X-ray Analyses of Polycyclic Hydrocarbon Metabolite Structures - ACS

7 X-ray Analyses of Polycyclic Hydrocarbon Metabolite Structures JENNY P. GLUSKER

Downloaded by UNIV QUEENSLAND on May 14, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch007

Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, PA 19111

The methods of X-ray diffraction analysis of crystal structures can be used to investigate steric effects in carcinogenic polycyclic aromatic hydrocarbons (PAHs) and their activated metabolites. For example, the presence of a methyl group adjacent to the bay region of a PAH greatly enhances its carcinogenicity; X-ray studies have shown the extent of the steric distortions of the bay region, in-plane and out-of -plane, caused by this methylation. In most PAHs the conformation of the molecule is determined by nonbonded H••••H interactions. If hydrogen atoms would approach each too closely if the molecule were planar, then bond angle and torsion angle changes are made to accommodate this strain. In addition, in more saturated ring systems such as PAH metabolites, this type of nonbonded interaction between hydrogen atoms may force a hydroxyl or other substituent to have an axial rather than an equatorial conformation. This has been analyzed for metabolites such as diols and diol epoxides. The mechanism of carcinogenesis by PAHs is believed to involve alkylation of an informational macromolecule in a critical, but at present unknown, manner. Such an interaction with a protein has been modelled by alkylation of a peptide; this showed a conformational change occurred on alkylation. It has not yet been possible to study the structure of DNA alkylated by an activated carcinogen; this is because DNA is a fiber and the structural order in it is not sufficient for a crystal structure determination. However the crystal structures of some alkylated portions of nucleic acids are described, particularly some nucleosides alkylated by chloromethyl derivatives of DMBA. In crystals of these alkylation products the PAH portion of the adduct shows a tendency to lie between the bases of other nucleoside 0097-6156/85/0283-0125$16.00/0 © 1985 American Chemical Society

In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1985.

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molecules in the crystal structure, although the more buckled part of the PAH does not take part in this stacking. There also appears to be an interaction between the oxygen of the ribose sugar and part of the PAH (the methyl group in a 12-methylbenz[a]anthracenyl derivative). Some preliminary studies in computer modelling experiments show that, at present, the X-ray data on adducts are consistent with both current models of interaction after covalent attachment - partial intercalation of the aromatic portion of the PAH between the bases of DNA or the situation where the aromatic group lies in a groove of DNA. The carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) was first deduced by Sir Percivall Pott in London in 1775 (J_) when he noted an appreciable incidence of scrotal cancer in young chimney sweeps. He correctly noted that this affliction was caused by an accumulation of soot on the skin. The work of his grandson, Henry Earle (2J, and of Curling (3_, 4) led to the ideas that, since scrotal cancer did not affect all chimney sweeps, another factor, such as an inherited predisposition, might also play a part in its occurrence, and that the disease could have an appreciable latent period. The fact that coal tar could, by itself, cause skin tumors was shown by Yamagiwa and Ichikawa (5J. Shortly after that Passey (6j extracted a single chemical from soot and it was demonstrated that it was a carcinogen. Finally the chemical formulae of many of the active carcinogenic components of coal tar and soot were established by the experiments of Kennaway, Heiger, Mayneord, Cook and Hewitt (7J. They showed that the carcinogens from soot and coal tar are PAHs with formulae such as I (benzo[a]pyrene), II (7,12-dimethylbenz[a]anthracene), III (3-methylcholanthrene), IV (benz[a]anthracene) and V (dibenz[ji,h]anthracene). It was PAHs like these that were responsible for the problems of the chimney sweeps. In fact, carcinogenic PAHs continue to be a problem and are environmental hazards found wherever pyrolysis of organic matter occurs, as in cooking and in cigarette smoking (8-10). 2

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V The f a t e o f such a p p a r e n t l y innocuous h y d r o p h o b i c c h e m i c a l s i n t h e body has been t h e s u b j e c t o f much s t u d y ( 1 1 ) . These compounds a r e f a i r l y i n s o l u b l e i n w a t e r , but t h e y a r e s o l u b l e i n f a t s and o i l s . The body, i n an e f f o r t t o s o l u b i l i z e , and t h e r e f o r e e x c r e t e , t h e s e f o r e i g n compounds, e p o x i d i z e s and h y d r o x y l a t e s them, o f t e n f o r m i n g c o n j u g a t e s w i t h amino a c i d s and p e p t i d e s such as g l u t a t h i o n e (j_2). Such metabolism o f PAHs proceeds v i a q u i n o n e s , a l c o h o l s , e p o x i d e s and p e p t i d e c o n j u g a t e s t o g i v e p r o d u c t s t h a t can be e l i m i n a t e d from t h e body. But o c c a s i o n a l l y , by a p r o c e s s known as " a c t i v a t i o n , " a d i o l e p o x i d e , w i t h f u n c t i o n a l groups i n v e r y s p e c i f i c p o s i t i o n s i n t h e m o l e c u l e , i s formed ( 1 3 ) . These d i o l epoxides are formed by t h e a c t i o n o f t h e enzyme cytochrome P-450 and e p o x i d e h y d r a s e . Init i a l l y an e p o x i d e i s formed and t h i s i s h y d r a t e d by e p o x i d e hydrase to a t r a n s - d i o l . F u r t h e r epoxidation gives the d i o l epoxide; t h i s i s an a l k y l a t i n g agent t h a t can i n t e r a c t w i t h a c e l l u l a r macromolec u l e such as DNA and d i s t o r t i t i n some manner a f t e r a l k y l a t i n g i t . T h i s i s b e l i e v e d t o be t h e b e g i n n i n g o f t h e c a r c i n o g e n i c p r o c e s s caused by c e r t a i n PAHs. The t h r e e - d i m e n s i o n a l s t r u c t u r e s o f t h e s e PAHs and t h e i r metabo l i t e s , o f t h e i r a c t i v a t e d p r o d u c t s and o f adducts w i t h p o r t i o n s o f DNA w i l l be t h e s u b j e c t o f t h i s c h a p t e r . While t h e f o r m u l a e I - V appear t o be o n l y t w o - d i m e n s i o n a l ( t h a t i s , f l a t ) , i t w i l l be shown t h a t t h e p r e s e n c e o f methyl g r o u p s , d i o l e p o x i d e g r o u p i n g s , e t c . i n c e r t a i n areas o f such m o l e c u l e s r e s u l t s i n t h r e e - d i m e n s i o n a l m o l e c u l a r s t r u c t u r e s t h a t may be b u c k l e d and s t r a i n e d . Some o f t h e t h r e e - d i m e n s i o n a l s t r u c t u r e s o f such PAHs and t h e i r m e t a b o l i t e s w i l l be d e s c r i b e d . In a d d i t i o n , t h e p o s s i b l e s t r u c t u r a l d i s t o r t i o n s t h a t may be caused i n DNA by s p e c i f i c a l k y l a t i o n s by a c t i v a t e d PAHs w i l l be d i s c u s s e d i n t h e l i g h t o f s t r u c t u r a l s t u d i e s o f adducts o f PAH a l k y l a t i n g agents w i t h n u c l e o s i d e s . From such i n f o r m a t i o n i t i s hoped e v e n t u a l l y t o be a b l e t o d e t e r m i n e what makes a PAH c a r c i n o geniC., and i n what way t h e d i s t o r t i o n s i n DNA, r e s u l t i n g from a l k y l a t i o n by an a c t i v a t e d c a r c i n o g e n i c PAH, make DNA behave d i f f e r e n t l y from normal so t h a t a c a r c i n o g e n i c p r o c e s s i s i n i t i a t e d . Geometry o f PAHs The t e c h n i q u e s o f X - r a y d i f f r a c t i o n a n a l y s e s o f c r y s t a l s o f compounds o f i n t e r e s t can be used t o d e t e r m i n e , w i t h high p r e c i s i o n , t h e t h r e e - d i m e n s i o n a l arrangement o f atoms, i o n s and m o l e c u l e s i n such c r y s t a l s ( 1 4 ) ; i n each case t h e r e s u l t i s r e f e r r e d t o as t h e "crystal structure." X - r a y d i f f r a c t i o n by c r y s t a l s was d i s c o v e r e d by von L a u e , F r i e d r i c h and K n i p p i n g (15) and t h e t e c h n i q u e was a p p l i e d by the Braggs t o t h e d e t e r m i n a t i o n o f t h e s t r u c t u r e s o f



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s i m p l e c r y s t a l s such as t h o s e o f sodium c h l o r i d e and diamond ( 1 6 , 1 7 ) , and by L o n s d a l e t o t h e s t r u c t u r e o f c r y s t a l s o f h e x a m e t h y l benzene ( 1 8 ) . T h i s l a t t e r study was t h e f i r s t e v i d e n c e t h a t t h e benzene r i n g i s a symmetrical hexagon o f carbon atoms w i t h each C-C bond l e n g t h t h e same w i t h i n e x p e r i m e n t a l e r r o r . The s t r u c t u r e s drawn by K e k u l e w i t h a l t e r n a t i n g s i n g l e and double bonds are not f o u n d , even at low t e m p e r a t u r e s where any i n t e r c o n v e r s i o n o f isomers would be e x p e c t e d t o be s l o w e r . Thus t h e benzene m o l e c u l e i s t r u l y " a r o m a t i c " w i t h s i x e q u i v a l e n t C-C bonds. D i f f r a c t i o n studies of benzene (J_9, 20) (which have n e c e s s a r i l y been done at low t e m p e r a t u r e s s i n c e benzene i s l i q u i d at room t e m p e r a t u r e s ) have shown t h a t t h e C-C bond l e n g t h i s 1.392(2) A , i n agreement with r e s u l t s from s p e c t r o s c o p i c d a t a (2J_). When two o r more benzene r i n g s are f u s e d t o g e t h e r t o g i v e n a p h t h a l e n e , a n t h r a c e n e , e t c . , X - r a y d i f f r a c t i o n s t u d i e s show t h a t some l o c a l i z a t i o n o f double bonds o c c u r s ( 2 2 - 2 4 ) ; t h i s a f f e c t s t h e chemical r e a c t i v i t i e s o f d i f f e r e n t r e g i o n s i n the m o l e c u l e . The e x p e r i m e n t a l l y measured bond l e n g t h s i n PAHs are t h o s e t h a t would be e x p e c t e d from a c o n s i d e r a t i o n o f t h e v a r i o u s t y p e s o f resonance h y b r i d s (25) t h a t are p o s s i b l e . The u n i t s by which c r y s t a l l o g r a p h e r s d e s c r i b e i n t e r a t o m i c d i s t a n c e s are Angstrom u n i t s (A = 1 0 ~ c m . ) . Normal v a l u e s f o r c a r b o n - c a r b o n i n t e r a t o m i c d i s t a n c e s are 1.34 A f o r a double bond (as i n e t h y l e n e ) and 1.54 A (as f o r * d i a m o n d ) f o r a s i n g l e bond. In a t r u l y a r o m a t i c compound (such as benzene) t h e C-C bond l e n g t h , as mentioned above, i s 1.39 A . C-C-C a n g l e s a r e 1 0 9 . 5 ° f o r a t e t r a h e d r a l c a r b o n atom (sp^) and 1 2 0 . 0 ° f o r a t r i g o n a l carbon atom ( s p ) . As shown i n F i g u r e 1 f o r BP and DMBA, measured C-C bond l e n g t h s , i n t e r b o n d a n g l e s and t o r s i o n a r e v a r i a b l e around t h e s e v a l u e s . Most c a r c i n o g e n i c PAHs c o n t a i n t h e phenanthrene g r o u p i n g i n them (see F i g u r e 2) and t h e r e a r e two i m p o r t a n t areas i n t h e s e m o l e c u l e s t h a t are o f t e n r e f e r r e d t o i n s t u d i e s o f PAH c a r c i n o g e n e s i s . One i s t h e " K - r e g i o n " which c o r r e s p o n d s t o t h e v e r y a c t i v e double bond between atoms 9 and 10 i n phenanthrene ( 2 6 ) . In BP (I) t h e " K - r e g i o n " i s t h e 4,5 bond and i n DMBA (II) i t i s t h e 5,6 bond. Bonds i n t h e " K - r e g i o n " are u s u a l l y s h o r t ( 1 . 3 4 - 1 . 3 5 A ) , near i n v a l u e s t o t h a t f o r a pure double bond, w h i l e most o t h e r C-C bonds i n PAHs are i n t h e range 1.36-1.44 A. The second area o f i n t e r e s t i s t h e "bay r e g i o n " (27) which c o r r e s p o n d s t o t h e h i n d e r e d r e g i o n between t h e 4 - and 5- p o s i t i o n s o f phenanthrene ( t h e a r e a between C10 and C l l o f BP (I) o r CI and C12 o f DMBA ( I I ) , f o r example). Some b a y - and K - r e g i o n s a r e i l l u s t r a t e d i n F i g u r e 2. While i t was o r i g i n a l l y thought t h a t t h e c o n d i t i o n f o r c a r c i n o g e n i c i t y o f a PAH i s t h e p r e s e n c e i n i t o f a K - r e g i o n , i t i s now c l e a r t h a t the p r e s ence o f a b a y - r e g i o n with a methyl group a t t a c h e d t o one s i d e o f i t i s a b e t t e r c r i t e r i o n (27). I n t e r e s t i n g l y the c o r r e l a t i o n s that were o r i g i n a l l y found between t h e p r e s e n c e o f a K - r e g i o n and c a r c i n o g e n i c i t y d e r i v e from t h e f a c t t h a t most c a r c i n o g e n i c PAHs c o n t a i n i n g a p h e n a n t h r e n e - l i k e group w i l l a l s o have a b a y - r e g i o n ; Pullman (28) s a i d t h a t " b a y - r e g i o n c o u l d be a k i n d o f 'back d o o r ' t o t h e K-region". E a r l y work on t h e s t r u c t u r e o f c a r c i n o g e n i c PAHs was done by John I b a l l at t h e U n i v e r s i t y o f Dundee, S c o t l a n d ( 2 9 ) ; he had s u g g e s t e d t h e use o f t h e " I b a l l i n d e x " (30) as a measure o f t h e 8

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F i g u r e 1. (a) Bond l e n g t h s , (b) i n t e r b o n d a n g l e s and (c) t o r s i o n a n g l e s f o r BP ( l e f t ) and DMBA ( r i g h t ) . Estimated standard d e v i a t i o n s a r e 0.002 A f o r d i s t a n c e s and 0 . 2 f o r a n g l e s . If the d i s t r i b u t i o n o f e r r o r s i s normal t h e r e i s a 99% chance t h a t a measurement w i l l d i f f e r by l e s s than 2.7 e . s . d . from t h e mean value of that quantity. A t o r s i o n angle i s the angle o f t w i s t o f a bond. In a s e r i e s o f f o u r bonded atoms, A - B - C - D , t h e t o r s i o n a n g l e about t h e B-C bond i s d e f i n e d as t h e angle o f r o t a t i o n about t h a t bond r e q u i r e d t o make t h e p r o j e c t i o n o f t h e l i n e A-B c o i n c i d e w i t h t h e p r o j e c t i o n o f t h e l i n e C-D, when viewed a l o n g t h e B-C d i r e c t i o n . In t h i s and a l l subsequent diagrams t h e l a r g e r c i r c l e s a r e carbon atoms, t h e s m a l l e r c i r c l e s a r e hydrogen atoms. In l a t e r diagrams oxygen atoms a r e s t i p p l e d and n i t r o g e n atoms a r e b l a c k .


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3-methy1cholanthrene

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11-methyl-15,16-di hydroeye 1 open ta[a_] phenanthrene

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F i g u r e 2. Views o f some c a r c i n o g e n i c m o l e c u l e s showing K- and bay- r e g i o n s . Views a r e g i v e n o f BP ( I ) , DMBA ( I I ) , 3 - m e t h y l cholanthrene ( I I I ) , ll-methyl-15,16-dihydrocyclopenta[a]phena n t h r a c e n e ( V I I ) and 5 - m e t h y l c h r y s e n e ( V I I I ) . These and a l l subsequent b a l l - a n d - s t i c k diagrams were drawn u s i n g t h e computer program VIEW ( 1 4 1 ) .



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r e l a t i v e c a r c i n o g e n i c i t y o f a g i v e n compound. T h i s index i n v o l v e d t h e p e r c e n t a g e o f c a n c e r i n mice t h a t have s u r v i v e d beyond t h e s h o r t e s t t i m e o f t h e l a t e n t p e r i o d v e r s u s t h e average l a t e n t p e r i o d . I b a l l s t u d i e d c h r y s e n e (31) and 1 , 2 - c y c l o p e n t e n o p h e n a n t h r e n e (32) s i n c e he was i n t e r e s t e d i n t h e s t r u c t u r a l s i m i l a r i t i e s between s t e r o l s , b i l e a c i d s , sex hormones, t o a d poisons and c a r c i n o g e n i c PAHsI b a l l a l s o s t u d i e d 3 - m e t h y l c h o l a n t h r e n e ( M C ) ( I I I ) ( 3 3 , 34) and b e n z o [ a ] p y r e n e (BP) (I) ( 3 5 , 3 6 ) . 7,12-Dimethylbenz[ajanthracene (DMBAT ( I I ) was s t u d i e d by Sayre and F r i e d l a n d e r ( 3 7 j , r e f i n e d by I b a l l (38) and remeasured at low t e m p e r a t u r e by Z a c h a r i a s and G l u s k e r (3|J. I n t e r e s t i n g l y , when t h e s t r u c t u r e was f i r s t r e p o r t e d t h e r e s u l t was assumed t o be e r r o n e o u s because t h e m o l e c u l e was so b u c k l e d ; i t was, o f c o u r s e , c o r r e c t ( 4 0 ) . Other PAHs s t u d i e d by c r y s t a l l o g r a p h i c t e c h n i q u e s i n c l u d e t h e weak c a r c i n o g e n b e n z [ a ] a n t h r a c e n e (BA) (IV) (41) and t h e c a r c i n o g e n i c PAH d i b e n z [ a , h ] a n t h r a c e n e (DBA) (V) ( 4 2 - 4 5 ) . It i s o f i n t e r e s t t o compare t h e shapes and s i z e s o f v a r i o u s molecules with appreciable carcinogenic a c t i v i t y . F o r example, a f l a t o x i n B] ( V I ) , found i n moldy peanuts and g r a i n , i s one o f t h e most powerful c a r c i n o g e n s known. I t s c r y s t a l s t r u c t u r e was determined by van S o e s t and Peerdeman ( 4 6 - 4 8 ) .

VI The mode o f a c t i o n o f t h i s c a r c i n o g e n i s b e l i e v e d t o i n v o l v e e p o x i d a t i o n o f a double bond ( 4 9 ) , as i n d i c a t e d i n F i g u r e 3; i n t h i s F i g u r e t h e s i m i l a r i t i e s o f t h e s h a p e s , and p a r t i c u l a r l y o f t h e s i t e s o f a c t i v a t i o n o f a f l a t o x i n , BP and DMBA a r e d e m o n s t r a t e d . Aflatoxin has f u n c t i o n a l groups at each end o f t h e m o l e c u l e , u n l i k e an a c t i v a t ed PAH which has f u n c t i o n a l groups o n l y at one end o f the m o l e c u l e . As shown i n F i g u r e 3, t h e s i z e s o f BP and DMBA a r e not o n l y s i m i l a r t o t h o s e o f a f l a t o x i n , but a l s o t o t h o s e o f base p a i r s i n DNA as noted by Haddow (50) and o f s t e r o i d s , such as e s t r a d i o l ( 5 1 ) , as noted by Huggins and Yang ( 5 2 ) . Such s t r u c t u r a l s i m i l a r i t i e s b e tween s t e r o i d s and c a r c i n o g e n i c PAH have i n t r i g u e d s c i e n t i s t s s i n c e Cook and Hazel wood c o n v e r t e d t h e s t e r o i d , d e o x y c h o l i c a c i d , t o the c a r c i n o g e n , 3 - m e t h y l c h o l a n t h r e n e . i n 1933 ( 5 3 ) . S i n c e then Coombs has demonstrated t h e c a r c i n o g e n i c i t y o f 1 1 - m e t h y l - 1 5 , 1 6 - d i h y d r o c y c 1 o p e n t a [ a ] p h e n a n t h r e n e (VII) which has an a r o m a t i z e d s t e r o i d - l i k e nucleus ( 5 4 j . The s i g n i f i c a n c e o f t h e s e s t r u c t u r a l s i m i l a r i t i e s i s not c l e a r at t h i s t i m e . In t h i s c h a p t e r we s h a l l c o n s i d e r d i s t o r t i o n s from p l a n a r i t y i n PAH systems. These d i s t o r t i o n s a r e i n t r o d u c e d by s u b s t i t u e n t methyl and o t h e r groups t h a t are t o o b u l k y t o f i t i n t h e a v a i l a b l e space and at t h e same time a l l o w t h e s u b s t i t u t e d PAH t o remain p l a n a r




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aflatoxin B

benzo[aj pyrene

7,12-di methylbenz[a] anthracene

F i g u r e 3. (a) S i m i l a r i t i e s i n t h e shapes o f BP, DMBA, a base p a i r o f guanine and c y t o s i n e , and t h e s t e r o i d , e s t r a d i o l . In (b) t h e shapes o f B and DMBA ( f i l l e d bonds) a r e compared w i t h t h a t o f a f l a t o x i n B] (open bonds) and t h e s i t e s o f a c t i v a t i o n o f each t o an e p o x i d e i s i n d i c a t e d by t h e a r r o w s . D


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YII ( a l t h o u g h i n m e t h y l - s u b s t i t u t e d PAHs a t l e a s t two o f t h e methyl hydrogen atoms must, o f c o u r s e , be out o f t h i s p l a n e ) . Most o f t h i s t y p e o f s t r a i n comes from r e p u l s i o n s between hydrogen atoms t h a t a r e not bonded t o a d j a c e n t carbon atoms. The minimum a l l o w a b l e nonbonded H...H d i s t a n c e a p p e a r s , from s t r u c t u r a l s t u d i e s , t o l i e i n t h e r e g i o n o f 1.6 A . X - r a y c r y s t a l l o g r a p h i c r e s u l t s show g r a p h i c a l l y how t h e s t r a i n t h a t o c c u r s orv methyl s u b s t i t u t i o n a d j a c e n t t o t h e b a y - r e g i o n i s accommodated w i t h i n t h e m o l e c u l e ; an i n s p e c t i o n o f F i g u r e 1 i s recommended. In t h e b a y - r e g i o n t h e C-C bond l e n g t h may be i n c r e a s e d t o 1.48 A , and i t i s usual t o see d i s t o r t i o n s o f i n t e r bond a n g l e s from t h e n o r m a l l y e x p e c t e d v a l u e s o f 1 1 8 - 1 2 2 ° . However, i n s p i t e o f t h i s s t r a i n t h e sum o f t h e a n g l e s around an sp2 carbon atom w i l l remain a t 3 6 0 ° , t h a t i s , t h e carbon r e t a i n s an e s s e n t i a l l y p l a n a r arrangement o f atoms around i t . The b u c k l i n g o f t h e m o l e c u l e from s t e r i c h i n d r a n c e a r i s e s from t o r s i o n about t h e bonds r a t h e r than from d i s t o r t i o n s from p l a n a r i t y o f s p ^ - h y b r i d i z e d carbon atoms. T h e r e f o r e an e x c e l l e n t measure o f s t r a i n i n PAHs i s p r o v i d e d by t h e C - C - C - C t o r s i o n a n g l e s , t h a t i s t h e a n g l e s o f t w i s t o f v a r i o u s C-C bonds. When a PAH i s c o m p l e t e l y p l a n a r a l l o f t h e t o r s i o n a n g l e s a r e e i t h e r 0 ° o r 1 8 0 ° . However i n the bay r e g i o n s o f some methylated PAHs, v a l u e s as h i g h as 2 3 ° i n DMBA (37-39) and 3 6 ° i n 1,12-dimethylbenzCi]anthracene (55) have been r e p o r t e d . F o r example, i n BP ( I ) , t h e r e i s s l i g h t o v e r c r o w d i n g between hydrogen atoms on CIO and CI 1 i n t h e bay r e g i o n . This overcrowding i s r e l i e v e d by a v e r y s l i g h t t w i s t o f 2 ° about t h e C17-C18 bond so t h a t t h e s e two hydrogen atoms on CIO and C l l l i e 0.04 A on e i t h e r s i d e o f t h e m o l e c u l a r p l a n e ( s e e F i g u r e 1 ) . A p a r t from t h i s the molecule i s f l a t . However, as shown i n F i g u r e 4 , DMBA i s much more b u c k l e d ; t h i s i s a r e s u l t o f t h e o v e r c r o w d i n g between CI and CI9 (where CI9 i s t h e carbon atom a t t a c h e d t o C 1 2 ) . Here t h e s t r a i n i s r e l i e v e d by a t w i s t o f 2 3 ° about t h e C13-C14 bond i n t h e bay r e g i o n , as w e l l as t w i s t s about o t h e r bonds i n t h e b a y - r e g i o n as shown i n F i g u r e 4. The a n g l e between t h e p l a n e s o f t h e two o u t e r r i n g s o f DMBA becomes 2 4 . 0 ° as a r e s u l t o f t h i s t w i s t i n g . Similar e f f e c t s a r e seen i n 5 - m e t h y l c h r y s e n e ( V I I I ) , t h e o n l y c a r c i n o g e n i c monomethylchrysene, and i t s d e r i v a t i v e s ( 5 6 - 5 8 ) . Here t h e r i n g system i s more amenable t o i n - p l a n e d i s t o r t i o n s and t h e r e f o r e t o r s i o n a n g l e s i n t h e bay r e g i o n a r e not as g r e a t as f o r DMBA (as shown i n a c o m p a r a t i v e way i n F i g u r e 5 ) . Thus we have a p i c t u r e o f a bay r e g i o n t h a t becomes d i s t o r t e d on s u b s t i t u t i o n w i t h a methyl g r o u p , a s u b s t i t u t i o n t h a t may cause i n - p l a n e or o u t - o f - p l a n e d i s t o r t i o n s . These a r e i l l u s t r a t e d i n F i g u r e 5 f o r 5 , 1 2 - d i m e t h y l c h r y s e n e ( i n - p l a n e d i s t r i b u t i o n s ) and DMBA



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5,6-dimethylchrysene F i g u r e 4. Views o f DMBA, BP, 5 , 6 - d i m e t h y l c h r y s e n e , 5 , 1 2 - d i m e t h y l c h r y s e n e and 5 - m e t h y l c h r y s e n e . These i l l u s t r a t e t h e d i s t o r t i o n s t h a t o c c u r as a r e s u l t o f s t e r i c e f f e c t s . These and many s u b s e quent r e p r e s e n t a t i o n s o f m o l e c u l a r s t r u c t u r e are s t e r e o v i e w s and may be viewed w i t h s t e r e o g l a s s e s ; a l t e r n a t i v e l y t h e r e a d e r can f o c u s h i s eyes on t h e two images u n t i l an image between them b e g i n s t o form and t h e n a l l o w h i s eyes t o r e l a x u n t i l t h e c e n t r a l image becomes t h r e e - d i m e n s i o n a l . This process c a l l s f o r patience and may t a k e a minute o r so. The r e a d e r who does not wish t o do t h i s may s i m p l y i n s p e c t one o f t h e two diagrams f o r each structure. C o n t i n u e d on next


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5-methylchrysene F i g u r e 4.

Continued.

F i g u r e 5. Bay r e g i o n geometry. The r e s u l t o f d o c k i n g t h e b a y r e g i o n o f DMBA ( f i l l e d bonds) on t o t h a t o f 5 , 1 2 - d i m e t h y l c h r y s e n e (broken bonds) i s shown ( 1 4 2 ) . In DMBA t h e 12-methyl group i s f o r c e d out o f t h e plane o f t h e r e s t o f t h e r i n g system. In 5 , 1 2 d i m e t h y l c h r y s e n e (used because more a c c u r a t e c o o r d i n a t e s a r e a v a i l a b l e than those o f 5 - m e t h y l c h r y s e n e ) t h e d i s t o r t i o n s a r e in-plane. Note t h e d i f f e r i n g o r i e n t a t i o n s o f t h e f o u r t h r i n g (upper r i g h t ) t h a t i n d i c a t e s d i f f e r e n t t y p e s o f f l e x i b i l i t y i n t h e r i n g systems o f DMBA and 5 - m e t h y l c h r y s e n e .


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1

12


VIII (out-of-plane distortions). Note how d i f f e r e n t l y t h e methyl groups (on p o s i t i o n s 5 and 12 r e s p e c t i v e l y ) l i e and a l s o t h e d i f f e r i n g l o c a t i o n o f the o u t e r r i n g s . Thus i t i s found t h a t a c a r c i n o g e n i c PAH may be a b u c k l e d m o l e c u l e , not t h e f l a t t w o - d i m e n s i o n a l m o l e c u l e d e p i c t e d i n t h e t e x t books. While BP i s c a r c i n o g e n i C . , i t s 11-methyl d e r i v a t i v e w i t h a methyl group i n t h e bay r e g i o n i s even more carcinogenic (59). Intercalation T h i s t y p e o f b u c k l i n g i s o f p a r t i c u l a r i n t e r e s t when we c o n s i d e r t h e i n t e r a c t i o n o f such c a r c i n o g e n s w i t h b i o l o g i c a l macromolecules such as DNA. DNA has a double h e l i c a l s t r u c t u r e with bases at d i s t a n c e s 3.4 A a p a r t , hydrogen-bonded i n planes p e r p e n d i c u l a r t o the h e l i x axis. By a s t r e t c h i n g o r t w i s t i n g o f t h e h e l i x i t i s p o s s i b l e t o extend t h e v e r t i c a l d i s t a n c e between t h e bases t o 6 . 8 - 7 . 0 A; a p l a n a r m o l e c u l e , with a t h i c k n e s s o f about 3 . 5 - 3 . 7 A , can then s l i p between t h e b a s e s . T h i s i s a common i n t e r a c t i o n between f l a t aromati c m o l e c u l e s such as p r o f l a v i n e o r 9 - a m i n o a c r i d i n e and DNA, and was f i r s t proposed by Lerman ( 6 0 ) . The f l a t m o l e c u l e becomes c o m p l e t e l y enveloped w i t h i n t h e h y d r o p h o b i c area o f the n u c l e i c a c i d , t h a t i s , between t h e hydrogen-bonded bases (6J_, 6 2 ) . P a u l i n g (63) l i s t s t h e van d e r Waals r a d i u s ( t h e d i s t a n c e o f c l o s e s t approach o f a n o t h e r m o l e c u l e ) o f hydrogen as 1.2 A , 2.0 A as t h e r a d i u s o f a methyl group and 1.85 A as the h a l f - t h i c k n e s s o f an a r o m a t i c m o l e c u l e . Thus t h e v e r t i c a l d i s t a n c e s between bases would have t o be f u r t h e r i n c r e a s e d i f a methyl group, which i s not f l a t and which has no i r - e l e c t r o n system, i s t o be i n t e r c a l a t e d . In p r a c t i c e , w h i l e v e r y small f l a t m o l e c u l e s such as water can i n t e r c a l a t e between bases ( 6 4 ) , so f a r no c a s e s where t h e more b u l k y methyl group i n t e r c a l a t e s have been f o u n d . S e v e r a l complexes t h a t i n v o l v e i n t e r c a l a t i o n o f an a c r i d i n e i n a p o r t i o n o f a n u c l e i c a c i d have been s t u d i e d by X - r a y c r y s t a l lographic techniques. These i n c l u d e complexes o f d i n u c l e o s i d e phosphates w i t h e t h i d i u m b r o m i d e , 9 - a m i n o a c r i d i n e , a c r i d i n e o r a n g e , p r o f l a v i n e and e l l i p t i c i n e ( 6 5 - 6 9 ) . A r e p r e s e n t a t i o n of the geometry o f an i n t e r c a l a t e d p r o f l a v i n e m o l e c u l e i s i l l u s t r a t e d i n F i g u r e 6 ( b ) ; t h i s i s a view o f t h e c r y s t a l s t r u c t u r e o f p r o f l a v i n e i n t e r c a l a t e d i n a d i n u c l e o s i d e phosphate, c y t i d y l y l - ( 3 ' - 5 ) guanos i n e (CpG) ( 7 0 , 7 J J . F o r comparison an example o f t h e s i t u a t i o n b e f o r e such i n t e r c a l a t i o n i s a l s o i l l u s t r a t e d i n F i g u r e 6 (a) by t h r e e a d j a c e n t base p a i r s found i n t h e c r y s t a l s t r u c t u r e o f a p o l y n u c l e o t i d e (72 7 3 ) . In t h i s l a t t e r s t r u c t u r e t h e v e r t i c a l d i s t a n c e ( p a r a l l e l t o t h e h e l i x a x i s ) between t h e bases i s a p p r o x i m a t e l y l

9



7.

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F i g u r e 6. (a) Views o f t h r e e base p a i r s i n t h e c r y s t a l s t r u c t u r e of a longer p o l y n u c l e o t i d e . No m o l e c u l e i s i n t e r c a l a t e d i n t h i s structure, (b) A view o f t h e i n t e r c a l a t i o n complex o f p r o f l a v i n e i n t h e s e l f - c o m p l e m e n t a r y complex o f c y t i d y l y l - ( 3 ' - 5 ' ) g u a n o s i n e .



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3.4 K compared w i t h a v a l u e o f a p p r o x i m a t e l y 6.8 A i n t h e i n t e r c a l a t e d complex. S i n c e CpG i s s e l f - c o m p l e m e n t a r y , two m o l e c u l e s may hydrogen bond t o each o t h e r i n a h e a d - t o - t a i l manner forming two s e t s o f W a t s o n - C r i c k base p a i r s , each i n v o l v i n g hydrogen bonds between c y t o s i n e and g u a n i n e . When p r o f l a v i n e i s i n t e r c a l a t e d , t h e p h o s p h o d i e s t e r backbone i s extended by i n c r e a s i n g two t o r s i o n a n g l e s [ t h o s e o f P - 0 5 * - C 5 - C 4 ' and 0 1 ' - C I ' - N 9 - C 8 i n g u a n o s i n e ] by about 6 0 ° ( 7 0 , TV). A s i m i l a r s i t u a t i o n has been found f o r p r o f l a v i n e i n t e r c a l a t e d i n CpA; a s e l f - c o m p l e m e n t a r y p a i r i s formed because c y t o s i n e i s p r o t o n a t e d i n t h i s s t r u c t u r e , and t h i s a l l o w s f o r base p a i r i n g by hydrogen bonding ( 7 4 ) . The p r o f l a v i n e t h e n i n t e r c a l a t e s i n t h e complex. However, such i n t e r c a l a t i o n i n v o l v e s t h e i n s e r t i o n o f a f l a t m o l e c u l e , w i t h a i r - e l e c t r o n s y s t e m , between t h e i r - e l e c t r o n systems o f the bases o f DNA. T h e r e f o r e a b u c k l e d m o l e c u l e a n d / o r a methyl group w i l l not f i t w e l l i n such an i n t e r c a l a t i o n mode. Thus, s i n c e t h e b u c k l e d m o l e c u l e s (with a methyl group i n t h e bay r e g i o n ) a r e more c a r c i n o g e n i C . , we c o n c l u d e d t h a t c o m p l e t e i n t e r c a l a t i o n o f the h y d r o c a r b o n between the bases o f DNA i s not a l i k e l y mechanism f o r c a r c i n o g e n i c i t y , s i n c e t h e l e s s p l a n a r m o l e c u l e s a r e more a c t i v e i n terms o f c a r c i n o g e n i c a c t i v i t y . However, as we s h a l l show l a t e r , i t i s p o s s i b l e t h a t t h e p l a n a r p o r t i o n o f t h e PAH may l i e between t h e bases o f DNA i n a s e m i - i n t e r c a l a t i o n mode; t h e methyl g r o u p s , i f p r e s e n t , p r o b a b l y do not t a k e p a r t i n t h i s s e m i - i n t e r c a l a t i o n . An example o f t h e s e i d e a s i s p r o v i d e d by c r y s t a l l o g r a p h i c s t u d i e s o f 7 - c h l o r o m e t h y l b e n z [ a ] a n t h r a c e n e (IX) and 1 2 - m e t h y l - 7 c h l o r o m e t h y l b e n z [ . a ] a n t h r a c e n e TX). These are c h l o r o m e t h y l a l k y l a t i n g agents t h a t , l i k e many o t h e r a l k y l a t i n g agents such as e p o x i d e s , a r e c a r c i n o g e n i c (75) ( a l t h o u g h t h e 12-methyl compound i s more c a r c i n o g e n i c than t h e one t h a t l a c k s a methyl group i n t h e bay r e g i o n (76, 7 7 ) ) ; t h i s i s i n l i n e with the idea that the r e a c t i v e species i s an e l e c t r o p h i l i c a g e n t , t h a t i s a p o s i t i v e charge i s d e v e l o p e d on i t . The compounds IX and X have minimal f l e x i b i l i t y ; t h e y a r e as p l a n a r as p o s s i b l e , but s t e r i c f a c t o r s cause them t o b u c k l e t o varying degrees. X - r a y d i f f r a c t i o n s t u d i e s (78-80) showed t h a t t h e compound IX, l a c k i n g a b a y - r e g i o n methyl g r o u p , i s a p p r o x i m a t e l y 9


,

IX

X

p l a n a r , a p a r t from t h e hydrogen and c h l o r i n e atoms o f the c h l o r o methyl group. This is i l l u s t r a t e d in Figure 7(a). However, f o r reasons a l r e a d y d e s c r i b e d ( t h e s t e r i c r e p u l s i o n between non-bonded hydrogen a t o m s ) , when t h e r e i s an a d d i t i o n a l methyl group at C12 (as i n X) t h e m o l e c u l e becomes markedly b u c k l e d as a r e s u l t o f



7.

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i n t e r a c t i o n s between t h e hydrogen atom on CI and t h o s e o f t h e methyl group a t t a c h e d t o C12. S i n c e t h e more b u c k l e d r i n g system i s found f o r t h e more c a r c i n o g e n i c compound i t must be c o n c l u d e d t h a t p l a n a r i t y o f t h e r i n g systems i n h a l o m e t h y l b e n z [ a ] a n t h r a c e n e s i s not a requirement f o r c a r c i n o g e n i c i t y . Thus complete i n t e r c a l a t i o n o f t h e a r o m a t i c PAH between t h e bases o f DNA, i n t h e manner d e s c r i b e d above f o r f l a t m o l e c u l e such as p r o f l a v i n e , d i d not seem t o be a l i k e l y mechanism f o r t h e c a r c i n o g e n i c a c t i o n o f t h e s e compounds. S i n c e a l k y l a t i o n and i n t e r c a l a t i o n a r e not s i m u l t a n e o u s l y p o s s i b l e f o r s t e r i c r e a s o n s , and s i n c e one m o l e c u l e i s wedge-shaped and t h e o t h e r i s f l a t t e r , i t was c o n s i d e r e d more l i k e l y t h a t t h e a c t i o n o f t h e s e compounds a r o s e from t h e i r a l k y l a t i n g a b i l i t y ; t h e y c o u l d a l k y l a t e a base o f DNA and t h e n , s i n c e t h e b u l k y a r o m a t i c h y d r o p h o b i c group would p o s s i b l y not remain p r o t r u d i n g i n t o the h y d r o p h i l i c environment, i t i s p o s s i b l e that the a r o m a t i c PAH group c o u l d then l i e i n one o f t h e grooves o f DNA. T h i s i s i l l u s t r a t e d i n F i g u r e 7(b) and ( c ) . K-region

Derivatives

O r i g i n a l l y i t was t h o u g h t , as mentioned above, t h a t t h e s i t e o f a c t i o n o f a c t i v a t e d c a r c i n o g e n i c PAHs was t h e K - r e g i o n ( 2 6 ) , a n d , i n d e e d , t h o s e PAHs w i t h e p o x i d e groups i n t h e K - r e g i o n a r e c a r c i n o genic. However, our main i n t e r e s t i s i n s t u d y i n g m e t a b o l i t e s o f t h e s e PAHs t h a t a r e i n t e r m e d i a t e s i n c a r c i n o g e n e s i s by t h e PAH, t h a t i s , the d i o l epoxides. B e f o r e t h e n o t i o n o f t h e d i o l e p o x i d e as t h e a c t i v e a g e n t , some c r y s t a l l o g r a p h i c s t u d i e s were done on K - r e g i o n d e r i v a t i v e s even though i t i s now c l e a r t h a t t h e m e t a b o l i t e s o f K - r e g i o n o x i d e s do not match t h o s e formed from t h e parent PAH i j i v i v o (81_). The s t r u c t u r a l s t u d i e s d o , however, throw some l i g h t on s t e r i c f a c t o r s t h a t a r e o p e r a t i v e f o r t h e b a y - r e g i o n d e r i v a t i v e s . At f i r s t t h r e e K - r e g i o n o x i d e s were s t u d i e d , t h o s e o f DMBA ( X I ) , BP (XII) and p h e n a n t h r e n e , t h e n o n - c a r c i n o g e n i c p a r e n t ( X I I I ) ; t h e s t r u c t u r e s a r e shown i n F i g u r e 8 . These e p o x i d e s , which were c o n s i d e r e d v e r y r e a c t i v e , were found t o remain s t a b l e both i n a i r and i n t h e X - r a y beam when i n t h e c r y s t a l l i n e s t a t e ( 8 2 , 8 3 ) .

XI

XII

XIII

The e p o x i d e r i n g i s an a p p r o x i m a t e l y symmetrical three-membered r i n g t h a t l i e s almost p e r p e n d i c u l a r t o t h e plane o f r i n g c o n t a i n i n g t h e d o u b l e bond t h a t has undergone an a d d i t i o n r e a c t i o n t o g i v e t h e e p o x i d e , and t h e hydrogen atoms s t i c k out above t h e r i n g . In gene r a l t h e bond l e n g t h s i n an e p o x i d e r i n g vary from 1.43 t o 1.46 A and t h e i n t e r b o n d a n g l e s a r e 6 0 ° . An averaged geometry o f an e p o x i d e r i n g i s shown i n F i g u r e 9. In F i g u r e 10 we show, from an




140

7-chloromethyl-12-methylbenz[a]anthracene

7-chloromethylbenz[a]anthracene (b)

base base

.CI

base base

CI

basebasebase"

(O CI

NH—base

J-HCl \—

base

F i g u r e 7. (a) Views o f 7 - c h l o r o m e t h y l - 1 2 - m e t h y l b e n z [ _ a ] a n t h r a c e n e showing t h e e x t e n t o f b u c k l i n g o f t h e m o l e c u l e and o f 7 - c h l o r o m e t h y l b e n z [ a ] a n t h r a c e n e which i s much more p l a n a r , (b) Diagram i l l u s t r a t i n g t h e e f f e c t o f i n t e r c a l a t i o n o f the c h l o r o m e t h y l PAH i n DNA. (c) Diagram i l l u s t r a t i n g t h e e f f e c t o f a l k y l a t i o n o f the e x t r a c y c l i c amino groups o f t h e bases o f DNA. S i n c e t h e more b u c k l e d m o l e c u l e i s more c a r c i n o g e n i c i t i s c o n c l u d e d t h a t a l k y l a t i o n r a t h e r than i n t e r c a l a t i o n i s t h e s i g n i f i c a n t mode o f i n t e r a c t i o n in t h i s carcinogenic process.



(d)

F i g u r e 8. The s t r u c t u r e s o f t h e K - r e g i o n o x i d e s o f DMBA (XI) (a, b and c ) , BP (XII) (d) and phenanthrene ( X I I I ) ( e ) . Views ( c ) , (d) and (e) are d i r e c t l y onto t h e p l a n e o f the e p o x i d e group. Note t h e asymmetry around t h e e p o x i d e r i n g and t h e n o n - p l a n a r i t y o f t h e a r o m a t i c r i n g system i n t h e K - r e g i o n o x i d e o f DMBA. Also note t h a t t h e 7-methyl and t h e 5 , 6 - o x i d e groups i n (a) a r e i n t h e r e l a t i v e p o s i t i o n s f o r a d i o l epoxide of a bay-region methylated d i o l epoxide. Perhaps t h e r o l e o f t h e b a y - r e g i o n methyl group i n such d i o l e p o x i d e s i s t o cause such a d i s t o r t i o n i n t h e o x i d e r i n g and so a f f e c t i t s a c t i v i t y .



142

Bond distances H n

• 1.47(2) A

X


T

/H /

O

Interbond angles

F i g u r e 9. Averaged geometry o f an e p o x i d e showing bond d i s t a n c e s and i n t e r b o n d a n g l e s . Values i n parentheses are estimated s t a n d a r d d e v i a t i o n s o f t h e s e average v a l u e s .

ketones

epoxides

ethers

F i g u r e 10. C o n t o u r s o f t h e p o s i t i o n s o f hydrogen bonded groups around k e t o n e , e p o x i d e and e t h e r g r o u p s . These c o n t o u r s are o b t a i n e d from s c a t t e r p l o t s o f such hydrogen bonding groups i n many crystal structures. Note t h a t hydrogen bonding groups approach e t h e r oxygen atoms i n a narrower range o f p o s i t i o n s than t h e y do f o r ketones o r e p o x i d e s .



7.

GLUSKER


143

a n a l y s i s o f a l l e p o x i d e s s t u d i e d by X - r a y d i f f r a c t i o n methods t o d a t e , t h e arrangement o f groups t h a t hydrogen bond t o an e p o x i d e ; such data a r e i m p o r t a n t f o r any model b u i l d i n g . An a n a l y s i s o f t h e s e d a t a showed t h a t hydrogen bonding groups tended t o l i e a l o n g t h e d i r e c t i o n s u s u a l l y a s c r i b e d t o t h e l o n e p a i r s o f t h e oxygen atom (84). In t h i s d i r e c t i o n a l i t y o f hydrogen bonding e p o x i d e s resemble ketones r a t h e r than e t h e r s , as shown i n F i g u r e 10. The a d d i t i o n o f an e p o x i d e group t o a PAH, e s p e c i a l l y i f t h e r e i s a nearby methyl g r o u p , i n c r e a s e s t h e o b s e r v e d b u c k l i n g o f t h e r i n g system. F o r example t h e angle between t h e o u t e r r i n g s o f BP i s i n c r e a s e d on e p o x i d a t i o n at t h e K - r e g i o n from 1° t o 5 ° , w h i l e t h e a n g l e between t h e o u t e r r i n g s o f DMBA i s , w i t h K - r e g i o n e p o x i d a t i o n , i n c r e a s e d from 2 4 ° t o 3 5 ° . The e p o x i d e bond C-0 bond l e n g t h s a r e 1.461(6) and 1.459(6) A f o r phenanthrene o x i d e ( t h e same w i t h i n e x p e r i m e n t a l e r r o r ) , and 1.481(4) and 1.478(4) A f o r BP o x i d e ( a g a i n symmetrical). Those f o r t h e K - r e g i o n o x i d e o f DMBA a r e 1.445(3) and 1.457(3) A ; t h e s e a r e u n e q u a l , p o s s i b l y f o r s t e r i c reasons s i n c e t h e methyl group on C7 causes s t e r i c problems i n t h i s a r e a , as shown i n Figure 11(a). It would be expected t h a t t h e l o n g e r C-0 bond, t h a t i s , C 6 - 0 , would be c l e a v e d more r e a d i l y than C 5 - 0 , and t h i s i s , i n f a c t , t h e case (see F i g u r e 1 1 c ) . F u r t h e r s t r u c t u r a l s t u d i e s on a 5 , 6 - c i s - d i o l o f DMBA (XIV) (85) showed t h e e x t e n t o f t h e s t e r i c e f f e c t t h a t t h e 7-methyl group was h a v i n g on t h e K - r e g i o n . It was found t h a t t h e 6 - h y d r o x y l group ( n e a r e s t t h e 7-methyl group) i s a x i a l w h i l e t h e 5 - h y d r o x y l group ( f u r t h e r from t h e 7-methyl group) i s e q u a t o r i a l , as shown i n F i g u r e 11(b). I f t h e 6 - h y d r o x y l group were e q u a t o r i a l i t would "bump" i n t o t h e 7-methyl group. S i n c e t h e r i n g b e a r i n g the d i o l group i s more f l e x i b l e than t h e r i n g b e a r i n g t h e 7-methyl group the s t r a i n i s accommodated by d i s t o r t i o n s at C5 and C6 as shown i n F i g u r e 1 1 ( b ) .

A n o t h e r c i s - d i o l (XV) d e r i v e d from BP was o b t a i n e d by t h e a c t i o n o f osmium t e t r o x i d e on 4 , 5 - d i hydroxy ben zo[ja] pyrene ( 8 6 ) . The h y d r o x y l group at C5a i s a x i a l and t h a t at C6 i s e q u a t o r i a l , i l l u s t r a t i n g t h e r e l a t i v e r i g i d i t y o f t h e BP r i n g system and t h e f l e x i b i l i t y at C6 o f the r i n g bearing the d i o l groups. Trans-Diols

and D i o l

Epoxides

C a r c i n o g e n e s i s by PAHs i s now b e l i e v e d t o proceed v i a " a c t i v a t i o n " t o a d i o l e p o x i d e t h a t i s c a p a b l e a l k y l a t i n g e i t h e r DNA ( 1 3 , 8 7 , 8 8 ) , o r , p o s s i b l y , a n o t h e r i n f o r m a t i o n - c o n t a i n i n g c e l l u l a r macrom o l e c u l e (such as a n u c l e a r p r o t e i n t h a t i n t e r a c t s w i t h DNA). The


144



(a)

F i g u r e 11. (a) View o f K - r e g i o n o x i d e o f DMBA. (b) View o f 5 , 6 - c i s d i o l o f DMBA. ( c ) Some c o n c l u s i o n s from t h e s t r u c t u r a l d a t a i n (a) and ( b ) . The p r e d i c t i o n i s t h a t t h e C-0 bond at C6 i s broken more r e a d i l y than t h a t at C5.


7.

GLUSKER

145



b a y - r e g i o n h y p o t h e s i s (27) suggested t h a t a d i o l e p o x i d e , w i t h an e p o x i d e group l o c a t e d a d j a c e n t t o t h e b a y - r e g i o n , may be t h e a c t i v a t e d m e t a b o l i t e o f a c a r c i n o g e n i c PAH t h a t i s i n v o l v e d i n a l k y l a t i n g the " c r i t i c a l target." We can now ask t h e q u e s t i o n s , how much d i s t o r t i o n i s t h e r e i n a b a y - r e g i o n d i o l e p o x i d e , and can any i n f o r m a t i o n on i t s c h e m i c a l r e a c t i v i t y and t h e s t e r e o c h e m i s t r y o f i t s i n t e r a c t i o n w i t h DNA be o b t a i n e d ? Our u n d e r s t a n d i n g o f t h e importance o f s t e r i c f a c t o r s was i n i t i a t e d by s t r u c t u r e d e t e r m i n a t i o n s by X - r a y d i f f r a c t i o n t e c h n i q u e s o f two t r a n s - d i o l s o f b e n z [ a ] a n t h r a c e n e ( 8 9 ) , XVI and X V I I , shown i n F i g u r e 12. The c r y s t a l s t r u c t u r e s showed d i e q u a t o r i a l

XVI

XVII

c o n f o r m a t i o n s f o r t h e h y d r o x y l groups i n t h e unhindered compound ( X V I ) , and d i a x i a l c o n f o r m a t i o n s i n t h e h i n d e r e d compound (XVII) w i t h a h y d r o x y l group a d j a c e n t t o t h e b a y - r e g i o n . In XVII t h e h y d r o x y l group would bump i n t o t h e hydrogen atom on CI2 i f t h e c o n f o r m a t i o n were d i e q u a t o r i a l ; t h i s s t e r i c e f f e c t would be f u r t h e r enhanced i f t h e r e were a methyl group on C I 2 . But what a r e t h e conformations i n s o l u t i o n ? NMR s t u d i e s showed t h a t i n XVII t h e h y d r o x y l groups were s t i l l 100% d i a x i a l i n s o l u t i o n , but t h a t f o r t h e l e s s h i n d e r e d copound, t h e h y d r o x y l groups were 30% d i a x i a l and 70% d i e q u a t o r i a l i n s o l u t i o n ( 8 9 ) . T h u s , s i m i l a r c o n c l u s i o n s , d e r i v e d from a c o n s i d e r a t i o n o f s t e r i c i n t e r a c t i o n s between non-bonded hydrogen atoms, a p p l y t o both c r y s t a l s t r u c t u r e ( i . e . , s o l i d s t a t e ) and s o l u t i o n d a t a . W h i l e t h e t r a n s d i o l s j u s t d e s c r i b e d were racemates i t has been p o s s i b l e t o use X - r a y c r y s t a l l o g r a p h i c methods t o d e t e r m i n e t h e a b s o l u t e c o n f i g u r a t i o n o f t h e isomer o f BP d i o l e p o x i d e t h a t has t h e h i g h e s t a c t i v i t y ( 9 0 ) . T h i s i s t h e method o f c h o i c e f o r t h e d e t e r m i n a t i o n o f a b s o l u t e c o n f i g u r a t i o n , and can be done u s i n g t h e anomalous s c a t t e r i n g e f f e c t o f a f a i r l y heavy atom ( i n t h i s case a b r o m o d e r i v a t i v e was s t u d i e d ) . T h i s method o f anomalous s c a t t e r i n g a n a l y s i s was o r i g i n a l l y used t o e s t a b l i s h t h e a b s o l u t e c o n f i g u r a t i o n o f t a r t a r i c a c i d (91) and o f many o t h e r c h i r a l m o l e c u l e s . The d e t e r m i n a t i o n o f t h e X - r a y m o l e c u l a r s t r u c t u r e and t h e a b s o l u t e c o n f i g u r a t i o n determination of (+)-trans-8-bromo-7-menthyloxyacetoxy7 , 8 , 9 , 1 0 - t e t r a h y d r o b e n z o [ a ] p y r e n e showed t h a t i t has t h e (7S,,8S) c o n f i g u r a t i o n ( 9 0 ) . T h i s i s shown i n F i g u r e 13; subsequent chemical r e a c t i o n s t o g i v e an e p o x i d e e s t a b l i s h t h a t (+)-benzo[a]pyrene 7 , 8 - o x i d e has t h e (7R_,8S) c o n f i g u r a t i o n . T h i s i n f o r m a t i o n then c o u l d be u s e d , w i t h chemical c o r r e l a t i o n s , t o e s t a b l i s h t h e a b s o l u t e c o n f i g u r a t i o n o f t h e most a c t i v e d i o l e p o x i d e o f BP ( X V I I I ) .



146


1,2-diol F i g u r e 12. The s t r u c t u r e s o f two t r a n s d i o l s o f b e n z [ a ] a n t h r a c e n e showing t h e d i e q u a t o r i a l c o n f o r m a t i o n o f t h e u n h i n d e r e d 1 0 , 1 1 - d i o l and t h e d i a x i a l c o n f o r m a t i o n o f t h e h i n d e r e d 1 , 2 - d i o l . These t r e n d s p e r s i s t i n s o l u t i o n where t h e 1 0 , 1 1 - d i o l e x i s t s as an e q u i l i b r i u m o f 30% a x i a l and 70% e q u a t o r i a l conformers ( t h a t i s , t h e r i n g i s f l e x i b l e ) ; on t h e o t h e r hand t h e 1 , 2 - d i o l i s 100% d i a x i a l even i n s o l u t i o n . I f t h e 1-hydroxyl group were e q u a t o r i a l i t would "bump" i n t o t h e hydrogen atom on CI2. C o n t i n u e d on next


page.


7.

GLUSKER


10,11-diol F i g u r e 12.

Continued.

American Chemical Society Library 1155 loth St. N. W. In Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R.; Washington. D. C.Society: 20036 ACS Symposium Series; American Chemical Washington, DC, 1985.

147


(0-7R.8S the more active isomer (0-BP-7,8-oxide F i g u r e 13. Absolute c o n f i g u r a t i o n of benzo[a]pyrene metabolites determined by anomalous d i s p e r s i o n ( X - r a y d i f f r a c t i o n ) s t u d i e s o f a bromoderivative.


7.

GLUSKER

149



There a r e f o u r s t e r e o i s o m e r s o f t h e d i o l e p o x i d e o f BP (XVIII XXI). Two (XVIII and XIX) have t h e e p o x i d e oxygen atom below t h e p l a n e o f BP and t h e o t h e r two (XX and XXI) have t h e oxygen above t h i s plane. In a d d i t i o n t h e 7 - h y d r o x y l group may be on t h e same (syn) s i d e (XIX and XXI) as t h e e p o x i d e oxygen atom o r on t h e oppos i t e s i d e ( a n t i ) (XVIII and X X ) . A l s o t h e t r a n s h y d r o x y l groups may be d i a x i a l o r d i e q u a t o r i a l , and t h i s may be demonstrated by c r y s t a l l o g r a p h i c and o t h e r , such as NMR, s t u d i e s ( 9 2 , 9 3 ) .

OH

OH (+>anti XVIII

(->anti XX

(+)-syn XIX

(->syn XXI

The s t r u c t u r e o f t h e a n t i - d i o l e p o x i d e o f BP ( X V I I I ) was d e t e r m i n e d by N e i d l e and c o - w o r k e r s ( 9 4 ) ; t h e s t r u c t u r e i s shown i n F i g u r e 14, t o g e t h e r w i t h t h e s t r u c t u r e o f t h e d i o l ( b e f o r e e p o x i d e f o r m a t i o n ) and t h e t e t r o l (opening o f t h e epoxide r i n g ) . The two h y d r o x y l groups i n t h e d i o l e p o x i d e a r e d i e q u a t o r i a l (as shown t o be t h e case i n s o l u t i o n by NMR s t u d i e s ) and t h e e p o x i d e r i n g l i e s i n a p l a n e n e a r l y p e r p e n d i c u l a r t o t h e PAH system. O t h e r analogous e p o x i d e s with one o r two h y d r o x y l groups i n t h e same r i n g ( X X I I , X X I I I ) have s i m i l a r c o n f o r m a t i o n s ( 9 5 , 96) ( F i g u r e 1 5 ) . The f i r s t s t r u c t u r e o f a syn-compound was done by X - r a y d i f f r a c t i o n on a naphthalene d e r i v a t i v e w i t h a methyl group i n p l a c e o f one o f t h e h y d r o x y l group (XXIV) ( 9 7 ) . Here t h e f o r m a t i o n o f an i n t e r n a l hydrogen bond, as p r e d i c t e d by H u l b e r t ( 9 8 ) , i s o b s e r v e d . However, t h i s hydrogen bond i s not t h e cause o f t h e presence o f a x i a l g r o u p s , but r a t h e r t h e r e s u l t , s i n c e i f t h e 7 - h y d r o x y group i s r e p l a c e d by a methoxy g r o u p , as i n a dimethoxy e p o x i d e (XXV), t h e c o n f o r m a t i o n o f t h e h y d r o x y l groups i s a l s o d i a x i a l ( 9 9 ) . These s t r u c t u r e s a r e i l l u s t r a t e d i n F i g u r e 16. In t h e ( ± ) - a n t i - t e t r a h y d r o b e n z o [ a ] p y r e n e d i o l e p o x i d e t h e r i n g b e a r i n g t h e e p o x i d e and d i o l has a C8 h a l f c h a i r c o n f o r m a t i o n (with C8 as t h e atom most out o f t h e p l a n e ) . In t h e s y n - h y d r o x y e p o x i d e s (XXIV and XXV) t h e r i n g has a s i m i l a r c o n formation. A s y n - d i o l e p o x i d e (XIX) s t u d i e d by N e i d l e ( 1 0 0 ) , i l l u s t r a t e d i n F i g u r e 17, was shown by X - r a y s t u d i e s t o have e q u a t o r i a l




150

F i g u r e 14. ( a , b ) Views o f t h e s t r u c t u r e o f an a n t i - d i o l e p o x i d e o f BP ( 9 4 ) . Note t h e d i e q u a t o r i a l c o n f o r m a t i o n o f t h e h y d r o x y l groups, ( c ) A d i h y d r o d i o l o f BP and (d) a t e t r o l o f BP, a l l d e t e r m i n e d by N e i d l e and c o - w o r k e r s .




7. GLUSKER

F i g u r e 16. S t r u c t u r e s o f a n a l o g s o f t h e s y n - d i o l e p o x i d e , (a) G l u s k e r , Z a c h a r i a s , Whalen and c o - w o r k e r s ( 9 7 ) , (b) K l e i n and Stevens ( 9 9 ) .



152

0

H0

X

equatorial OH

OH

equatorial


equatorial XXII

XXIII

0

o

CH 0 3

axial OH

OCH

axial

axial

XXIV

3

XXV

h y d r o x y l g r o u p s , even though p o t e n t i a l energy c a l c u l a t i o n s had i n d i c a t e d t h a t t h i s might be a h i g h e r energy form. The e p o x i d e s i n F i g u r e s 14 t o 16 show two g e n e r a l c o n f o r m a t i o n s , o f which t h e more common i s shown i n F i g u r e 18 ( a ) . Since the r e s t o f t h e PAH i s p l a n a r , or n e a r l y s o , t h e f i r s t bonds i n t h e s a t u r a t e d r i n g b e a r i n g t h e d i o l e p o x i d e must a l s o l i e a p p r o x i m a t e l y i n t h a t plane. The e p o x i d e w i l l a l s o l i e n e a r l y i n t h e p l a n e , but w i t h C9 ( o r i t s e q u i v a l e n t ) s l i g h t l y above o r below t h i s p l a n e . However, o n l y t h e carbon atom e q u i v a l e n t t o C8 i n BP d i o l e p o x i d e i s pushed f a r out o f t h e p l a n e o f t h e PAH (see F i g u r e 18 ( b ) ) . As d i s c u s s e d by Whalen and c o - w o r k e r s ( 1 0 1 , 1 0 2 ) , i n t h e a n t i - and s y n - d i o l e p o x i d e s o f BP, t h e major d i f f e r e n c e s i n t h e c o n f o r m a t i o n s o f d i o l e p o x i d e s i n v o l v e o r i e n t a t i o n o f t h e e p o x i d e group w i t h r e s p e c t t o the p l a n a r PAH p a r t o f t h e m o l e c u l e ; they used t h e r e l a t i v e o r i e n t a t i o n o f t h e b e n z y l i c C-0 bond t o t h e i r - o r b i t a l s o f t h e a r o m a t i c r i n g s (101) as a measure ( r e f e r r e d t o as " a l i g n e d " o r " n o n a l i g n e d " ) . These i r - o r b i t a l s l i e p e r p e n d i c u l a r t o t h e p l a n e o f t h e PAH. X-ray d a t a g i v e a more d e t a i l e d measure o f t h i s f o r t h e d i o l e p o x i d e s o f BP, as shown i n T a b l e I. In t h e s y n - i s o m e r ( " a l i g n e d " ) t h e 0-C10 bond i s n e a r l y p e r p e n d i c u l a r t o t h e p l a n e o f t h e PAH ( t o r s i o n a n g l e 9 8 ° ) ; i n t h e a n t i - i s o m e r " n o n a l i g n e d " i t i s at about 4 0 ° t o t h i s p l a n e ( t o r s i o n a n g l e 4 9 ° ) (see F i g u r e 18 ( c ) ) . These r e s u l t s can now be used t o c o n s i d e r what happens when a d i o l e p o x i d e a t t a c k s DNA. The e p o x i d e group w i l l open and t r a n s addition w i l l occur. The p r o d u c t (XXVI) w i l l have t h e DNA s u b s t i t u t e d a d j a c e n t t o t h e bay r e g i o n ( p a r t i c u l a r l y i f i t i s h i n d e r e d so t h a t t h e e p o x i d e group i s made more r e a c t i v e ) and w i l l l i e a x i a l t o t h e PAH r i n g system. T h i s means t h a t t h e p l a n e o f t h e PAH and t h e a l k y l a t e d base o f DNA must have a p e r p e n d i c u l a r r e l a t i o n s h i p t o each o t h e r as i n d i c a t e d i n F i g u r e 19. In t h i s F i g u r e t h o s e s i t e s i n



7.

GLUSKER


F i g u r e 17. Views o f t h e s t r u c t u r e o f a s y n - d i o l d e t e r m i n e d by N e i d l e and c o - w o r k e r s ( 1 0 0 ) .

e p o x i d e o f BP

(a)

anti-isomer

syn-isomer

syn-isomer

F i g u r e 18. C o n f o r m a t i o n s o f d i o l e p o x i d e s o f BP. (a) Diagrams o f t h e a n t i - and s y n - i s o m e r s o f a d i o l e p o x i d e showing t h e p o s s i b i l i t y f o r i n t e r n a l hydrogen-bonding i n t h e s y n - d i o l e p o x i d e . (b) C o n f o r m a t i o n s o f t h e r i n g s i n a p o r t i o n o f t h e a n t i - and s y n - d i o l e p o x i d e s o f BP showing t h e d i f f e r e n t r i n g c o n f o r m a t i o n s . ( c ) R e l a t i v e o r i e n t a t i o n o f t h e e p o x i d e group and t h e PAH system i n d i o l e p o x i d e s o f BP. „ C o n t i n u e d on next page.




154

F i g u r e 18.

Continued.



7.

T a b l e I.

Torsion

155


GLUSKER

Angle

0-9-8-7 0-10-10A-6A 8-9-10-10A 9-10-10A-6A 10-10A-6A-7 10A-6A-7-8 6A-7-8-9 7-8-9-10 8-9-10-10A

T o r s i o n Angles

in Diol

Anti-BPDE 35 -49* 2 19 -7 -25 44 -34 2

Epoxides

Syn-BPDE -111 98** -1 29 -12 -33 59 -45 1

o f BP

T

aromatic dihydroxy epoxide

*"aligned" **"nonaligned" Source:

Adapted from R e f s .

93 and 99.


10A

ring group


156


F i g u r e 19. P e r p e n d i c u l a r i t y o f t h e PAH and t h e b a s e . Conformations of t r a n s - d i o l s . The s t e r i c h i n d r a n c e i n a bay r e g i o n w i l l cause h y d r o x y l o r o t h e r s u b s t i t u e n t s (such as DNA) t o l i e i n an a x i a l o r i e n t a t i o n . S i t e s where t h i s o c c u r s a r e marked "a". Those s i t e s where t h e r e i s no s t e r i c h i n d r a n c e are marked " a e " ( a x i a l or e q u a t o r i a l ) . The c o n c l u s i o n i s t h a t when a d i o l e p o x i d e a l k y l a t e s DNA t h e base on DNA w i l l be bonded a x i a l l y t o t h e PAH group.


7.

GLUSKER

157


c a r c i n o g e n i c m o l e c u l e s t h a t a r e h i n d e r e d a r e i n d i c a t e d by " a " ( a x i a l ) , w h i l e t h o s e t h a t a r e unhindered a r e d e s i g n a t e d " a e " ( e i t h e r conformer p o s s i b l e ) ,

DNA

I


OH XXVI Interactions

of A c t i v a t e d Carcinogens

with

Polypeptides

S i n c e our i n t e r e s t i s i n t h e s t e r e o c h e m i c a l r e s u l t o f a l k y l a t i o n o f a b i o l o g i c a l m a c r o m o l e c u l e , we have s t u d i e d models o f both p r o t e i n s and n u c l e i c a c i d s . In p r o t e i n s t h e r e a r e s e v e r a l ways i n which t h e p o l y p e p t i d e c h a i n can f o l d ; o f t h e s e t h e most common o r g a n i z e d f o l d i n g i s t h e a l p h a - h e l i x (103) and t h e b e t a - p l e a t e d sheet ( 1 0 4 ) . Other p a r t s o f t h e p o l y p e p t i d e may f o l d i n a random manner. The s t e r e o c h e m i c a l e f f e c t o f i n t r o d u c i n g a l a r g e h y d r o p h o b i c group i n t o a p r o t e i n has been demonstrated (105) by t h e d e t e r m i n a t i o n s o f t h e c r y s t a l structures of the t r i p e p t i d e s a r c o s y l g l y c y l g l y c i n e , CH NH2CH C0NHCH2C0NHCH C02 ( X X V I I ) , and i t s a l k y l a t e d p r o d u c t (XXVIII). The a r o m a t i c a l k y l a t i n g agent (XXIX) c r y s t a l l i z e d w i t h 3

2

2

XXVIII

XXIX

i t s a r o m a t i c r i n g systems s l i g h t l y o v e r l a p p i n g . The t r i p e p t i d e (XXVII) c r y s t a l l i z e d with an i n t r i c a t e t h r e e - d i m e n s i o n a l network o f hydrogen bonds, but no water o f c r y s t a l l i z a t i o n . The a l k y l a t e d t r i p e p t i d e ( X X I X ) , however, c r y s t a l l i z e d i n a " b i l a y e r " t y p e s t r u c t u r e as shown i n F i g u r e 20. The a r o m a t i c groups packed i n much t h e same way t h a t t h e y d i d i n t h e s i m p l e a l k y l a t i n g agent ( X X I X ) , and t h e p e p t i d e groups formed s h e e t s h e l d t o g e t h e r by hydrogen bonds. Hydrogen bonding between t h e s e s h e e t s o f p e p t i d e s i n v o l v e d a d d i t i o n a l water o f c r y s t a l l i z a t i o n . W h i l e t h e s i m p l e t r i p e p t i d e had t o r s i o n a n g l e s near t h o s e expected f o r an a l p h a - h e l i x , on a l k y l a t i o n t h e t o r s i o n a n g l e s changed t o l i e more n e a r l y t h o s e e x p e c t e d f o r a b e t a pleated sheet. Thus a s i m p l e model was p r o v i d e d o f t h e p o s s i b l y l o s s o f a l p h a - h e l i c i t y on a l k y l a t i o n by a b u l k y h y d r o p h o b i c group.



F i g u r e 20. C r y s t a l s t r u c t u r e o f an a l k y l a t e d p e p t i d e , (a) A l k y l a t e d p e p t i d e s t r u c t u r e showing t h e n o n - p l a n a r p e p t i d e group (upper r i g h t o f d i a g r a m ) , (b) P a c k i n g i n t h e c r y s t a l s t r u c t u r e of the a l k y l a t e d peptide. T h i s diagram shows t h e s e g r e g a t i o n o f t h e v a r i o u s p o r t i o n s o f t h e s t r u c t u r e i n t o areas o c c u p i e d by a r o m a t i c g r o u p s , a r e a s o c c u p i e d by t h e more h y d r o p h i l i c p e p t i d e g r o u p s , and t h e i n t r o d u c t i o n o f water o f c r y s t a l l i z a t i o n i n t o the s t r u c t u r e .



7.

GLUSKER


159

I t might a l s o be e x p e c t e d t h a t the a r o m a t i c p o r t i o n o f the PAH a l k y l a t i n g agent w i l l tend t o l i e i n t h e i n t e r i o r o f the p r o t e i n , as f a r as p o s s i b l e from t h e h y d r o p h i l i c s u r f a c e and i t s aqueous e n v i r o n ment. Thus t h e r e s u l t o f a l k y l a t i o n can be e x t e n s i v e and can l e a d t o a l o s s o f any f u n c t i o n , such as c a t a l y t i c a c t i v i t y ( i f the p r o t e i n i s an enzyme) a n d / o r l o s s o f a b i l i t y t o r e c o g n i z e an attachment s i t e or another macromolecule. A n o t h e r i n t e r e s t i n g p o i n t a r i s i n g from t h i s s t r u c t u r e d e t e r m i n a t i o n was t h a t t h e p e p t i d e became d i s t o r t e d on a l k y l a t i o n ; t h e p e p t i d e group f a r t h e s t removed from t h e s i t e o f a l k y l a t i o n had a n o n - p l a n a r p e p t i d e group ( t o r s i o n angle o f 1 5 9 ° i n s t e a d o f t h e expected v a l u e o f 1 8 0 ° ) . T h i s may have been caused by t h e c r y s t a l p a c k i n g , s i n c e i f t h a t p e p t i d e group were p l a n a r a c a r b o x y l group would p r o j e c t i n t o an a r e a o f t h e c r y s t a l o c c u p i e d by t h e a r o m a t i c groups. Presumably l e s s energy was l o s t i n d i s t o r t i n g t h e p e p t i d e group than i n r e v i s i n g t h e p a c k i n g o f t h e a r o m a t i c g r o u p s . A s i m i l a r s i t u a t i o n c o u l d be e n v i s i o n e d t o o c c u r i n b i o l o g i c a l systems where t h e r e a r e e x t e n s i v e h y d r o p h o b i c areas t h a t might s e q u e s t e r a PAH g r o u p ; such a d i s t o r t i o n might a l s o r e n d e r t h e p e p t i d e group more l i a b l e t o a t t a c k by o t h e r a g e n t s . However, u n t i l t h e n a t u r e o f t h e r e l e v a n t a l k y l a t e d p r o t e i n i s known, i t i s not p o s s i b l e t o deduce t h e d i r e c t e f f e c t s o f a l k y l a t i o n by an a c t i v a t e d c a r c i n o g e n . Interactions

of A c t i v a t e d Carcinogens

with

Nucleotides

In a s i m i l a r way t h e e f f e c t o f a l k y l a t i o n o f DNA by an a c t i v a t e d PAH may be d r a s t i C . , as t h e h y d r o p h o b i c a r o m a t i c group o f t h e a l k y l a t i n g agent t r i e s t o a v o i d the aqueous environment o f the n u c l e i c a c i d . The e x a c t n a t u r e o f t h e l e s i o n i n DNA i s unknown, and so i s t h e t y p e o f DNA t h a t i s a t t a c k e d . Recent X - r a y c r y s t a l l o g r a p h i c s t u d i e s , as w e l l as o t h e r p h y s i c o c h e m i c a l s t u d i e s , have made i t c l e a r t h a t DNA i s not s i m p l y a p o l y n u c l e o t i d e , f o l d e d as Watson and C r i c k (106) proposed. There a r e t h r e e main c o n f o r m a t i o n a l t y p e s o f DNA; they each keep t h e hydrogen-bonded bases i n t h e c e n t e r o f t h e h e l i x , but may t i l t them by a " p r o p e l l o r t w i s t , " may s l i d e them from t h e c e n t e r o f t h e h e l i x i n t h e p l a n e o f t h e base p a i r s , and may v a r y t h e amount o f r o t a t i o n from one base p a i r t o t h e next up t h e h e l i c a l a x e s . These v a r i a t i o n s a r e l i s t e d i n T a b l e II and r e p r e s e n t d a t a from t h e X-ray structures of various polynucleotides. The t h r e e main c o n f o r m a t i o n a l t y p e s a r e B-DNA (which i s l i k e Watson and C r i c k ' s r i g h t handed h e l i c a l model and was demonstrated i n t h e s t r u c t u r e o f a dodecamer CGCGAATTCGCG, 107, 108, F i g u r e 21 ( a ) ) , A-DNA ( a n o t h e r r i g h t - h a n d e d h e l i c a l form o f DNA t h a t has some o f t h e c h a r a c t e r i s t i c s o f RNA, 109, 110, F i g u r e 21 (b)) and Z-DNA (a c o n f o r m a t i o n found f o r n u c l e i c a c i d s c o n s i s t i n g o f a l t e r n a t i n g p u r i n e - p y r i m i d i n e sequences and at h i g h s a l t c o n c e n t r a t i o n s , with a l e f t - h a n d e d h e l i c a l c o n f o r m a t i o n , 111, 112, F i g u r e 21 ( c ) ) . Other i m p o r t a n t c o n f o r m a t i o n a l parameters o f n u c l e i c a c i d s are t h e s u g a r pucker and t h e c o n f o r m a t i o n about t h e g l y c o s i d i c l i n k a g e . The p u c k e r i n g o f t h e five-membered sugar r i n g i n r i b o s e or deoxyr i b o s e d e r i v a t i v e s , i s u s u a l l y d e s c r i b e d with r e s p e c t t o t h e plane through t h e f o u r most p l a n a r atoms and then d e f i n i n g how t h e f i f t h atom (one o f 0 1 ' , C I , C 2 ' , C3' o r C 4 ' ) l i e s with r e s p e c t t o t h i s p l a n e ; i f t h i s f i f t h atom l i e s on t h e same s i d e o f the p l a n e as does 1



160


T a b l e II.

Three Major C o n f o r m a t i o n s

Handedness

of

Base p a i r s

per t u r n

Axial rise residue

per n u c l e o t i d e

Degrees

helix

per

van d e r Waals d i a m e t e r Sugar pucker

Glycosidic

B-DNA

A-DNA

Z-DNA

Right

Right

Left

10

11

12

3.37 Jt

of winding

bond

36° 19.3

K

3.7 &

32.7°

-30°

23

K

18.2 X

C3'-endo

C3'-endo or CI'-exo

anti

anti

anti a l t e r nating with syn

groove

deep and wide

Minor

groove

shal1ower

See R e f s .

K

2.56

C2'-endo

Major

Note:

o f DNA

113 and 114 f o r more

v e r y deep

very

shallow

shallow

v e r y deep

details.



7.

X-ray Analyses of Polycyclic

GLUSKER

161

Hydrocarbons

C 5 ' , then t h e atom i s s a i d t o be endo, and i f i t i s on t h e o p p o s i t e s i d e i t i s d e f i n e d as e x o . The c o n f o r m a t i o n about t h e s u g a r - b a s e l i n k a g e i s d e f i n e d as a n t i when t h e t o r s i o n a n g l e ( c h i , 0 4 ' - C T N T - C 2 f o r p y r i m i d i n e s and 0 4 ' - C I ' - N 9 - C 4 f o r p u r i n e s ) l i e s near 180° and syn when i t l i e s near 0 ° . These s i t u a t i o n s a r e i l l u s t r a t e d i n F i g u r e 22. The g l y c o s i d i c l i n k a g e a r e a l l a n t i i n B-DNA and A-DNA, but i n Z-DNA t h e guanine bases a r e i n t h e syn c o n f o r m a t i o n . In F i g u r e 23 t h e s i t e s on DNA a c c e s s i b l e t o an a l k y l a t i n g agent a r e shown. In t h e major groove o f B-DNA 06 and N7 o f g u a n i n e , N4 o f c y t o s i n e , N6 and N7 o f a d e n i n e and 04 o f thymine a r e a v a i l a b l e f o r alkylation. In t h e minor groove o f B-DNA N2 o f g u a n i n e , 02 o f c y t o s i n e and o f thymine and N3 o f adenine a r e a v a i l a b l e f o r a l k y l a t i o n . The amino groups N2 on guanine and N6 on adenine a r e t h e commonest s i t e s o f i n t e r a c t i o n w i t h halomethyl PAH a l k y l a t i n g agents ( 7 5 , 7 6 ) ; t h i s i m p l i e s a t t a c k o f guanine i n t h e minor groove and adenine i n t h e major groove o f B-DNA. However t h e groove s i z e v a r i e s with o t h e r c o n f o r m e r s o f n u c l e i c a c i d s ( 1 1 3 , 114) as shown i n T a b l e II. In B-DNA t h e major groove i s deep and wide and t h e minor groove i s narrower and s h a l l o w ; i n A-DNA t h e major groove i s d e e p e r ; i n Z-DNA o n l y t h e minor groove r e m a i n s . So t h e r e a r e d i f f e r e n t p o s s i b l i t i e s f o r accommodating a bulky PAH group f o r each c o n f o r m a t i o n a l type o f DNA. Very i n t e r e s t i n g i n f o r m a t i o n r e l e v a n t t o t h e s t e r e o c h e m i c a l r e s u l t s o f a l k y l a t i o n o f DNA comes from s t u d i e s o f n u c l e o s i d e s a l k y l a t e d by a c t i v a t e d PAHs. E i g h t such s t r u c t u r e s have been r e p o r t e d (115-118). Three are products o f the i n t e r a c t i o n o f chloromethyl PAHs w i t h N6 o f a d e n o s i n e XXX-XXXII, two w i t h deoxyadenosine ( X X X I I I , XXXIV), two a r e p a r a - s u b s t i t u t e d benzyl d e r i v a t i v e s o f g u a n o s i n e , a l k y l a t e d at 0-6 (XXXV, XXXVI) (117) and one i s an a c e t y l ami n o f l uorene d e r i v a t i v e o f g u a n o s i n e , a l k y l a t e d at C8 (XXXVII)

(Hi). The general scheme f o r p r e p a r a t i o n o f t h e a l k y l a t e d a d e n o s i n e and deoxyadenosine i n v o l v e d t h e i n t e r a c t i o n o f t h e a p p r o p r i a t e aminohydrocarbon with a 6 - c h l o r o p u r i n e r i b o s i d e (116). This allowed f o r t h e most s p e c i f i c i n t e r a c t i o n w i t h o u t t o o many b y p r o d u c t s . The m o l e c u l a r s t r u c t u r e s d e t e r m i n e d from d i f f r a c t i o n s t u d i e s a r e shown i n F i g u r e s 24 and 25. In F i g u r e 24 t h e shape o f an a l k y l a t e d n u c l e o s i d e (115) i s compared w i t h t h a t o f t h e u n a l k y l a t e d f o r m ,

XXX

XXXI

XXXII




162



7.

GLUSKER


F i g u r e 21.

Continued.

C3'-endo

2- "

5-

C2'-endo

4' 3' 5'

3

V

'

N C3'-endo,

4'

anti

2'

C2'-exo

r

syn

F i g u r e 22. (a) R i b o s e sugar c o n f o r m a t i o n s i l l u s t r a t i n g the meanings o f C 3 ' - e n d o , C 2 ' - e n d o and C2'-_endo, C 3 ' - e x o . (b) G l y c o s i d i c bond c o n f o r m a t i o n s o f r i b o s i d e s showing a n t i and s y n - c o n f o r m a t i o n s .


164


major groove


\

—N2

\

minor groove

major groove

minor groove F i g u r e 2 3 . S i t e s on DNA a c c e s s i b l e t o a l k y l a t i n g a g e n t s . Oxygen and n i t r o g e n atoms i n t h e major and minor grooves a r e i n d i c a t e d .



7.

GLUSKER


F i g u r e 24. Comparison o f t h e s t r u c t u r e s o f (a) deoxyadenosine and (b) an a l k y l a t e d d e r i v a t i v e . Note t h e syn c o n f o r m a t i o n o f t h e product o f a l k y l a t i o n .


166



(a)

F i g u r e 25. Shapes o f f i v e a l k y l a t e d d e r i v a t i v e s o f adenosine and d e o x y a d e n o s i n e . (a) Adenosine d e r i v a t i v e o f DMBA ( X X X I I ) . (b) Deoxyadenosine d e r i v a t i v e o f DMBA (XXXIV). No hydrogen atom p o s i t i o n s f o r t h i s d i a g r a m , ( c ) Adenosine d e r i v a t i v e o f d i m e t h y l a n t h r a c e n e (XXXI). Note t h e d i f f e r e n t c o n f o r m a t i o n . (d) Deoxyadenosine d e r i v a t i v e o f d i m e t h y l a n t h r a c e n e ( X X X I I I ) . (e) A d e n o s i n e d e r i v a t i v e o f methyl a n t h r a c e n e (XXX). C o n t i n u e d on next page.



Figure

25.

Continued. C o n t i n u e d on next


page.



F i g u r e 25.

Continued.


GLUSKER


169


7.

XXXVII deoxyadenosine ( 1 1 9 ) . Note t h a t i n t h e a l k y l a t e d n u c l e o s i d e t h e adenine and PAH r e s i d u e s l i e n e a r l y p e r p e n d i c u l a r t o one a n o t h e r . I t can be seen t h a t a l k y l a t i o n has changed t h e s u g a r - b a s e c o n f o r m a t i o n ( d i s t a n t from t h e s i t e o f a l k y l a t i o n ) from a n t i (as i n deoxya d e n o s i n e and i n B-DNA) t o s,y_n (as i n a l k y l a t e d deoxyadenosine and i n some bases i n Z-DNA). The sugar puckers a r e e i t h e r C 2 ' - e n d o o r C3'-endo. A second s t r u c t u r a l f e a t u r e o f g r e a t i n t e r e s t i n t h e s e c r y s t a l s i s t h a t t h e more p l a n a r a n t h r a c e n e p o r t i o n o f t h e PAH i s s t a c k e d between adenine r e s i d u e s o f o t h e r m o l e c u l e s t h r o u g h o u t t h e crystal. The h i g h l y b u c k l e d r e g i o n o f t h e PAH does not t a k e p a r t



170


i n t h i s s t a c k i n g , but i s p o s i t i o n e d out o f t h e way, as shown i n F i g u r e 26. T h i s general scheme o f p a c k i n g i s found i n a l l f i v e adenine d e r i v a t i v e s s t u d i e d by d i f f r a c t i o n methods; t h e PAH and adenine a r e i n t e r l e a v e d , but t h e hydrogen bonding arrangements i n t h e c r y s t a l s o f d i f f e r e n t d e r i v a t i v e s (XXX-XXXIV) a r e d i f f e r e n t . T h i s l e d us t o suppose t h a t t h i s s t a c k i n g i s a s i g n i f i c a n t i n t e r a c t i o n . Interesti n g l y i n f o u r o f t h e f i v e compounds t h e r e i s t h e i n t r a m o l e c u l a r hydrogen bond between t h e 5 ' - h y d r o x y l group and a d e n i n e , but i n t h e f i f t h compound t h i s hydrogen bond i s not found i n t h e c r y s t a l s t u d i e d , even though t h e c o n f o r m a t i o n i s s t i l l s y n . This indicates that the f o r m a t i o n o f t h i s i n t e r n a l hydrogen bond i s not n e c e s s a r y f o r the a d o p t i o n by t h e m o l e c u l e o f t h e s y n - c o n f o r m a t i o n . F i n a l l y there seems t o be a weak (but p o s s i b l y s i g n i f i c a n t ) i n t e r a c t i o n between t h e r i b o s e r i n g oxygen atom o f one m o l e c u l e and t h e carbon atom o f a 12-methyl group i n t h e 12-methyl b e n z [ a ] a n t h r a c e n y l d e r i v a t i v e ; such i n t e r a c t i o n s have been noted i n o t h e r c r y s t a l s t r u c t u r e s (84, 108, 111, 1 2 0 ) . Such an i n t e r a c t i o n , i l l u s t r a t e d i n F i g u r e 27, may be i m p o r t a n t i n a i d i n g i n t h e p o s i t i o n i n g o f t h e PAH p o r t i o n o f t h e adduct w i t h i n DNA. The two O ^ - a l k y l a t e d guanosine d e r i v a t i v e s ( 1 1 7 ) , shown i n F i g u r e 2 8 , do not demonstrate a p e r p e n d i c u l a r i t y o f hydrocarbon and b a s e , presumably because t h e hydrocarbon i s much s m a l l e r . However, a g a i n t h e n u c l e o s i d e has t h e s y n - c o n f o r m a t i o n . This perpendicul a r i t y , however, i s found f o r t h e a c e t y l a m i n o f l u o r i n e d e r i v a t i v e . Computer M o d e l l i n g

of DNA-Activated Carcinogen

Interactions

The computer has p r o v i d e d an e x c e l l e n t medium f o r m o d e l l i n g t h e i n t e r a c t i o n s o f such a c t i v a t e d PAHs w i t h p o r t i o n s o f DNA. As ment i o n e d e a r l i e r , i t i s g e n e r a l l y b e l i e v e d t h a t t h e e x t r a c y c l i c base amino g r o u p s , N2 o f g u a n i n e , and more r a r e l y N6 o f a d e n i n e , a r e a t t a c k e d by a c t i v a t e d PAHs. T h e r e are two g e n e r a l c l a s s e s o f models f o r DNA a l k y l a t e d by a d i o l e p o x i d e . In t h e f i r s t t h e PAH l i e s i n one o f t h e grooves o f DNA but on t h e p e r i m e t e r o f t h e h e l i x (1211 2 8 ) ; t h i s i s r e f e r r e d t o as " e x t e r n a l b i n d i n g . " The p y r e n e - l i k e chromophore o f b e n z o [ a ] p y r e n e d i o l e p o x i d e l i e s at about 3 5 ° t o t h e DNA h e l i x a x i s . T h i s i s i n l i n e w i t h model b u i l d i n g e x p e r i m e n t s by us i n which we " d o c k e d " t h e a l k y l a t e d deoxyadenosine compounds s t u d i e d (XXX-XXIV) onto t h e s^n-base (guanine) i n Z-DNA. We used Z-DNA because we had found a s y n - c o n f o r m a t i o n f o r t h e a l k y l a t e d adenosines and deoxyadenosines and Z-DNA c o n t a i n s guanine r e s i d u e s i n t h e s y n conformation. It meant t h a t an adenine group was " d o c k e d " onto a guanine r e s i d u e ; t h e r e s u l t i s t h a t major groove a l k y l a t i o n (on 06 o f guanine o r N6 o f a d e n i n e ) i s m o d e l l e d . The r e s u l t i s shown i n F i g u r e 29. A r o t a t i o n o f 180° about t h e C6-N6 bond (which i s f r e e t o r o t a t e ) t o t h e PAH p l a c e d t h e PAH on t h e s u r f a c e o f t h e h e l i x and d i d not d i s r u p t normal i n t e r b a s e hydrogen bonding w i t h i n t h e h e l i x i n any way. Some o t h e r r o t a t i o n a n g l e s about t h i s bond l e d t o unacceptable s t e r i c i n t e r a c t i o n s . The i n t e r e s t i n g f e a t u r e o f t h e r o t a t e d model i s t h a t t h e c u r v a t u r e o f t h e d i m e t h y l b e n z [ j a ] a n t h r a c e n e m o i e t y conforms t o some e x t e n t t o t h e shape o f t h e h e l i x . Attempts t o dock t h e a l k y l a t e d bases i n t o B-DNA l e d t o more s t e r i c problems. Such model b u i l d i n g by o t h e r s (129-131) has l e d t o t h e s u g g e s t i o n


GLUSKER


171


7.

F i g u r e 26. S t a c k i n g o f PAHs and bases i n a l k y l a t e d adenosine and deoxyadenosine d e r i v a t i v e s , (a) The m e t h y l a n t h r a c e n y l d e r i v a t i v e of adenosine, (b) The DMBA d e r i v a t i v e o f deoxyadenosine viewed onto t h e planes o f adenine g r o u p s , ( c ) S i d e view o f t h i s , (d) Diagram o f o v e r l a p o f a DMBA d e r i v a t i v e i n t h e adenosine and deoxyadenosine adducts A r denotes t h e a r o m a t i c r i n g and pu indicates purine. Areas o f o v e r l a p a r e b l a c k . C o n t i n u e d on next


page.



172

F i g u r e 27. I n t e r a c t i o n o f a r i b o s e oxygen atom w i t h t h e methyl group i n a n o t h e r m o l e c u l e o f an a l k y l a t e d n u c l e o s i d e . Two s h o r t l i n e s i n d i c a t e the i n t e r a c t i o n .



7.

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173


F i g u r e 28. guanosine.

The s t r u c t u r e o f an 06 a l k y l a t e d

derivative of


174



(a)

F i g u r e 29. Docking o f an a l k y l a t e d base i n t o Z-DNA and t o r s i o n o f t h e PAH group, ( a , b) Two views o f t h e a l k y l a t e d base (as d e t e r m i n e d by X - r a y d i f f r a c t i o n t e c h n i q u e s ) docked onto the s y n guanine group i n Z-DNA. (C., d) Two views o f the same m o d e l l i n g experiment with a t o r s i o n a l r o t a t i o n o f 180 about C6-N6. Note how t h e c u r v a t u r e o f t h e PAH group conforms t o t h a t o f the h e l i x . T h i s i s a model o f t h e c a r c i n o g e n l y i n g i n a groove o f DNA. C o n t i n u e d on next


page.

GLUSKER



7.

F i g u r e 29.

Continued.


175


176


t h a t t h e b e n z o [ a ] p y r e n e adduct f o r m a t i o n c o u l d promote a l o c a l B - t o - A DNA t r a n s i t i o n , s i n c e A-DNA has a w i d e r but l e s s deep minor groove i n t o which t h e a r o m a t i c p o r t i o n c o u l d f i t . An a l t e r n a t i v e m o d e l , i n which t h e PAH i s i n t e r n a l l y bound ( " q u a s i - i n t e r c a l a t e d " ) , i s shown i n F i g u r e 30. Presumably some d i s t o r t i o n must o c c u r i f a PAH i s both c o v a l e n t l y bound t o a base and l i e s between t h a t base and i t s n e i g h b o r i n g base up t h e DNA h e l i x axis. T h i s i s i l l u s t r a t e d i n F i g u r e 31 which shows f i r s t a d i o l e p o x i d e docked onto B-DNA and then t h e e f f e c t o f t r y i n g t o b r i n g t h e a r o m a t i c group between t h e b a s e s ; because i t i s c o v a l e n t l y a t t a c h e d some d i s t o r t i o n o f bases from planes a p p r o x i m a t e l y p e r p e n d i c u l a r t o t h e h e l i x a x i s must o c c u r . One attempt t o r e s o l v e t h i s problem l e d t o t h e s u g g e s t i o n t h a t t h e DNA becomes k i n k e d at t h i s p o i n t ( 1 3 8 ) . However t h e r e i s a l s o p h y s i c a l e v i d e n c e f o r t h i s k i n d o f b i n d i n g (132-138). Our X - r a y r e s u l t s on the i n t e r l e a v i n g o f adenine and PAH a l s o a r e i n l i n e w i t h t h i s m o d e l , and suggest t h a t t h e r e are f o r c e s t h a t f a v o r at l e a s t p a r t i a l i n t e r c a l a t i o n . To date we do not have enough i n f o r m a t i o n t o be a b l e t o t e l l what t h e l e s i o n i n DNA would be on a l k y l a t i o n i n t h e a p p r o p r i a t e p o s i t i o n o f DNA ( t h a t i s , t h e one t h a t i s c a r c i n o g e n i c a l l y a c t i v e ) . Attempts t o overcome t h i s a r e i n p r o g r e s s . A s y n t h e t i c heptameric d u p l e x d(GTCA*GAC):d(GTCTGAC) p r e p a r e d by Stezowski and c o l l e a g u e s (139) has an a d e n o s i n e group (A*) i n t h e c e n t e r o f one s t r a n d a l k y l a t e d by a PAH group. Attempts a r e i n p r o g r e s s t o make a minor groove a d d u c t . The r e p o r t o f t h e c r y s t a l s t r u c t u r e o f a s y n t h e t i c o l i g o n u c l e o t i d e duplex w i t h a T : T mismatch (140) shows no l o s s o f h e l i c i t y but m o d i f i e d (wobble) base-pairing. So we a r e s t i l l l e f t w i t h two models o f t h e s t e r e o c h e m i s t r y o f DNA a l k y l a t e d by a PAH d i o l e p o x i d e ; t h e PAH e i t h e r l i e s i n a groove o f DNA o r e l s e t r i e s t o i n t e r c a l a t e between t h e bass o f DNA. Since i t i s c o v a l e n t l y bonded t o a base i t must cause c o n s i d e r a b l e d i s t o r t i o n i f i t t r i e s t o l i e between t h e b a s e s . However, t h e s t a c k i n g observed i n t h e c r y s t a l l i n e s t a t e seems t o argue f o r partial intercalation. We w i l l need c r y s t a l s t r u c t u r e s o f at l e a s t one a p p r o p r i a t e l y a l k y l a t e d p o l y n u c l e o t i d e b e f o r e t h i s problem can be r e s o l v e d . And when t h i s i s done i t w i l l be j u s t t h e b e g i n n i n g o f t h e answer t o t h e problem o f a l k y l a t i o n o f DNA by a c t i v a t e d c a r c i n o gens. The subsequent q u e s t i o n i s , what i s t h e l e s i o n i n DNA t h a t i s i m p o r t a n t i n c a r c i n o g e n e s i s , and then what does i t cause t o happen so t h a t tumor f o r m a t i o n i s i n i t i a t e d ? Acknowledgments I thank D r s . H. L. C a r r e l l , A. D i p p l e , R. G. Harvey, S. H e c h t , R. M o s c h e l , J . J . S t e z o w s k i , and D. E. Z a c h a r i a s f o r many h e l p f u l d i s c u s s i o n s and c o l l a b o r a t i o n s . Most o f t h e diagrams were drawn u s i n g t h e computer programs VIEW (141) and DOCK ( 1 4 2 ) . F i g u r e 21 was drawn w i t h d a t a o b t a i n e d from t h e P r o t e i n Data Bank, Brookhaven. The work o f t h e a u t h o r was s u p p o r t e d by g r a n t s CA-10925, CA-22780, CA-06927, RR-05539 from t h e N a t i o n a l I n s t i t u t e s o f H e a l t h , BC-242 from t h e American Cancer S o c i e t y , and by an a p p r o p r i a t i o n from t h e Commonwealth o f P e n n s y l v a n i a .


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(a)

(b)

sugar

1

1

sugar

sugar

(c)

sugar

sugar

sugar

F i g u r e 30. Schematic diagrams o f o v e r l a p o f r i n g systems i n (a) t h e a l k y l a t e d n u c l e o s i d e d e s c r i b e d h e r e , (b) daunomycin i n t e r c a l a t e d i n a h e x a n u c l e o t i d e ( 1 4 3 ) , (c) p r o f l a v i n e i n t e r c a l a t e d i n a d i n u c l e o s i d e phosphate (70)TTd) a model o f a d i o l e p o x i d e o f DMBA s e m i - i n t e r c a l a t e d i n a manner analogous t o t h a t i n (a) so t h a t N2 o f guanine may be a l k y l a t e d , (e) a model o f a d i o l e p o x i d e o f DMBA s e m i - i n t e r c a l a t e d i n a manner analogous t o t h a t i n (c) so t h a t 06 o f guanine may be a l k y l a t e d . C o n t i n u e d on next


page.



sugar

F i g u r e 30.

Continued.


7.

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(a)

F i g u r e 3 1 . Schematic diagrams o f a l k y l a t i o n o f DNA by a d i o l e p o x i d e by (a) s e m i - i n t e r c a l a t e d (viewed from a b o v e ) , (b) s e m i - i n t e r c a l a t e d (viewed from t h e s i d e ) . The guanine i n t h e lower r i g h t o f t h e diagram w i l l be a l k y l a t e d when t h e e p o x i d e r i n g opens, ( c ) a l k y l a t i o n o f a guanine w i t h t h e PAH l y i n g i n a groove o f DNA, and (d) t h e problem t h a t r e s u l t s when a c o v a l e n t bond i s formed. The base and PAH group cannot both l i e i n p l a n e s perpendicular to the helix axis. T h e r e f o r e , i f t h e PAH i s s e m i - i n t e r c a l a t e d , as shown, t h e base must d i s t o r t out o f t h e p l a n e o f t h e base p a i r w i t h subsequent d i s t o r t i o n s along t h e helix. V a r i o u s models have suggested k i n k s o r c o n f o r m a t i o n a l changes t o a c c o u n t f o r t h i s . One way i n which t h i s might o c c u r i s i l l u s t r a t e d i n t h i s schematic diagram. The c o n f o r m a t i o n t h a t a c t u a l l y r e s u l t s i n r e a l i t y on such a l k y l a t i o n i s s t i l l unknown. C o n t i n u e d on next


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1985


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