 kHz. The diagonalized forms of the hyperfine tensors calculated by LSF, along with t h e i r d i r e c t i o n cosines, are presented i n Tables 1 and 2 f o r the r a d i c a l s I and I I , res p e c t i v e l y . 5.4 Assignment of the couplings and Radical I d e n t i f i c a t i o n : 5.4.1 R a d i c a l ( I ) : The assignment of the hyperfine coupling tensors to s p e c i f i c protons i n r a d i c a l ( I ) i s indicated i n Table 1. At the outset, we should note that these coupling tensors are t y p i c a l of an aromatic T r - r a d i c a l . The d i r e c t i o n s of a l l the intermediate couplings are c l o s e l y p a r a l l e l to each other and perpendicular to the plane of the molecule as expected for a r a d i c a l with the unpaired electron i n a ir-molecular o r b i t a l . We attempted to r e l a t e the d i r e c t i o n s of the largest p o s i t i v e component of the anisotropic part of the coupling tensor to various C-H bond d i r e c t i o n s i n the undamaged c r y s t a l s . For protons attached to carbon atoms expected to have p o s i t i v e spin d e n s i t i e s , the d i r e c t i o n s of the largest p o s i t i v e component of the dipolar tensor are very nearly p a r a l l e l to the C-H bond d i r e c t i o n s . For other protons, deviations from the C-H bond d i r e c t i o n s were observed. A l l the hyperfine tensors could, however, be assigned to s p e c i f i c protons without much d i f f i c u l t y on the basis of the d i r e c t i o n cosines, the sign of the i s o t r o p i c part, symmetry consider-ations and comparison with the INDO r e s u l t s . It must be noted that because the unpaired electron i s extensively d e l o c a l i z e d over the whole molecule, the d i r e c t comparison of small coupling tensors to bond d i r e c t i o n s cannot y i e l d unequivocal conclusions. Four types of r a d i c a l s are encountered i n s o l i d organic matter as TABLE 1 PROTON HYPERFINE INTERACTION TENSORS FOR RADICAL(I) IN X-IRRADIATED SINGLE CRYSTAL OF PHENAZINE 2 Coupling (MHz) D i r e c t i o n Cosines Proton P r i n c i p a l Value 1 Isotropic Dipolar 1,8 + 2.64 -10.50 +13.14 +0.697 ±0.249 -0.673 -14.22 - 3.72 -0.634 ±0.652 -0.415 -19.91 - 9.41 -0.336 ±0.716 -0.612 2 + 3.38 + 7.58 - 4.20 -0.552 ±0.003 +0.834 + 9.21 + 1.63 -0.641 ±0.639 -0.425 +10.15 + 2.57 -0.533 ±0.769 -0.353 3 -4.01 -9.46 +5.45 -0.032 ±0.483 +0.875 - 9.43 + 0.03 -0.589 ±0.698 -0.407 -14.93 - 5.47 -0.808 ±0.529 +0.262 4,5 + 2.42 + 6.19 - 3.77 +0.574 ±0.643 -0.508 + 7.75 + 1.56 -0.799 ±0.576 -0.175 + 8.39 + 2.20 -0.179 +0.506 -0.844 6 - 4.13 - 9.62 + 5.49 +0.700 ±0.704 +0.121 - 9.58 + 0.04 -0.603 ±0.673 -0.428 -15.15 - 5.53 -0.383 ±0.226 +0.896 7 + 3.12 + 7.20 - 4.08 +0.782 ±0.385 -0.490 + 8.83 + 1.63 -0.583 ±0.730 -0.357 +9.64 +.2.44 -0.220 ±0.565 -0.796 10 - 2.93 - 8.50 + 5.57 +0.286 ±0.367 -0.885 - 9.79 - 1.29 -0.799 ±0.418 -0.432 -12.79 - 4.29 +0.529 ±0.831 -0.174 1. Signs of the p r i n c i p a l elements are assumed on the basis of INDO r e s u l t s . Uncertainty i n the p r i n c i p a l values i s ±0.01 MHz. 2. The two sign combinations chosen con s i s t e n t l y r e l a t e one distinguishable c r y s t a l s i t e to the other. -75-TABLE 2 PROTON HYPERFINE INTERACTION TENSORS FOR RADICAL(II) IN X-IRRADIATED SINGLE CRYSTAL OF PHENAZINE1 Coupling (MHz) D i r e c t i o n Cosines Proton E —- i — P r i n c i p a l value Isotropic Dipolar a b c - 3.97 +5.43 +0.697 ±0.712 +0.091 2- - 9.42 -9.40 -0.02 -0.639 ±0.673 -0.372 -14.81 -5.41 -0.326 ±0.200 +0.924 + 3.09 -4.08 +0.754 ±0.400 -0.520 3' + 8.79 +7.17 +1.62 -0.628 ±0.671 -0.394 + 9.62 +2.45 -0.192 ±0.624 -0.758 + 2.26 +12.55 +0.636 ±0.229 -0.737 4',5' -12.60 -10.29 - 2.31 -0.658 ±0.659 -0.364 -20.52 -10.23 -0.402 ±0.717 -0.570 + 3.27 - 4.12 -0.507 ±0.004 +0.862 6' + 9.01 +7.39 + 1.62 -0.651 ±0.658 -0.379 + 9.88 + 2.49 -0.566 ±0.753 -0.337 Bond d i r e c t i o n s from c r y s t a l structure C.-H., C.-H., C c-H c, C 0-H 0 : +0.678 ; ±0.210 ; -0.704 1 1 4 4 J J o o C 3-H 3 and C ?-H 7 : -0.005 ; +0.489 ; +0.872 C -H„ and C,-H, : ±0.685 ; ±0.714 ; ±0.143 L 2. O O 1. See footnotes to Table 1. -76-a r e s u l t of i o n i z i n g r a d i a t i o n involving either removal or attachment of an electron, d i s s o c i a t i o n of covalently bonded atomic hydrogen or as s o c i a t i o n of atomic hydrogen on unsaturated positions of the molecules. 1^ 1 Of these, the i o n i c r a d i c a l s r e s u l t i n g from an electron removal or attach-ment have been found to be stable only at low temperatures and can, therefore, be ruled out i n the present study. Of the remaining two p o s s i b i l i t i e s , namely the r a d i c a l s produced either by the add i t i o n or removal of a hydrogen atom to the undamaged molecule, only the former i s relevant i n the present case because we observe more intramolecular proton couplings than could be accounted for without an extra hydrogen added to the phenazine molecule. Therefore i t can be concluded with reasonable c e r t a i n t y that the r a d i c a l i s formed as a r e s u l t of the attachment of a hydrogen atom to one of the r i n g p o s i t i o n s . Now i f the hydrogen atom i s attached to any of the carbon atoms (other than at posi t i o n s 11-14), the r e s u l t i n g r a d i c a l would have a 39 40 methylene group with a large spin density on the adjacent carbon atom. ' These two protons of the methylene group (B-protons) should then have f a i r l y large i s o t r o p i c coupling. Our r e s u l t s do not indi c a t e the presence of such a large i s o t r o p i c coupling. The next p o s s i b i l i t y i s , of course, the attachment of a hydrogen atom to one of the nitrogen atoms, r e s u l t i n g i n a large spin density on the other ( c f . fi g u r e 10). For example, i f a hydrogen atom i s attached at NQQ)> then the r e s u l t i n g r a d i c a l would have a large spin density on the i r - o r b i t a l of N^g^. In an e a r l i e r study of N-hydroacridinyl r a d i c a l ^ i n molecule A and B of a c r i d i n e , the large spin density (^0.5) on was v e r i f i e d by the determination of the hyperfine tensor of proton at p o s i t i o n 9. This was the largest coupling having an i s o t r o p i c value of ^30.2 MHz Fig. 10. Spin density d i s t r i b u t i o n i n (a) N.- r a d i c a l s i n molecule A and B of a c r i d i n e (b) n Q 0 ) - H a n d N f q - ) ~ H r a d i c a l s i n phenazine. The INDO spin densities are given i n parentheses. -78-and assigned to C^-H proton. Based on s t r u c t u r a l and chemical s i m i l a r i t i e s between a c r i d i n e and phenazine, N-hydrophenazinyl r a d i c a l should be the f i r s t choice to be considered. A large Tr-spin density on would give 14 r i s e to large anisotropic hyperfine coupling with the N-nucleus. Although we have not observed any ENDOR l i n e s due to nitrogen, the o r i e n t a t i o n dependence of the ESR linewidth and s p e c t r a l spread ind i c a t e the highly anisotropic nature of the coupling. None of the proton hyperfine couplings observed i n t h i s r a d i c a l was large enough to account for the ESR s p e c t r a l spread. These observations point to a large Tr-spin density on the nitrogen atom. However, on account of the poor r e s o l u t i o n of the ESR spectra, the 14 N coupling tensor could not be determined. 5.4.2 R a d i c a l ( I I ) : In f i g u r e 7(b), the weaker l i n e s with primed numbers do not belong to the same r a d i c a l as the other l i n e s . This was v e r i f i e d by s e l e c t i v e l y saturating d i f f e r e n t portions of ESR spectrum. In a l l o r i e n t a t i o n s , these l i n e s were about f i v e times weaker than the main l i n e s . Although only three such weak l i n e s are seen i n Figure 7(b), a l l the main l i n e s had such accompanying weaker l i n e s but they were c l e a r l y resolved only i n some orien t a t i o n s . Only four of these l i n e s could be followed unambigu-ously i n a l l the three planes. The r e s u l t s obtained from the analysis of these l i n e s are presented i n Table 2. Comparison of hyperfine tensors presented i n Table 2 with those i n Table 1 show that they belong to a r a d i c a l which i s almost i d e n t i c a l i n structure and o r i e n t a t i o n to the N-hydrophenazinyl r a d i c a l . Because of the center of inversion of the phenazine molecule, the two nitrogen atoms are completely equivalent i n the undamaged c r y s t a l . Addition of hydrogen to either N ^ or N ^ r j ^ should, therefore, y i e l d -79-r a d i c a l s i d e n t i c a l i n a l l respects. The atomic positions 1 and 8 of the phenazine molecule are equivalent to positions 4 and 5; so also 2 and 3 to 6 and 7, res p e c t i v e l y . Thus the ENDOR l i n e s from the symmetry-related protons should, therefore, be indis t i n g u i s h a b l e for the two r a d i c a l s . But i f there are some s l i g h t conformational differences between the two ra d i c a l s because of constraints i n the s o l i d state, they would y i e l d d i f f e r e n t set of ENDOR l i n e s for the corresponding protons. We propose, therefore, that these weaker l i n e s belong to the same type of r a d i c a l , but with the hydrogen atom added to N ( g ) - T n e c r y s t a l surrounding of N ^ - H d i f f e r s from that of N^^-H r e s u l t i n g i n a s l i g h t conformational change of N^^-hydrophenazinyl r a d i c a l with respect to r a d i c a l formed by the addition of hydrogen to N ^ Q ^ . I t i s d i f f i c u l t , however, to understand the i n t e n s i t y differences between these two sets of ENDOR l i n e s . Since the structure of the two ra d i c a l s are i d e n t i c a l , the rel a x a t i o n pathways i n both the r a d i c a l s must be s i m i l a r leading to s i m i l a r ENDOR enhancements. The d i f f e r e n t i n t e n s i t i e s , therefore, ind i c a t e d i f f e r e n t concentrations of the two r a d i c a l s i n the c r y s t a l . As the two nitrogen atoms are completely equivalent i n the undamaged c r y s t a l , i t i s hard to understand why the addition of a hydrogen atom to one of them i s more favourable than to the other. Similar i n t e n s i t y 40 differences have been observed by Bohme and Wolf between two sets of ENDOR l i n e s i n the a-hydronaphthyl r a d i c a l s produced by X - i r r a d i a t i o n of naphthalene sing l e c r y s t a l s . They ascribe the i n t e n s i t y difference to the d i f f e r e n t rates of formation of the two symmetry related r a d i c a l s . 5.5 Spin Densities: The spin d e n s i t i e s on the r i n g atoms were deduced from the experimental -80-i s o t r o p i c couplings by using the f a m i l i a r McConnell r e l a t i o n . ' In order to confirm that our r a d i c a l i d e n t i f i c a t i o n i s correct INDO-MO cal c u l a t i o n s were performed on several possible r a d i c a l structures. Of the many r a d i c a l s considered, only the one with the hydrogen atom added to the nitrogen yielded good agreement for the spin d e n s i t i e s . Moreover, t h i s r a d i c a l has s i g n i f i c a n t l y lower t o t a l energy (INDO) as compared to a l l other structures considered. Thus we are f a i r l y c e r t a i n of the i d e n t i t y of the r a d i c a l . The experimental and INDO spin d e n s i t i e s for N-hydro-phenazinyl r a d i c a l s are presented i n table 3 along with those reported f o r N-hydroacridinyl r a d i c a l s for comparison. Inspection of table 3 reveals that the spin density d i s t r i b u t i o n i n the N-hydrophenazinyl r a d i c a l i s quite s i m i l a r to that found i n the a c r i d i n y l r a d i c a l . The large spin density on the second nitrogen atom could not be experimentally v e r i f i e d because there was no proton coupling at t h i s p o s i t i o n . On the whole the agreement can be deemed excellent considering the INDO approximations. The carbon atoms expected to have negative 2p spin d e n s i t i e s have unusually large spin populations. Similar large negative spin d e n s i t i e s were also 92 40 found i n N-hydroacridinyl and a-hydronaphthyl r a d i c a l s . 5.6 D i r e c t i o n Cosines: As mentioned e a r l i e r , f o r the r a d i c a l s reported i n t h i s study, a l l tensors have an axis i n common i n d i c a t i n g that the unpaired electrons i n these r a d i c a l s occupy molecular o r b i t a l s with Tf-symmetry. However, i n the course of discussion, mention must be made of a s i g n i f i c a n t deviation from c o l l i n e a r i t y of the common p r i n c i p a l d i r e c t i o n . As can be v e r i f i e d from tables 1 and 2, a l l the couplings are approximately of a-proton nature with f a i r l y small spin d e n s i t i e s on the respective r i n g atoms. TABLE 3 SPIN DENSITIES ON THE RING ATOMS OF N-HYDROACRIDINYL AND N-HYDROPHENAZINYL RADICALS P o s i t i o n N-hydroacridinyl r a d i c a l N-hydrophenazinyl r a d i c a l ' - .. p (INDO) TT p (Observed) TT p (INDO) TT p (Observed) TT Molecule A Molecule B Ref.103 Radical(I) Radical(II) Ref.103 1,8 +0.144 +0.114 +0.120 - +0.152 +0.165 +0.162 -2 -0.087 -0.157 -0.152 - -0.095 -0.119 -0.116 -3 +0.142 +0.165 +0.164 - +0.146 +0.149 — — 4,5 -0.089 -0.145 -0.144 - -0.082 -0.097 - -6 +0.161 +0.163 +0.156 - +0.146 +0.151 +0.148 -7 -0.101 -0.138 -0.147 - -0.095 -0.113 -0.113 -9 +0.473 +0.475 +0.475 0.45 +0.584 - - 0.37 10 +0.208 +0.138 +0.137 0.15 +0.171 +0.108 — 0.22 11,14 -0.127 - - - -0.127 - - -12,13 +0.171 - — — +0.129 ENDOR l i n e assigned to the N-H proton has anomalously low i n t e n s i t y and large linewidth as compared to a l l other l i n e s . This may be due to 14 an i n d i r e c t e f f e c t of the fast spin l a t t i c e r e l a x a t i o n of N nucleus. The d i p o l a r component of smallest magnitude for the N-H protons (designated as proton 10 i n table 1) shows a deviation of approximately 20°, i n d i c a t i n g that the N-H bond may be bent with respect to the molecular plane. This i s further confirmed by comparing the d i r e c t i o n of the larges t p o s i t i v e d i p o l a r component expected to be along the N-H bond d i r e c t i o n with those calculated from c r y s t a l l o g r a p h i c data. Our r e s u l t s thus ind i c a t e that the hydrogen atom that i s added on to nitrogen i s out of the plane by approxi-mately 20°. The largest couplings are assigned to protons at positions 1,8 i n r a d i c a l ( I ) and 4',5' i n r a d i c a l ( I I ) . These two tensors are very s i m i l a r i n nature with a spin density of about 0.163 on the r i n g atom. The d i r e c t i o n of the p o s i t i v e anisotropic component i s within 3° of the C^-H^ d i r e c t i o n i n the undamaged molecule. Interactions 2, 6 and 7 of r a d i c a l ( I ) correspond to i n t e r a c t i o n s 6',2' and 3' of r a d i c a l ( I I ) , r e s p e c t i v e l y . In the case of protons 3 and 6 of r a d i c a l ( I ) which are bonded to carbons with p o s i t i v e spin d e n s i t i e s , the d i r e c t i o n s of the p o s i t i v e d i p o l a r components coincide with the bond d i r e c t i o n s within 2°. However, deviation up to about 15° can be seen at protons 2 and 7. These deviations are caused by the fact that these protons are bonded to carbons with negative spin density. This negative spin density r e s u l t s from a p o l a r i z a t i o n of the a-electrons of the carbon skeleton whose influence on the l o c a l f i e l d must be accounted f o r . Although i n a l l the cases we have assumed that the unpaired electron i s only l o c a l i z e d -83-i n a carbon p - o r b i t a l , t h i s i s not the case with carbon with negative spin density because of the p o l a r i z a t i o n of a-electrons. Similar notable 40 deviations have been reported by Bohme and Wolf i n X- i r r a d i a t e d naphthalene s i n g l e c r y s t a l s . A s i g n i f i c a n t deviation from c o l l i n e a r i t y i s observed for the tensors associated with protons 4 and 5. No relevant explanation for the deviation can be given, except that the ENDOR data for these two protons are l e s s accurate because of very low si g n a l i n t e n s i t y (cf. f i g u r e 7(b)). 5.7 Discussion: As can be v e r i f i e d from tables 1, 2 and 3, there are small differences i n the corresponding values f o r the two types of r a d i c a l s produced from phenazine. The i s o t r o p i c couplings of r a d i c a l ( I ) are co n s i s t e n t l y higher than those of r a d i c a l ( I I ) . Hence the dipolar parts of these coupling tensors are also s l i g h t l y d i f f e r e n t . These differences have been at t r i b u t e d to the f a c t that the r a d i c a l s occur i n s l i g h t l y d i f f e r e n t conformations. The d i p o l a r part of the tensors for a l l the protons were calculated 68 a n a l y t i c a l l y by the method of McConnell and Strathdee for the two r a d i c a l s separately. However, the accuracy of these c a l c u l a t i o n s i s not good enough to compare such small d i f f e r e n c e s . Therefore, no further e f f o r t was made to explain them. As expected, the spin density d i s t r i b u t i o n i n the N-hydrophenazinyl r a d i c a l i s quite s i m i l a r to that found i n the a c r i d i n y l r a d i c a l studied 92 e a r l i e r i n t h i s laboratory. Although the spin density on i n the phenazinyl r a d i c a l was not experimentally determined, the INDO c a l c u l a t i o n s indicate that i t i s s l i g h t l y larger than on i n the a c r i d i n y l r a d i c a l (cf. table 3). This d i f f e r e n c e can probably be j u s t i f i e d by the differe n c e - 8 4 -i n the e l e c t r o n e g a t i v i t i e s of the two atoms. Accordingly, the spin d e n s i t i e s on the other atoms i n the two r a d i c a l s are also s l i g h t l y d i f f e r e n t . 102 103 Subsequent to the completion of t h i s work, Furrer et a l . ' have reported the ESR studies of r a d i c a l p a i r formation from excited states i n the guest-host systems ( i ) acridine-fluorene ( i i ) phenazine-fluorene c r y s t a l s . Their r e s u l t s indicate that a r a d i c a l p a i r i s formed by hydrogen abstraction from fluorene and hydrogen addition to a c r i d i n e or phenazine. Table 3 l i s t s the c e n t r a l spin d e n s i t i e s f o r a c r i d i n y l and 103 phenazinyl r a d i c a l s as reported by these authors for comparison. While excellent agreement can be stated for a c r i d i n y l r a d i c a l , the ce n t r a l spin d e n s i t i e s i n the phenazinyl case f o r these authors are quite d i f f e r e n t from those of ours and INDO, although the sum of the two c e n t r a l spin d e n s i t i e s seems comparable. This d i f f e r e n c e has been a t t r i b u t e d by them to the larger angle /_(Z*,b) observed for phenazinyl and corresponding move of the charge density center towards the NH fragment o f the phenazinyl r a d i c a l . The work of these authors provides a substantial a d d i t i o n a l support to the i d e n t i f i c a t i o n of our r a d i c a l . Whereas there i s l i t t l e doubt about the i d e n t i t y and nature of the ra d i c a l s discussed here, the mechanism of t h e i r formation cannot be as-certained from the present study. Most probably i o n i c species are produced as primary r a d i c a l s as a r e s u l t of the impact of X-rays. As these i o n i c r a d i c a l s are unstable at room temperature, they undergo proton addition to form the r a d i c a l i d e n t i f i e d i n t h i s study. However, t h i s can be v e r i f i e d only by low temperature i r r a d i a t i o n studies. There i s l i t t l e we can say about the source of the hydrogen atoms. They are probably extracted from other molecules. However, no hydrogen -85-abstraction r a d i c a l s are i d e n t i f i e d i n the present work. In some orientations there were some weak ENDOR l i n e s which did not belong to the hydrogen addition r a d i c a l s . The experimental data were eit h e r incomplete or u n r e l i a b l e , so that t h e i r i d e n t i f i c a t i o n was not se r i o u s l y attempted. These weak l i n e s most c e r t a i n l y belong to another r a d i c a l the nature of which we cannot a s c e r t a i n i n the present study. CHAPTER SIX 14 1 N AND H ENDOR STUDIES OF RADIATION DAMAGE IN HIPPURIC ACID: 6.1 Introduction: Hippuric acid i s one of the few n a t u r a l l y occurring amino acids found i n the urine of most mammals including humans. The compound was is o l a t e d from horse urine i n 1829 and i t s composition determined i n 1839 104 by L i e b i g . M e t a b o l i c a l l y produced benzoic acid i s rendered harmless, by reaction with glycine to give hippuric a c i d , which i s then excreted. The reaction takes place i n the l i v e r and may be used as a diagnostic C i • r . 1 0 5 test of lxver function. Although ESR and ENDOR techniques have been extensively used i n the study of r a d i a t i o n chemistry of many b i o l o g i c a l l y important amino acids and t h e i r d e r i v a t i v e s , very l i t t l e a t tention has been focussed on the study of r a d i a t i o n damage i n hippuric a c i d . V o t i n o v 1 ^ has reported an ESR study of y - i r r a d i a t e d p o l y c r y s t a l l i n e hippuric a c i d . He has suggested that the r a d i c a l i s formed by the loss of the N-H hydrogen atom. Our r e s u l t s , however, indicate that the stable paramagnetic species formed by room temperature X - i r r a d i a t i o n of hippuric acid i s the N-Benzoyl-amino-methyl r a d i c a l which i s thought to be the end product of oxidation, caused by i o n i z i n g r a d i a t i o n i n hippuric acid. In addition to detecting ENDOR l i n e s due to a-protons and weakly 14 coupled distant protons, we have observed N ENDOR l i n e s i n t h i s r a d i c a l . 14 Although the N nucleus i s present i n many b i o l o g i c a l l y important compounds, i t s ENDOR spectrum has been detected i n only a few of these compounds. This i s due i n part to i t s small nuclear magnetic moment and rapid nuclear spin -87-23 l a t t i c e r e l a x a t i o n . Box, Freund and Budzinski were the f i r s t to report hyperfine constants obtained from an ENDOR study of x - i r r a d i a t e d v a l i n e . R i s t and Hyde"^ 7 observed ligand "^N ENDOR signals from a study of Cu-8-hydroxyquinolinate doped i n c r y s t a l s of phthalimide and 8-hydroxyquinoline. Rustgi and Box"*"^ have reported the "^N hyperfine and 2-quadrupole coupling tensors of the inorganic species, N0 2 and NO^ , formed 14 i n x - i r r a d i a t e d glycylglycine-HNO^. An i s o t r o p i c N ENDOR with a low 109 hyperfine coupling has been obtained by Deigen and coworkers i n y-i r r a d i a t e d glycine si n g l e c r y s t a l s . This unique observation i s consistent with the fi n d i n g that the maximum spin density i s found not on the nitrogen, but on the adjacent carbon atom i n the r a d i c a l , NHgCHCOO proposed by these workers. Nelson, Atwater and Gordy 1 1^ obtained the ^ ^N hyperfine and quadrupole coupling tensors from an ENDOR analysis of free r a d i c a l s trapped i n y - i r r a d i a t e d dimethylglyoxime si n g l e c r y s t a l s . Nitrogen hyperfine and quadrupole data have also been presented by Schweiger and Gunthard11"'" from the sing l e c r y s t a l ESR and ENDOR of b i s - ( s a l i c y l a l d o x i m a t o ) -Cu(II) doped i n b i s - ( s a l i c y l a l d o x i m a t o ) N i ( I I ) . More recently ENDOR l i n e s have been detected and analyzed by a number of workers from t h i s laboratory 112 i n x - i r r a d i a t e d c a f f e i n e hydrochloride dihydrate and Cu(II) impurity com-plexes i n a - g l y c i n e 1 1 3 and a l a n i n e . A d e t a i l e d analysis of hyperfine and quadrupole coupling of a ligand nitrogen i n the system of Cu(II) i n L-alanine has been presented by Calvo, Oseroff and Abache.1''""' 14 1 In t h i s chapter we report for the f i r s t time N and H ENDOR studies of a stable r a d i c a l formed by room temperature x - i r r a d i a t i o n of si n g l e c r y s t a l s of hippuric a c i d . 6.2 Experimental: Single c r y s t a l s of hippuric acid were grown by slow evaporation of i t s -88-saturated aqueous s o l u t i o n maintained at 30°C. Deuterated c r y s t a l s were obtained s i m i l a r l y from heavy water so l u t i o n s . Well developed c o l o r l e s s c r y s t a l s of approximate dimensions 8x5x3 mm were e a s i l y obtained. The c r y s t a l morphology i s shown i n F i g . 11 along with the c r y s t a l l o g r a p h i c axes. In t h i s f i g u r e , the c-axis i s perpendicular to the plane of the paper. There have been three d i f f e r e n t c r y s t a l structure determinations 116—118 of hippuric a c i d , including one by neutron d i f f r a c t i o n . These reports are i n subs t a n t i a l agreement regarding the structure, while d i f f e r i n g i n t h e i r choice of the cr y s t a l l o g r a p h i c axes. We have used 118 the neutron d i f f r a c t i o n r e s u l t s . The c r y s t a l i s orthorhombic with o a = 10.586, b = 9.123, c = 8.880 A, Z = 4 and space group V 2 1 2 1 ^ 1 - T n e four molecules i n the unit c e l l are magnetically equivalent along the cry s t a l l o g r a p h i c axes; they are pairwise equivalent i n the cr y s t a l l o g r a p h i c planes; i n a l l other orientations they are d i s t i n c t . The molecular structure of hippuric acid i s depicted i n F i g . 12. The c r y s t a l l o g r a p h i c axes were located p r i o r to i r r a d i a t i o n by x-ray d i f f r a c t i o n measurements. The c r y s t a l s were x - i r r a d i a t e d at room temperature using a Machlett type OEG—60 x-ray tube operating at 40 kV, 20 mA. Optimum ENDOR signals were obtained a f t e r about 8 hours of i r r a d i a t i o n . On increasing the r a d i a t i o n dose, noticeable l i n e broadening occurred. The i r r a d i a t e d c r y s t a l s can be stored i n d e f i n i t e l y at ambient conditions i n closed v i a l s . ENDOR measurements were made at 77 K with our X-band superheterodyne spectrometer described i n chapter four. Spectra were recorded at i n t e r v a l s of 5° as the magnet was rotated about each of the cr y s t a l l o g r a p h i c axes; -89-F i g . 1 1 . C r y s t a l m o r p h o l o g y and a x i s sys tem o f a s i n g l e c r y s t a l o f h i p p u r i c a c i d . F i g . 12. Molecular structure of hippuric acid. -91-however, i n the v i c i n i t y of free proton NMR frequency, spectra were recorded at 2° i n t e r v a l s because of very high s p e c t r a l density. 6.3 Experimental Results: A preliminary ESR i n v e s t i g a t i o n revealed a considerably anisotropic 1:2:1 t r i p l e t along the c r y s t a l l o g r a p h i c axes (the four molecules i n the unit c e l l are equivalent along these d i r e c t i o n s ) , i n d i c a t i n g the presence of two nearly equivalent protons strongly i n t e r a c t i n g with the unpaired e l e c t r o n . ESR spectra of an x - i r r a d i a t e d hippuric acid c r y s t a l along the three c r y s t a l l o g r a p h i c axes are presented i n F i g . 13. Each of these t r i p l e t l i n e s showed further i l l - r e s o l v e d structures i n d i c a t i n g the existence of other weaker hyperfine i n t e r a c t i o n s , however, poor r e s o l u t i o n i n most o r i e n t a t i o n s , combined with the presence of s i t e s p l i t t i n g , pre-vented us from reaching any quantitative conclusions regarding the nature of the r a d i c a l . Examination of the ESR spectra of c r y s t a l s grown from heavy water indicated that these i n t e r a c t i o n s were not due to exchangeable protons. A t y p i c a l ENDOR spectrum of the x - i r r a d i a t e d hippuric a c i d c r y s t a l can be divided into three sections: ( i ) a high frequency region of 25-55 MHz co n s i s t i n g of highly anisotropic ENDOR l i n e s from two nearly equivalent protons (Fig. 14) ( i i ) a r e l a t i v e l y small region of 10-17 MHz around vp, con s i s t i n g of many c l o s e l y spaced proton ENDOR l i n e s with considerably 14 smaller anisotropy ( F i g . 15) and ( i i i ) a region of 1-7 MHz of N ENDOR l i n e s (Figure 16). Analysis of the spectra i n regions ( i ) and ( i i i ) was straightforward; however, the region around vp was crowded and only l i n e s from f i v e d i f f e r e n t proton couplings could be followed i n a l l the three planes. For proton tensor c a l c u l a t i o n s and graphical presentation vp was - 9 2 -F i g . 13. T y p i c a l ESR spectra of X - i r r a d i a t e d s i n g l e c r y s t a l of hippuric a c i d . • i . i I I I 1 1 I -L- - I 48 49 50 51 33 34 35 36 37 37 38 39 ENDOR FREQUENCY, MHz F i g . 14. ENDOR spectra due to the a-protons in the N-benzoylaminomethyl r a d i c a l i n an X- i r r a d i a t e d s i n g l e c r y s t a l of hippuric acid at 77 K. -94-A l/p IB r i i i — i — i 12 13 14 15 16 ENDOR FREQUENCY,MHz F i g . 15. ENDOR spectra due to the weakly coupled protons i n an X-i r r a d i a t e d s i n g l e c r y s t a l of hippuric a c i d . -95-I I I I I I 2 3 4 5 6 7 ENDOR FREQUENCY,MHz 14 F i g . 16. ESR and ENDOR spectra of N i n X - i r r a d i a t e d s i n g l e c r y s t a l of hippuric acid f o r the magnetic f i e l d p a r a l l e l to a-axis. Three ENDOR spectra correspond to three d i f f e r e n t points of saturation of ESR l i n e . -96-set equal to 13.906 MHz and the observed t r a n s i t i o n frequencies were corrected accordingly. 14 F i g . 16 shows the N ENDOR spectra for the magnetic f i e l d along the a-axis. The e f f e c t of saturating d i f f e r e n t ESR t r a n s i t i o n s i s i l l u s t r a t e d i n the three spectra i n t h i s f i g u r e . The angular v a r i a t i o n s of proton and nitrogen ENDOR frequencies as measured i n three orthogonal planes are depicted i n F i g . 17, 18 and 19. For strongly coupled protons, only the high frequency branch of the ENDOR spectra was used i n the an a l y s i s , whereas for weakly coupled protons around vp, both v + and v_ were used. For nitrogen coupling, however, data from a l l four sets of l i n e s were analyzed. The experimentally observed frequencies belonging to the same i n t e r a c t i o n were f i t t e d to the spin Hamiltonian with the help of the computer program discussed e a r l i e r . Approximately 120 data points were used to f i t each tensor and the rms error was t y p i c a l l y l e s s than 30 kHz for the large couplings and 10 kHz for the others. The diagonalized forms of the hyperfine and quadrupole tensors are presented i n Tables 4, 5 and 6. 6.4 Discussion: 6.4.1 a-proton Interactions: Two large couplings observed i n the x - i r r a d i a t e d s i n g l e c r y s t a l s of hippuric acid are both markedly ani s o t r o p i c . These coupling tensors c l e a r l y i n d i c a t e a r a d i c a l structure ending at -CR^ i n which the two protons attached to the carbon atom having large spin density are responsible for the anisotropic couplings. The hyperfine coupling tensors i n Table 4 represent these very nearly i d e n t i c a l i n t e r a c t i o n s . Small differences between the two tensors can be ascribed to the environmental differences -97-ENDOR FREQUENCY, MHz CRYSTAL ORIENTATION F i g . 17. Angular v a r i a t i o n of the high frequency branch of the ENDOR frequencies of the a-protons i n X - i r r a d i a t e d s i n g l e c r y s t a l of hippuric a c i d . -98-ENDOR FREQUENCY, MHz i i i ! i i i i i i a 30° 60° b 30° 60° C 30° 60° a CRYSTAL ORIENTATION F i g . 18. Angular v a r i a t i o n of the ENDOR frequencies of the weakly coupled protons i n X - i r r a d i a t e d hippuric a c i d . The experimental points are omitted from the f i g u r e f o r the sake of c l a r i t y . Data points were c o l l e c t e d at 2° i n t e r v a l s and excellent agreement was obtained with the calculated values. -99-ENDOR FREQUENCY, MHz a 30° 60° b 30° 60° C 30° 60° a CRYSTAL ORIENTATION F i g . 19. Angular v a r i a t i o n of N ENDOR frequencies i n an X-i r r a d i a t e d s i n g l e c r y s t a l of hippuric a c i d . -100-TABLE 4 HYPERFINE COUPLING TENSORS FOR THE ot-PROTONS OF N-BENZOYLAMINOMETHYL RADICAL 2 Coupling (MHz) D i r e c t i o n Cosines Proton P r i n c i p a l Value"*" Isotropic Dipolar a U± -24.22 -51.37 +27.15 +0.496 ±0.792 +0.357 -48.70 + 2.67 ±0.072 +0.372 +0.926 -81.20 -29.83 ±0.866 ±0.484 +0.128 H 2 -25.46 -51.17 +25.71 +0.413 +0.847 -0.334 -48.98 + 2.19 ±0.081 +0.400 +0.913 -79.06 -27.89 +0.908 ±0.350 +0.234 Sign of the p r i n c i p a l values i s assumed on the basis of theory. Uncertainty i n the p r i n c i p a l value i s ±0.03 MHz. 2 The four d i f f e r e n t sign combinations r e f e r to the four inequivalent s i t e s i n the c r y s t a l . -101-118 of the two protons In the c r y s t a l . The neutron d i f f r a c t i o n r e s u l t s i n d i c a t e unequal C-H bond lengths for the two hydrogen atoms i n the 119 120 undamaged c r y s t a l . In x - i r r a d i a t e d N-acetylglycine Box et a l . ' observed s i m i l a r differences for the two nearly equivalent protons. In a planar - C ^ r a d i c a l the hyperfine tensors of both the protons should have a common p r i n c i p a l d i r e c t i o n , which i s perpendicular to the plane containing the three atoms. This was v e r i f i e d from the fact that the angle between the two intermediate couplings of a-protons was found to be ^2°. For the tensors reported i n Table 4, the d i r e c t i o n s of the smallest p r i n c i p a l values make an angle of 125.8° with each other. From 121 theory these d i r e c t i o n s are expected to be along the C-H bond d i r e c t i o n s . These observations lead us to the conclusion that the r a d i c a l under i n -v e s t i g a t i o n has a planar - C ^ group, with a f a i r l y large spin density on the terminal carbon pu o r b i t a l . The structure of the r a d i c a l and numbering of atoms are shown i n Fi g . 20. Now, such a r a d i c a l structure i s possible i f the carboxyl group i s homolytically cleaved from a molecule of hippuric a c i d . The most probable pathway leading to such a process could be deprotonation of the oxidized hippuric acid followed by the loss of a molecule of carbon dioxide. Supporting evidence for the r a d i c a l structure comes from the following observations. From chemical analysis of amino acids i r r a d i a t e d i n the dry state, ' 122 Gott s c h a l l and Tolbert concluded that oxidation of glycine and a-alanine causes decomposition accompanied by the production of carbon dioxide. Recent 123 X 2 4 studies on the oxidation products i n x - i r r a d i a t e d carboxylic acids ' , 120 119 amino acids and N-acetylglycine suggest that the o v e r a l l process -103-i n l t i a t e d by oxidation i s decarboxylation. From the f a m i l i a r McConnell r e l a t i o n ^ , we c a l c u l a t e a spin density of 0.809 on the pu-orb i t a l of the a-carbon atom using a Q-value of -63.5 MHz. The experimental spin d e n s i t i e s along with the spin d e n s i t i e s obtained from INDO-MO c a l c u l a t i o n s are given i n Table 7. A spin density of 0.842 on the a-carbon was obtained from INDO-MO c a l c u l a t i o n s on the N-benzoylaminomethyl r a d i c a l . The agreement can be deemed excellent considering INDO approximations. Thus we are almost c e r t a i n about the i d e n t i t y of the r a d i c a l . If the r a d i c a l was formed by the d i s s o c i a t i o n of the N-H bond as suggested by Votinov"^ 6, the r a d i c a l would have a methylene group with a large spin density on the adjacent nitrogen atom. These two protons of the methylene group (8-protons) should then have f a i r l y large i s o t r o p i c hyperfine coupling. Moreover, large spin density on the T T - o r b i t a l of the nitrogen atom would give r i s e to large anisotropic coupling with the 14 N nucleus. Our experimental r e s u l t s do not support these p o s s i b i l i t i e s . 14 6.4.2 N Hyperfine and Quadrupole Interactions: S e l e c t i v e saturation study on the ESR line-shape showed that a l l the 14 four l i n e s i n F i g . 16 are due to coupling of N. The r e s u l t s reported 14 i n Table 5 show that the N hyperfine coupling tensor i s f a i r l y i s o t r o p i c m character. This unique observation i s consistent with the f i n d i n g that maximum spin density i s found not on nitrogen, but on the adjacent 109 carbon atom. A s i m i l a r observation has been noted by Deigen et a l . i n y- i r r a d i a t e d glycine. The nearly i s o t r o p i c coupling i s probably caused by the small spin density induced i n the s - o r b i t a l of nitrogen by the unpaired electron i n the T T - o r b i t a l of the carbon atom, through the spin -104-TABLE 5 NITROGEN HYPERFINE AND QUADRUPOLE COUPLING TENSORS IN N-BENZOYLAMINO-METHYL RADICAL Di r e c t i o n Cosines P r i n c i p a l Values (MHz) a b Hyperfine Coupling A ^ -7.38±0.01 ±0.185 +0.432 +0.883 A22 A33 -8.77±0.01 +0.983 ±0.057 +0.178 -9.44±0.01 ±0.027 +0.900 ±0.435 Quadrupole Coupling x__ -0.843±0.005 ±0.195 +0.398 +0.896 X22 +0.582±0.005 +0.980 ±0.128 +0.157 X33 +0.26110.005 ±0.050 +0.909 ±0.415 C2-N bond d i r e c t i o n : +0.996 +0.064 -0.058 The four d i f f e r e n t sign combinations r e f e r to the four inequivalent s i t e s i n the c r y s t a l . - 1 0 5 -p o l a r i z a t i o n mechanism. The unpaired electron density i n the T r - o r b i t a l of nitrogen can be estimated from the anisotropy of the nitrogen hyperfine coupling. The 1 4 N hyperfine tensor can be treated as approximately a x i a l l y symmetric with the d i r e c t i o n of the p o s i t i v e dipolar component p a r a l l e l to the T r - o r b i t a l . The deviation from a x i a l symmetry i s probably caused by the influence of the large spin density on the adjacent carbon atom. The d i p o l a r part of the tensor has the form - B Q , - B Q » 2 B Q , where B Q i s r e l a t e d to the unpaired electron density on the T r - o r b i t a l of nitrogen. The value of B Q i n the present case can be taken as the mean of two smaller d i p o l a r 1 2 5 values i . e . 0 . 5 8 MHz. The t h e o r e t i c a l value of B Q for unit spin density i s 4 8 MHz. The spin density on the T r - o r b i t a l of nitrogen i s calculated to be 0 . 0 1 2 . This i s i n excellent agreement with the value of 0 . 0 1 3 calculated from the i s o t r o p i c part of the N-H proton coupling using McConnell r e l a t i o n . ^ 5 (Table 7 ) From symmetry considerations the p r i n c i p a l axes of the quadrupole tensor are expected to coincide with those of the hyperfine coupling tensor. Inspection of table 5 shows that the p r i n c i p a l element of hyperfine ( A ^ ) and quadrupole (x__) tensors are p a r a l l e l to pir-orbital of nitrogen w i t h i n ^ 7 ° . A^ a n d a r e P a r a l l e l t o C 2-N bond d i r e c t i o n i n the undamaged c r y s t a l within ^ 7 ° and i s p a r a l l e l to X33 within ^ 2 ° . A rigorous i n t e r p r e t a t i o n of the quadrupole coupling would be very d i f f i c u l t because of the complex dependence of the f i e l d gradient at the nitrogen nucleus on a l l the extra nuclear charges. However, reasonable r e s u l t s have been obtained using an approximate method developed by Townes 7 8 — 8 0 8 1 — 8 3 and Dailey and l a t e r extended by Gordy. In i t s simplest form, -106-the p r i n c i p a l values of the t h e o r e t i c a l quadrupole coupling tensor are given by: X u = [ n x " % ( * y + n z)]eqQ X22 = [ n y "  h ( n z  + n x > ] e q Q Xoo = [n - h(n + n )]eqQ (6-1) 33 z x y where the symbols have t h e i r usual meanings and n^, n y, n z are the occupation numbers of the 2p o r b i t a l s of the nitrogen atom. Employing the electron d e n s i t i e s obtained from our INDO-MO c a l c u l a t i o n s and using a 84 value of -10 MHz for eqQ as suggested by Gordy and Cook , we obtained 2.35, 3.16 and -5.51 MHz for the p r i n c i p a l values of the quadrupole coupling tensor. These values are much larger than the experimental values of 0.261, 0.582 and -0.843 MHz (Table 5). Since the l e a s t squares f i t t i n g program for the assigned t r a n s i t i o n s converged with rms error of less than 5 kHz for 120 input data points, the disagreement must have resulted from the assumptions leading to equations (6-1). Similar disagreement has also been reported i n the hydrogen addition r a d i c a l trapped i n an X- i r r a d i a t e d single c r y s t a l of ca f f e i n e hydrochloride ... . , 112 dihydrate. The signs of the p r i n c i p a l values of the quadrupole tensor r e l a t i v e to those of the hyperfine coupling tensor were determined by the method 12 6 outlined by Cook. The energy l e v e l diagram shown i n F i g . 3 i s drawn for the case i n which a and P were both p o s i t i v e . I f the low f i e l d ESR l i n e , corresponding to the |-%,1> to \h,l> t r a n s i t i o n i s saturated, then the two observed ENDOR t r a n s i t i o n s have frequencies given by: -107-A l = a/2 + v„ - P (6.2) N A4 = a/2 - v„ + P N I f , however, the r e l a t i v e sign of a and P i s reversed, saturation of the same ESR l i n e would give r i s e to two ENDOR t r a n s i t i o n s with frequencies: A l = a/2 + v„ + P (6.3) N A4 = a/2 - v„ - P N As i l l u s t r a t e d i n f i g . 16, saturating d i f f e r e n t portions of the ESR 14 s i g n a l resulted i n dramatic i n t e n s i t y differences of the various N ENDOR l i n e s . Since by saturating the low f i e l d ESR l i n e , the two outer-most l i n e s become prominent, the hyperfine and quadrupole couplings have opposite signs along a-axis. Similar observations along other d i r e c t i o n s led to the choice of signs indicated i n Table 5. 6.4.3 Weakly coupled Protons: From among the large number of c l o s e l y spaced ENDOR signals i n the v i c i n i t y of vp, only f i v e could be followed unambiguously i n a l l the three planes. The r e s u l t s obtained from the analysis of these l i n e s are presented i n Table 6. The assignment of these tensors to s p e c i f i c protons i n the c r y s t a l was accomplished on the basis of d i r e c t i o n cosines, symmetry considerations and comparison of INDO-MO r e s u l t s . In support of our assignment of ENDOR l i n e s to exchangeable protons, we c a r r i e d out a few ENDOR measurements on p a r t i a l l y deuterated samples. Figure 21 shows a t y p i c a l ENDOR spectrum of x - i r r a d i a t e d hippuric acid s i n g l e c r y s t a l s grown from heavy water for the magnetic f i e l d p a r a l l e l to the a-axis. A consistent reduction i n the i n t e n s i t y of l i n e s A and B r e l a t i v e to other prominent l i n e s -108-TABLE 6 HYPERFINE COUPLING TENSORS FOR THE WEAKLY COUPLED PROTONS IN N-BENZOYLAMINO-METHYL RADICAL Proton Coupling (MHz) 2 Di r e c t i o n Cosines P r i n c i p a l Value Isotropic Dipolar a b c (B) N-H a: +0.45 -1.00 +1.45 ±0.661 +0.622 -0.420 TT : +3.30 +4.03 ±0.160 +0.428 +0.889 1: -6.47 -5.47 +0.733 +0.655 -0.185 N-H bond d i r e c t i o n : -0.433 -0.823 -0.369 (D) H4 a: 2.25 2.54 -0.29 ±0.549 +0.712 -0.436 TT : 4.02 +1.48 ±0.117 +0.451 +0.885 1= 1.34 -1.20 ±0.828 ±0.537 +0.165 C.-H. bond d i r e c t i o n : 4 4 -0.505 -0.731 -0.457 (E) H 6 a: 1.05 2.92 -1.87 ±0.993 ±0.085 -0.086 TT : 3.03 +0.11 ±0.119 +0.548 +0.829 1: 4.69 +1.77 ±0.023 +0.833 -0.554 C,-H, bond d i r e c t i o n : o 6 +0.996 +0.067 -0.048 (C) H 8 a: 1.83 2.88 -1.05 +0.444 ±0.707 +0.551 TT : 3.86 +0.98 ±0.074 +0.583 +0.809 1: 2.94 +0.06 +0.893 +0.399 -0.207 C 0-H 0 bond d i r e c t i o n : o o -0.482 +0.686 +0.546 (A) H.B. a: -1.97 -0.07 -1.90 +0.205 +0.854 -0.479 TT : -1.68 -1.61 ±0.121 +0.508 +0.853 +3.44 +3.51 ±0.972 +0.116 -0.207 N-H...0g bond d i r e c t i o n : -0.143 -0.764 -0.529 Signs are assumed on the basis of INDO spin d e n s i t i e s , except for the hydrogen bonded (H.B.) proton. Uncertainty i n the p r i n c i p a l value i s ±0.01 MHz. The four d i f f e r e n t sign combinations r e f e r to the four inequivalent s i t e s . -109-I I I I I 12 13 14 15 16 ENDOR FREQUENCY, MHz F i g . 21. ENDOR spectrum i n the v i c i n i t y of vp from an X - i r r a d i a t e d s i n g l e c r y s t a l of hippuric a c i d grown from heavy water for the magnetic f i e l d p a r a l l e l to a-axis. -110-i n the spectrum of deuterated sample suggests that they belong to exchangeable protons. The ENDOR l i n e B i s f a i r l y a n isotropic and i s r e a d i l y assigned to the coupling of the N-H proton. A r e l a t i v e l y small spin density on the nitrogen atom induces a small negative i s o t r o p i c component. The corres-pondingly large anisotropy of the tensor a r i s e s from the large spin density on the a-carbon. From the i s o t r o p i c part of the tensor, a spin density of 0.013 i s calculated on the T r-orbital of nitrogen, from McConnell r e l a t i o n ^ using a Q-value of 78.5 MHz. This i s i n excellent agreement with the spin density calculated from the anisotropy of the nitrogen coupling. The d i r e c t i o n of the intermediate component of the tensor i s within 5° of the common p r i n c i p a l d i r e c t i o n of the a-protons. However, the d i r e c t i o n of a-component of the tensor does not match very w e l l with the N-H bond d i r e c t i o n calculated from c r y s t a l structure. The disagreement goes up to the extent of ^ 18° which might have resulted from the e f f e c t of the large spin density on a-carbon. That the tensor i s associated with an exchangeable proton was v e r i f i e d from the spectrum of the deuterated sample (figure 21). Out of f i v e phenyl protons, only three could be i d e n t i f i e d i n our study. In the frequency range of 10-17 MHz around vp, there were so many overlapping l i n e s , that i t was hard to pick up ENDOR l i n e s of the weakly coupled metaprotons. Besides, an attempt to study a l l the l i n e s does not shed any a d d i t i o n a l l i g h t on the e l e c t r o n i c structure of the r a d i c a l . Therefore, we did not make any further attempt to analyze these l i n e s . Interactions C, D and E are assigned to the para-and two ortho- protons on the basis of t h e i r d i r e c t i o n cosines and the larger spin densities expected at these positions as compared to the meta p o s i t i o n s . Inspection -111-of tensors H,, H, and H„ i n Table 6 reveals that they a l l have one p r i n c i p a l axis i n common which i s perpendicular to the phenyl r i n g . The o-components of the tensors C, D and E are p a r a l l e l to Cg-Hg, C.-H. and C,-H, bonds i n the undamaged c r y s t a l within 3° and hence 4 4 o o assigned to ortho-(Hg,H^) and para-(Hg) protons r e s p e c t i v e l y . Furthermore, the d i r e c t i o n of the a-component of para proton i s found to be close to ^120° with respect to those of two ortho protons within experimental error. From the i s o t r o p i c parts of these tensors, spin d e n s i t i e s of -0.046 and -0.043 are calculated f o r the Tr-orbitals of the para- and the ortho-positions, r e s p e c t i v e l y , from McConnell r e l a t i o n with a Q value of 63.5 MHz. These are i n good agreement with the r e s u l t s of INDO c a l c u l a t i o n s ( c f . Table 7). The remaining hyperfine coupling tensor (A) i n Table 6 i s assigned to an intermolecular hydrogen bonded proton. That the tensor belongs to an exchangeable proton was further v e r i f i e d by the inspection of spectrum of a p a r t i a l l y deuterated sample ( c f fi g u r e 21). Just as the ENDOR l i n e B, the i n t e n s i t y of l i n e A i s also reduced r e l a t i v e to other strong l i n e s . This tensor i s nearly d i p o l a r i n nature with an i s o t r o p i c component of -0.07 MHz. The a x i a l symmetry of t h i s tensor i s also obvious. These 127-129 c h a r a c t e r i s t i c s are t y p i c a l of hydrogen bonded protons. Of the two possible hydrogen bonded protons i n hippuric a c i d , N-H proton of the neighbouring molecule hydrogen bonded to the amide oxygen i s thought to be responsible for the i n t e r a c t i o n reported here, on the basis of the d i r e c t i o n cosines. This i s consistent with our observation that the carboxyl group i s removed on r a d i c a l formation, thus the second hydrogen bond which was present i n the undamaged molecule i s no longer present i n -112-TABLE 7 COMPARISON OF EXPERIMENTAL AND CALCULATED (INDO) UNPAIRED ELECTRON DENSITIES IN N-BENZOYLAMINOMETHYL RADICAL ATOM C l N C2 0 C3 C4,8 C5,7 C6 pTT (INDO) 0.842 0.094 -0.054 0.124 0.007 -0.008 0.004 -0.007 P1T (obs.) 0.809 0.013 - - - -0.043 - -0.046 -113-the r a d i c a l . The calculated N-H ... 0 bond d i r e c t i o n agrees with the d i r e c t i o n of o-component within ^6°. 6.5 Conclusion: The hyperfine tensors for two a-protons, f i v e weakly coupled 14 protons and hyperfine as we l l as quadrupole tensors for N have been determined. Our r e s u l t s demonstrate that the superior r e s o l u t i o n of the ENDOR technique i s very useful i n the study of r a d i c a l s trapped i n i r r a d i a t e d s i n g l e c r y s t a l s of organic compounds. In the present study the ESR spectrum only exhibited a p a r t i a l l y resolved t r i p l e t s p l i t t i n g i n an approximate r a t i o of 1:2:1 i n d i c a t i n g the presence of two equivalent protons attached to a carbon atom with large spin density. Detailed s t r u c t u r a l information about the r a d i c a l could not be obtained from the ESR study, because important intramolecular hyperfine s p l i t t i n g s were buried w i t h i n the ESR linewidths. Using the ENDOR technique, the e l e c t r o n i c structure of the r a d i c a l along with i t s i d e n t i f i c a t i o n could be unambiguously accomplished. 14 N ENDOR l i n e s belonging to such a low hyperfine coupling have been 109 reported only once before i n the free r a d i c a l trapped i n the y - i r r a d i a t e d glycine s i n g l e c r y s t a l . For the r a d i c a l analogous to ours i n N-acetyl-119 14 glyci n e , Box and coworkers did not observe N ENDOR. This i s not 14 p a r t i c u l a r l y s u r p r i s i n g because detection of N ENDOR signals i s often hampered by the r a p i d i t y of i t s spin l a t t i c e r e l a x a t i o n and the small s i z e 14 of i t s nuclear magnetic moment. Often the N signals l i e below the observation range of most ENDOR spectrometers. In a survey of r a d i a t i o n e f f e c t s on organic compounds, i t has been _ . .. , ,119,120,123,124 _ _ , . ^ . established that the carboxyl group i s the s i t e of the -114-primary oxidation with subsequent decarboxylation. However, i t has been reported that the decarboxylated r a d i c a l i s usually unstable at room temperature, and decays with hydrogen abstraction processes. The present study establishes that i n hippuric acid, the decarboxylated r a d i c a l i s stable at room temperature. A similar observation was made i n the case 130 of creatine monohydrate containing only one carboxyl group. In a s t r u c t u r a l l y related compound, N-acetylglycine, the decarboxylated r a d i c a l 119 131 i s stable only at low temperatures ' , indicating that phenyl group plays a c r i t i c a l role i n the unusual s t a b i l i t y of the decarboxylation r a d i c a l i n hippuric acid. CHAPTER SEVEN ENDOR STUDY OF X-IRRADIATED SINGLE CRYSTALS OF N-ACETYLGLYCINE - A  REINVESTIGATION 7.1 Introduction: Radiation induced radicals in proteins have been studied for a long 132 time using the electron spin resonance (ESR) technique. In the f i r s t 133 13A ESR papers by Gordy, Ard and Shields and Gordy and Shields , the protein spectra were divided into the sulfur pattern due to a cysteine sulfur radical and a doublet resonance. The nature of the doublet has been discussed thoroughly and there have been lot of controversies over the identity of the radical. The original suggestion that the unpaired electron was localized on an oxygen atom in a hydrogen bond was replaced by a model where the unpaired spin density is localized mainly in a T r-orbital on an a-carbon atom in the protein back bone. H 0 I Jl • • • v N C \ / \ - / \ C C • • » II J 0 H A sensible approach to the study of radiation damage in exceedingly complex protein molecule begins with an investigation of simpler model compounds. The main objective of these studies is to examine the effects of ionising radiation on the peptide linkage (-C0-NH-) that joins amino acids together in proteins. We believe that these studies also w i l l have -116-some bearing with regard to the processes taking place i n i r r a d i a t e d proteins. N-acetylglycine i s one of the simplest compounds with a peptide 135 bond. When i r r a d i a t e d i n powders, the ESR absorption shows a doublet which i s approximately of the same spacing as the doublets of proteins. In subsequent years, the i r r a d i a t e d s i n g l e c r y s t a l of N-acetylglycine has been the subject of several ESR and ENDOR s t u d i e s . 2 7 " 2 9 , 1 1 9 ' 1 3 1 ' 1 3 6 ' 1 3 7 119 In a recent ENDOR study, Box and coworkers i d e n t i f i e d three d i f f e r e n t r a d i c a l s i n X- i r r a d i a t e d s i n g l e c r y s t a l s of N-acetylglycine at 4.2K, two of which were reduction products and the other an oxidation product. The oxidized species decompose l o s i n g a proton and a molecule of carbondioxide; the two reduced species are formed by electron a d d i t i o n to the peptide oxygen and the carboxyl group, the l a t t e r product occurring i n two conformations. In t h i s chapter we deal with the stable r a d i c a l produced by room temperature X - i r r a d i a t i o n of N-acetylglycine. The room temperature r a d i c a l I , u -A J -L n F , 27,28,131,136,137 has been i d e n t i f i e d by several groups of workers as CH3-CO-NH-CH-COOH II 28 Miyagawa and coworkers reported the a-proton tensor for t h i s r a d i c a l i n 136 an ESR study. Mangiaracina observed a small nitrogen hyperfine s p l i t t i n g 27 i n the ESR spectra of deuterated a c e t y l g l y c i n e . Later Piazza and Patten detected the ENDOR l i n e s due to a r o t a t i n g methyl group i n the same r a d i c a l . In a d e t a i l e d ESR observations and MO c a l c u l a t i o n s on the same r a d i c a l , 137 Saxebol et a l . found that the unpaired spin density could migrate through the peptide group even upto 6-proton. The Sextet pattern of ESR -117-spectra of N-acetylglycine s i n g l e c r y s t a l s grown from heavy water was explained to be due to the i n t e r a c t i o n of the unpaired electron with the methyl protons and the nitrogen i n the peptide bond. S i n c l a i r and 131 Codella i n a d e t a i l e d v a r i a b l e temperature ESR study, established the pathways leading to the stable r a d i c a l from the i n i t i a l r a d i c a l s produced at low temperatures. In a more recent 'negative' ENDOR study Helms, Suzuki and Miyagawa* (hereafter referred to as HSM) raised doubts about the structure of the r a d i c a l produced by room temperature X - i r r a d i a t i o n of N-acetylglycine. They proposed that the r a d i c a l i s e s s e n t i a l l y a cation: CH„-C0-NH-CH-C00H I II They also asserted that the hyperfine coupling due to the methyl protons of the r a d i c a l was i d e n t i c a l to the methyl proton coupling from the neighboring molecule, which i n the undamaged c r y s t a l i s re l a t e d by a centre of inversion. These proposals were based on i n t e n s i t y measurements of the ENDOR l i n e s . These workers also detected ENDOR l i n e s due to several weakly coupled protons which were not observed i n the e a r l i e r ENDOR work 27 of Piazza and Patten. The hyperfine tensors reported for these weakly coupled protons have considerable i s o t r o p i c parts, i n d i c a t i n g the presence of non-negligible spin d e n s i t i e s at the respective atoms. Based on these observations, they concluded that the wave function of the unpaired electron extends s i g n i f i c a n t l y over the neighboring molecules. 14 We started ENDOR investigations on t h i s r a d i c a l i n search of N-ENDOR 14 1 l i n e s , a f t e r completing a d e t a i l e d N and H ENDOR study of the r a d i a t i o n damage i n a rela t e d compound, hippuric acid (N-benzoylglycine). Although -118-14 we were unsuccessful i n our attempt to f i n d N-ENDOR s i g n a l s , we r e a l i z e d that a r e i n v e s t i g a t i o n of the "'"H-ENDOR of t h i s r a d i c a l was i n order. This was due i n part because unlike the ENDOR spectra reported by Piazza and 27 Patten, we obtained many well-resolved l i n e s i n the v i c i n i t y of the free proton N.M.R. frequency. We also observed several l i n e s due to the a-proton coupling, instead of the expected si n g l e l i n e . These preliminary findings prompted us to undertake a de t a i l e d i n v e s t i g a t i o n . In the course of t h i s study we have been able to gather s u f f i c i e n t evidence to propose a d e f i n i t e p i c t u r e of the r a d i a t i o n damage i n N-acetylglycine, at variance 29 with the 'negative' ENDOR r e s u l t s of HSM. 7.2 Experimental: Single c r y s t a l s of N-acetylglycine were grown by slow evaporation of i t s saturated aqueous s o l u t i o n at room temperature. Deuterated c r y s t a l s were grown i n a s i m i l a r fashion from heavy water. Well-developed c r y s t a l s of approximate dimensions 8x5x4 mm were e a s i l y obtained. "^N-acetylglycine was prepared by condensing glycine ("^N-99%) with a c e t i c anhydride i n the 138 presence of a small amount of water. The c r y s t a l s were c o l o r l e s s and exhibited a very regular external form which i s shown i n figu r e 22 together with the orthogonal reference system used i n these experiments. 139 The c r y s t a l structure of N-acetylglycine i s known to be monoclinic o with a tetramolecular unit c e l l of dimensions a = 4.86, b = 11.54, c = 14.63A with 8 = 138.2° and space group P2^/c. The four molecules i n the unit c e l l are pairwise equivalent, and therefore, only two s i t e s can be distinguished by ESR or ENDOR spectroscopy. When the magnetic f i e l d i s eit h e r p a r a l l e l or perpendicular to the b-axis, only one s i t e can be observed. A l l measure-ments were c a r r i e d out i n the reference coordinate system a*, b, c (a* = bxc). -119-F i g . 22. C r y s t a l l i n e f o r m a n d c r y s t a l l o g r a p h i c a x e s o f N - a c e t y l g l y c i n e . -120-The molecular structure of N-acetylglycine i s shown i n fi g u r e 23, along with the numbering system. The c r y s t a l s were X - i r r a d i a t e d at room temperature using the device described i n the e a r l i e r chapters. The optimum concentration of r a d i c a l s was obtained a f t e r about two hours of i r r a d i a t i o n . ENDOR measurements were made at 77K with our X-band superheterodyne spectrometer described previously, using an f.m. frequency of "VL.1 kHz. Spectra were recorded at i n t e r v a l s of 2° i n the v i c i n i t y of vp and at 5° i n t e r v a l s for the a-proton l i n e s . Preliminary ESR measurements were made on a Varian E-3 spectrometer. Second d e r i v a t i v e ESR spectra were recorded on a home-built ESR instrument using 50 kHz f i e l d modulation and detecting the second harmonic. No ENDOR l i n e s due to the hyperfine coupling of the unpaired electron with the nitrogen atom were observed i n our study. A l l the observed l i n e s a r i s e from i n t e r a c t i o n s with protons. The experimentally observed f r e -quencies belonging to the same proton coupling were f i t t e d to the spin Hamiltonian with the least-squares-adjustment program LSF. Approximately 120 data points were used to f i t each tensor and the rms error was t y p i c a l l y less than 20 kHz. For the a-proton tensor only the high frequency branch of the ENDOR spectra was used i n the an a l y s i s , while both the branches were used for the weakly coupled protons. 7.3 Results and Discussion: 7.3.1 Experimental Results: Although the hyperfine coupling tensor f o r the a-proton i n t e r a c t i o n 28 has been reported from an ESR study , we decided to redetermine i t from our ENDOR data i n order to obtain more accurate r e s u l t s . When we recorded F i g . 23. Molecular structure and numbering system of N-acetylglycine. -122-the ENDOR spectrum i n the frequency region expected for the a-hydrogen coupling, we discovered that, instead of the expected s i n g l e l i n e f o r each dis t i n g u i s h a b l e c r y s t a l s i t e , there were three to four c l o s e l y spaced l i n e s e x h i b i t i n g s i m i l a r angular v a r i a t i o n s . Figure 25 shows the high frequency branch of the a-proton ENDOR l i n e s for an o r i e n t a t i o n where the magnetic f i e l d makes f i v e degrees with the b-axis i n the bc-plane. The separation between the two groups of l i n e s i s due to s i t e s p l i t t i n g . When the magnetic f i e l d i s oriented along the b-axis, the two sets of l i n e s merge together to y i e l d a c l o s e l y spaced i l l - r e s o l v e d f o u r - l i n e spectrum. From the doublet pattern i n the ESR spectrum (figure 24), i t i s evident that the unpaired electron i s strongly coupled only to one proton. This apparent c o n t r a d i c t i o n between the ESR and ENDOR observations prompted us to pursue the study further. Of these four ENDOR l i n e s , we have been able to follow only three i n a l l the three planes. For s i m p l i c i t y , the angular v a r i a t i o n of the high frequency branch of the ENDOR frequencies belonging to one l i n e only i s shown i n figu r e 26. The diagonalized forms of the a-proton hyperfine tensors for these three ENDOR l i n e s are presented i n Table 8. It i s evident from these data that these small differences could not be resolved i n the ESR spectra. Figure 27 shows the ENDOR spectrum i n the v i c i n i t y of vp f o r the magnetic f i e l d p a r a l l e l to the a*-axis. On comparing t h i s spectrum with 29 F i g . 1 of HSM , one notices several s a l i e n t differences between the two, the foremost among them being the better r e s o l u t i o n of our spectrum. The i 29 broad l i n e r e f e r r e d to as Hc 2 and Hc 2 by HSM and assigned to two equivalent methyl groups by them, i s c l e a r l y resolved i n our spectrum. These two l i n e s along with the one designated by Hc^ have very s i m i l a r angular v a r i a t i o n s , analogous to the three l i n e s due to the a-proton -123-F i g . 24. F i r s t d e r i v a t i v e ESR spectrum of X - i r r a d i a t e d s i n g l e c r y s t a l of N-acetylglycine f o r the magnetic f i e l d p a r a l l e l toa*-axis. -124-I I I I I I l_ 33 35 37 39 ENDOR FREQUENCY, MHz 25. High frequency branch of the ENDOR spectrum due to the a-proton i n t e r a c t i o n i n X- i r r a d i a t e d s i n g l e c r y s t a l of N-acetylglycine f or the magnetic f i e l d making 5° with b-axis i n the bc-plane. -125-ENDOR FREQUENCY, MHz CRYSTAL ORIENTATION F i g . 26. Angular v a r i a t i o n of the high frequency branch of the ENDOR frequencies for the a-proton i n an X - i r r a d i a t e d s i n g l e c r y s t a l of N-acetylglycine. -126-F i g . 27. ENDOR spectra i n the v i c i n i t y of vp from an X-i r r a d i a t e d s i n g l e c r y s t a l of N-acetylglycine f o r the magnetic f i e l d p a r a l l e l to a*-axis: (a) c r y s t a l grown from H_0 (b) c r y s t a l grown from D_0. TABLE 8 HYPERFINE COUPLING TENSORS FOR a-PROTON AND METHYL PROTONS OF HYDROGEN ABSTRACTION RADICAL IN N-ACETYLGLYCINE 2 PROTON Coupling (MHz) Direct i o n Cosines  P r i n c i p a l V alue 1 I s o t r o p i c Dipolar a* b e -23.65 +25.59 -0.053 ±0.815 +0.577 oH(A) -48.64 -49.24 + 0.60 -0.999 +0.038 -0.038 -75.42 -26.18 +0.009 ±0.579 -0.816 -23.31 +25.60 -0.059 ±0.814 +0.578 ctH(B) -48.47 -48.91 + 0.44 -0.998 +0.046 -0.037 -74.96 -26.05 +0.004 ±0.579 -0.815 -23.16 +25.38 -0.053 ±0.815 +0.577 aH(C) -48.02 -48.54 + 0.52 -0.998 +0.035 -0.043 -74.44 -25.90 +0.014 ±0.578 -0.816 TABLE 8 (cont'd) PROTON Coupling (MHz) 2 Directio n Cosines P r i n c i p a l Value 1 Isotropic Dipolar a* b c CH 3(A) - 7.35 - 7.77 -10.94 - 8.69 + 1.34 + 0.92 - 2.25 -0.792 -0.602 -0.100 ±0.431 +0.435 +0.791 +0.432 -0.670 +0.604 CH 3(B) - 6.98 - 7.37 -10.58 - 8.31 + 1.33 + 0.94 - 2.27 -0.780 -0.618 -0.099 ±0.440 +0.429 +0.789 +0.445 -0.659 +0.607 CH 3(C) - 6.81 - 7.15 -10.39 - 8.12 + 1.31 + 0.97 - 2.27 -0.803 -0.589 -0.091 ±0.417 +0.447 +0.792 +0.425 -0.673 +0.605 1 Sign of the p r i n c i p a l values i s assumed on the basis of theory. Uncertainty i n the p r i n c i p a l value i s ±0.02 MHz. 2 The two sign combinations chosen consistently r e l a t e on distinguishable c r y s t a l s i t e to the other. -129-i n t e r a c t i o n . In some orientations a fourth l i n e i s evident, but i t could not be followed throughout. The hyperfine coupling tensors obtained from the analysis of these three l i n e s are included i n Table 8. A c a r e f u l examination of f i g u r e 27 reveals that most of the l i n e s 29 designated as Hx^-Hx^ show s i m i l a r s p l i t t i n g s . HSM reported that the s p l i t t i n g s observed for Hx^ and Hx^ by them was due to a s l i g h t deviation of the or i e n t a t i o n from the a*-axis. However, we f i n d that the number of l i n e s double as we rotate the magnetic f i e l d d i r e c t i o n away from the axis, thus confirming that the s p l i t t i n g seen i n the spectrum along a*-axis i s not due to misalignment of the c r y s t a l . In our spectrum the l i n e s due to Hx^ i n fact are resolved into three l i n e s along a*-axis, and i n some other orientations even a fourth l i n e appears. Besides the l i n e s due to Hx^ and Hx^, a l l the other l i n e s also show s i m i l a r s p l i t t i n g s i n most or i e n t a t i o n s , although some of them appear as s i n g l e l i n e s i n the spectrum shown i n figu r e 27. The angular v a r i a t i o n of the ENDOR frequencies i n the v i c i n i t y of vp i s shown i n figu r e 28. Although we have c o l l e c t e d data at two degree i n t e r v a l s i n the three planes, f i g u r e 28 shows data points only at every 10°, t h i s d isplay being chosen for the sake of c l a r i t y . On comparing t h i s 29 diagram with fi g u r e 2 of HSM , i t i s apparent that at least i n the ac-plane the v a r i a t i o n of most of the ENDOR l i n e s has been misassigned by them; th i s no doubt was due to the complex angular v a r i a t i o n of these l i n e s and the need for many data points. It i s a l s o to be noted that, except f o r the l i n e s a t t r i b u t e d to the methyl group, the ENDOR l i n e s of a l l the weakly coupled protons cross vp at le a s t i n one plane, thus i n d i c a t i n g that not a l l the p r i n c i p a l values of t h e i r hyperfine tensors would be of the same -130-ENDOR FREQUENCY, MHz F i g . 28. Angular v a r i a t i o n of the ENDOR frequencies of the weakly coupled protons i n an X- i r r a d i a t e d s i n g l e c r y s t a l of N-acetylglycine. -131-sign. The hyperfine tensors obtained from these data are presented i n Table 9. For the sake of br e v i t y only one tensor per proton i s included i n t h i s table although two or three were determined for each. 29 Although HSM reported p a r t i a l or complete tensors for several a d d i t i o n a l non-exchangeable protons, we have chosen not to analyze those c l o s e l y spaced ENDOR l i n e s i n the immediate v i c i n i t y of vp. We f e e l that such an analysis would not y i e l d r e l i a b l e data. Moreover, study of these extremely weak couplings, would not shed any a d d i t i o n a l l i g h t on the r a d i c a l structure or the nature of the wavefunction of the unpaired electron. 7.3.2 Discussion: In order to explain the appearance of more ENDOR l i n e s than expected from the c r y s t a l symmetry, several p o s s i b i l i t i e s were considered. Mis-alignment of the c r y s t a l could not give r i s e to the a d d i t i o n a l s p l i t t i n g s because of the presence of the inversion centre i n the monoclinic unit c e l l . Twinning of the c r y s t a l was ruled out by checking several c r y s t a l s ; a l l of them gave the same number of ENDOR l i n e s with the same i n t e n s i t y r a t i o . The s p l i t t i n g could occur i f the symmetry of the c r y s t a l was changed on cooling to 77 K, where our ENDOR measurements were made. However, when we measured the spectra at room temperature, e s s e n t i a l l y the same type of spectra was observed except for poorer s i g n a l to noise r a t i o . Therefore, any temperature e f f e c t can sa f e l y be ruled out as the cause of the observed s p l i t t i n g s . Another l i k e l y explanation i s that the s p l i t t i n g s a r i s e because of very small differences i n the precise geometrical shape or the or i e n t a t i o n 140 of the r a d i c a l i n the c r y s t a l l a t t i c e . H o r s f i e l d et a l . observed a -132-s i m i l a r phenomenon i n an ESR study of y - i r r a d i a t e d s i n g l e c r y s t a l s of g l u t a r i c acid and postulated several reasons for the e f f e c t . They could not obtain s u f f i c i e n t experimental evidence to e s t a b l i s h the mechanism 14 producing the s p l i t t i n g . Later Kwiram re-investigated t h i s system using the ENDOR technique. On the basis of the weakly coupled proton i n t e r a c t i o n s , he postulated that the s p l i t t i n g resulted from two d i f f e r e n t conformations of the same r a d i c a l species, d i f f e r i n g i n the o r i e n t a t i o n of the carbonyl group r e l a t i v e to the skeleton of the r a d i c a l \ C // H C H I C i H O—H IV In our ENDOR study of X - i r r a d i a t e d s i n g l e c r y s t a l of phenazine (chapter 5) we have observed two r a d i c a l s which were chemically i d e n t i c a l but d i f f e r i n g i n t h e i r precise geometrical shape. It i s not uncommon to fi n d r a d i c a l s produced by i o n i z i n g r a d i a t i o n occurring i n more than one 22 38 119 123 141 conformation. » > > » Based on these examples i t i s reasonable to assume that the s p l i t t i n g s we observe i n N-acetylglycine are caused by very small conformational di f f e r e n c e s of the same r a d i c a l . Now, i f the s p l i t t i n g of the ENDOR l i n e s of the a-proton and the protons designated by Hx^-Hx^ a r i s e s from small conformational differences of the same r a d i c a l , i t would seem u n l i k e l y that the l i n e s due to the methyl protons are s p l i t f o r a d i f f e r e n t reason. Therefore, we have assigned the three l i n e s at ^ 17.4 MHz i n figu r e 27 to the methyl protons. -133-29 The previous assignment of these l i n e s by HSM to two methyl groups and a proton i n the v i c i n i t y of the r a d i c a l was based on i n t e n s i t y measure-ments of the ENDOR l i n e s . Our contention i s that the i n t e n s i t y measure-ment of these l i n e s would be u n r e l i a b l e because the l i n e s are not w e l l separated. Our r e s u l t s show that the l i n e they measured as a s i n g l e l i n e f o r Hc^ and Hc^ i s i n f a c t a composite of two c l o s e l y spaced l i n e s separated by ^100 kHz. In support of t h e i r assignment, HSM presented the second d e r i v a t i v e ESR spectrum of a c r y s t a l grown from heavy water. They analyzed the m u l t i p l e t s t r u c t u r e i n the ESR spectrum ( f i g u r e 3 of HSM) as eight e q u a l l y spaced l i n e s w i t h b i n o m i a l i n t e n s i t y r a t i o . The eight l i n e s , they argued, a r i s e from seven e q u a l l y coupled protons: s i x methyl protons and another i n the v i c i n i t y of the r a d i c a l . F i g . 29(a) shows the ESR spectrum under i d e n t i c a l c o n d i t i o n s w i t h a s t i c k diagram immediately beneath the spectrum. We s t r o n g l y b e l i e v e that the m u l t i p l e t s t r u c t u r e i n the spectrum can be analyzed as s i x e q u a l l y spaced l i n e s w i t h i n t e n s i t y r a t i o 1:4:7:7:4:1. Such a s i x l i n e spectrum i s p o s s i b l e i f we assume that the hyp e r f i n e coupling of the three protons of a r o t a t i n g methyl 14 group i s approximately equal to the c o u p l i n g due to the N nucleus. 125 HSM probably missed the important paper of Saxebol et a l . who have s a t i s f a c t o r i l y analyzed the ESR spectra on the assumption that the sextet p a t t e r n a r i s e s from hyp e r f i n e i n t e r a c t i o n w i t h the methyl protons and the N nucleus. In order to confirm that the a d d i t i o n a l l i n e s i n the ESR spectrum 14 are due to N i n t e r a c t i o n and not a second methyl group and an e x t r a proton, we prepared s i n g l e c r y s t a l s of "^N l a b e l l e d a c e t y l g l y c i n e from heavy water. The ESR spectrum f o r such a c r y s t a l under i d e n t i c a l -134-15 , H a". a NQ = 0.360 mT Cl£H =0.257 mT-^ F i g . 2 9 . Second d e r i v a t i v e ESR s p e c t r a o f d e u t e r a t e d N - a c e t y l g l y c i n e f o r t h e m a g n e t i c f i e l d p a r a l l e l t o a * - a x i s : ( a ) - ' - ^ N - a c e t y l g l y c i n e (b ) 1 % - a c e t y l g l y c i n e . -135-conditions i s shown i n f i g . 29(b). Now, i f the a d d i t i o n a l l i n e s were due to protons, no change should be noticed between the two spectra. However, one notices that the si x l i n e spectrum has changed to a poorly resolved f i v e l i n e spectrum. ^ N has a nuclear spin of 1/2 and i t s magnetic 14 moment i s s l i g h t l y higher than that of N with 1=1. The s t i c k diagram 14 beneath f i g . 29(b) i s calculated on the assumption that a(CHg)~a( N), 14 and taking i n t o account the dif f e r e n c e i n the magnetic moments of N and "^N. I t i s to be noted that the ESR spectra of F i g . 29 are i n very good agreement with the calculated ones. Based on these observations we can safel y r u l e out a second methyl group and a proton i n the v i c i n i t y of the r a d i c a l as being responsible f o r the a d d i t i o n a l structure i n the ESR 136 spectrum. In an ESR study of i r r a d i a t e d a c e t y l g l y c i n e , Mangiaracina 14 observed w e l l resolved N hyperfine s p l i t t i n g i n a c r y s t a l where the methyl group was deuterated. This observation supports our ana l y s i s of the ESR spectra. In a d e t a i l e d v a r i a b l e temperature ESR study of X- i r r a d i a t e d N-131 a c e t y l g l y c i n e , S i n c l a i r and Codella have established that the room temperature r a d i c a l i s produced from an undamaged molecule by a hydrogen atom transfer to r a d i c a l s stable only at lower temperatures. The o v e r a l l scheme, they have suggested f o r a c e t y l g l y c i n e i s : CHoC0NDCHoC00D . I o n ^ z ^ n g > CH-CONDCH-C*' +e~+D+ (1) 3 2 r a d i a t i o n 3 6 CH,CONDCH ?-/ jLt*ZL2L± C 0 2 + CH3COND-CH,, (2) 3 z x 0 -136-CH.-C -NDCH0COOD (3a) y  3 2 e + CH-CONDCH-COOD CH CONDCH -C (3b) J OD 0" 0 • / —isn°r ' CH„CONDCH -C > CH„-C-NDCH„COOD (4) X0D 0" 0 .i -so°r H CH3-C-NDCH2COOD — > CH3~C-ND + CH^COOD (5) CH3-COND-CH2 + CH3CONDCH2COOD ~ 8 ° C CH„COND-CH„ + CH-COND-C-COOD (6) 3 3 3 | H >-SO°C •CH2-COOD + CH3CONDCH2COOD > CH3COOD + CH3CONDCH-COOD (7) It i s evident from the above steps that there would be no free proton a v a i l a b l e i n the v i n c i n i t y of the r a d i c a l to form a weak bond with i t as 29 15 argued by HSM. Our r e s u l t s on i r r a d i a t e d N-acetylglycine have established beyond any reasonable doubt that the existence of such a weakly bonded proton i n the v i c i n i t y of the r a d i c a l i s highly improbable. From Table 9 i t should be noted that the tensors associated with Hxn TABLE 9 HYPERFINE COUPLING TENSORS FOR THE WEAKLY COUPLED PROTONS OF HYDROGEN ABSTRACTION RADICAL IN N-ACETYLGLYCINE.1 PROTON Coupling (MHz) Dire c t i o n 2 Cosines P r i n c i p a l value Isotropic Dipolar a* b c +2.87 +7.93 -0.006 +0.923 -0.384 Hx1(NH) -8.93 -5.06 -3.87 -0.519 ±0.331 -0.787 -9.12 -4.06 -0.854 (-0.021 +0.195 +0.877 +0.481 -0.482) -1.81 +5.34 -0.026 ±0.193 +0.981 Hx2(OH) +5.78 +3.53 -2.25 -0.999 +0.048 -0.017 +6.61 -3.08 +0.044 (+0.018 +0.980 -0.096 +0.194 +0.995) +1.61 +1.61 -0.066 ±0.463 +0.884 Hx 3 ( 0 2 HN) -3.88 0.00 -3.88 +0.997 ±0.072 +0.038 +2.26 +2.26 +0.046 (+0.021 +0.883 -0.877 +0.467 +0.482) TABLE 9 (cont'd) P R 0 T 0 N Coupling (MHz) Di r e c t i o n Cosines P r i n c i p a l value Isotropic Dipolar a* b +1.34 +1.28 +0.187 +0.283 -0.941 -3.52 +0.06 -3.58 +0.920 ±0.385 +0.067 +2.37 +2.31 -0.344 ±0.883 -0.332 -1.21 -1.36 +0.023 +0.489 -0.872 Hx 5 -1.56 +0.15 -1.71 +0.999 +0.018 +0.021 +3.21 +3.06 0.007 +0.872 +0.489 -1.74 -1.71 -0.059 +0.998 +0.000 Hx 6 ( 0 3 HC-p -1.56 -0.03 -1.53 +0.998 +0.059 +0.037 +3.21 +3.24 -0.037 -0.002 +0.999 (-0.002 +0.068 +0.998) 1 See footnotes to Table 8. 2 Numbers i n parentheses represent bond d i r e c t i o n s . -139-and have considerable i s o t r o p i c parts. Based on t h i s and t h e i r d i r e c t i o n cosines these two tensors are assigned to the N-H and 0-H protons i n the r a d i c a l . Both the protons have one p r i n c i p a l axis i n common which i s also p a r a l l e l to the intermediate coupling of the a-proton. On com-paring the d i r e c t i o n s of the largest couplings with the bond d i r e c t i o n s , i t i s found that the agreement i s within ^6°. The remaining four exchangeable proton tensors are e s s e n t i a l l y d i p o l a r i n nature, i n d i c a t i n g that they belong to intermolecular protons. The tensor Hx^ has one component perpendicular to molecular plane and another component p a r a l l e l to C^-O^ bond. Hence t h i s was assigned to NH proton of the neighboring molecule hydrogen bonded to 0 2 of parent molecule. S i m i l a r l y the i n t e r a c t i o n Hx^ has one component which i s p a r a l l e l to C^-N bond d i r e c t i o n which i n turn p a r a l l e l to 0^ H-0. Thus both the hydrogen bonded protons are accounted f o r . The remaining two exchangeable proton tensors belong to intermolecular exchangeable protons i . e . N-H or 0-H protons of neighboring molecules not hydrogen bonded to the parent molecule. From the i s o t r o p i c part of the a-proton tensor we calculated an unpaired electron density of 0.75 on the pu o r b i t a l of the a-carbon, using the McConnell relation^"* with a Q-value of -65 MHz. This i s i n very good agreement with the r e s u l t s of INDO c a l c u l a t i o n s (pir INDO = 0.698). The calculated (INDO) and observed spin d e n s i t i e s are given i n Table 10. From the N-H proton i s o t r o p i c coupling, a spin density of 0.065 on the pTr o r b i t a l of nitrogen was calculated using a Q-value of 78 MHz (pn INDO = 0.111). Our 137 INDO r e s u l t s i n agreement with s i m i l a r c a l c u l a t i o n s by Saxebol et a l . indicate that the remaining spin density i s de l o c a l i z e d over the enti r e molecule. TABLE 10 COMPARISON OF EXPERIMENTAL AND INDO UNPAIRED ELECTRON DENSITIES IN THE HYDROGEN ABSTRACTION RADICAL OF N-ACETYLGLYCINE ATOM C l ° 1 °2 C2 N s ° 3 C4 pn(INDO) -0.146 -0.024 +0.299 +0.698 +0.111 -0.064 +0.130 +0.003 pTT (obs . ) +0.752 +0.065 +0.129 -141-In an attempt to f i n d out the reason for the f a i l u r e of Piazza and 27 Patten to f i n d a well-resolved ENDOR spectrum i n the v i c i n i t y of vp, we c a r r i e d out ENDOR measurements as a function of i r r a d i a t i o n dose. The optimum s i g n a l to noise r a t i o was obtained with i r r a d i a t i o n time of about two hours. Longer i r r a d i a t i o n resulted i n rapid decrease i n the i n t e n s i t y and r e s o l u t i o n of the main ENDOR spectrum while s h i f t i n g i n t e n s i t y to the 'distant' ENDOR l i n e . F i n a l l y a f t e r about 10 hours of i r r a d i a t i o n a 27 spectrum s i m i l a r to that reported by Piazza and Patten was obtained. ESR spectra of the heavily i r r a d i a t e d c r y s t a l s showed a s i g n i f i c a n t i n -crease i n i n t e n s i t y , i n d i c a t i n g that the r a d i c a l concentration increased 142 with i r r a d i a t i o n dose. These observations suggest that cross r e l a x a t i o n i s a dominant r e l a x a t i o n route i n i r r a d i a t e d N-acetylglycine. Our observation that the i r r a d i a t i o n dose and hence the r a d i c a l concentration i s rather c r i t i c a l for a well-resolved spectrum may we l l explain the frequent f a i l u r e s to detect ENDOR signals i n molecular c r y s t a l s . 7.4 Concluding Remarks: In conclusion i t should be noted that our r e s u l t s give no i n d i c a t i o n that the wavefunction of the unpaired electron s i g n i f i c a n t l y extends over the neighboring molecules. A l l the intermolecular hyperfine i n t e r a c t i o n s we have observed are e s s e n t i a l l y d i p o l a r i n nature and hence do not indicate 29 any appreciable spin density at the respective n u c l e i . The r e s u l t s of HSM showed considerable spin density on the neighboring molecules, but t h e i r r e s u l t s were obtained most l i k e l y from erroneous analyses of the ENDOR data. There i s also no evidence that an atom or a group which i s broken from a molecule upon i r r a d i a t i o n remains trapped i n the immediate v i c i n i t y of the damaged molecule i n the c r y s t a l . We have conclusively shown that the -142-m u l t i p l e t structure i n the ESR spectrum of i r r a d i a t e d N-acetylglycine a r i s e s from the i n t e r a c t i o n of the unpaired electron with protons of 14 the methyl group and the N nucleus. Although we have not been able 14 14 to obtain N ENDOR, i t should be noted that the f a i l u r e to f i n d N ENDOR does not exclude the presence of nitrogen hyperfine i n t e r a c t i o n . While i t may be true that 'negative' ENDOR technique has some advantages over i t s conventional counterpart, however, the f a i l u r e to detect conventional ENDOR may be due to too large a concentration of the r a d i c a l i n the c r y s t a l . We note that a c a r e f u l l y c o n t r o l l e d ENDOR experiment should y i e l d at least as good r e s u l t s as negative ENDOR, thus avoiding the need f o r an intense r . f . f i e l d , and hence, the r e s u l t i n g heat generation. CHAPTER EIGHT CONCLUSIONS: In the foregoing chapters, the ENDOR investigations on three room temperature X - i r r a d i a t e d compounds, namely,phenazine, hippuric acid and N-acetylglycine have been described. Phenazine i s ex c l u s i v e l y aromatic, hippuric acid i s aromatic with an a l i p h a t i c side chain and N-acetyl-glycine i s purely a l i p h a t i c . Impressive consistencies have been observed while going from f u l l y aromatic to f u l l y a l i p h a t i c compounds. For example, phenazine requires about 80 hours, hippuric acid about 8 hours and N-acetyl-glycine about 2 hours of i r r a d i a t i o n for an optimum ENDOR si g n a l i n t e n s i t y . The c r y s t a l structure of phenazine i s monoclinic. The two molecules i n the unit c e l l are magnetically equivalent along b-axis and i n ac-plane; i n a l l other orie n t a t i o n s they have two d i s t i n c t s i t e s . A l l the ENDOR l i n e s a r i s e from i n t e r a c t i o n of unpaired electron with protons and no l i n e s due to 14 the hyperfine coupling of unpaired electron with N were observed i n our study, although the ESR sp e c t r a l spread indicates large T r-spin on nitrogen. The ESR spectrum exhibited very much poorer r e s o l u t i o n i n most ori e n t a t i o n s , because the unpaired electron i s extensively d e l o c a l i z e d and coupled to large number of n u c l e i i n aromatic systems. Our well resolved ENDOR spectra of phenazine have enabled us to characterize a l l the protons of an aromatic molecule which i s otherwise impossible by ESR studies. The r e s u l t indicates that the r a d i c a l i s formed by the addit i o n of hydrogen atom to N ^ Q ^ of phenazine. Analysis of some weaker accompanying l i n e s suggests that they belong to the same type of r a d i c a l but with the hydrogen atom added on with a s l i g h t conformational change with respect to the previous r a d i c a l . -144-Th e c r y s t a l of hippuric acid i s , on the other hand, orthorhombic. The four molecules i n the unit c e l l are magnetically equivalent along the c r y s t a l l o g r a p h i c axes and they have two d i s t i n c t s i t e s i n a l l the cry s t a l l o g r a p h i c planes. A preliminary ESR spectrum revealed a highly anisotropic 1:2:1 t r i p l e t along the cr y s t a l l o g r a p h i c axes i n d i c a t i n g only two nearly equivalent protons strongly i n t e r a c t i n g with the unpaired electron. However, poor r e s o l u t i o n combined with the presence of s i t e s p l i t t i n g prevented us from reaching any quantitative conclusion regarding the nature of the r a d i c a l . ENDOR inve s t i g a t i o n s on t h i s system f u l l y c l a r i f i e d the i d e n t i t y and nature of the r a d i c a l . In addi t i o n to strong a-proton coupling and some weak i n t r a - and intermolecular proton i n t e r -actions, we have detected 1 4 N ENDOR l i n e s belonging to a low hyperfine and quadrupole coupling which are r a r e l y observed. The r a d i c a l i s believed to be formed by deprotonation of oxidized hippuric acid followed by the loss of a molecule of carbon dioxide. Among weakly coupled protons, N-H proton, three r i n g protons and one hydrogen bonded proton have been assigned. The exchangeable protons have been confirmed by p a r t i a l deuteration. Although the decarboxylated r a d i c a l s i n carboxylic acids are usually unstable at room temperature, the present study establishes that i n hippuric acid at l e a s t , i t i s stable at room temperature. In a s t r u c t u r a l l y r e l a t e d compound N-acetylglycine the decarboxylated r a d i c a l i s stable only at low tem-peratures i n d i c a t i n g that the phenyl group probably plays a c r i t i c a l r o l e i n the unusual s t a b i l i t y of decarboxylated r a d i c a l i n hippuric a c i d . The ENDOR i n v e s t i g a t i o n on X- i r r a d i a t e d N-acetylglycine i s a r e i n v e s t i -gation of an e a r l i e r 'negative' ENDOR study by Helms, Suzuki and Miyagawa (HSM) Based on t h e i r r e s u l t s , they proposed that the room temperature r a d i c a l was -145-e s s e n t i a l l y a c a t i o n , and t h a t t h e w a v e f u n c t i o n o f t h e u n p a i r e d e l e c t r o n e x t e n d s s i g n i f i c a n t l y o v e r t h e n e i g h b o r i n g m o l e c u l e s . On r e i n v e s t i g a t i o n we d i s c o v e r e d s e v e r a l i n c o n s i s t e n c i e s i n t h e r e s u l t s o f H S M . F i r s t o f a l l , p r o b a b l y t h e r a d i a t i o n d o s e was n o t p r o p e r l y c o n t r o l l e d f o r t h e d e t e c t i o n o f w e l l - r e s o l v e d s p e c t r a . L o n g e r i r r a d i a t i o n r e s u l t e d i n t h e p r o d u c t i o n o f a l a r g e c o n c e n t r a t i o n o f t h e r a d i c a l s most l i k e l y l e a d i n g t o c r o s s r e l a x a t i o n , t h u s r a p i d l y d e c r e a s i n g t h e i n t e n s i t y and r e s o l u t i o n o f t h e ENDOR s p e c t r u m w h i l e s h i f t i n g i n t e n s i t y t o t h e ' d i s t a n t ' ENDOR l i n e . B e c a u s e o f p o o r r e s o l u t i o n t h e y w e r e n o t a b l e t o f o l l o w u n a m b i g u o u s l y t h e c l o s e l y s p a c e d ENDOR l i n e s i n t h e v i c i n i t y o f f r e e p r o t o n NMR f r e q u e n c y . T h i s l e d t o m i s a s s i g n m e n t o f s e v e r a l l i n e s i n t h e a n g u l a r v a r i a t i o n p l o t s , l e a d i n g t o e r r o n e o u s v a l u e s f o r t h e h y p e r f i n e t e n s o r s . T h e i r r e s u l t s show a p p r e c i a b l e i s o t r o p i c p a r t s f o r t h e t e n s o r s o f s e v e r a l i n t e r m o l e c u l a r p r o t o n s . On t h e o t h e r h a n d , w e w e r e a b l e t o o b t a i n w e l l - r e s o l v e d ENDOR s p e c t r a b y c a r e f u l l y c o n t r o l l i n g t h e i r r a d i a t i o n d o s e . Upon c a r e f u l a n a l y s i s o f t h e d a t a we f i n d t h a t t h e h y p e r f i n e t e n s o r s o f i n t e r m o l e c u l a r p r o t o n s a r e a l l n e a r l y d i p o l a r i n n a t u r e w i t h n e g l i g i b l e i s o t r o p i c p a r t s . B a s e d on i n t e n s i t y m e a s u r e m e n t s o f ENDOR l i n e s HSM c o n c l u d e d t h a t a m e t h y l g r o u p on t h e n e i g h b o r i n g m o l e c u l e h a d i d e n t i c a l c o u p l i n g t o t h a t on t h e r a d i c a l i t s e l f . They a l s o c l a i m e d t h a t one o f t h e ENDOR l i n e s t h e y o b s e r v e d b e l o n g e d t o a w e a k l y bonded p r o t o n i n t h e v i c i n i t y o f t h e r a d i c a l . T h e s e c l a i m s w e r e ' s u b s t a n t i a t e d ' by p r e s e n t i n g t h e s e c o n d d e r i v a t i v e ESR s p e c t r u m a l o n g t h e a * - a x i s . They a n a l y z e d t h e e x t r a h y p e r f i n e s t r u c t u r e i n t h e s p e c t r u m a s e i g h t l i n e s w i t h b i n o m i a l i n t e n s i t y r a t i o , a r i s i n g f r o m s e v e n n e a r l y e q u i v a l e n t p r o t o n s - two m e t h y l g r o u p p r o t o n s and a w e a k l y -146-bonded proton. We have unequivocally shown that the extra hyperfine structure i n the ESR spectrum a r i s e s from in t e r a c t i o n s with a methyl group 14 (that on the r a d i c a l i t s e l f ) and the N nucleus present i n the r a d i c a l . 14 15 This was achieved by replacing N by N and demonstrating the accompanying change i n the ESR spectrum. Our analysis of the ESR spectra i s supported by the reports of Saxebol et a l . and Mangiaracina. Based on our r e s u l t s we have established that the stable r a d i c a l i n room temperature X - i r r a d i a t e d N-acetylglycine i s CHgCONHCHCOOH. We also f i n d that the r a d i c a l i s present i n the c r y s t a l i n four s l i g h t l y d i f f e r e n t conformations giving r i s e to the complex ENDOR spectra. There i s no evidence for extensive d e r e a l i z a t i o n of the unpaired electron beyond the bounds of the r a d i c a l i t s e l f . One of the fundamental aspects of these studies i s the control of r a d i c a l concentration i n the c r y s t a l s for the success of an ENDOR i n v e s t i g a t i o n . It has been demonstrated that the optimum s i g n a l to noise r a t i o i s obtained with i r r a d i a t i o n time of d e f i n i t e hours depending on the system. Longer i r r a d i a t i o n r e s u l t s i n rapid decrease i n the i n t e n s i t y and r e s o l u t i o n because of cross r e l a x a t i o n . This i s probably the main reason why the previous workers on N-acetylglycine have f a i l e d to detect well-resolved ENDOR spectra of i r r a d i a t e d N-acetylglycine. It i s d i f f i c u l t to r e l a t e the r a d i c a l s trapped i n si n g l e c r y s t a l s with r a d i a t i o n damage i n l i v i n g organisms as a r e s u l t of the f a r greater complexity of the l i v i n g system. However, the r a d i c a l s which can be trapped i n single c r y s t a l s , can also be formed i n the l i v i n g organisms, although t h e i r l i f e times are too short to allow t h e i r analyses by conventional ENDOR techniques. As a r e s u l t of b i o l o g i c a l action of r a d i a t i o n , f l a v o p r o t e i n undergoes reduction -147-and i t i s thought that the s i t e of the r a d i c a l i s the f l a v i n mononucleotide moiety i n the f l a v o p r o t e i n . The r a d i c a l anion i s formed by electron a d d i t i o n to a neutral f l a v i n and the r a d i c a l c a t i o n i s formed by e f f e c t i v e addition to a neutral f l a v i n ; so both anion and cation are the reduced forms of f l a v i n molecules. Under some conditions,a neutral f l a v i n r a d i c a l i s formed by hydrogen addition to nitrogen atom of f l a v i n moiety; t h i s i s s i m i l a r to cation r a d i c a l and has a s i m i l a r spin density d i s t r i b u t i o n . Since si n g l e c r y s t a l s of f l a v o p r o t e i n are d i f f i c u l t to prepare and s i n g l e c r y s t a l analysis gives the most complete information about a r a d i c a l , i t i s easier to s t a r t with f l a v i n moiety as a model compound to study the e f f e c t of r a d i a t i o n on flavoproteins. Our work on phenazine which resembles f l a v i n moiety shows that the r a d i c a l i s e s s e n t i a l l y formed by hydrogen addition at one of the nitrogen atoms of the molecule. Hippuric a c i d and N-acetylglycine are the simplest compounds with peptide bonds that unite i n d i v i d u a l amino acids into proteins. Since s i n g l e c r y s t a l s of proteins are d i f f i c u l t to prepare, i t has been u s e f u l to s t a r t with the s i n g l e c r y s t a l s of these model compounds i n order to i n t e r p r e t the r a d i a t i o n damage i n proteins. A v a r i e t y of chemical agents are known that can protect organisms against the e f f e c t s of r a d i a t i o n and s e n s i t i z e tumour c e l l s to r a d i a t i o n improving the effectiveness of r a d i a t i o n therapy i n the treatment of cancer. It has long been known that i o n i z i n g r a d i a t i o n i s considerably more l e t h a l to c e l l s . In view of compelling need for increasing r a d i a t i o n therapy i n the treatment of cancer, the use of s e n s i t i z i n g agents has been receiving increased a t t e n t i o n . Moreover the enhancement of r a d i a t i o n damage by c e r t a i n compounds could lead to new methods for the treatment of cancers. -148-If these compounds could be s e l e c t i v e l y introduced into cancer ti s s u e , r a d i a t i o n damage and subsequent c e l l death would be enhanced i n t h i s t i s s u e . Thus the dosage required to destroy a cancer could be reduced, thereby reducing r a d i a t i o n damage to healthy t i s s u e s . These compounds could also exhibit a remarkable capacity to protect l i v i n g organisms from the e f f e c t s of r a d i a t i o n . Mechanismsof r a d i a t i o n protection are of fundamental i n t e r e s t i n r a d i a t i o n biology. The r a d i a t i o n protectors intervene i n the r a d i a t i o n damage process by scavenging some of the primary r e a c t i v e species produced i n the r a d i o l y s i s of body f l u i d . Thus the organism i s protected, at le a s t p a r t i a l l y , against i n d i r e c t e f f e c t s of r a d i a t i o n . A bio-molecule having been oxidised by removal of hydrogen at some e a r l i e r stage of r a d i a t i o n damage process, i s subsequently repaired by a donation of hydrogen from a protector molecule. -149-BIBLIOGRAPHY 1. E. Zavoisky, J . Phys., U.S.S.R., 10, 197 (1946). 2. W. Gordy, W. B. Ard and H. Shields, Proc. Nat. Acad. S c i . 41, 983 (1955). 3. J. Uebersfeld and E. Erb, Compt. Rend. 242, 478 (1956). 4. G. Feher, Phys. Rev. 103, 834 (1956). 5. W. C. Holton, H. Blum and C. P. S l i c h t e r , Phys. Rev. Letters 5_, 197 (1960). 6. A. L. Kwiram, Ann. Rev. Phys. Chem. 22_, 133 (1971). 7. L. Kevan and L. D. Kispert, 'Electron Spin Double Resonance Spectroscopy', Wiley, New York (1976). 8. H. C. Box, 'Radiation E f f e c t s , ESR and ENDOR Analysis', Academic Press, Inc., New York (1977). 9. K. Mobius, i n 'Electron Spin Resonance', ed. R.O.C. Norman ( S p e c i a l i s t P e r i o d i c a l Reports), The Chemical Society, London, _, 16 (1977). 10. N. M. Atherton, i n 'Electron Spin Resonance' ed. R.O.C. Norman ( S p e c i a l i s t P e r i o d i c a l Reports), The Chemical Society, London, 1, 32 (1973); 2, 36 (1974); 3, 23 (1976). 11. T. Cole, C. He l l e r and J. Lambe, J . Chem. Phys. 34, 1447 (1961). 12. A. L. Kwiram, Ph.D. t h e s i s , C a l i f o r n i a I n s t i t u t e of Technology, Pasadena (1962). 13. A. L. Kwiram and J. S. Hyde, J . Chem. Phys. 42, 791 (1965). 14. A. L. Kwiram, J . Phys. Chem. 55, 2485 (1971). 15. J . W. Wells and C. L. Ko, J . Chem. Phys. 69(5), 1848 (1978). -150-16. L. L. Finch, J . E. Johnson and G. C. Moulton, J. Chem. Phys. 70 3662 (1979). 17. H. C. Box, E. E. Budzinski and H. G. Freund, J . Chem. Phys.. 50^ 2880 (1969). 18. M. Welter, T. Krober, S. Wartewig and W. Windsch, Molecular Physics 36 , 1385 (1978). 19. D. M. Close, G. W. Fouse and W. A. Bernhard, J . Chem. Phys. 70 , 2131 (1979). 20. F. 0. Ngo, E. E. Budzinski and H. C. Box, J . Chem. Phys. 60 , 3373 (1974). 21. D. M. Close, G. W. Fouse and W. A. Bernhard, J. Chem. Phys. 66 , 1534 (1977). 22. J. W. Wells and H. C. Box, J. Chem. Phys. M_ , 2935 (1967). 23. H. C. Box, H. G. Freund and E. E. Budzinski, J . Chem. Phys. 46 , 4470 (1967). 24. D. J. Whelan, J. Chem. Phys. 49 , 4734 (1968). 25. B. W. Castleman and G. C. Moulton, J . Chem. Phys. 57_, 2762 (1972). 26. D. A. Hampton and G. C. Moulton, J. Chem. Phys. 63 . , 1078 (1975). 27. R. E. Piazza and R. A. Patten, Molecular Physics 17_ , 213 (1969). 28. I. Miyagawa, Y. Ku r i t a and W. Gordy, J . Chem. Phys. 33' 1 5 9 9 U 960). 29. H. A. Helms, I. Suzuki and I. Miyagawa, J. Chem. Phys. J59_ , 5055 (1973). 30. D. A. Hampton and C. Alexander, J r . , J . Chem. Phys. 58, 4891 (1973). 31. D. M. Close, G. W. Fouse and W. A. Bernhard, J . Chem. Phys. , 4689 (1977). 32. H. C. Box, E. E. Budzinski and W. R. Porter, J. Chem. Phys. 61 , 1136 (1974). -151-33. H. Oloff and J . Hutterman, J. Mag. Res. 27^ , 197 (1977). 34. E. Haindl and J . Huttermann, J . Mag. Res. 30, 13 (1978). 35. J . N. Herak, D. K r i l o v and C. A. McDowell, J . Mag. Res. 23_, 1 (1976). 36. J . N. Herak and C. A. McDowell, J . Mag., Res. 16, 434 (1974). 37. J. N. Herak and C. A. McDowell, J. Chem. Phys. 61 , 1129 (1974). 38. J . N. Herak, D. R. Lenard and C. A. McDowell, J. Mag. Res. _26 , 189 (1977). 39. U. R. Bohme and G. W. Jesse, Chem. Phys. L e t t . 3^  » 3 2 9 (1969). 40. U. R. Bohme and H. C. Wolf, Chem. Phys. L e t t . 17_ , 582 (1972). 41. B. Lamotte and P. Gloux, J . Chem. Phys. 5_9 , 3365 (1973). 42. P. Gloux and B. Lamotte, Moi. Phys. 25 , 161 (1973). 43. B. Bleany and K. W. H. Stevens, Reports Progr. Phys. 16, 108 (1953). 44. H.S. J a r r e t t i n "Advances i n So l i d State Physics", 14, 215 (1963). 45. M. H. L. Pryce, Nuovo Cimento, Supp. 6, 817 (1957). 46. S. Geschwind i n "Hyperfine Interactions", ed. A. J . Freeman and R. B. Frankel, (Academic Press, N.Y.) 225 (1967). 47. K. A. Thuomas and L. Lund, J. Mag. Res. 18, 12 (1975). 48. C. P. S l i c h t e r , " P r i n c i p l e s of Magnetic Resonance", Harper and Row, New York (1963). 49. A. Carrington and A. D. McLachlan, "Introduction to Magnetic Resonance", Harper and Row, New York (1967). 50. A. Abragam and B. Bleaney, "Electron Paramagnetic Resonance of Tr a n s i t i o n Ions", Clarendon Press, Oxford (1970). 51. N. M. Atherton, "Electron Spin Resonance", Wiley, New York (1973). 52. G. E. Pake, "Paramagnetic Resonance", Benjamin, New York (1962). -152-53. J . N. M u r r e l l , S. F. A. K e t t l e and J . M. Tedder, "Valence Theory", Wiley, London, p.84 (1970). 54. H. F. Hameka i n "The T r i p l e t State", (A. B. Zahlen, ed.), Cambridge University Press, p . l (1967). 55. M. H. L. Pryce, Proc. Phys. Soc. (London) A63, 25 (1950). 56. A. Abragam and M. H. L. Pryce, Proc. Roy. Soc. (London) A205, 135 (1951). 57. S. I. Weissman, J . Chem. Phys. 2J2, 1378 (1954). 58. E. Fermi, Z. Physik 60, 320 (1930) 59. J . R. Dickinson, Ph.D. Thesis, U.B.C. (1974). 60. N. S. D a l a i , J . R. Dickinson and C. A. McDowell, J. Chem. Phys. 57_, 4254 (1972). 61. J . A. Hebden, Ph.D. Thesis, U.B.C. (1970). 62. H. S. J a r r e t t , J . Chem. Phys. 25, 1289 (1956). 63. S. I. Weisman, J . Chem. Phys. 25., 890 (1956). 64. R. Bersohn, J . Chem. Phys. 24, 1066 (1956). 65. H. M. McConnell, J . Chem. Phys. 24, 764 (1956). 66. H. M. McConnell, C. H e l l e r , T. Cole and R. W. Fessenden, J. Amer. Chem. Soc. 8j_, 766 (1960). 67. D. K. Ghosh and D. H. Whiffen, Moi. Phys. 2, 285 (1959). 68. H. M. McConnell and J . Strathdee, Moi. Phys. 2, 129 (1959). 69. J . P. Colpa and E. DeBoer, Moi. Phys. ]_, 333 (1964). 70. A. D. McLachlan, Moi. Phys. 1, 233 (1958). 71. W. Derbyshire, Moi. Phys. ,5» 2 2 5 (1962). 72. F. C. Adam and F. W. King, J. Chem. Phys. 58, 2446 (1973). 73. D. H. Levy and R. J . Myers, J. Chem. Phys. 43, 3063 (1965). -153-74. J . R. Bolton, A. Carrington and A. D. McLachlan, Moi. Phys. 5_, 31 (1962). 75. J . W. Wells, J . Chem. Phys. 52, 4062 (1970). 76. A. Carrington and J . dos Santos-Viega, Moi. Phys. 21 (1962). 77. A. H o r s f i e l d , J. R. Morton, J. R. Rowlands and D. H. Whiffen, Moi. Phys. _, 241 (1962). 78. C. H. Townes and B. P. Dailey, J . Chem. Phys. 17_, 782 (1949). 79. C. H. Townes and B. P. Dailey, J . Chem. Phys. 20, 35 (1952). 80. C. H. Townes and B. P. Dailey, J . Chem. Phys. 23, 118 (1955). 81. W. Gordy, J . Chem. Phys. 19, 792 (1951). 82. W. Gordy, J . Chem. Phys. 2_2, 1470 (1954). 83. W. Gordy, Disc. Far. Soc. 19, 9 (1955). 84. W. Gordy and R. L. Cook, "Microwave Molecular Spectra", Wiley-Interscience, New York, Ch. 14 (1970). 85. J. A. Pople, D. L. Beveridge and P. A. Dobosh, J. Chem. Phys. 47, 2026 (1967). 86. J . A. Pople, D. L. Beveridge and P. A. Dobosh, J . American Chem. Soc. 90, 4201 (1968). 87. J. A. Pople, D. P. Santry and G. A. Segal, J . Chem. Phys. 42, 5129 (1965) . 88. J . A. Pople and G. A. Segal, J . Chem. Phys. 43, 5136 (1965). 89. J. A. Pople and G. A. Segal, J . Chem. Phys. 44, 3289 (1966). 90. J . A. Pople and D. L. Beveridge, "Approximate Molecular O r b i t a l C alculations", McGraw-Hill, New York (1970). 91. N. S. D a l a i , Ph.D. t h e s i s , The University of B r i t i s h Columbia (1971). 92. V. P. Chacko, C. A. McDowell and B. C. Singh, Moi. Phys. 38 , 321 (1979). -154-93. T. G. Castner and A. M. Doyle, Rev. Scient. Instrum. 39, 1090 (1968). 94. N. M. Atherton and R. S. F. Harding, J . Chem. Soc. A, 5587 (1967). 95. N. M. Atherton and R. S. F. Harding, J . Chem. Soc. A, 1490 (1967). 96. H. Blum, P. L. Mattern, and R. A. Arndt, Moi. Cryst. L i q . Cryst. 3, 269 (1967). 97. H. A. Harper, "Review of P h y s i o l o g i c a l Chemistry" Ch. 6, p.73 (1963). 98. R. G. Wood and G. Williams, P h i l . Mag. 31, 115 (1941). 99. F. H. Herbstein and G. M. J . Schmidt, Acta. Cryst. 8, 399 (1955); Ibid , 8, 406 (1955). 100. F. L. H i r s h f e l d and G. M. J . Schmidt, J. Chem. Phys. _26» 9 2 3 ( 1 9 5 7>-101. A . M i l l e r and J. Huttermann, Ann. N.Y. Acad. S c i . 222, 411 (1973). 102. R. Furrer, M. Heinrich, D. S t e h l i k and H. Zimmermann, Chem. Phys. 3_6, 27 (1979). 103. R. Furrer, J . Petersen and D. Stehlik, Chem. Phys. 44, 1(1979). 104. V. Von Richter, 'The Chemistry of the Carbon Compounds' Vol. I l l , E l s e v i e r , New York, p.301 (1946). 105. G. H. B e l l , J . N. Davidson and H. Scarborough, 'Textbook of Physiology and Biochemistry', Livingston, Edinburgh and London, 3rd Edn., p.347 (1956). 106. M. P. Votinov, Radio b i o l o g i y a (1), No. 1, 149 (1961). 107. G. H. Rist and J. S. Hyde, J . Chem. Phys. 50 , 4532 (1969). 108. S. N. Rustgi and H. C. Box, J. Chem. Phys. 59_ , 4763 (1973). 109. M. F. Deigen, V. G. Krivenko, M. K. Pulatova, M. A. Ruban, V. V. Teslenko and L. P. Kayushin, B i o f i z i k a 18_ , 235 (1973). 110. W. H. Nelson, F. M. Atwater and W. Gordy, J. Chem. Phys. 61 , 4726 (1974). -155-111. A. Schweiger and Hs. H. Gunthard, Chem. Phys. 32, 35 (1978). 112. D. R. Lenard, Ph.D. t h e s i s , U.B.C. (1977). 113. M. Fujimoto, C. A. McDowell and T. Takui, J . Chem. Phys. 70_ , 3694 (1979). 114. C. A. McDowell and A. Naito, unpublished r e s u l t s . 115. R. Calvo, S. B. Oseroff and H. C. Abache, J . Chem. Phys. 1_2 1 760. (1980). 116. H. Ringertz, Acta Cryst. B27, 285 (1971). 117. W. Harrison, S. Rettig and J . Tro t t e r , J. Chem. Soc. Perkin I I , 1036 (1972). 118. M. Currie and A. L. Macdonald, J . Chem. Soc. Perkin I I , 784 (1974). 119. H. C. Box, E. E. Budzinski and K. T. L i l g a , J. Chem. Phys. _5_7, 4295 (1972). 120. H. C. Box and E. E. Budzinski, J . Chem. Phys. 55, 2 ^ 4 6 (1971). 121. T. Cole, C. He l l e r and H. M. McConnell, Proc. Natl. Acad. S c i . , U.S.A. 45, 525 (1959). 122. C. G o t t s ' c h a l l and B. Tolbert, Advan. Chem. Ser. 1, 374 (1968). 123. H. C. Box, H. G. Freund, K. T. L i l g a and E. E. Budzinski, J. Phys. Chem. 74, 40 (1970). 124. M. Iwasaki, B. Eda and K. Toriyama, J . Am. Chem. Soc. 92_, 3211 (1970). 125. A. H o r s f i e l d , J . R. Morton, J . R. Rowlands, and D. H. Whiffen, Moi. Phys. 5, 241 (1962). 126. R. J . Cook, J . S c i . Instrum. 43, 548 (1966). 127. H. Muto and M. Iwasaki, J . Chem. Phys. 59, 4821 (1973). 128. H. Muto and M. Iwasaki, J . Chem. Phys. 61, 5315 (1974). -156-129. H. Muto, K. Nunome and M. Iwasaki, J . Chem. Phys. 61, 1075, 5311 (1974). 130. J . W. Wells and C. L. Ko, J. Chem. Phys. 69, 1848 (1978). 131. J. S i n c l a i r and P. Codella, J . Chem. Phys. 59, 1569 (1973). 132. T. Henriksen, 'Solid State Biophysics' Ed. S. J. Wyard, pp.203-241, McGraw-Hill (1969). 133. W. Gordy, W. B. Ard and H. Shields, Proc. Nat. Acad. S c i . U.S.A. 41, 983 (1955). 134. W. Gordy and H. Shields, Radiat. Res. _, 611 (1958). 135. G. McCormick and W. Gordy, J . Phys. Chem. 62_, 783 (1958). 136. R. S. Mangiaracina, Rad. Res. 32.* 27 (1967). 137. G. Saxebol, T. B. Melo and T. Henriksen, Rad. Res. 51, 31 (1972). 138. A. B l a t t , 'Organic Synthesis', Wiley, New York, 2, 11 (1943). 139. G. Carpenter and J . Donohue, J . Am. Chem. Soc. 7_2, 2315 (1950). 140. A. H o r s f i e l d , J . R. Morton and D. H. Whiffen, Moi. Phys. 4_, 169 (1961) 141. J. W. S i n c l a i r and M. W. Hanna, J. Phys. Chem. 71, 84 (1967). 142. L. R. Dalton, A. L. Kwiram and J. A. Cowen, Chem. Phys. L e t t . 14, 77 (1972). 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0047164/manifest

Comment

Related Items