Quantum-mechanical study on the photodimerization of aromatic molecules

J. Theoret. Biol. (1965) 9, 357-365

Quantum-mechanical Study on the Photodimerization of Aromatic Molecules C. NAGATA,

A. IMAMURA,

Y. TAGASHIRA

AND M. KODAMA

Division of Biophysics, National Cancer Center ResearchInstitute, Tstrkiji, 5-chome. Chuo-ku. Tokyo, Japan AND

N. FUKUDA National Institute of Radiological Science, Chiba, Japan (Received 17 August 1964, and in revisedform 7 December 1964) Distinct parallelismis found betweenthe quantum-mechanicalindex, the R delocalization energy and the degreesof dimer-forming ability of thymine, uracil and other aromatic compounds.It is surprisingthat the R delocalization energiesfor thymine and uracil are larger than those of well-known photodimer-forming compounds such as anthracene and acenaphthacene. The possiblestructuresof thymine dimer isomersin vitro and in viva, are postulated from considerationsof, for example, the z delocalization energy and steric properties. Biological inactivation by photo-irradiation is discussedin relation to the energy liberation due to the thymine dimer formation.

Introduction Beukers & Berends (1960) found that the ultraviolet irradiation of thymine in frozen solution led to the formation of thymine dimer and this finding stimulated the studies in the field of photobiology. Since then a lot of information has accumulated, and the important role of dimer formation in biological action has been ascertained. For example, Wacker et al. (1962) and Smith (1962a,b) isolated thymine dimer from deoxyribonucleic acid (DNA) of irradiated bacteria, and Setlow & Setlow (1962) found that about 50 % of the biological inactivation of the transforming activity of Hemophilus inpUenza DNA irradiated in vitro can be accounted for by thymine dimer formation.

With the increase of data on the dimer formation pyrimidines, the following questions have arisen: 357

of thymine and other

358

NAGATA,

IMAMURA,

TAGASHIRA,

KODAMA

AND

FUKUDA

(i) Why is dimer formation restricted to the pyrimidine bases? Furthermore, of the pyrimidine bases, why are thymine and uracil apt to form photodimer whereas cytosine and bromouracil are resistant to forming photodimer? (ii) Many experiments have been carried out concerning the photodimerization of polycondensed aromatic hydrocarbons such as anthracene. In this connection, it may be interesting to know the relative degree of reactivity of dimer formation of pyrimidine bases in comparison with that of aromatic hydrocarbons. (iii) There are four possible isomers of thymine dimer (Wulff & Fraenkel, 1961). Many attempts have been made to clarify which of these isomers is really produced, in Go and in vitro, but no conclusive evidence has been obtained. In the present paper we have attempted to clarify these points from the quantum-mechanical point of view. That is, we have assumed that photodimer formation by pyrimidine bases is no more than a chemical reaction in an excited state, and have applied the same theoretical treatment as was used in the case of anthracene photodimerization (Fukui, Morokuma & Yonezawa, 1961). We have found that the 71delocalization energy resulting from photodimer formation explains well the differences in photodimerforming ability of pyrimidine derivatives and other compounds observed experimentally. The most probable structure of thymine dimer is discussed, considering the sterical circumstances and the n delocalization energy. Furthermore, it is pointed out that the energy liberated due to dimer formation in DNA would be of great importance. This might have some connection with the inactivation of biological entities and also with the occurrence of mutation or initiation of tumor production by ultraviolet irradiation. Method In the present paper, the linear combination of atomic orbital-molecular orbital (LCAO-MO) method is used (Chalvet & Mason, 1962). The 7~electronic energy and electron density can easily be obtained by means of the variation method. The n delocalization energy used in discussing dimer-forming ability has already been derived by Fukui et al. (1961), in the case of the photodimerization of aromatic hydrocarbons. They assumed that the photodimer of aromatic hydrocarbons might be formed through the following mechanism : (T...T*) T+T*

----+

(7-T)* II

--f b

(7-T)

where T and T* are aromatic hydrocarbons in ground and excited states,respectively, (2’:. . T)* is transition state, (TT)* is an intermediate unstable complex

PHOTODIMERIZATION

OF

AROMATIC

359

MOLECULES

between Tand T*, and (TT) is the stable photodimer (Fig. 3). If the rate-determining step of this reaction is assumti as step a, the rate of reaction would be determined by the degree of lowering of activation energy and this lowering of activation energy may be measured by the I[ delocalization energy. The K delocalization energy was defined as the 11energy difference between the transition state$T. . . T)* and the initial one (T+T*); therefore the larger the z delocalization energy is, the smaller is the activation energy. The I delocalization energy, SH, for the photo-excited reaction was derived by means of the perturbation method (Fukui ef al., 1961) and was given by the following formula: (ci dj)2 $ (1) j i j i > 2(&i where cj, di, etc., are the coefficients of rth or sth atomic orbital of the jth molecular orbital and E, is the energy of the ith molecular orbital. Y< is the number of II electrons in the ith molecular orbital and y is the resonance integral between the occ vat atomic orbitals concerned with the dimer formation. C and I: mean the summation of the occupied and vacant molecular orbital, respectively. Meanings of superscripts of the coefficients of the atomic orbital, ho, etc., can be seen in Fig. 1 ; 6H

=

,/($‘”

&2

+ ($l

d;“)2

y +

‘;

‘;

vi -

“f;’

(2 -

;

,vi)

-Is

Ej)

;=

j= I I

I

2:

, I

2’

1’

1’

02 -

IV

01

-

-

2-

ho

-2 I A*

0I I

A

FIG. 1. Numbering of molecular orbitals. A and A* represent the ground and excited compounds, respectively.

ho and Iv denote the highest occupied and lowest vacant orbitals, respectively, of the ground state molecule A, and 01 and 02 denote the lower and the higher halfoccupied (occupied with one electron) levels, respectively, of the photo-excited molecule A*.

360

NAGATA,

IMAMURA,

TAGASHIRA,

KODAMA

AND

FUKUDA

In calculating the 7~delocalization energy, the first order term alone in equation (I) is sufficient, because the second order term is negligible compared with the first one owing to smallness of the value of y.

Results and Discussion DIMER

FORMATION

AND

In Table 1, the theoretical pyrimidine

THE

7? STABILIZATION

ENERGY

indices and the 71 delocalization

bases and related compounds TABLE

energies for

(calculated from (1)) are given and 1

Comparison of the II delocalization energies of pyrimidine bases and other aromatic compounds with their dimer forming abilities Compound Uracil Thymine Acenaphthylene Anthracene 2-Methyl-anthracene Stilbene 9-Methyl-anthracene 9,10-Dimethyl-anthracene Naphthalenet Acenaphthene Tetracene Naphthoquinone Cytosine Pentacene Pyrenet

Positions of dimer formation 4-4 5-5 44

5-5 l-l 2-2 9-9 la-10 9-9 lo-10 5-5 6-6 9-9 10-10 9-9 10-10 l-l 44 l-l 8-8 5-5 12-12 2-2 3-3 44 5-5 ai 13-13 3-3 8-8

Delocalization energy Dimer-forming (in unit of y) ability 0.761 0.701 0.568 0.547 0.544 0.543 0.541 0.534 0.512 0.483 0.417 0.414 0406 0.399 0.384

t Excimer formation. + and - indicate that the compound is active and inactive, respectively, in photodimer formation; very slightly active compounds are designated by the sign f.

PHOTODIMERIZATION

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compared with experimental results. The second column of the table shows the positions in the molecule participating in dimer formation. It is surprising that the A delocalization energy of pyrimidine bases such as uracil and thymine is larger than that of anthracene, which has been known as a typical example of photodimerization. From these results the ease of dimer formation by these pyrimidine bases is clear. It is known that the photodimerization of anthracene is markedly influenced by meso-substitution, and when both the 9 and 10 positions are substituted by methyl groups the formation of the stable dimer is known to be prevented (Birks & Aladekomo, 1963). These facts have previously been explained as due to the steric hindrance of the substituents. But the facts may also be explained by taking into consideration the n delocalization energy; that is (Table I), the values of the n delocalization energy for anthracene, g-methylanthracene and 9,10-dimethyl-anthracene become progressively smaller, in accordance with the experiments. Cytosine has been found to produce a small amount of photodimer, to the extent of 3 to 5% (Smith, 1963). The delocalization energy for cytosine is very small (Table 1) in accordance with this fact. In this connection it is of note that tetracene and pentacene, the theoretical values of which are near to that of cytosine, have not been known to produce photodimer until recently, when these compounds were found to be slightly active in dimer formation (Birks, Appleyard & Pope, 1963). The photodimerization product of pyrene was known to be unstable compared with that of other photodimer compounds, such as anthracene, and this product was named as “excimer”. The x delocalization energy for pyrene is rather smaller than those of the dimer-forming compounds (Table I), and in this connection the excimer formation seems to occur in the compound whose n delocalization energy lies below that of the active compounds. But naphthalene is an exception to our theory if we assume dimer formation at the l-l and 4-4 positions. The n delocalization energy for 5-bromouracil is calculated as 0*715y, assuming dimer formation in the 4-4 and 5-5 positions. But it is not tabulated in Table 1, because the response of bromouracil to photo-irradiation is known to be different from those of the other pyrimidine analogs, probably owing to the heavy atom substitution. Thus, it has been demonstrated by the tracer experiment that at least five photoproducts of bromouracil were produced and these products were all known to be debrominated. Therefore, it may not be adequate to compare the experimental dimer-forming ability with the theoretical values, which are obtained on the assumption that the bromine atom is not liberated during the photochemical reaction. In addition to the above-mentioned characteristics of bromouracil it may be of interest

362

NAGATA,

IMAMURA,

TAGASHIRA,

KODAMA

AND

FUKUDA

that phage, bacteria and mammalian cells that have incorporated 5-bromouracil into their DNA in place of thymine are particularly sensitive to photodimerization. As was pointed out by Smith (1962c), the mechanism by which bromouracil sensitizes bacteria and phages to photo-irradiation has been considered from many points of view, but it is still unexplained. STEREO-ISOMER

OF

THYMINE

DIMER

There exist four possible isomers of thymine dimers, according to the relative orientation of methyl groups and mode of superposition of the planar rings (Fig. 2) (Wulff & Fraenkel, 1961).

0

FIG. 2. Possible structure of isomers of thymine dimer.

To determine which type of isomer is really produced by photo-irradiation is very interesting and has been studied from many points of view. Thus, the nuclear magnetic resonance spectrum of the thymine dimer has been obtained (Beukers & Berends, 1960), and the absorption bands found to consist of a high and a low peak; the former is caused by the methyl groups and the latter by the hydrogen at carbon 6. From the fact that no splitting of the bands is observed it was assumed that no hydrogen atoms would occur at adjacent carbon atoms and the structure of the thymine dimer was postulated to be

PHOTODIMERIZATION

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363

the one in which the methyl groups are trans. But later Wulff & Fraenkel (1961) pointed out that the argument concerning the splitting of the bands was invalid. They insisted that the hydrogens are in equivalent magnetic environments, and consequently that no spin-spin splitting is to be expected. According to their discussion, therefore, it is impossible to tell from the nuclear magnetic resonance studies alone which of the four isomers is really produced. From the measurement of the absorption maximum of ultraviolet of the in vitro thymine dimer, Smietanowska & Shugar (1961) concluded that the dimer consists of one or a mixture of isomers I and III (Fig. 2). It seems, however, premature to determine the most likely structure of the isomer from the standpoint of the ultraviolet absorption spectrum alone. As is stated above, the stereo-structure of the thymine dimer has not yet been determined unambiguously, therefore it may be worth investigating from the theoretical point of view. We have previously pointed out the importance of the n delocalization energy in dimer formation. From this point of view isomers I and II are expected to predominate, because the II delocalization energy of both isomers is 0.7008y, whereas that of the isomers III and IV is calculated as 0.5678~. The validity of this conclusion must be tested. Up to the present, the discussion has concentrated mainly on the structure of the thymine dimer formed in the frozen solution or film of thymine. However, in connection with its biological activity, thymine dimer formation in DNA may be of importance. But it is more difficult to determine the stereo-structure of thymine dimer in DNA and no conclusive evidence has been obtained to date. Wulff & Fraenkel (1961) assumed from inspection of molecular models that only isomer I (Fig. 2) will arise from photodimerization of adjacent thymine moieties in DNA. They also suggested that interchain cross-linkage in irradiated DNA film might arise from formation of one or both of the truns isomers It and IV (Fig, 2). Concerning the structure of the thymine dimer in free base, Beukers & Berends (1961) made a suggestion that it might be tram, but they suggested the possibility of it being a cis isomer under special conditions, e.g. in DNA. The present authors concluded from the inspection of the molecular models that in the case of thymine dimer formation in DNA, only isomer I (Fig. 2) is possible in the case of intrachain dimer formation, whereas isomers II and III (Fig. 2) are possibly produced in the case of interchain cross-linkage. With regard to the possible structure in intrachain dimer formation, our conclusion coincides with that of Wulff & Fraenkel (1961); namely, that all of the four except isomer I are supposed to be impossible in intrachain dimer

364

NAGATA,

IMAMURA,

TAGASHIRA,

KODAMA

AND

FUKUDA

formation. On the other hand, isomer I is supposed to be formed easily because only 36” of twisting of the adjacent thymine moieties is needed. From this point of view, the ease of thymine dimer formation and the quantity of dimer formed seems to depend on the existence of the adjacent thymine molecule in DNA. With regard to the interchain dimer formation, however, the situation is more complicated and the conclusions of Wulff & Fraenkel(l961) contradict those of the present authors on some points. Thus, the former regarded isomers II and IV as possible ones, whereas II and III are adopted by the latter. It is easily understood from inspection of the Watson-Crick stereo model of DNA that isomer IV (Fig. 2) cannot be produced without inversion of the molecular plane of the bases, and such a drastic change would probably lead to the breakage of the bonds of bases and main chain of DNA. The situation is similar for isomer I. On the other hand, isomer 111, which was regarded by Wulff & Fraenkel(l961) as unlikely, is clearly shown to be formed with the twisting of about 70”. About 45” of twisting is needed for the formation of isomer II; therefore, from this viewpoint alone, it is likely that in the case of interchain dimer formation isomer II is more easily formed. ENERGY

LIBERATION

DUE

TO

THE

THYMINE

DIMER

FORMATION

It has been conclusively established that thymine dimer formation in DNA is responsible for the inactivation of biological activity, though the mechanism remains unexplained. From quantum-mechanical considerations, a great deal of energy liberation is expected in dimer formation; the present authors regard this energy liberation as one of the causes of biological inactivation. Thymine dimer formation is represented schematically in Fig. 3, where T and T* stand for the thymine and the photo-excited thymine, respectively. (TT) and (TT)* designate the thymine dimer and unstable intermediate dimer. respectively, and (T. . .T)* the transition state of the dimer.

(T-TIC -;.:1i Reaction

path

--f

FIG. 3. Reaction path of photodimer

formation of aromatic compounds.

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The energy liberation due to thymine dimer formation will be the sum of the absorbed energy, AE,, and the stabilization energy, AE, due to the formation of covalent bonds at the positions of dimer formation (Fig. 3). It has been known that a thymine dimer is effectively produced by light of 3750 A; therefore the magnitude of AE, will amount to 4.5 eV (+ 100 kcal./ .mole). On the other hand, the magnitude of AE, is difficult to estimate, for we must calculate not only the energy of the n electron but also that of the CTelectron. However, it is almost certain that the magnitude of AE, is not hmall enough to be neglected. The above-stated consideration indicates that about 100 kcal.fmole or more will be liberated in the course of thymine dimer formation, and it is permissible to postulate that this energy causes biological inactivation. Skin cancer production by ultraviolet irradiation is well known. Although the complete action spectrum has not yet been obtained, mercury arc source experiments have shown that photo-irradiation with wavelengths of 254 to 313 rnp is most effective in inducing tumours (Blum, 1959). It is interesting that the effective dimer forming radiation of 275 m/c is included in this region. in view of this it may be of value to search for the correlation between cancer production by photo-irradiation and energy liberation due to dimer formation. In this connection it is also noteworthy that Nakahara & Fukuoka (1959) and Nakahara (1961) have pointed out the importance of energy liberation due to the chemical reaction between chemical carcinogens and tissue components. REFERENCES DEUKERS,R. & BERENDS,W. (1960). Biochim. biophys. Acta, 41, 550. BEUKERS,R. & BERENDS,W. (1961). Biochim. biophys. Acta, 49, 181. BIRKS, J. B. & ALADEKOMO, J. B. (1963). Phorochem. & Photobiof. 2, 415. BIRKS, J. B., APPLEYARD, J. H. & POPE, R. (1963). Photochem. & Photobiol. 2, 493. BLUM, H. F. (1959). “Carcinogenesis by Ultraviolet Light”, p. 198. New Jersey: Princeton. CHALVET, 0. & MASON, R. (1962). J. Theoret. Biol. 3, 51. FUKUI, K., MOROKUMA, K. & YONEZAWA, T. (1961). Bull. them. Sot. Japan, 34,117s. NAKAHARA, W. (1961). Prog. exp. Tamor. Res. 2, 158. NAKAHARA, W. & FUKUOKA, F. (1959). Gum?, 50, 1. SETLOW, R. B. & SETLOW, J. K. (1962). Proc. natn. Acad. Sk., U.S.A. 48, 1250. SMIETANOWSKA, A. & SHUGAR, D. (1961). Bull. Acad. pol. Sci. cl. II, 9, 375. SMITH, K. C. (1962a). Biochem. biophys. Res. Commun. 8, 157. SMITH, K. C. (19626). Biochem. biophys. Res. Commun. 6, 458. SMITH, K. C. (1962~). “Photobiology: Action of Light on Living Materials” (A. C. Giese, ed.), p. 53. New York: Academic Press Inc. SMITH, K. C. (1963). Photochem. & Photobiol. 2, 503. WACKER, A., DELLWEG, H. & JACHERTS, D. (1962). J. molec. Biol. 4, 410. WULFF, D, L. & FRAENKEL, G. (1961). Biochim. biophys. Acta, 51, 332.