THE IDENTIFICATION OF FLAVIN AND
PTERIDINE FREE RADICALS BY
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THE IDENTIFICATION OF FLAVIN AND
PTERIDINE FREE RADICALS BY
ISBN 90 6231 047 8 soft-bound edition ISBN 90 6231 050 8 hard-bound edition
THE IDENTIFICATION OF FLAVIN A N D
PTERIDINE FREE RADICALS BY
ELECTRON SPIN RESONANCE
PROEFSCHRIFT
ter verkrijging van de graad van Doctor in de
Technische Wetenschappen aan de
Techni-sche Hogeschool Delft, op gezag van de Rector
Magnificus Prof. Ir. L. Huisman, voor een
commissie aangewezen door het college van
dekanen te verdedigen op woensdag
26 april 1978 te 16.00 uur
door
JACOB WESTERLING
scheikundig Ingenieur geboren te Barendrechtyc9^
/ v5" / 1978Dit proefschrift is goedgekeurd door de promotoren
Prof.Drs. W. Berends
en
Het in dit proefschrift beschreven onderzoek werd uitgevoerd op het Laboratorium voor Biochemie en op het Laboratorium voor Fysische Chemie van de Technische Hogeschool Delft onder auspicien van de Stichting Scheikundig Onderzoek Nederland (SON) en met financiele hulp van de Nederlandse
Organisatie voor Zuiver-Wetenschappelijk Onderzoek (ZWO).
Aan alien,die op enigerlei wijze hebben bijgedragen tot de totstandkoming van dit proefschrift,betuig ik hierbij mijn dank.
Met name dank ik alle wetenschappelijke medewerkers en het personeel van het Laboratorium voor Biochemie, in het bijzonder Dr.Ir.H.I.X.Mager, Ir.R.Addink en Ir.J.A.Jongejan die mij steeds met raad en daad ter zijde hebben gestaan.
De ESR metingen werden uitgevoerd op het Laboratorium voor Fysische Chemie. De aldaar genoten gastvrijheid heb ik bijzonder op prijs gesteld, evenals de stimulerende discussies met Drs.P.J.J.M.van der Put , Ir.J.J.M.Potters, Drs.G.A.Korteweg en Ir.G.van Veen.
Drs.A.H.Huizer van de vakgroep Organische Chemie der Rijksuniversiteit Leiden heeft mij meerdere malen de juiste weg gewezen op het gebied van de interpretatie van isotrope ESR spectra.
Mijn dank gaat ook uit naar Professor Dr.Th.J.de Boer en zijn medewerkers Dr.A.H.M.Kayen en Dr.Ih.A.B.M.Bolsman voor hun hulp en adviezen, die vooral in de beginfase van het onderzoek onontbeerlijk waren.
Tenslotte ben ik dank verschuldigd and het personeel van de Algemene Diensten van het gebouw voor Scheikunde en het gebouw voor Biologische Chemie voor de in verband met het onderzoek uitgevoerde werkzaamheden.
CONTENTS
CHAPTER I. GENERAL INTRODUCTION. 1 1.1. The purpose of the investigation. 1 1.2. The redox properties of flavins, isoalloxazines and pteridines. 2
1.2.1. Flavins and isoalloxazines. 2
1.2.2. Pteridines. 6 1.3. References of chapter I. 9
CHAPTER II. THE INTERPRETATION OF ISOTROPIC ESR SPECTRA IN TERMS OF
HYPERFINE COUPLING CONSTANTS AND NUCLEAR SPINS. 10
11.1. Introduction. 10 11.2. Interpretation by visual inspection. 12
11.3. The method of Newton et. al.. 13 11.4. Analysis of the sine Fourier transform. 16
11.5. The method of Balaban et. al.. 17
11.6. Autocorrelation. 17 11.7. Crosscorrelation. 18 11.8. Transformation to the cepstrum. 20
11.9. Isotope substitution. 20 11.10. Miscellaneous methods. 21
11.11. Simulation. 22 11.12. References of chapter II. 23
CHAPTER III. ESR OF FLAVIN AND 10-ALKYLATED ALLOXAZINE RADICALS. 25
111.1. Introduction. 25
111.2. I n t e r p r e t a t i o n of the ESR spectra of cationic f l a v i n and
10-alkylated alloxazine r a d i c a l s . 27 111.3. Free radicals i n hydroxylating systems containing
1,3,10-trimethyl-5,10-dihydroalloxazine. 41
111.3.1. I n t r o d u c t i o n . 41 111.3.2. Oxyradicals. 43 111.3.3. Hydroxycyclohexadienyl radicals and a l l o x a z i n y l
-oxy-cyclohexadienyl r a d i c a l s . 44
CHAPTER IV. ESR OF PTERIN AND LUMAZINE RADICALS. 48
I V . 1 . Introduction. 48 I V . 2 . Oxidation of 5-alkyl-tetrahydropteridines i n formic acid. 49
IV.3. Oxidation of 5,8-dialkyl-tetrahydrolumazines i n formic acid. 58 IV.4. Oxidation of 5-alkyl-tetrahydrolumazines in chloroform. 62 IV.5. Oxidation of 5,8-dialkyl-tetrahydrolumazines in chloroform. 67 IV.6. Conversion of neutral t r i h y d r o and monohydro radicals to
cationic tetrahydro and dihydro radicals by protonation. 71
IV.7. Temperature dependence of the ESR spectra. 71 IV.8. I n t e r p r e t a t i o n of the spectra of the p t e r i d i n e r a d i c a l s . 72
IV.9. References of chapter IV. 79
CHAPTER V. MATERIALS AND EXPERIMENTAL METHODS. 80
V . l . Materials. 80 V.2. Sample preparation and ESR measurements. 82
V.3. References of chapter V. 85
CHAPTER VI. CONCLUSIONS AND FINAL RESULTS. 86 V I . 1 . The i n t e r p r e t a t i o n of i s o t r o p i c ESR spectra in terms of
HFCC's and nuclear spins. 86 V I . 2 . Cationic f l a v i n and 10-alkylated alloxazine r a d i c a l s . 87
V I . 3 . Hydroxylations by l,3,10-trimethyl-5,10-dihydroalloxazine 3.4j
and oxygen or hydrogen peroxide. 87 V I . 4 . Cationic and neutral pteridine r a d i c a l s . 88
V I . 5 . References of chapter V I . 89
SUMMARY 90 SAMENVATTING 9 1
ABBREVIATIONS
DMF dimethylformamide
ENDOR electron nuclear double resonance ESR electron spin resonance
FAD flavin-adenine dinucleotide FMN flavin mononucleotide G gauss
HFCC hyperfine coupling constant TFA trifluoroacetic acid
CHAPTER I
GENERAL INTRODUCTION
I . l . The purpose of t h e i n v e s t i g a t i o n .
Flavins** and p t e r i d i n e s play an important r o l e in the l i v i n g c e l l as c o f a c t o r s of enzymes. Flavin c o f a c t o r s a r e found in hydrogenases,
dehydrogenases, h y d r o x y l a s e s , oxidases and e l e c t r o n - t r a n s f e r r i n g enzymes. Enzymes with a p t e r i d i n e c o f a c t o r a r e involved in the metabolism of one-carbon compounds, t h e hydroxylation of p h e n y l a l a n i n e and t y r o s i n e , the g l y c i n e - s e r i n e i n t e r c o n v e r s i o n and the s y n t h e s i s of purine and pyrimidine n u c l e o t i d e s which a r e e s s e n t i a l f a c t o r s in the formation of n u c l e i c a c i d s .
ESR and ENDOR experiments have been c a r r i e d out to answer the q u e s t i o n whether f l a v i n and p t e r i d i n e r a d i c a l s are formed during the enzymatic conversions mentioned above.
Flavin r a d i c a l s a r e e s s e n t i a l i n t e r m e d i a t e s in the r e a c t i o n of several dehydrogenases with t h e i r s u b s t r a t e s . They have a l s o been d e t e c t e d in
hydroxylases, but t h e i r role is not quite clear in this case.
Up t i l l now no free radicals have been found in enzymes with a pteridine cofactor. This means either that no radicals are formed or that their steady-state concentration is low.
ESR is also used for the elucidation of the chemical structures of co-factors. The only prerequisite for this method is that the cofactor can be reduced or oxidized to a free radical after being disconnected from the enzyme. It has been applied to the identification of flavo-coenzymes ' and
2) naturally occurring benzo- and naphthoquinones .
It is the main purpose of this investigation to study the relationship between the chemical structures of flavins, isoalloxazines and pteridines and the ESR spectra of their radicals, thus providing information that could be useful for future structure-elucidations of naturally occurring flavins and pteridines.
In addition a study was made of the radicals occurring in a model system 3 4) for hydroxylases that is based on l,3,10-trimethyl-5,10-dihydroalloxazine ' '
1.2. The redox properties of flavins, isoalloxazines and pteridines.
1.2.1. Flavins and isoalloxazines.
The isoalloxazine ring system (Fig. 1.1) constitutes the redox-active
Fig. 1.1
part of the cofactors r i b o f l a v i n 1.1a ( F i g . 1.2), f l a v i n mononucleotide 1.2a (FMN) and flavin-adenine dinucleotide 1.3a (FAD) ( F i g . 1.3).
H H H H^C-C—C—C-CHoOH
OH OH OH
Fig. 1.2 Riboflavin
Covalently bound flavin cofactors are also known. The flavin is bound to the amino acid chain of the enzyme through its 8a position. In succinate dehydrogenase, for instance, the 8a position of FAD is linked to the imidazole ring of histidine (Fig. 1.4). ESR played an important role in the determination of the structures of these cofactors .
H3C H3C H2C- 'N-H 'N-H 'N-H 0 0 I I I II II •C —C — C — C H 2 - G - P - O - P - O - C H 2 I I I OH OH OH N^O I OH OH
Y
1.3aFig. 1.3 Flavin-adenine dinucleotide
H H ? l ' N - C - C ' CH9
V"3
R N ^ \ ^ ^ - H Fig. 1.4The isoalloxazine structure requires a substituent at N,„ in order to be stable. In the absence of such a substituent the molecule has the alloxazine structure (Fig. 1.5).
The compounds obtained by one or two electron reduction of isoalloxazines are named as 10-substituted alloxazines because they no longer contain the C. -N(- and C.p, -N, double bonds of the isoalloxazine ring system.
Two electron reduction of isoalloxazines and flavins leads to 10-substituted -5,10-dihydroanoxazines and 1,5-dihydroflavins respectively (Fig. 1.6).
Fig. 1.6
This reduction i s thermodynamically r e v e r s i b l e , as can be demonstrated by polarography.
When an isoalloxazine or f l a v i n (Fl ) and i t s dihydro form (Fl J H „ ) are present in an aqueous solution together, they enter i n t o an equilibrium reaction that produces 10-substituted alloxazine or f l a v i n free radicals ( F I H . ) :
Fl + Fl ,H„ = 2 FIH. ox red 2
This mode of radical formation is referred to as comproportionation. The same radicals are formed by one electron oxidation of Fl ,H„ or one electron reduction of Fl
ox
The redox behaviour of isoalloxazines and flavins is complicated by the fact that each of the three redox states has acidic and/or basic properties. The various stages of protonation of the three redox states are shown in Fig. 1.7 together with the corresponding pK values. The three protonation states of the free radical are: the cationic dihydro radical F1H„. , the neutral monohydro radical FIH. and the anionic radical FIT .
Attention is drawn to the fact that the three types of radicals are in the same redox state, although different amounts of hydrogen (dihydro-, monohydro-) appear in the names of the radicals.
R H R R
0 pKa=±0 0 pKa=±10 o .
R H R ^ R ' ^ ^ ^ ^ ^ N ^ N Y ^ - H ^ . R V ^ ^ ^ ^ N ^ N Y N ^ O - H ^ ^ R ' Y ^ : ^ N Y N Y O f r e e
^!^ 0 pKa=i2.5 ^I^ 0_ pKa=i8.5 • 0_
F I H j t FIH- FIT
R H R H R
H H 0 pKa=i1 H 0 pKa=±7 H 0 .
F'redHs ^ke6»2 F l f e d H "
CATION NEUTRAL ANION
Fig. 1.7 Aaid-base behaviour of the three redox states of (iso)alloxazines and flavins.
1.2.2. Pteridines.
The naturally occurring pteridines are either pterins or lumazines (Fig. 1.8). H
pteridine
^ N ^ N H 2
0
pterin
Fig . 1.80
lumazine
HThus tetrahydrobiopterin i s a cofactor of phenylalanine hydroxylase and 6 , 7 - d i m e t h y l - 8 - r i b i t y l - l u m a z i n e ( F i g . 1.9) i s a precursor i n the biosynthesis of r i b o f l a v i n . OH OH H-I H-I H H-:>C-C—C-•^ I I H H H N-I H N ^ N H 2 -N. •H 0 H3C-HoC CH2OH H - C - O H H - C - O H H - C - O H CH2 • N \ ^ N \ ^ 0 •N^
'Y
•H 0tetrahydrobi opteri n 6,7-dimethyl-8-ribityl-lumazine
Fig. 1.9
K Pterin= 2-amino-lt(3H)-pteridinone. K3« Lumazine= 2 ,U( 1H,3H)-pteridinedione.
Chemical reduction of pterins and lumazines leads to the 7,8-dihydro- or 5,6,7,8-tetrahydro-derivatives (Fig 1 10) or mixtures of both depending on the structure of the parent compound and on the reducing agent.
H H .1. H - / N \ H - ^ N - ^ ^ N ^ N H 2
Kf"-"
0 H H 1 H - / N v . ^ N : ^ N H 2 H ^ N ^ / N ^ H H 0 7,8-dihydropterin 5,6,7,8-tetrahydropterin Fzg. 1.10The first diamagnetic product in the oxidation of tetrahydropterins or tetrahydrolumazines is an unstable quinoid dihydropterin or dihydrolumazine, which could be detected in a number of cases The quinoid intermediate shown
in Fig. 1.11 was detected during the oxidation of tetrahydrobiopterin by phenylalanine hydroxylase. OH OH H-I H-I H3C-C-C H H
H
H 1 . ^ N-v^^NH ' ^ Fig. 1.11Quinoid dihydroptenns and dihydro!umazines tautomenze rapidly to the 7,8-dihydro form. Further oxidation of the 7,8-dihydro form leads to f u l l y oxidized pterins and lumazines.
The chemical, polarographic and biochemical oxidations of tetrahydro-pterins are believed to proceed through p t e r i n free radicals ' ' .
oxidation of tetrahydropterins with hydrogen peroxide i n TFA ' ' . The 5,6-dihydropterin structure is found in 5-methyl-6,7-diphenyl-5,6-dihydropterin. Oxidation with hydrogen peroxide in TFA leads to a cationic
7 91
dihydropterin r a d i c a l , which was detected by ESR ' . The HFCC's of t h i s radical indicate that i t has a 5,8-dihydro structure ( F i g . 1.12) rather than a 5,6-dihydro s t r u c t u r e .
H
CH3 0
Fig. 1.12
NH2
Clearly, pterins and lumazines have two paramagnetic oxidation levels, whereas flavins and isoalloxazines have only one. Each of the two paramagnetic oxidation levels has acidic and basic properties.
7 91 Of the radicals obtained by one-electron oxidation of tetrahydropterins ' ' only the cationic form in TFA has been reported. Neutral and anionic forms of these radicals are unknown.
The protonation steps of the pterin and lumazine radicals obtained by one-electron reduction of the fully oxidized compounds in water were studied spectrophotometrically by Moorthy and Hayon ' ' '(Fig. 1.13 and 1.14). The radical structures are tentative because the precise tautomeric forms of the radicals are not known. The radicals are extremely unstable and react to diamagnetic products with a second order rate constant varying from SxlO'^ to 5.4x10^ M'-^s'^
H +H
pKa=2.3 pKa=6.6
Fig. 1.13
pKa=2.9 pKa=8.5 pKa=12.6
Fig. 1.14
1.3. References of chapter I.
1) J.Salach, W.H.Walker, T.P.Singer, A.Ehrenberg, P.Hemmerich, S.Ghisla and U.Hartmann, Europ.J.Biochem.,26, 267 (1972).
2) J.A.Pedersen, Abstracts of the Sixth International Symposium on Magnetic Resonance, Banff, Alberta, Canada, (F.H.A.Rummens and J.A.Weil Eds.),1977, p. 351.
3) H.I.X.Mager and W.Berends, Tetrahedron,30, 917 (1974).
4) H.I.X.Mager, Flavins and Flavoproteins, (Editor T.P.Singer), pp. 23-37, Elsevier Scientific Publ. Co., Amsterdam, Oxford, New York (1976). 5) M.C.Archer and K.G.Scrimgeour, Can.J.Biochem. ,48, 526 (1970).
6) D.J.Vonderschmitt and K.G.Scrimgeour, Biochem.Biophys.Res.Commun.,28, 302 (1967).
7) A.Ehrenberg, P.Hemmerich, F.Mueller, T.Okada and M.Viscontini, Helv.Chim. Acta, 50, 411 (1967).
8) A.Bobst, Helv.Chim.Acta, b\, 607 (1968).
9) A.Ehrenberg, P.Hemmerich, F.Mueller and W.Pfleiderer, Europ.J.Biochem., 26, 584 (1970).
10) P.Moorthy and E.Hayon, J.Org.Chem.,41, 1607 (1976). 11) P.Moorthy and E.Hayon, J.Phys.Chem.,79, 1059 (1975). 12) P.Moorthy and E.Hayon, Indian J.Chem.,MB, 206 (1976).
CHAPTER II
THE INTERPRETATION OF ISOTROPIC ESR SPECTRA IN TERMS OF HYPERFINE COUPLING CONSTANTS AND NUCLEAR SPINS.
II.1. Introduction.
In the present thesis, ESR spectra of organic free radicals in solution are interpreted using a spinhamiltonian with isotropic parameters (eq. 2.1)
n
X = gBHS, + ( l A ^ I „) S equation 2.1 ^ p=l P ^P ^
The symbols in equation 2.1 have the usual meaning:
g: the g-factor of the free radical. 6: the Bohr magneton.
S : the operator for the electron spin component in the direction (z) of the external magnetic field (H).
n: the number of nuclei in the radical molecule with a spin greater than zero.
A : the hyperfine coupling constant for nucleus p.
I : the operator for the spin of nucleus p in the direction (z) of the magnetic field (H).
Second or higher order effects are neglected. This is a reasonable
approximation provided the HFCC's (A,,Ap, ,A _i.A ) are small with respect to the Zeeman term.
Considering allowed ESR transitions only, the resonance fields H of the lines in the spectrum can be calculated using equation 2.2,
H, = H^ - gig i^Apmp equation 2.2
and H is the resonance f i e l d of the centre of the spectrum (H =(hv)/(gB) ). The HFCC A may be replaced by a (eq. 2 . 3 ) , which i s measured i n the same u n i t s as the magnetic f i e l d H.
. =i
equation 2.3Substituting a gB for A in equation 2.2 we obtain equation 2.4.
H^ = H^ - I a„m„ p=l
equation 2.4
The function F (H), representing the f i r s t - d e r i v a t i v e i s o t r o p i c ESR spectrum, may be calculated using equation 2 . 5 , in which F (H) is the f i r s t -d e r i v a t i v e lineshape function with i t s centre of symmetry at H .
^^^^'- A L — Z . ?^ ^o("-
?IVP^'^"''^•°" 2-^
m, m„ m 1 m n-1 n
Alternatively, the function F (H) may be calculated using n equations, each of which adds a HFCC to the spectrum (equations 2.6). The order in which
Fi(H)= /^ FjH-a^.mJ m, F,(H)= / Fi(H-a,.m, "2 m^
I
eq. 2.6.1 eq. 2.6.2 V l ( H ) V . V2(H-an-i-Vl) ^^- ^-^-n-l %-l equations 2.6the HFCC's are added to the spectrum does not influence the final result. According to equations 2.5 and 2.6 an isotropic ESR spectrum is completely defined by the lineshape function, the g-value, the number of nuclei
interacting with the free electron, the HFCC's and the nuclear spins. When trying to interpret a spectrum we are confronted with the reverse problem. The function value F (H) is known for any value of H, whereas the lineshape function, the number of nuclei, the HFCC's and the nuclear spins are unknown. For this problem there exists no straightforward algebraic solution.
Several methods for the interpretation of isotropic ESR spectra have been published. The scope and limitations of each method are discussed briefly in the following sections of this chapter.
II.2. Interpretation by visual inspection.
The analysis of the spectrum is started from one of the two outer lines, usually from the low-field outer line. The distance in magnetic field units between this line and the one closest to it is the smallest distinguishable HFCC (a,). The nucleus or nuclei having a HFCC equal to a, give rise to a pattern (P.) of equidistant lines in the spectrum which includes the outer line and the one closest to it.
The number of equidistant lines in P, and their intensity ratios depend on the number of nuclei involved and on their nuclear spins. In the case of one nucleus with spin I the equidistant lines have the same intensity and their number equals 21+1. Two equivalent protons (1=1) produce a pattern with intensity ratios 1:2:1, three protons give a 1:3:3:1 pattern and one proton plus one nitrogen atom (1=1) with the same HFCC give a 1:2:2:1 pattern. For any nucleus or combination of nuclei with the same HFCC the theoretical number of lines and their intensity ratios can be calculated.
When the pattern P., caused by the nuclei with HFCC a,, has been identified we search for the next line in the spectrum that is not part of P,. The
distance between this line and the outer line is the smallest but one HFCC (a„). The corresponding pattern P^ is examined and interpreted in the same way as P,.
The next line in the spectrum that does not belong to P,p, the cartesian product of P, and P„, gives the smallest but two HFCC (a,). The remaining
HFCC's are found in an analogous way.
If the largest HFCC (a ) is much larger than all the other HFCC's it is immediately apparent in the spectrum. In that case the spectrum consists of a number of symmetrical groups with equidistant symmetry centres. The number of nuclei having a HFCC equal to a and their spins can be determined from the intensity ratios between the groups and the number of groups.
The analysis of the spectrum is complete when spectrum simulation (section II.11) with the aid of equation 2.5 or 2.6 gives a theoretical spectrum reconstruction that is identical with the experimental spectrum.
The HFCC's that have been found must be in agreement with the width (W) of the experimental spectrum as measured between the low-field and high-field outer lines (eq. 2.7). In equation 2.7 each nucleus is taken separately, therefore the number of nuclei n is greater than or equal to the number of different HFCC's (m).
n
2 y a I = W equation 2.7 p=l P P
Visual analysis is the most widely used method for the interpretation of isotropic ESR spectra. It is obvious that it can only be applied to spectra that are well resolved.
Several mathematical procedures for the interpretation of spectra with strongly overlapping lines have been developed (sections II.3 to 11.11). The number of computations involved in such procedures is very great and requires the use of a digital computer.
11.3. The method of Newton et al. ' •'
This method provides an answer to the question whether an experimental ESR spectrum contains a ramdomly chosen HFCC (a ) belonging to a nucleus with a randomly chosen spin (I )• In order to answer this question the spectrum- represented by the function F (H)- is transformed using the values of a and I and the symmetry of the transformed spectrum is evaluated.
As an example we shall consider the case when I equals I. If the
assumption that the radical contains a nucleus with spin I and HFCC a is
^n'^'^ ^ - 1 ^ ^ ^ ^ - % ) + ^-l(^"^-^p) equation 2.8
According to equation 2.8 the experimental spectrum is built up from two symmetrical functions of identical shape, the symmetry centres of which are
situated at la upfield and downfield from the centre of symmetry of the
spectrum The unknown function F , is calculated from F using the
^ n-1 n transformation of equation 2.9 (Fig. 2.1).
Fn.i(H+iap)= FjH)-F^(H-ap)+F^(H-2ap)-F^(H-3ap)+ equation 2.9
The number of terms in equation 2.9 is infinitely large.
If the assumption that the radical producing the experimental spectrum
contains a nucleus with spin l and HFCC a is correct, the transformation
of equation 2.9 produces a function that is symmetrical relative to a point
CS situated at la downfield from the original symmetry centre of the
experimental spectrum (Fig. 2.1). Because the symmetry is evaluated in a limited interval (IN) around C S , the transformation requires only a limited number of terms of equation 2.9.
If the assumption is incorrect the function resulting from the
transformation will not be symmetrical relative to CS in most cases(Fig. 2.2) If F (H) represents a first-derivative ESR spectrum, the type of symmetry for which F _, should be tested is inversion symmetry. The deviation from symmetry (DS) is calculated using equation 2.10 in which H-,. and H. are the magnetic fields corresponding to the point CS and to the low-field limit of IN respectively.
•^^^CS'^A
/ ( ^-l^^CS+^-^p^'^^ + ^ - l ^ ^ C S + ^ - V ^ ^ ^^•''^ equation 2.10 DS =
R=0
DS i s calculated f o r a large number of values of a and displayed graphically as a function of a . The x-coordinates of the minima in t h i s curve represent potential HFCC's for nuclei with spin I.
Similar curves are calculated f o r spin values other than I, taking i n t o account that the form of equation 2.9 and the p o s i t i o n of CS depend on the spin of the nucleus.
experimental spectrum
first three terms of equation 2.9
CS H„ °p/2
"TiT
sum of the f i r s t three terms of equation 2.9
Fig. 2.1. Transformation for correct HFCC. A vertical line represents a first-derivative ESR line.
experimental spectrum
f i r s t three terms of equation 2.9
CS Hj < ^ Op/2 IN
sum of the first three terms of equation 2.9
Fig. 2.2. Transformation for incorrect HFCC. A vertical line represents a first-derivative ESR line.
The effect of the second and following terms of equation 2.9 can be described as a shift to higher field of the high-field component ^ j ^ . A ^ ' i ^ n ) °'^ the
spectrum F (H). The high-field component is shifted to higher field so far that it does no longer have a measurable intensity in the interval IN. Then the symmetry test of the low-field component can be carried out without interference from the high-field component.
Since the function F contains experimental errors, the final errors in F , increase with the number of terms of equation 2.9 that are needed to effect the required shift. This means that the errors in F , increase with decreasing
^ n-1 ^ a . Values of a smaller than the top-top linewidth of the experimental
spectrum- i.e. unresolved HFCC's- cannot be determined with sufficient accuracy.
The method of Newton et. al. yields all the HFCC's that are actually present in the experimental spectrum. In addition several HFCC's are found that are not present in the spectrum. As an example we consider the spin J case. If a is a HFCC that is present in the experimental spectrum, the method of Newton et. al. provides not only a =a as a solution but also a =a / 3 , a =a / 5 ,
•^ p q p q ' ' p q' '
a =a /7 etcetera. p q
3 41 I I . 4 . Analysis of the sine Fourier transform ' '.
The sine Fourier transform M(a) of a f i r s t - d e r i v a t i v e ESR spectrum F (H) is defined as:
M(a)= j F^(H).sin(2TTa(H-Ho))dH equation 2.11
If a is a HFCC of the experimental spectrum belonging to a nucleus of spin I , the sine Fourier transform is zero at values aj given by equation 2.12.
a..
k.
^ equation 2.12 •^ (21 +l).a ^ q ' q
k. is any positive integer that is not a multiple of 21 +1.
The lowest value of a at which the transform is zero (a . ) is found for ^ min'
determined from the transform and a can thus be calculated for each qmax
possible value of I . The a values are useful as upper limits in any
'^ q qmax ^'^
search for HFCC's.
Since we want to compare the results of the Fourier transform method to those of other methods, a function FF(a) is required which has the property that the x-coordinates of i t s minima are potential HFCC's.
r , / k.
FF(a) = I M J j = ll M 2 I + l ) . a
equation 2.13
For each value of I a different function FF(a) is calculated. The meaning of the symbols I, a, k. and M has been discussed previously. Theoretically the number r (eq. 2.13) should be infinitely large but in practice only that part of the sine Fourier transform which differs significantly from zero is considered. This means that the Fourier transform is taken to be zero for values of a larger than a value a . The value r is the highest value of j ^ max for which a. (eq.2.12) is less than a .
II.5. The method of Balaban et. al. 5,6)
This method will not be discussed in detail because in our opinion the assumptions on which it is based are incorrect.
The method involves a stick-diagram representation of the first-derivative ESR spectrum. For spectra with strongly overlapping lines stick-diagrams are at best a crude approximation.
In addition the assumption is made that all HFCC's in the experimental spectrum are exact multiples of the smallest resolved HFCC. There exists no theoretical or experimental basis for this assumption.
II.6. Autocorrelation^'^'^'^°^
The autocorrelation function A(a) of an ESR spectrum F (H) is defined as:
It was not recognized until recently that the autocorrelation function is identical with the high-field half of a second-derivative ESR spectrum (SD) of a hypothetical radical containing twice the number of nuclei of the actual radical with spectrum F (H) and having the same HFCC's .
The spectrum SD contains all HFCC's in even numbers and therefore has a centre line. In the autocorrelation the centre line is situated at a=0. Consequently all HFCC's are measured as distances between the centre line and other lines of the autocorrelation. This is correct for well resolved spectra giving a well resolved autocorrelation function. In the case of spectra with strongly overlapping lines each of the observed lines in the central portion of the spectrum is an envelope of a number of actual resonance lines which do not coincide exactly. It is clear that distances between such enveloping lines are not related to HFCC's in the simple way in which the distances between actual resonance lines are related to HFCC's. Since an autocorrelation function with strongly overlapping lines is in fact a second derivative ESR spectrum with strongly overlapping lines, the distances between the centre line and other lines are not necessarily equal to the HFCC's.
II.7. Crosscorrelation ' '.
The crosscorrelation function C(a) is defined as:
C(a) = j F^(H).Q(H,a) dH equation 2.15
0
Q(H,a) is the testfunction of the crosscorrelation. A theoretical first-derivative ESR spectrum with a variable HFCC (a) and the estimated lineshape of the experimental spectrum is used for this purpose.
The testfunction must have the same multiplicity (M) as the HFCC for which the search is being carried out.
M = 2.n.I+l equation 2.16
For example, when we are looking for a HFCC of three equivalent protons,
n equals 3 and I equals I. According to equation 2.16 M equals 4. The
1:3:3:1, as obtained when three equivalent nuclei of spin I interact with a free electron.
The method fails when more than one group of nuclei in the radical has an even multiplicity. The simplest case is a spectrum with two non-equivalent nuclei of spin I (Fig. 2.3).
-> experimental spectrum testfunction for 3 = 32 testfunction f o r a=a,
Fig. 2.3. Complete failure of the crosscorrelation method. No maxima are found for a=a^ or a=a„. Maxima are found for a=a^-a^ and a=a^+a^. A vertical line represents a first-derivative ESR line.
When the spectrum under i n v e s t i g a t i o n has strongly overlapping resonance l i n e s , the lines that can be observed i n the spectrum are envelopes of the actual resonance lines (section I I . 6 ) . The function C(a) reaches maxima when
a (eq. 2.15) is equal to the distances between such enveloping lines. The HFCC's, which are found as the x-coordinates of the maxima in the
crosscorrelation function, are not necessarily correct in that case.
121 11.8. Transformation to the cepstrum .
The cepstrum CP(H) of a spectrum F (H) is defined as:
CP(H)= r^ I In /iF^(H) equation 2.17
The symbol f denotes the complex Fourier transformation and is defined for a function Z(x) as:
/(a,) =Jz(x).e-i"^ dx equation 2.18
A HFCC (a) gives rise to a maximum in CP(H) at H=a.
The method is very sensitive to experimental errors in F (H) ', which
lead to a high level of noise in CP(H).
II.9. Isotope substitution.
In most organic radicals there is interaction between hydrogen ( H) nuclei and the free electron. Substitution of H by deuterium leads to a different ESR spectrum. The deuteron has a nuclear spin equal to 1 and a
1 1
different HFCC than H. The HFCC's of H and deuterium (D) in the same position in the same radical are related according to equation 2.19.
a^ = 0.1535.a^ equation 2.19
The HFCC 3^, can be C3lculated from the difference between the total width 1
(Wn) of the spectrum of the H containing radic3l (HR) and the total width (Wp|) of the spectrum of the D containing radical (DR). From equations 2.7 and 2.19 it follows that:
a^ = 1.443.(W^-Wp) equation 2.20
However, equation 2.20 is only valid when both HFCC's an and a^ are resolved (i.e. each one is greater than the top-top linewidth of the spectrum in which it appears). If both HFCC's are unresolved the two spectra are equally wide and the HFCC's can not be calculated. If an is resolved and a^, is not, equation 2.21 should be used instead of 2.20.
a^= W^-W„ equation 2.21
An alternative approach involves the interpretation of the spectrum HR followed by simulation of HR and DR. If both simulations agree with the corresponding experimental spectra the HFCC of the substituted nucleus is thereby confirmed.
Radicals that are deuterated in specific positions are prepared in two ways. Firstly, deuterated reagents may be used at certain stages of the synthesis of the parent compound. Secondly, the radic3l generstion from the parent compound may be effected in a solvent in which all exchangesble protons 3re repl3ced by deuterium, thus le3ding to replacement of the exchangeable protons of the radical.
14 15 In nitrogen containing radicals N (1=1) can be replaced by N (I=i)
15
through the use of N labelled reagents in the synthesis of the parent compound. Theoretically all nitrogen and hydrogen HFCC's can be determined by selective isotope substitution of each of the nuclei that have interaction with the free electron. In practice the applicability of the method is limited by the synthetic difficulties encountered in the introduction of isotopes at specific positions.
11.10. Miscellaneous methods.
Least-squares optimization using the steepest descent method and other
methods has been suggested by a number of authors ' ' ' '.
151 The required initial V3lues of the HFCC's must be accurate to AH/2 ' (AH is the top-top linewidth of the experimental spectrum). Consequently, those values must be obtained by other methods before least-squ3res
optimization is carried out.
ENDOR (Electron Nuclear Double Resonance) of free radicals in liquid samples has the advantage that the HFCC's are immediately apparent in the spectrum.
The sensitivity is at least a factor ten less than for ESR '.
Resolution enhancement ' ' is useful for spectra with a very high signal
to noise ratio.
Quantummechanical calculations of HFCC's require knowledge of the geometry of the free radical.
The ratio between the total intensity (S) of an isotropic ESR spectrum and the intensity (L) of one of the two outer lines is a function of the number of nuclei (n-) with resolved HFCC's and their nuclear spins (I-).
' "i
S/L = n (2I-+1) equation 2.22 i = l
p is the number of groups of nuclei with different spins. S and L are
calculated by double numerical integration of the first-derivative ESR spectrum and of the outer line. If there is only one type of nuclei (p=l), their number (n,) is the only unknown quantity in equation 2.22 and can therefore be
calculated. If p ^ 2 there is more than one solution. Since the double integrals S and L cannot be calculated with very great accuracy, this method does not always provide the correct number of nuclei.
Finally, conclusions can be drawn from the absence of a central line in an ESR spectrum. If such a line is absent there is at least one group of
equivalent nuclei in the radical that consists of an odd number of nuclei with non-integer spin ( 1= 1/2,3/2,5/2,7/2 or 9/2).
11.11. Simulation.
Spectrum simulation is the final step in any spectrum interpretation process. It can be performed in three different ways.
In the first place the value of the function F (H) representing the simulated spectrum can be calculated for any value of H using equation 2.5. When the function F (H) has been calculated for a sufficiently large number of different H-values it is represented graphically on the same scale as the experimental spectrum. The H-coordinates for which F (H) is calculated are usually equidistant but this is not necessary.
The second method is also based on equation 2.5. The function F (H) is calculated for a sufficiently large number of equidistant H-coordinates and stored in a memory block FO(I). A memory block FN(I) in which the function values of F (H) are to be stored is set to zero. The subscript values I represent equidistant H-coordinates. The H-coordinate of the symmetry centre of each of the lines that make up the spectrum is calculated and rounded to the nearest corresponding value of I. The values of FO(I) are added to the values of FN(I) in the appropriste storage locations around the the symmetry centre of each line. When this has been done for all the lines that make up the spectrum, F (H) is represented graphically.
n 201
The third simulation method ' uses equations 2.6. Two memory blocks A(I) and B(I) are employed. The function F (H) is calculated for a number of equidistant H-coordinates and stored in A(I). F,(H) is then calculated from F (H) and stored in B(I). A(I) is set to zero and F2(H) is calculated from F,(H) and stored in A(I). This process is repeated until F (H) is obtained in one of the two memory blocks.
11.12. References of chapter II.
1) R.Newton, K.F.Schulz and R.M.Elofson, Can.J.Chem. ,44, 752 (1966). 2) Y.S.Lebedev and S.N.Dobryakov, J.Struct.Chem.(USSR), 8, 757 (1967). 3) S.N.Dobryakov, J.Struct.Chem.(USSR), 6, 30 (1965).
4) R.H.Silsbee, J.Chem.Phys.,45, 1710 (1966).
5) A.T.Balaban, Rev.Roum.Chim., ]J_, 9 (1972).
6) C.Pomponiu and A.T.Balaban, Rev.Roum.Chim., ^ S , 1173 (1973). 7) K.D.Bieber and T.E.Gough, J.Magn.Reson., 2J., 285 (1976).
8) E.Ziegler and E.G.Hoffmann, Fresenius' Z.Anal.Chem., 240, 145 (1968). 9) L.C.Allen, Nature(London), 1962, 663.
10) J.D.Swalen and H.M.Gladney, IBM J.Res.Develop., 8, 515 (1964). 11) A.H.Huizer, J.Magn.Reson., to be published.
12) D.W.Kirmse, J.Magn.Reson., U, 1 (1973).
13) D.W.Marquardt, R.G.Bennett and E.J.Burrell, J.Mol .Spectrosc., _7, 269 (1961). 14) L.Newman, Comput.Chem.Instrum., 1973, 3.
16) A.Bauder and R.J.Myers, J.Mol.Spectrosc., 27, 110 (1968). 17) M.Plato and K.Mobius, Messtechnik, 80, 224 (1972).
18) L.C.Allen, H.M.Gladney and S.H.Glarum, J.Chem.Phys., 40, 3135 (1964) 19) A.Hedberg and A.Ehrenberg, J.Chem.Phys., 48, 4822 (1968).
CHAPTER III
ESR OF FLAVIN AND 10-ALKYLATED ALLOXAZINE RADICALS
III.l. Introduction. 1 21
Ehrenberg ' ' was the first to obtain ESR spectra of flavin radicals with hyperfine structure. The cationic, neutral and anionic radicals of FMN (1.2a) and FAD (1.3a) were investigated in water at various pH's. The spectra are poorly resolved and cannot be interpreted.
Better resolution was achieved using flavins like lumiflavin, which have a methyl group at N,^ instead of the long side-chains of FMN and FAD. The ESR spectrum in water at pH 12 of the anion radical 3.1a (Fig. 3.1) of
3 51
lumiflavin could be interpreted ' '. HFCC's were found for N,-, N.^,, the methyl group on N.^,, one proton (H,) and the methyl group on C^ (table 3.1).
3.1a
Fig. 3.1
Table 3.1 HFCC's (in gauss) of the anionic lumiflavin radical and the neutral 3-alkylated lumiflavin radical.
N,(H) N^Q N^Q(r-H) Cg(l'-H) Hg Reference anion neutral 7.0 8.0 -7.6 3.5 3.6 3.5 3.9 3.5 2.4 3.5 1.7 3 7
The anion radical of 3-alkylated lumiflavin in DMF has the same HFCC's as lumiflavin in water '. The linewidth is smaller in DMF and an additional HFCC of a single proton is visible (0.9 G ) . The assignment of the HFCC's to
specific positions in the radical molecule was made possible by the use of derivatives of lumiflavin and by isotope substitution.
15
Selective substitution with N in positions 1,3,5 and 10 revealed that Nr has the higher of the two nitrogen HFCC's and N-^Q has the lower one. The single proton HFCC of 0.9 gauss must be assigned to Hg as shown by selective
deuteration '. The single proton HFCC of 3.5 gauss must therefore originate
from H,. Selective substitution of the Cj and C Q methyl groups by chlorine
atoms proves that the HFCC of the Cj methyl protons is unresolved and the
one of the Cg methyl protons equals approximately 4.0 gauss. The ENDOR spectrum of the lumiflavin anion radical in a frozen solution shows peaks corresponding to the Nig and Cg methyl protons .
The neutral monohydro radical (3.2a)(Fig. 3.2) of 3-alkylated lumiflavin was obtained in a buffered aqueous solution at pH 7.5 '.
H 3 H ^^^3
H H 0. H (1^3 0.
Fig. 3.2
The neutral radicals of 3,5-dialkyl-flavins (3.3a)(Fig. 3.2) and iso-alloxazines were generated in chloroform. The ESR spectra are well resolved and suitable for interpretation (Table 3.1). The assignment of HFCC's to the corresponding positions of the radical molecules was carried out using
2 15
derivatives substituted with H(D), N or CI in the same way as for the anionic radical 3.1a.
The fact that Nr has the highest HFCC and carries a proton with an equally high HFCC indicates that the zwitterionic mesomeric structures of Fig. 3.2 make a large contribution to the electronic structure of the neutral radical. The ENDOR spectrum of a frozen solution of 3.3a shows peaks for the methyl
Q \
by ESR.
91
Crespi et. al. •' isolated FMN from algae grown on heavy water, in which all hydrogen atoms are replaced by deuterium. The neutral radical of perdeuterated FMN was generated in neutral aqueous solution. Its ESR spectrum is moderately well resolved unlike the one of the neutral FMN radical. The HFCC's of N5, Nr(H) and N,Q are of the same magnitude as in the neutral lumiflavin radical (3.2a).
In view of the r e l a t i v e ease with which the ESR spectra of the anionic and neutral radicals could be i n t e r p r e t e d , i t is surprising that the spectra of the cationic f l a v i n and 10-alkylated alloxazine radicals could not be interpreted c o r r e c t l y . Two attempts to i n t e r p r e t these spectra have been published ' ' . Ehrenberg ' showed that the c a t i o n i c radicals of l u m i f l a v i n
15
and l u m i f l a v i n - 1 , 3 - N, have identical ESR spectra. This means that T o l l i n ' s 101
i n t e r p r e t a t i o n , involving a high HFCC f o r N,, cannot be correct. H I
Mueller et. a l . ' assigned the following HFCC's to a number of c a t i o n i c isoalloxazine r a d i c a l s : Ng:8.5, Hg:11.5, Hg:3.4, N ^ Q : 4 . 3 and Njj^(l'-H) :4.7 (gauss). We tested these values with a computer program f o r the simulation
131
of ESR spectra ' . The simulated spectra bear no resemblance to the published 141 experimental spectra of the cationic r a d i c a l s . Simulation of the spectrum ' of the 3-alkylated l u m i f l a v i n cation radical was equally unsuccesful. We therefore regard Mueller's i n t e r p r e t a t i o n as erroneous.
I I I . 2 . Interpretation of the ESR spectra of cationic f l a v i n and 10-alkylated alloxazine r a d i c a l s .
The cationic radicals of the following compounds were prepared i n 6N HCl by half-reduction with sodium d i t h i o n i t e : 1,3,10-trimethylalloxazinium-perchlorate 3.4a, l,3-dimethyl-10-methyl-D.,-alloxazinium1,3,10-trimethylalloxazinium-perchlorate 3.4d, 1,3,10-trimethyl-Dg-alloxaziniumperchlorate 3.4g, 10-methylisoalloxazine 3.5a and l u m i f l a v i n 3.6a ( F i g . 3 . 3 ) . Each radical was also prepared in 6N DCl, thus leading to the analogues deuterated i n position 5. The spectra were
recorded at 4, 22, 45 and 90°C. The spectra were independent of the temperature except f o r a s l i g h t line-broadening e f f e c t at 90°. The formation of c a t i o n i c radicals from 3.5a and 3.6a requires the addition of one electron and two protons to the parent compound. Since 3.4a, 3.4d and 3.4g are cations, they require one electron and only one proton.
Figure 3.3. Formation of cationic flavin and 10-alkylated alloxazine radicals by one-electron reduction in 6N HCl and 611 DCl.
CH3 CH3 CD3CH3 ,-<:v^Nv^N«.(.0 ^ ^ ^ ^ . H 3 ^ 0 ^ CD3 CD3 - N ^v- N S4O ,<:>v-N>,-NsfrO 9H3 0 e H * -> e D^ •> e H-* -^ e D' e H * •> -^ e D+ e H * -> CH3 CH3 H 0 CH3 CH3 D 0 CD3 CH3
(6t-Xt„3-H 0 CD3 CH3 D 0 CD3 CD3 H 0 CD3 CD3
@ ; ; 0 : 3
-D 0 CH3H H 0 3.5b CH3Z^Xt. ^
e 0-^ 9H3P e H-^ D 0 CH3H H 0 e D CH3D D 0Table 3.2. HFCC's(in gauss) of the cationic flavin and 10-alkylated alloxazine radicals in 6N HCl and 6N DCl. 3.4b 3.4c 3.4e 3.4f 3.4h 3.4i 3.5b 3.5c 3.6b ^5 7.15 7.15 7.15 7.15 7.15 7.15 7.39 7.39 7.27 ^10 5.20 5.20 5.20 5.20 5.20 5.20 4.85 4.85 4.66 N^W 7.55 7.55 7.55 7.75 7.68 N5(D) 1.16 1.16 1.16 1.19
ho
( I ' - H ) 4.61 4.61 4.92 4.92 4.90 ^ 0 ( I ' - D ) 0.71 0.71 0.71 0.71 ^8 2.66 2.66 2.66 2.66 2.66 2.66 2.91 2.91 ( ^8 ( I ' - H ) 3.40 ^^6 1.60 1.60 1.60 1.60 1.60 1.60 1.67 1.67 1.62I «
10 G
Figure 3.4. ESR spectrum of 3.4b in 6N HCl (top) and simulation (bottom).
Figures 3.4 to 3.11 show the ESR spectra of the radicals and their
simulations. The spectra of 3.4e and 3.4h and those of 3.4f and 3.4i(Fig.3.3)are identical, as expected in case of a low spin density on N^, N and the protons of their methyl groups. For the same reason there is a strong resemblance
Figure 3.5. ESR spectrum of 3.4o in 6N DCl (top) and simulation (bottom).
The comparison of total widths (section II.9) provides an estimated value for the HFCC of the exchangeable proton on N5. The spectrum of 3.4b is 5.0 G wider than the one of 3.4c (Fig. 3.5). The same difference in total widths exists between the spectra of 3.5b and 3.5c (Fig. 3.9). Using equation 2.20
(section II.9) HFCC's of 7.22 G for Ng(H) in 3 ^ and 1.11 G for Ng(D) in 3.4c are found.
Detailed examination of the shape of the outer lines of the spectrum of 3.4b reveals the presence of a HFCC (1.6 G) of approximately the same magnitude as the top-top linewidth (1.52 G ) . The shape of the low-field outer line and the line closest to it cannot be simulated when the assumption is made that the distance between those lines (2.13 G) represents the smallest resolved
Figure 3.6. ESR spectrum of 3.4e in 6N HCl (top) and simulation (bottom).
HFCC. Two HFCC's of 1.60 and 2.66 G are required to simulate the outer lines correctly.
The ENDOR spectrum of frozen solutions of 3.6b has peaks corresponding to the methyl groups on Cg and N^o" Their HFCC's are 3.4 and 4.8 G respectively 6,15)_
3) 71 Ehrenberg et.al. interpreted the spectra of anionic ' and neutral ' flavin and alloxazine radicals using comparison of total widths of spectra with
different isotopic substitution (section II.9) and spectrum simulation (section 11.11). Apparently this combination of methods failed in the case of the cationic radicals '.
At the earlier stages of our attempts to interpret the spectra of the cationic radicals we used the same methods and were equally unsuccessful. Clearly a different approach was called for.
The most promising of the existing interpretation methods (chapter II) are: 1. Determination of the upper limits of the HFCC's for I=J and 1=1 (section
Figure 3.7. ESR spectrum of 3.4f in 6N DCl (top) and simulation (bottom).
2. Analysis of the sine Fourier transform (section II.4). 3. The method of Newton et.al.(section II.3).
4. Autocorrelation (section II.6). 5. Crosscorrelation (section II.7).
6. Transformation to the cepstrum (section II.8)
All six methods were applied to the spectra of 3.4b (Figures 3.12 to 3.16 and table 3.3), 3.5b (table 3.4) and 3.6b (table 3.5) and the HFCC's found by each method were tabulated.
The curves obtained by the method of Newton (Fig.3.14) and Fourier analysis (Fig. 3.13) are very similar. Small differences between the x-coordinates of the minima result in slightly different HFCC's (table 3.3).
In order to be able to proceed with the interpretation process the method giving the most reliable HFCC's must be selected. The method of Newton was preferred, a choice which will be justified below.
CH3 N •+I N 1 H H 1
-"v
1 -~r^ II 0Figure 3.8. ESR spectrum of 3. 5b in 6tl HCl (top) and simulation (bottomj.
As the method of Newton produces both physically relevant and unrelevant HFCC's , a criterion for the selection of the correct HFCC's is required. Therefore it was assumed that the HFCC's of the cationic radicals are of the same order of magnitude as those of the neutral and anionic radicals. This
Figure 3.9. ESR spectrum of 3.5a in 6N DCl (top) and simulation (bottom).
assumption is based on the striking similarity between the HFCC's of the neutral and anionic radicals of lumiflavin (table 3.1). The HFCC values which were selected are underlined in tables 3.3, 3.4 and 3.5.
Figure 3.10. ESR spectrum of 3.6b in 6N HCl (top) and simulation (bottom).
Table 3.3. HFCC's found f o r 3.4b by various d i f f e r e n t methods.
method upper limits Fourier analysis method of Newton crosscorrelation autocorrelation cepstrum
final result after squares optimizati least-3n spin 1/2 1 1/2 1 1/2 1 1/2 1 1/2 1 9.6 6.4 1.5 1.7 1.4 1.8 1.5 1.7 1.3 1.8 2.5 7.4 1.2 3.7 2.6 5.0 2.6 4.5 1.60 2.f 5.20 7. 2.1 2.5 2.1 2.5 6.1 7.4 4.9 56 4 15 HFCC's(G) 2.5 2.7 4.0 4.6 4.8 5.0 7.4 9.9 3.6 5.0 7.1 2.5 2.7 4.1 4.7 4.8 7.5 10.0 3.6 5.0 7.2 8.5 9.9 5.1 7.3 7.5 61 7.55
Figure 3.11. ESR spectrum of 3.6a in 6N DCl (top) and simulation (bottom).
Table 3.4. HFCC's found for 3.5b by various different methods. method upper limits Fourier analysis method of Newton crosscorrelation autocorrelation cepstrum
final result after squares optimizati least-on spin 1/2 1 1/2 1 1/2 1 1/2 1 1/2 1 9.6 6.4 1.4 1.7 1.5 1.9 1.4 1.7 1.2 1.9 2.5 7.4 1.2 3.7 2.5 5.0 2.6 4.7 1.67 2. 4.85 7. 2.2 2.3 2.2 2.4 6.2 7.5 5.0
n 4
39 HFCC's(G) 2.8 3.4 4.2 4.5 5.0 5.5 7.1 7.6 9.f 3.0 3.7 4.8 7.3 2.6 2.8 3.3 4.2 5.0 5.5 7.1 7.6 9./ 3.0 3.7 4.8 7.4 9.9 7.5 7.7 7.9 92 7.75A 0—'
JL
^s/s/vTL-,^
— 1 — 0.5 — I 1 — 1 1.5 K (gauss"^) — 1 2.5Figure 3.12. Sine Fourier Transform (section II. 4, equation 2.11) of the ESR spectrum of 3.4b. (absolute value).
Table 3.5. HFCC's method upper limits Fourier analysis method of Newton crosscorrelation autocorrelation cepstrum
final result after
found for 3. • least-squares optimization spin 1/2 1 1/2 1 1/2 1 1/2 1 1/2 1 6b by 9.6 6.4 1.5 1.9 1.4 1.9 7.7 3.8 3.9 1.6 1.62 4.66 various 2.2 2.2 1.6 2.3 7.8 7.8 3.3 3.^ 7.< 2.6 2.9 2.2 2.9 3.6 10 4 17 different methods. HFCC's (G) 3.2 3.5 4.2 4.7 5.0 3.7 3.9 4.5 5.8 7.3 2.6 2.8 3.3 3.6 4.4 3.7 4.0 4.6 5.8 7.3 4.6 4.9 5.1 7.4 7.6 90 7.68 6.6 7.7 8 4.7 5.0 6 7.8 8.1 9 2 6 7.7 8
1 = 1/2 — I r-5 10 a (gauss) — I 15 1 = 1 1 5 a (gauss) 10 15
Figure 3.13. Analysis of the Sine Fourier Transform of the ESR spectrum of 3.4b. (section II. 4, equation 2.13).
Q 1=1/2
J
I 1 1 1 0 5 10 15 a (gauss) > I 1 1 1 0 5 10 15 a (gauss) >Figure 3.15. Cross'-orrelation functions(section II. 7) of the ESR spectrum of 3.4b,calculated with the spectrum of one nucleus as testfunction.
< 1 1 1 5 10 15 a (gauss) > Q-o 0 H (gauss) 10 15 ->
Figure 3.16. Left:Autocorrelation function(section II.6) of the ESR spectrum of 3.4b. Right:Cepstrum(section II.8) of the ESR spectrum of 3.4b.
Using the selected HFCC's as an initial guess, a least-squares optimization (section 11.10) was carried out by the method of Powell '. The HFCC's which gave the best fit are not greatly different from the initial guess (tables 3.2
, 3.3, 3.4 and 3.5). The spectra of 3.4b, 3.5b and 3.6b were simulated using the optimized HFCC's and top-top linewidths of 1.52, 1.44 and 1.72 gauss respectively.
Finally the HFCC's (table 3.2) of the deuterium atoms in the deuterated radicals were calculated (equation 2.19, section II.9) and the spectra of the deuterated radicals 3.4c (Fig. 3.5), 3.4e (Fig. 3.6), 3.4f (Fig.3.7), 3.4h, 3.4i, 3.5c (Fig.3.9) and 3.6c (Fig. 3.11) were simulated successfully, proving
that the HFCC's of N ^ Q ( I ' - H ) in 3.4b and Ng(H) in 3.4b, 3.5b and 3.6b are
correct.
Comparison of the final interpretations of 3.4b, 3.5b and 3.6b with the results obtained by autocorrelation, crosscorrelation and cepstrum
transformation shows that those methods do not give all the correct HFCC's and are therefore unsuitable as sources for the initial guess required by least-squares optimization. Fourier analysis should give equally good results as the method of Newton et.al..
III.3. Free radicals in hydroxylating systems containing 1,3,10-trimethyl-5,10-di hydroal1oxazi ne.
III.3.1. Introduction.
The hydroxylation of aromatic compounds in aqueous acid by model systems consisting of l,3,10-trimethyl-5,10-dihydroalloxazine 3.4j (Fig. 3.17) and
CH3 C H 3
H 0 -^
(A'^H)
oxygen or hydrogen peroxide has been studied by Mager. The stoichiometry of the hydroxylation reaction ' ' proves that a free radical mechanism is operative.
Indirect evidence for a free radical mechanism was obtained by Lindsay 19)
Smith et. al. . Oxene hydroxylations, which proceed through a non-radical mechanism, are accompanied by a characteristic proton shift in the aromatic substrate. This is commonly referred to as the NIH-shift. Lindsay Smith et. al found that the NIH shift does not occur in the hydroxylation of phenylalanine by 3.4j and oxygen, thereby excluding an oxene mechanism.
The free radical intermediates ' ' involved in hydroxylations by 3.4j and oxygen or hydrogen peroxide are shown in Figure 3.18. Detection of these
radicals would provide valuable confirmation of the proposed mechanism ' '.
M e A M e I 0 I
1 (A^Lo.)
N^^^'^-Me
3.7a 3.8a Me I N I H 3.4b M e -N\.^,0T
(A^i-Ht)
-Me
3.9aHO'
Fig. 3.1iThe literature dealing with ESR detection of radicals similar to the ones of Figure 3.18 will be summarized here briefly.
Hydroxy1 radicals have not been detected by ESR in solution at room temperature.
The free electron in the alloxazinyl-oxyradical 3.7a is localized on an oxygen atom attached to a tertiairy carbon atom. Therefore 3.7a should be
compared with alkoxy radicals and not with the well known aryloxy r a d i c a l s . Relatively few ESR studies on alkoxy radicals have been carried out. ESR detection of oxyradicals derived from alloxazines or f l a v i n s has not been
201
reported. Weiner and Hammond ' found a g-value of 2.004 and a top-top l i n e w i d t h of 12 G f o r the ESR spectrum of the t b u t y l o x y r a d i c a l in d i t -butylperoxide at room temperature. The signal has no hyperfine s t r u c t u r e .
Addition of the hydroxyl radical to the aromatic substrate (R'-0) gives the hydroxy-cyclohexadienyl radical 3.9a ( F i g . 3.18). ESR spectra of
t r a n s i e n t hydroxy-cyclohexadienyl radicals have been reported by a number of 21-271
authors . The spectra are centered around g=2.003 and are well resolved. Addition of the alloxazinyl-oxyradical 3.7a to the aromatic substrate y i e l d s the a l l oxazinyl-oxy-cyclohexadienyl radical 3.8a ( F i g . 3.18). Alloxazinyl-oxy-cyclohexadienyl radicals or flavinyl-oxy-cyclohexadienyl r a d i c a l s have not been studied by ESR previously. The k i n e t i c s t a b i l i t y of t h i s type of radicals is unknown. The spectroscopic properties are expected to be the same as those of hydroxy-cyclohexadienyl r a d i c a l s .
Unlike the other radicals of Figure 3.18, radical 3.4b i s very stable i n water a t low pH. The ESR spectrum of 3.4b has been presented in section
I I I . 2 . Radical 3.4b is formed continuously in the hydroxylating system by one electron oxidation of 3.4j and by a comproportionation reaction of 3.4j with the 1,3,10-trimethyl-alloxazinium i o n , which is the f i n a l oxidation product resulting from 3 . 4 j . Because a l l organic radicals have t h e i r ESR absorption in the same spectral region, i t is obvious that the spectrum of 3.4b w i l l i n t e r f e r e strongly with the detection of other r a d i c a l s .
111.3.2. Oxyradicals.
In the hydroxylating system (H / H , 0 / 3 ^ 4 j / 0 , or H,O„/R'0) the a l l o x a z i n y l -R 181 oxyradical 3.7a i s formed from the intermediate peroxide A -OOH ' . The presence of the aromatic substrate R'-0 i s not required f o r the formation of 3.7a. In the absence of the substrate the radicals 3.8a and 3.9a ( F i g . 3.18), which would i n t e r f e r e with the detection of the oxyradical 3.7a, cannot be
formed. Therefore we decided to study 3.7a i n the absence of substrate. This approach has the additional advantage that other solvents than water may be used. Whereas the hydroxylation of aromatic substrates by 3.4j/0p or
be formed upon oxidation of 3.4j, regardless of the nature of the solvent. A solution of 3.4j in dry benzene was flushed alternately with oxygen (5 minutes) and argon (5 minutes) for two hours. The ESR spectrum was recorded continuously. The alternate flushing with oxygen and argon serves to vary the oxygen concentration. This is necessary because on the one hand oxygen is needed for the formation of 3.7a and on the other hand it causes
line-broadening which decreases the amplitude of the ESR signal. No ESR signal was observed when the solution was flushed with oxygen. During the flushing with argon a broad (± 50 G between the outer lines), unsymmetrical spectrum showing hyperfine structure was observed at g=2. This spectrum is attributed to a mixture of radicals derived from 3.4j, in which the free electron is distributed over the heterocyclic ring system. A single peak at g=2.004 or any other g-value, which might be attributed to oxyradicals, was not observed.
The same experiment was carried out in toluene at -60 C and -30 C giving a spectrum of low intensity with the same characteristics as the one in benzene.
Oxidation of 3.4j in aqueous acid (HCl, H„SO. or HCIO., 0.5 or 2 N) with oxygen or hydrogen peroxide, followed by deoxygenation with argon, gave the spectrum of the cationic radical 3.4b (Fig. 3.4). Other signals were not detected.
III.3.3. Hydroxy-cyclohexadienyl radicals and alloxazinyl-oxy-cyclo-hexadienyl radicals.
The hydroxylation of aromatic compounds by 3.4j and oxygen or hydrogen peroxide gives high yields of hydroxylated products in water at low pH. In water at neutral pH the yields are lower. Hydroxylation does not take place
in water at alkaline pH or in nonaqueous solvents.
At low pH the ESR signal of 3.4b formed by oxidation of 3.4j is relatively strong by comparison with the poor signal intensity that is usually obtained for transient hydroxy-cyclohexadienyl radicals (3.9a). The spectra of 3.4b and 3.9a have approximately the same g-value and the spectrum of 3.4b is both intenser and broader than the one of 3.9a. Under those circumstances 3.9a and also 3.8a would remain undetected. Conditions in which the signal of 3.4b is weak or absent must be used.
oxidized in 0.5 N HpSO. with a f i f t e e n - f o l d molar excess of H„0„. Oxidation with a i r or oxygen gives high concentrations of 3.4b and is therefore not s u i t a b l e . ESR spectra of radicals derived from 3.4j were not detected during oxidation with an excess HpOp in 0.1 N acetic acid or i n unbuffered water.
During the hydroxylation of phenylalanine- the substrate that was used by Mager in the majority of his hydroxylation experiments- by 0.5 N Hp|S0./3.4j/ HpOp no cyclohexadienyl radicals could be observed. The same experiment i n 0.1 N acetic acid or in unbuffered water was also negative.
Compound 3.4j is only p a r t l y soluble under the conditions used and dissolves slowly as the reaction proceeds. This means that the reaction does not go to completion very rapidly and that the use of a rapid flow system f o r mixing of the reactants in the cavity of the ESR spectrometer offers no advantages.
Indeed i t is l i k e l y that the dissolution of 3.4j is a l i m i t i n g step i n the r e a c t i o n , thus decreasing the steady-state concentration of any cyclo-hexadienyl radicals formed. Solvents in which 3.4j is better s o l u b l e , l i k e nonaqueous solvents or concentrated solutions of strong acids i n water, can not be used f o r reasons outlined above.
Higher steady-state concentrations of cyclohexadienyl radicals are expected when s t e r i c a l l y hindered aromatic compounds l i k e p y r o m e l l i t i c a c i d , which give r e l a t i v e l y stable hydroxy-cyclohexadienyl r a d i c a l s , are used as substrates.
No cyclohexadienyl radicals were observed, however, when 1,2,4,5-benzene-tetracarboxylic acid ( p y r o m e l l i t i c a c i d ) , 1,3,5-benzenetricarboxylic acid (trimesic a c i d ) , 1,3,5-benzenetrisulphonic a c i d , benzylalcohol, benzoic a c i d , benzene, pyridine or s - t r i a z i n e were hydroxylated by 3.4j and H„0„ in 0.5 N H„SO.. Experiments with p y r o m e l l i t i c a c i d , trimesic acid or 1,3,5-benzene-trisulphonic acid i n 0.1 N acetic acid/3.4j/Hg0g and in Hp0/3.4j/Hp0p were equally negative.
The formation of hydroxylated products in the system 0.5 N H S0./3.4J/ HpOp/pyromellitic acid was detected with Folin-Ciocalteu reagent.
In order to establish that the experiments were performed with the proper spectrometer settings f o r the detection of cyclohexadienyl r a d i c a l s ,
2+
p y r o m e l l i t i c acid was hydroxylated with Fenton's reagent (Fe /EDTA/HpO„), both at pH 7 and in 0.5 N H,SO.. The spectrum of the hydroxy-cyclohexadienyl
24)
radical of p y r o m e l l i t i c acid ' was observed c l e a r l y in both cases. The i n t e n s i t y of the spectrum was greater at pH 7 than i n 0.5 N H„SO..
system 0.5 N H„S0./3^4j/H„0,/pyromellitic acid is consistent with Mager's
181 <- 4 c c
view ' that most of the hydroxylation proceeds via alloxazinyl-oxy-cyclo-hexadienyl radicals and that hydroxy-cycloalloxazinyl-oxy-cyclo-hexadienyl radicals play a minor role. Our failure to detect any alloxazinyl-oxy-cyclohexadienyl radicals with ESR indicates that these radicals are extremely unstable under the experimental conditions of the hydroxylation reaction.
Finally an effort was made to detect transient radicals in the hydroxylating system by means of the spin trapping technique.
281
Lagercrantz and Forshult ' used dimethylsulphoxide as a substrate for hydroxyl radicals generated by photolysis of H„0„:
CH, OH
I ^ I
HO. + S=0 ^ CH,. + S=0
I ^ I
CH3 CH3
The methyl radicals were spin-trapped by a tertiary nitroso compound and the resulting nitroxide radical was detected by ESR.
We used dimethylsulphoxide as a substrate in the hydroxylating system 0.1 N AcOH/3.4j/H,0, in the presence of the water-soluble spin trap
2-methyl-'91
2-nitrosopropanol 3.10a " '. The methyl radical adduct of 3.10a was detected by ESR. When dimethylsulphoxide-d^ was the substrate the spectrum of the corresponding trideuteromethyl nitroxide was obtained, proving that the intermediate methyl radical originates from dimethylsulphoxide and not from 3.4j or 3.10a. However, the spectrum of the methyl nitroxide radical was also obtained when 3.4j was omitted from the reaction mixture. Consequently the intermediate hydroxyl radicals must be the product of a reaction between 3.10a and H„0„ and no conclusions can be drawn regarding the formation of hydroxyl radicals from 3.4j and H„0„.
III.4. References of chapter III.
1) A.Ehrenberg, Acta Chem.Scand.,14, 766 (1960).
2) A.Ehrenberg, Electron.Aspects.Biochem., Proc.Int.Symp.Ravello Italy, (Editor B.Pullman), pp.379-396, Academic Press, New York and London(1964) 3) L.E.G.Eriksson and A.Ehrenberg, Acta Chem.Scand.,^8, 1437 (1964).
5) L.E.G.Eriksson and A.Ehrenberg, Arch.Biochem.Biophys. , n O , 628 (1965). 6) L.E.G.Eriksson, J.S.Hyde and A.Ehrenberg, Biochim.Biophys.Acta,192, 211
(1969).
7) F.Mueller, P.Hemmerich, A.Ehrenberg, G.Palmer and V.Massey, Europ.J. Biochem.,14, 185 (1970).
8) L.E.G.Eriksson and W.H.Walker, Acta Chem.Scand.,24, 3779 (1970).
9) H.L.Crespi, J.R.Norris and J.J.Katz, Biochim.Biophys.Acta,253, 509 (1971). 10) A.V.Guzzo and G.Tollin, Arch.Biochem.Biophys.,103, 231 (1963).
11) F.Mueller, P.Hemmerich and A.Ehrenberg, Flavins and Flavoproteins,(Editor H.Kamin), pp.107-122, University Park Press, Butterworths, Baltimore and Londen(1971).
12) A.Ehrenberg and L.E.G.Eriksson, Arch.Biochem.Biophys.,105, 453 (1964). 13) J.Westerling, H.I.X.Mager and W.Berends, Tetrahedron,31^, 437 (1975). 14) W.H.Walker and A.Ehrenberg, FEBS Letters,3, 315 (1969).
15) W.H.Walker, J.Salach, M.Gutman, T.P.Singer, J.S.Hyde and A.Ehrenberg, FEBS Letters,5, 237 (1969).
16) M.J.D.Powell, Computer Journal,7, 155 (1965).
17) H.I.X.Mager and W.Berends, Tetrahedron,30, 917 (1974).
18) H.I.X.Mager, Flavins and Flavoproteins, (Editor T.P.Singer), pp. 23-37, Elsevier Scientific Publ. Co., Amsterdam, Oxford, New York (1976).
19) J.R.Lindsay Smith, D.M.Jerina, S.Kaufman and S.Milstein, J.Chem.Soc.Chem. Commun.,1975, 881.
20) S.Weiner and G.S.Hammond, J.Am.Chem.Soc. ,91^, 2182 (1969). 21) T.Shiga, T.Kishimoto and E.Tomita, J.Phys.Chem.,77, 330 (1973). 22) W.G.Filby and K.Guenther, J.Chem.Phys.,60, 3355 (1974).
23) P.Neta and R.W.Fessenden, J.Phys.Chem.,78 523 (1974). 24) G.Filby and K.Guenther, J.Phys.Chem. ,78, 1521 (1974). 25) C.R.E.Jefcoate and R.O.C.Norman, J.Chem.Soc.B,1968, 48. 26) W.T.Dixon and R.O.C.Norman, J.Chem.Soc. ,1964, 4857. 27) W.T.Dixon and D.Murphy, J.Chem.Soc.Perkin 11,1974, 1630. 28) C.Lagercrantz and S.Forshult, Acta Chem.Scand.,23, 811 (1969).
29) J.J.M.C.de Groot, G.J.Garssen, J.F.G.Vliegenthart and J.Boldingh, Biochim. Biophys.Acta,326. 279 (1973).
CHAPTER IV
ESR OF PTERIN AND LUMAZINE RADICALS
I V . 1 . Introduction.
Pterins and lumazines have three diamagnetic redox levels (section 1.2.2). Addition of two electrons and two protons to the f u l l y oxidized state leads to a diamagnetic dihydropteridine, whereas addition of four electrons and four protons gives a t e t r a h y d r o p t e n d i n e . I t is clear that there must be two d i f f e r e n t paramagnetic redox l e v e l s . Addition of one electron and one proton to the f u l l y oxidized state gives a neutral monohydropteridine radical and addition of three electrons and three protons gives a neutral t r i h y d r o -pteridine r a d i c a l . Depending on the s i t e of attachment of the protons vai'ious tautomeric forms of each paramagnetic redox state are possible. Furthermore each redox state has both acidic and basic p r o p e r t i e s .
1 21
Moorthy and Hayon ' ' investigated the one-electron adducts of pteridines by UV spectroscopy and found that this redox state has at least three d i f f e r e n t protonation states i . e . the cationic dihydro r a d i c a l , the neutral monohydro radical and the anionic r a d i c a l . In addition to that a di-cation was detected in the case of pterins and a di-anion in the case of lumazines. The tautomeric forms of the protonation states could not be determined with c e r t a i n t y .
Since there are four protonation states of the one-electron adduct of pteridines and since pteridines can be subdivided i n t o pterins and lumazines, there are eight d i f f e r e n t types of one-electron adduct radicals. Only two of those the cationic dihydropterin and c a t i o m c dihydrolumazine r a d i c a l s
-3 41 were studied by ESR ' ' .
S i m i l a r l y , at least three protonation states ( c a t i o n i c tetrahydro r a d i c a l s , neutral t n h y d r o radicals and anionic dihydro r a d i c a l s ) are expected for the three-electron adduct of oxidized p t e r i d i n e . Detection of the neutral and anionic forms was not reported. Cationic tetrahydropterin radicals have poorly resolved ESR spectra ' ' ' . They were generated by one-electron oxidation of tetrahydropterins in TFA. Cationic tetrahydrolumazine radicals have not been reported.
