Electronic structures of the free radicals derived from ascorbic acid and α-hydroxytetronic acid

Journal of Molecular Structure, 67 (1980) 133-140 Elsevier Scientific Publishing Company, Amsterdam

-

Printed in The Netherlands

ELECTRONIC STRUCTURES OF THE FREE RADICALS DERIVED FROM ASCORBIC ACID AND a!-HYDROXYTETRONIC ACID

COLIN

THOMSON

Regional Workshop of the National Foundation for Cancer Research, Department Chemistry, University of St. Andrews, St. Andrews KYi6 9ST (Ct. Britain) (Received

4 February

of

1980)

ABSTRACT Calculations on several free radicals derived from ascorbic acid, and ol-hydroxytetronic acid are reported. The calculations have been carried out both with the INDO method and the ab initio UHF method. The cakufated spin densities are only consistent with the assignment of the structure of the predominant radicaI derived from these molecules to the anion radical.

INTRODUCTION

Ascorbic acid (vitamin C) (Fig_lA), which was discovered by A. SzentGyiirgyi in 1930 [ 11, plays an important role in various biochemical processes such as the hydroxylation of procollagen [l] , but despite much research over the last 45 years its biochemical functions are still not completely ~derstood. It is present in high concentrations in various tissues in the body, especially in the adrenal glands, in leucocytes, in the brain, and in blood plasma [ 11. There has been, and there continues to be, much controversy concerning the amounts of the vitamin which are optimal for good health in human beings, who must obtain this vitamin from the diet, and especially controversial has been the use of large quantities (ea. 5-10 g/day) in treating various illnesses inciuding viral infections, heart disease and cancer [ 2, 31. It is well established that ascorbic acid is very easily oxidized to a relatively stable free radical under a variety of conditions and various authors have proposed a physiological role for this radical Cl] , The precise structure of the radical has been the subject of some dispute until relatively recently 111, but recent work with C13-Iabelled compounds and ESR spectroscopy has provided conclusive evidence for the structure [4] (see Fig. 1). However, there have been no detailed theoretical studies of the radical which should enable the ESR assignments to be verified. Fir&y, the recent evidence that ascorbic acid, and possibly the ascorbic acid free radical, are involved in cancer [ 3, S] , and the idea that electronic 0022-2860/80/0000-0000/$02.25

Q 1980 Elsevier Scientific Publishing Company

134

A

B

Fig. 1. (A) Ascorbic acid; hydrogen atoms 15 and 16 are attached to Cll, is attached to C9. (9) Ascorbic acid radical anion.

hydrogen 18

processes such as charge transfer are important in this context [ 5,6 3, has stimulated renewed experimental and theoretical interest in this molecule and its metabolites. A more detailed knowledge of the electronic structure is clearly desirable, and this paper describes the results of theoretical calculations on the free radicals derived from ascorbic acid and the related molecuie a-hydroxytetronic acid. Calculations on the neutral molecules are described in detail in a separate publication, which also contains a more detailed discussion of the role of ascorbic acid in the charge-transfer processes and their relevance to the cancer problem [63. PREVIOUS

THEORETICAL

STUDIES

The first theoretical study of ascorbic acid was by Pullman and Pullman [ 71, who discussed the 7r-molecular orbitals. More recently Flood and Skancke [ 81 have reported the results of calculations on ascorbic acid and u-hydroxytetronic acid and their related ions, using a semi-empirical SCF method for the n-electrons. In addition, a modified CNDO method with CI was used to study the effect of the side chain on the resultant orbit&. The effect was shown to be smah and the emphasis in this work was on the calculation of the wavelengths of the UV absorption bands which are due to .IT* t- II transitions. The authors obtained good agreement with experiment. There has only been one previous ab initio calculation on the ascorbic acid molecule [9] and none on a-hydroxytetronic acid. Carlson et al. [9] investigated the conformation of the neutral molecule and the ion using the STO-3G basis set in SCF-MO calculations. Agreement with experiment was good and there was some discussion of the charge distribution and the molecuIar orbitals in terms of the known chemistry. A limited amount of geometry optimization was carried out in this work. EXPERIMENTAL

ESR STUDIES

OF THE ASCORBIC

ACID

FREE

RADICAL

The earliest ESR observation of a free radical from ascorbic acid (I) was that of Yamazaki et al. in 1959 IlO, 111. The numerous subsequent studies

135

B

A

Fig. 2. (A) or-Hydroxytetronic

C

acid; (B) wbromotetronic

acid; (C) reductic acid.

have been reviewed by Laroff et al. [ 4 ] who generated the radicals by in situ radiolysis of aqueous solutions, and, by using ‘3C-labelled compounds, were able to confirm the conclusions of Kirino and Kwan [ 123 that the radical exists as the anion (Fig. 1B) with the unpaired electron delocalized over the tricarbonyl system. Additional support for this assignment came from studies of the radicals derived from ar-hydroxytetronic acid (Fig. 2A), cu-bromotetronic acid (Fig. ZB), D-araboascorbic acid, the diastereoisomer of ascorbic acid, and reductic acid (Fig. 2C). There have been numerous reports of the observation of the ascorbate radical ESR spectrum in biological studies. These have been reviewed by Lewin [ 11, and more recently by Cameron and Pauling [3]. There is good evidence for a variation in the ascorbate radical concentration, as measured by ESR techniques, between normal tissue and cancerous tissue, although the biological significance of this is not at all clear [3, 131. It is clear that in view of the undoubted importance of ascorbic acid and its metabolites in biological processes, a thorough theoretical study is of some importance. The present study was undertaken firstly to clarify the nature of the free radicals derived from ascorbic acid, and secondly, to examine the possible charge-transfer interactions between the ascorbic acid molecule and other small molecules. The present paper deals with the theoretical calculation of the structure and hyperfine coupling constants in the radicals, and a later paper wi.lI discuss in more detail the charge-transfer properties of this molecule [6]. METHODS

OF CALCULATION

Two theoretical methods were used in this work. In the first, the isotropic hyperfine coupling constants in various radicals derived from ascorbic acid and related molecules were computed by the INDO semi-empirical SCF method [ 141 which is designed to yield reliable theoretical isotropic hyperfine coupling constants (hfcc). The calculated ‘H and 13C coupling constants are of particular interest. The geometry of the radicals has not been optimized, but it has been assumed that the basic ring geometry is the same as is found

136

experimentally for ascorbic acid itself ]15] . Geometry optimization should not affect the main qualitative conclusions of this work. Ab initio LCAO-MO-SCF wave functions for these molecules, from a minimal S’I’O-3G basis set, have also been computed_ The wave functions were calculated with the Gaussian 70 program system [16] , and spin densities were also obtained routinely, together with the charge distribution. A limited geometry optimization of the radical from a-hydroxytetronic acid is currently being carried out and the results will be described in a subsequent publication [S] . The SCF section of the Gaussian 70 program was extensively modified, since earlier work has shown that serious convergence difficulties often occur with open shell systems, especially for molecules as large as these. The calculations were carried out on an IBM 360/44 and on an IBM 3’70/165 at the Universities of St. Andrews and Cambridge, respectively. RESULTS

OF CAL~~ATIONS

It is apparent from the earlier work of Flood and Skancke [S] that the highest molecular orbitals of ascorbic acid are n-orbitals and hence are little affected by the existence of the side chain. Hence a suitable model compound to investigate in detail the hfcc is ff-hydroxytetronic acid (Fig. ZA), and indeed it was used in the ESR studies in order to help assign the spectrum of the radical [4,12] . Hence the calculations on radicals derived from this molecule will be discussed first, and then the more limited calculations carried out on the radicals derived from ascorbic acid itself will be described.

Figure 3 depicts the various free radicals which might be derived from a-hydroxytetronic acid and which have been studied in the present work. These are the cation radical (3A) formed by loss of an electron from 2A, the two possible radicals formed by loss of an I-Iatom from the OH groups (3B, 3C) and the anion radical formed by loss of H’ (to give the anion) and then loss of an H atom (333). The ESR evidence indicates that the last radical is dominant, since the spectrum consists of a single triplet with CL~= 2.32 G. The two protons are equivalent. The ‘3C-labelled molecule was also studied by Laroff et al. [4] leading to the values for the four 13C hfcc given in Table 1. The assignment to specific atoms was not completely unambiguous, but seemed reasonable in the light of the results for the radical derived from I. The computed values for the hfcc from the four radiedls 3A-3D are also given in Table 1. It can be seen at once that only in the case of the anion radical (313) is the magnitude of the proton hfcc low enough to agree with experiment. In all the other radicals a large (ca. 12-16 G) splitting is predicted which is not observed. Hence it is reasonably certain that the assignment is

137

H

H

R

R

HO

Fig. 3. Radicals derived From ascorbic acid (R = CH(OH)CH,OH) acid (R = II). (A) Radical cation; (B) and (C) neutral radicals;(D) TABLE

and a-hydroxytetronic the anion radical

1

Calculated ‘INDO hyperfine coupling constants for radicals 3A-3D a-hydroxytetronic acid (R=H) Hfcc=

a SO

a7

a, a3

a2 a

11

a

13

al a4

afi

Experimental hfcc

Radical 3A

‘9,

(Fig. 3) derived from

11.45, -12.68 -3.00 2.50 9.82 -4.39 -4.74 7.64 6.67 -2.04

3B 12.08

13.63, 3.16 -4.61 10.40 -12.43 -1.21 13.57 1.76 0.12

3c 13.28

-15.1, 14.5 -10.1 4.37 -16.03 16.11 -0.005 2.62 13.34 -0.98

3D -2.61, -0.43 -0.06 -3.51 -5.29 11.6 5.8 -0.09

-2.60

2.32 5.72b 2.85 3.65 1.03b -

aai = Isotropic hyperfine coupling constant for nucleus i in Gauss. bAssignments a2 and a, p robably reversed (see text).

correct. However it is also clear that the values for the 13C hfcc are in rather poor agreement with experiment except for C3. The assignments of C2 and C7 are probably reversed, but even if this is the case the very small computed value at C5 is surprising since the hydrogen coupling constants at H9 and HlO are in good agreement with experiment. The spin densities in the four radicals computed from the ab initio SW wave functions are given in Table 2. These are the total spin densities on the atoms, but more detailed analysis shows that the s-orbital spin densities on

138 TABLE Atomic derived

2 spin densities from ab initio UHF from cu-hydroxytetronic acid

3A 9, 10 7 5 3 2 11 12

+ 0.007, -0.58 -0.010 + 0.122 + 0.42 -0.013 -0.017

calculations

3c

3B f 0.007

+ 0.03, to.54 -0.01 +0.70 -0.72 -0.006

on the. radicals

f 0.03

-0.03, -0.03 -0.57 + 0.096 -0.77 + 0.75 -0.002 -

3A-3D

(Fig.

3)

3D -0.014, -0.014 + 0.0016 0.039 -0.37 -0.22 -

-0.042, 0.008 and 0.002, respectively. It is C2, C3, C5 and C7 are -0.029, thus clear that the 13C hyperfine splittings at C5 and C7 are much lower than those from C2 and C3, confirming the conclusion from the INDO results that the assignments suggested by Laroff et al. [4] may be incorrect. Although the magnitudes of the s-orbital densities are of course quite different in the two sets of calculations, only for radical 3D are the values consistent with the low l3C hfcc observed. Both the INDO and SCF spin densities are in agreement about the expected low hfcc from C5 and C7. The calculated INDO coupling constants predict a(&) > a{&). Assuming that the assignments of a(C2) and a(C7) are reversed, this is in agreement with the experimental order, but the calculated value of a(C5) is considerably less than the remaining experimental value. Put in a different way, the calculations predict the ratio of smallest to the largest 13C coupling to be much greater than observed. This difference is almost an order of magnitude. The differences in the ab initio results are less marked but the theoretical ratio remains about a factor of four too large. It is possible that the source of this discrepancy lies in the use of a nonoptimized geometry for the radical. This possibility is currently being investigated. However, it seems more likely that the smallest coupling constant of Laroff.et al. (1.03 G) is too high, and these authors point out that its value was not unambiguously determined. The possibility of the generation of the neutral radicals 3B and 3C by protonation of 3D in acid solution was suggested in the earlier ESR studies [4, 12]_ However, the present calculations suggest that such radicals would have a large proton hfcc from the protons H9 and HlO attached to C5, and the spectra obtained in acid solution are not consistent with this prediction. Radicals derived from ascorbic

acid

Both INDO and ab initio calculations have also been carried out on the radicals 3A’-3D’ derived from ascorbic acid itself (Fig. 3), the geometry of the radical being that of the neutral molecule. This assumption is clearly an approximation, but it should not affect the qualitative conclusions.

139 TABLE

3

Calculated INDO hyperfine coupling constants For radicals 3A’-3D’ ascorbic acid (R = -CH(OH)CH,OH) Hfcc

al3

a,, a16

a lb

a2 a3 a.5 a7 a9 alI

019 a20

Radical 3A’

3B’

3C’

3D’

13.19 0.22 1.95 0.01 10.61 2.29 -2.75 -13.58 1.98 -0.26 -4.15 -3.86

13.75 0.07 0.61 -0.32 -12.04 10.27 -3.88 2.88 +3.15 -0.10 -

-15.99 -0.05 -0.59 0.41 16.26 -16.17 3.65 -10.13 -3.71 0.09 -0.13 -

-2.08 + 0.01 -0.04 0.05 -5.39 -2.85 -0.37 -0.46 -0.63 -0.05 -

-1.13

(Fig. 3) derived from

Experimental hfcc 1.76 0.19 0.19 0.07 0.96 3.62 2.78 5.74 -

TabIe 3 gives the computed coupling constants. Once again, it is clear that only radical 3D’ is compatible with experiment. However, the assignment of the largest 13C hfcc to atom C2 as in 3D seems clear. The remaining coupling constants are similar to those of 3D except the 13C hfcc at C5 which is substantially increased. It is clear that the computed hfcc are in qualitative agreement with the order observed experimentally, but the couplings to H15 and H16 in the side chain are different; whereas experimentally these are almost equal in ascorbic acid, they differ appreciably in araboascorbic acid [4]. The precise values will clearly depend on the precise stereochemistry of the side chain, which has not been varied in this work. The limited number of ab initio calculations carried out on these radicals gave results similar to those found for a-hydroxytetronic acid, although these are not reproduced here. Convergence difficulties were experienced in the SCF in each case, and despite the use of damping in the present version of Gaussian 70, the spin densities converged to low4 onIy. However, the spin densities on H15 and H16 were two orders of magnitude smaller than on H13. This is not reflected in the observed hfcc. Clearly these discrepancies are the result of using an SCF wave function, but more accurate spin densities are not feasible at present for a molecule of this size. DISCUSSION

The calculations reported in this paper show quite clearly that of the four possible radicals derived from ascorbic acid, (or cr-hydroxytetronic acid), only the anion radical 3D is compatible with the ESR spectrum of the

140

dominant radical. All other radicals would be expected to show at least one hfcc of 10-16 G and this is not observed. The calculations also strongly suggest that the experimental 13C hfcc for C2 and C7 should be reversed. The calculated value for C5 is, however, much lower than the experimental value, and the reason for this requires further study. It is possible that geometry optimization is required for the resolution of this discrepancy. It would clearly be of some interest to study 170-substituted radicals, in view of the large differences between the “0 hfcc. ACKNOWLEDGEMENTS

The author is indebted to the National Foundation for Cancer Research for partial financial support, to Dr. Albert Szent-Gyargyi and Dr. P. Gascoyne for useful discussions, and to the staff of the University of St. Andrews computing laboratory for running the calculations on the IBM 360/44. REFERENCES 1 S. Lewin, Vitamin C; its Molecular Biology and Medical Potential, Academic Press, New York, 1976. 2 L. Pauling, Vitamin C, the Common Cold and the Flu, W. H. Freeman, San Francisco, 1976. 3 E. Cameron and L. Pauling, Cancer Res., 39 (1979) 663. 4 G. P. Laroff, R. W. Fessenden and R. H. Schuler, J. Am. Chem. Sot., 94 (1972) 9062. 5 Ciba Symposium No. 67, Submolecular Biology and Cancer, Excerpta Medica, Amsterdam, 1979. 6 C. Thomson, to be published. 7 B. Pullman and A. Pullman, Quantum Biochemistry, Interscience, New York, 1963. 8 E. Flood and P. N. Skancke, Acta Chem. Stand., 27 (1973) 3069. 9 G. L. Carlson, H. Cable and L. G. Pederson, Chem. Phys. Lett., 38 (1976) 75. 10 I. Yamazaki, H. S. Mason and L. H. Piette, Biochem. Biophys. Res. Commun., 1 (1959) 336. 11 I. Yamazaki, H. S. Mason and L. H. Piette, J. Biol. Chem., 235 (1960) 2444. 12 Y. Kirino and T. Kwan, Chem. Pharm. Bull., 20 (1972) 2651. 13 N. F. J. Dodd and J. M. Giron-Conland, Brit. J. Cancer, 32 (1975) 451. 14 J. A. Pople, D. L. Beveridge and P. A. Dobosh, J. Chem. Phys., 47 (1967) 2026. 15 J. Hvosief, Acta Crystallogr., Sect. B, 24 (1968) 23. 16 W. J. Hehre, W. A. Lathan, R. Ditchfield, M. D. Newton and J. A. Pople, Gaussian 70. Program No. 236, Quantum Chemistry Program Exchange, University of Indiana, Bloomington.