Theoretical investigation of lipid peroxidation mechanism

Journal of Molecular Structure (Theochem), 204 (1990) 379-388 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

THEORETICAL MECHANISM

INVESTIGATION

379

OF LIPID PEROXIDATION

G. SABA Dipartimento (Italy)

di Scienze Chimiche, UniversitZl di Cagliari, Via Ospedale 72,09124 Cagliari

P. SCANO, P.A. SEDDA and C. THOMSON Department

of Chemistry,

University

of St. Andrews, St. Andrews KY16 9ST (Ct. Britain)

(Received 4 January 1989; in final form 20 January 1989) ABSTRACT The semiempirical quantum mechanical MNDO and AM1 methods are employed to model Porter’s scheme for lipid peroxidation. The results obtained by the two methods, while supporting the proposed reaction pathway, show the expected differences in the structure of the investigated molecules.

INTRODUCTION

A characteristic property of polyunsaturated fatty acids (PUF’A) is that they undergo oxidation in radical chain reactions [ 11.This process, known as lipid peroxidation, has been suggested to be responsible for numerous effects observed in biological systems and it has also been thought to be related to cancer pathology [ 2,3]. Studies on the tumor-promoting and/or antitumor activity of the lipid peroxidation secondary products have been the subject of many reviews [ 2,3]. In fact it has been shown that peroxidizing PUFA produce a wide spectrum of secondary, highly reactive and/or toxic degradation products [ 41 some of which have been shown to be strong inhibitors of protein synthesis [ 51, to form DNA adducts [ 61, and to be mutagenic [7-l 11. On the other hand, it is also known that the membranes of many neoplastic cells exhibit a profound resistance to lipid peroxidation [ 12-141. Although much work has been published on lipid peroxidation, details of the chemical mechanisms involved in the process are still unknown. While for many years the classical scheme proposed by Dahle [ 151 has been taken as the basis of the lipid peroxidation, in the last decade the hypothesis due to Porter’s group has become widely accepted [ 16,171; it is also the rationale at the molecular level, for this biological process. Although partial confirmation of this scheme has come from the isolation and characterization, in chemical model systems, both in vitro and in vivo [ 1618] of some of the most stable compounds, the less stable intermediates in the scheme have not yet been characterized. This is due mainly to the transient 0166-1280/90/$03.50

0 1990 Elsevier Science Publishers B.V.

380

nature of the molecules involved, especially the radical species. However, if the mechanistic aspects of the reaction pathway are to be fully understood, both structural and energetic data on all the molecules in the scheme as well as those of the transition states connecting the “reactants” and “products” are needed. Methods of computational chemistry have now developed to the point where it is possible to locate energy minima of both stable and unstable polyatomic molecules together with their associated structural data. These computations have been shown to be usually in good agreement with experimental data [ 19,201. THEORY AND STRATEGY

For the quantum-mechanical calculation of molecular properties, two strategies are generally employed: the ab initio [ 191 and the semi-empirical methods [21,22]. Ab-initio calculations are non-empirical and can be carried out using SCF methods, or including electron correlation, but the computational complexity is such that they are confined to the calculation of the properties of small molecules containing about 20 atoms. Semi-empirical SCF methods, however, can be used in calculations on medium to large molecular systems, i.e. the vast majority of the systems of biological interest. During the last few years, the semi-empirical method MNDO [21,23] has been extensively used in such studies; this method and the more recent AM1 method [ 241 were developed by Dewar’s group [ 221. On the basis of the welldocumented successes in many chemical applications, it was decided to use these methods in this study to test the ability of approximate quantum-mechanical methods in modelling the molecular events comprising Porter’s scheme of lipid peroxidation. Since the molecules in the real system are quite large in terms of quantummechanical calculations, even for these methods, 2,5-heptandienoic acid was chosen as a model compound. In fact, this molecule contains the same key functional groups as the actual molecules, but is still small enough to allow full geometry optimizations. All the calculations were carried out using the MNDO and AM1 Hamiltonians as implemented in the MOPAC system [ 251. The final results were refined using the PRECISE keyword which increase the criteria for terminating all optimizations (electronic and geometric) by a factor of 100. Final calculations on the optimized geometry were run with the FORCE option to ascertain that real minima or maxima in the potential-energy surface were obtained. As already indicated in the previous section, a number of open-shell molecules need to be investigated. For semi-empirical studies of these molecular systems, the best quantum-mechanical approach would be the UHF formalism. Unfortunately, an extremely high spin contamination was found in all the radical species, and this formalism was abandoned in favour of the RHF-HE scheme as implemented in the MOPAC package.

381

Finally, since no structural data were available in the literature for the molecules under investigation or similar compounds, standard bond lengths, bond angles and dihedral angles were taken for the starting geometries [ 261. RESULTS

A representation of Porter’s scheme is given in Fig. 1. The scheme comprises the following steps: (i) hydrogen abstraction from the bis-allylic carbon of the acid (1)yields the W cis,cis radical (2) [17,27]; (ii) molecular oxygen reversibly reacts with this radical to give the truns,ck peroxy radical isomers 3 and S [ 28,291; (iii) /I-fragmentation (oxygen abstraction) competes with the capacity of a proton donor to form hydroperoxides with cis,trum conformations 4 and 6 [30]; and (iv) a new process of oxygen addition and proton abstraction finally leads to the tram, tram hydroperoxides 9 and 11. According to Porter the formation of trunqcis hydroperoxides is kinetically controlled, while the trum,truns products are under thermodynamic control t171. As stated in the previous section, an understanding of the mechanistic aspect of this pathway relies on its energetics. This, in turn, requires a knowledge of the energies of all the “stable species” and of the transition states. Calculation of these properties was carried out at both the MNDO and AM1 levels, _

LO.

.i_,,. m COOH CH3 3 AH=-67.60(-62.67)

RH AH--g&22(-93.64)

AH--93.70(-92.66)

AH:-67.91(-62.96)

c~cooH~~oo-&-+cooH~

HO&&__/oH

-02 AH'- 53.72(-52.69)

AH*-73.00(-64.95)

AH=-95.0 C-92.17)

E,-1.9(4.6) Ii .OO.:p_/cOOHT(H

1 lEa= 0.76t2.7) ,,?WH

10

II

AH*-72.93~65.35)

AH--94S4C92.50)

Fig. 1. AM1 heats of formation for the reaction pathway of the R isomers. The MNDO values are given in parentheses.

one of the aims of this work being the comparison of the two methods in modelling lipid peroxidation. A selected range of internal coordinates of 2,5-heptandienoic acid, the cis,cis W radical and the 2,5-heptandienoic hydroperoxide are given in Tables l-3, respectively. From the analysis of the results, it is seen that both methods for the closed-shell systems give similar bond lengths and the bond angles, the maximum deviations obtained being less than 0.04 A and 4 ’ for the former and latter, respectively. The major difference between the results obtained with the two methods is seen in the torsion angles. In particular, the AM1 method always leads to planar geometries while the MNDO method invariably gives distorted conformations. This result seems to be related to the overestimation of the core-core repulsions by MNDO which are mostly corrected for in the AM1 Hamiltonian [ 241. TABLE 1 Comparison of internal coordinates calculated using the MNDO and AM1 methods for 2,5-heptandienoic acid

MNDO

AM1

Bond length (A) c4-c7 c7-c9 c9-Cl1 Cll-012 Cll-013 013-H14

1.507 1.344 1.493 1.230 1.358 0.949

1.480 1.340 1.460 1.238 1.367 0.971

Interbond angles ( ’ ) C16-Cl-C2 Cl-C2-C4 C2-C4-C7 c4-c7-c9 c7-c9-Cl1 c9-Cll-012 c9-Cll-013

128.7 127.4 113.6 129.2 127.7 126.6 114.3

125.7 124.3 113.6 124.5 123.5 130.1 113.9

59.5

7.7 9.1 -2.0

Torsion angles ( ’ ) H19-C16-Cl-C2 C2-C4-C7-H8 c7-c9-Cll-012

79.5 -89.2

383 TABLE 2 Comparison of selected internal coordinates calculated using the MNDO and AM1 methods for the W radical

MNDO

AM1

Bond lengths (A) C4-C6 C6-C8 C8-Cl0 ClO-011 ClO-012

1.435 1.359 1.492 1.231 1.359

1.407 1.359 1.456 1.239 1.369

Interbond angles ( ’ ) C15-Cl-C2 Cl-C2-C4 C2-C4-C6 C4-C6-C8 C6-C8-Cl0 C8-ClO-011 CS-ClO-Cl2

128.8 127.7 123.8 127.8 127.5 126.7 114.3

124.8 123.9 122.1 125.6 124.7 131.1 113.5

- 179.8

- 178.3 0.0 0.0

Torsion angles ( a ) H16-C&Cl-C2 C2-C4-C6-H7 c6-c8-c10-011

0.1 - 86.3

This overestimation could also be the reason for the slightly larger bond angle values found in the AM1 results as compared with MNDO. As far as the radicals are concerned, similar general results were obtained and, consequently, similar conclusions can be drawn (cf. Table 2). The next step was to obtain the values of the rotational barriers connecting the rotamers of the same hydroperoxide. These were obtained by taking the rotation around the C-C bond (C64!8 in Table 3) as the reaction coordinate, and then letting the geometry relax to the equilibrium for each value of the coordinate. In one case (the R-truns,cis-hydroperoxide) a similar calculation was done without geometry optimization for comparison. This procedure was dictated by the impossibility of obtaining rotational barriers for the radicals by the first method within a reasonable time. The comparison of the two methods at the AM1 level for the R-trurw,ck-

384 TABLE 3 Comparison of selected internal coordinates 2,Sheptandienoic acid hydroperoxide

MNDO Bond lengths c4-C6 C6-C8 C8-Cl0 ClO-011 ClO-012 C8-014 014-015

calculated

using the MNDO and AM1 methods

for

AM1

(A)

Interbond angles ( ’ ) C2-C4-C6 C4-C6-C8 C6-C8-014 C6-C8-Cl0 C8-014-015 Torsion angles ( ’ ) H19-C18-Cl-C2 H3-C2-C4-C6 H9-C8-C6-C4 015-014-C8-H9 011-ClO-C8-H9

1.349 1.522 1.555 1.229 1.356 1.426 1.292

1.340 1.488 1.511 1.235 1.358 1.456 1.285

124.4 124.5 106.3 110.3 114.2

122.4 122.9 105.8 114.1 112.2

75.3 -51.2 0.8 - 44.5 137.7

112.6 -5.2 62.9 -53.3 112.4

hydroperoxide (Fig. 2) shows how closely the two barriers are calculated, and therefore suggests that the quantities obtained for the radicals with the more approximate method are reasonably good. The activation energies between the different rotamers are shown in Figs. 1 and 3, and indicate that the values computed with MNDO are consistently larger than those calculated with AMl. It must be pointed out that, in the MOPAC package, the geometry optimization is made in Cartesian space, independently of the geometry input. Therefore, problems could arise for the optimization of the torsional angles, as the force constants for these parameters in Cartesiancoordinates are generally very small and, consequently, the energy decrease may be negligible. However, the results of the overall activation-energy calculations (using both MNDO and AM1

385

0.02

72.00

I++.00 216.02 288.00

Fig. 2. Dependence of the heat of formation on the dihedral angle H7-C6-C&H9 for R-tranwishydroperoxide. Results were obtained at AM1 level with ( + ) and without (*) geometry relaxation.

moon%

~cooH

Ali.-77.42(-74.50,

AH- -6:40(-62.17)

g

&y-Q&

-E-

AH=-67.06(-62.99) E,-o.sC3.1) II

c&“Y_ 3

AH.-94.22(-92.99)

e

I4

AH= -72.91(-95.00) E..Cm(4.43 .,,$q IS AH=-73.19(6627)

E,=0.7C3.0) II

IS

AH--97.60t92.693

AH= -93.72(-92.99)- -

cdG;

RH

AH- -937Ot9266)

AH--95.49~9292~ ~~E,-l.O~4.6, HOO$ro" 19 AH*-99.79(-93.30

Fig. 3. AM1 heats of formation for the reaction pathway of the S isomers. The MNDO values are given in parentheses.

Hamiltonians) make us confident that the energy barriers for the different rotamers are meaningful. As far as other transition states are concerned, there are some serious and as yet unresolved problems in the calculations. Specifically, in the reaction of molecular oxygen addition or abstraction, the spin multiplicity of the whole system changes when moving from the reactant (doublet + triplet ) to the prod-

386

ucts (doublet), or vice versa. No methods at the RHF level can handle systems like these. On the other hand, the high spin contamination introduced by the UHF method made this method unsuitable for the present aims. Therefore, no activation energy for this process could be calculated. However, it has been shown experimentally that these processes are extremely fast and so very low activation barriers must be suspected for these reactions [ 291. The other unsurmountable problem was found in the study of the proton addition from a donor to the peroxy radicals. This study would require calculations extended to bimolecular systems which, again, was beyond the computational resources available. However, Figs. 1 and 3 (for the R isomers and for the S isomers, respectively) clearly show that this is the final stage which involves the stabilization of radicals which are either rotational or geometrical isomers the activation energies of which are, therefore, expected to be very much the same. In the light of these findings, we can now turn our attention to the reaction pathway reported in Fig. 1 for the R isomers. A similar analysis can be made for the S isomers reported in Fig. 3. The analysis of the AM1 formation energies in both pathways clearly indicates that the trans,tram products are slightly more stable than the corresponding truns,cis isomers. However, the energy difference between the final trum,ci.s (4 and 6) and tram, truns hydroperoxides (9 and 11) (less than 1 kcal mol-l ) is in no way sufficient to overcome the energy gap separating radicals 6 and 7 in the j?-fragmentation pathway. This means that the truns,ci.s products (4 and 6) which are formed via a fewer number of transition states, are favoured in comparison with the trum,truns compounds. On the other hand, Figs. 1 and 3 also show that if sufficient energy is given from the outside to the system to overcome the transition state connecting radicals 6 and 7, the B-fragmentation pathway should be promoted, thus leading to the final tram, tram hydroperoxides 9 and 11.Thus an increase in temperature would lead to the most stable dienyl radical, 7, and from this to the truns,truns compound. These findings agree with the experimental observation that in linoleate autoxidation at higher temperatures, more tram, truns product is formed [ 171. It is important to note that the rotational barrier between radicals 3 and 6 has the same value as the energy gap between them, so that the conversion of 3 to the stereochemically more productive radical 5 is an easy process. A further observation regarding the peroxides is the relatively lower heats of formation found in the truns,trun.s compounds (8 and 10) as compared with those found in the cis,trum analogues (3 and 6). In the analysis of the eigenvectors of these open-shell molecules, it was found that one of the molecular orbit& is consistently more delocalized in the truns,trum peroxides than in the cis,tram ones. This is mainly due to the participation, in the trams,truns compounds, of the double bond C-O of the acid group in extending this orbital. It is our suggestion that a consequence of this is that the odd electron can

381

delocalizeits charge over the whole molecule and, thereby,better stabilizethe tram,trans moleculesthan the ci.s,trans ones. As far as the comparison of the two methods is concerned, the values reported in bracketsfor the MNDO calculationssupportthe overallanalysisand conclusions.The major differenceis seen in the R-trun.s,cis-hydroperoxide being calculated slightly more stable than the correspondingS ones. However, the smallnessof this difference makes this result of minor importance. It should be pointed out that the resultsobtained in this work should not be emphasizedtoo much because other effects may occur which could push the reactionpathwaytowards some other direction.For example,steric hindrance and the terminalgroupshave not been consideredin this work. In fact, possible steric effects were investigatedwith the substitution of the terminal methyl group by a heavier ethyl group. At this level of hindrance no difference was found in eitherthe geometryor the relativeformation energiesfor some of the most stable compounds.However,heaviersubstitutionson the extremesof the molecule as in the case of linoleic or arachidonic acid autoxidation are likely to be of primary importance in deciding which of the extremes of the dienilic bond the oxygen attack can occur. Even more caution should be used when transferringthis model calculation to the biologicalenvironment.In this case enzymaticprocessescan occur which hold the molecule in some suitable conformation, not necessarilythe most stable one, and thus allow new possible reactions. Nevertheless,the resultsof the present investigationstrongly support the consistency of the scheme proposed by Porter, and show that these methods of molecularmodelling can be of invaluableuse in the study of biological system. ACKNOWLEDGEMENTS

We would like to thank both the National Foundation for Cancer Research (U.S.A.), and the Association for InternationalCancer Research (U.K.) for financial support, and Professor F. P. Corongiufor suggestingthis problem to us.

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