pyrene Diol Epoxides to DNA - American Chemical Society

10 A Mechanism for the Stereoselectivity and Binding of Benzo[a]pyrene Diol Epoxides to D N A Downloaded by UNIV QUEENSLAND on May 14, 2013 | http://pubs.acs.org Publication Date: July 19, 1985 | doi: 10.1021/bk-1985-0283.ch010

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KENNETH J. MILLER, ERIC R. TAYLOR , and JOSEF DOMMEN Department of Chemistry, Rensselaer Polytechnic Institute, Troy, NY 12181

A molecular model is proposed for stereoselectivity during the process of covalent binding of (+)-trans7,8- dihydroxy- anti-9,10- epoxy-7,8,9,10- tetrahydro benzo[a]pyrene, denoted by (+)-anti-BPDE or BPDE I(+); (-)-trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene denoted by (-)-anti-BPDE or BPDE I I ( - ) , and their mirror images (-)-anti-BPDE or BPDE I ( - ) , and (+)-syn-BPDE or BPDE II(+) to DNA. Inter calation of the BPDEs places the reactive epoxide near N2 on guanine (G), 06 on G, N6 on adenine (A), and N4 on cytosine (C). A proton catalyzed nucleophilic S 2 reaction between C10 of the BPDEs and base atoms and thus trans addition is favored during this step. These positive H atoms reside in the most negative regions of the DNA, the grooves. The DNA must kink for proper hybridization to occur during intercalative covalent binding between C10 and the base atoms. Stereoselectivity occurs during this step. The I(+) and II(+) isomers are selected by N2(G), the I(-) and II(-) isomers by 06(G), N6(A), and II(+) and I(-) by N4(C). Reorganization of the DNA places BPDE I(+) and II(+) outside the helix (most l i k e l y the minor groove for the I(+)-N2(G) adduct), whereas the I(-) and II(-) remain intercalatively covalently bound. A less favored proton addition via an SN1 reaction forms the carbonium ion of the BPDEs which allows both trans and cis base adducts. Cis addition accounts for minor products which are the I(-) and II(-) isomers with N2(G), the I(+) and II(+) isomers with N6(A) and 06(G) and II(-) and I(+) by N4(C). A combined analysis of experimental results and theoretical modeling calcula tions is presented with detailed stereographic molecular structures. N

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1Currentaddress: Department of Chemistry, University of Southwestern Louisiana, Lafayette, LA 70504 0097-6156/85/0283-0239S13.00/0 © 1985 American Chemical Society

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

POLYCYCLIC HYDROCARBONS AND CARCINOGENESIS

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A combined e x p e r i m e n t a l and t h e o r e t i c a l approach has been undertaken i n many r e s e a r c h l a b o r a t o r i e s t o u n d e r s t a n d t h e r e l a t i o n s h i p between t h e c a r c i n o g e n i c i t y , m u t a g e n i c i t y , and s t r u c t u r e o f t h e p o l y n u c l e a r a r o m a t i c hydrocarbons (PAHs). A l t h o u g h any r e l a t i o n s h i p may be more c o m p l i c a t e d than t h e emerging models, i t appears t h a t t h e s t u d i e s o f one p a r t i c u l a r well-known c a r c i n o g e n , b e n z o [ a ] p y r e n e , BP, a r e ex t e n s i v e enough t o permit a somewhat comprehensive p r o p o s a l f o r t h e p h y s i c a l b i n d i n g o f i t s d i o l e p o x i d e s , BPDEs, t o DNA. One t o f o u r o f t h e d i a s t e r e o i s o m e r s have been i n v o l v e d i n e x t e n s i v e r e s e a r c h . They a r e denoted by BPDE l ( + ) f o r t h e ( + ) - t r a n s - T , 8 - d i h y d r o x y - a n t i 9 , 1 0 - e p o x y - T , 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 o r (+)-anti-BPDE and by BPDE I I ( - ) f o r t h e ( - ) - t r a n s - T , 8 - d i h y d r o x y - s y n - 9 , 1 0 - e p o x y - 7 , 8 , 9 , 1 0 tetrahydrobenzo[a]pyrene o r (-)-syn-BPDE i n F i g u r e 1. The BPDE i ( - ) and BPDE I l ( + ) a r e m i r r o r images o f t h e l ( + ) and I l ( - ) i s o m e r s , r e s p e c t i v e l y . The CIO p o s i t i o n i s i n v o l v e d i n adduct f o r m a t i o n w i t h N2(G) and i t i s assumed t o be i n v o l v e d w i t h N6(A), 06(G) and NMc).

BPDE I(+)

BPDE I I ( - )

(+)-anti-BPDE

(-)-syn-BPDE F i g u r e 1.

BPDE i s o m e r s .

T h i s paper p r e s e n t s a u n i f i c a t i o n o f t h e o r e t i c a l c a l c u l a t i o n s and e x p e r i m e n t a l r e s u l t s i n a proposed model f o r t h e s t e r e o s e l e c t i v i t y o f t h e BPDEs by DNA. I t i s d i v i d e d i n t o f i v e major s e c tions: The E x p e r i m e n t a l Background p r e s e n t s a l i t e r a t u r e r e v i e w o f t h e c u r r e n t s t a t u s o f t h e s t r u c t u r a l and c o n f o r m a t i o n a l i n t e r p r e t a t i o n o f t h e c h e m i c a l and p h y s i c a l r e s u l t s f o r BPDE-DNA adduct f o r m a t i o n . The Approach t o t h e T h e o r e t i c a l M o d e l i n g o u t l i n e s t h e o v e r a l l c o n s i d e r a t i o n s o f t h e problem. The e n e r g e t i c s o f forming r e c e p t o r s i t e s , b i n d i n g o f t h e BPDEs t o DNA and d e f i n i t i o n s o f t h e energy e x p r e s s i o n s used i n t h e c a l c u l a t i o n s a r e d i s c u s s e d i n Steps i n t h e Binding Process. The Mechanism f o r BPDE-DNA Adduct Formation con t a i n s an a n a l y s i s o f each s t e p i n t h e proposed model. I t consti t u t e s t h e major p a r t o f t h e paper. R e s u l t s from t h e l i t e r a t u r e , as w e l l as from o u r l a b o r a t o r y , a r e used t o support t h e mechanism and t o i n t e r p r e t the experimental r e s u l t s a t a molecular l e v e l . This



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major s e c t i o n , w h i c h c o n t a i n s t h e study o f a l l s t e p s i n t h e mecha nism, i s subdivided i n t o s i x subsections: benzo-ring conformations and h y b r i d i z a t i o n o f BPDEs and adducts r e p o r t t h e dominant local conformations w h i c h must be c o n s i d e r e d . The p o s s i b i l i t y of r e h y b r i d i z a t i o n o f amino groups i s c o n s i d e r e d t o i n s u r e t h a t t h e p r o p e r h y b r i d i z a t i o n about CIO o f t h e BPDE and t h e amino n i t r o g e n atoms o f t h e bases a r e used. Quantum m e c h a n i c a l c a l c u l a t i o n s are r e p o r t e d i n t h e s e two s u b s e c t i o n s and t h e y a r e compared w i t h e x p e r i mental r e s u l t s . Then t h e m o d i f i c a t i o n s o f t h e DNA a r e examined i n r e c e p t o r s i t e s i n which c l a s s i c a l i n t e r c a l a t i o n , k i n k e d DNA f o r s t e r e o s e l e c t i v i t y and r e a r r a n g e d DNA t o t h e f i n a l states o f e x t e r n a l l y and i n t e r n a l l y bound adducts a r e c o n s i d e r e d . Molecular mechanics methods a r e used t o o b t a i n t h e r e s u l t s r e p o r t e d i n t h e s e four subsections. A summary o f t h e e n t i r e mechanism, t h e s t e r e o s e l e c t i v i t y o f BPDEs f o r t r a n s and c i s a d d i t i o n p r o d u c t s and p r e d i c t i o n s and suggestions f o r e x p e r i m e n t a l i s t s are presented i n the f i n a l major s e c t i o n D i s c u s s i o n and C o n c l u s i o n . Stereographic pro j e c t i o n s o f adducts i n v o l v i n g a l l f o u r BPDE isomers are presented. They i l l u s t r a t e t h e p r o c e s s o f s t e r e o s e l e c t i v i t y by k i n k e d DNA and t h e f i n a l o r i e n t a t i o n o f t h e pyrene moiety w h i c h depends on t h e p a r t i c u l a r BPDE-isomer i n v o l v e d .

E x p e r i m e n t a l Background The c a r c i n o g e n i c i t y o f PAHs i n v o l v e s t h e i r c o n v e r s i o n t o a c t i v e m e t a b o l i t e s ( l _ ) , and t h e i r subsequent r e a c t i o n w i t h n u c l e i c a c i d s (2-22). An organism o r organ may produce a unique s e t o f m e t a b o l i t e s , each o f w h i c h may be c a r c i n o g e n i c t o t h e host o r t o o t h e r systems. These m e t a b o l i t e s may be c l a s s i f i e d as f o l l o w s (2325.): P r i m a r y o r u l t i m a t e c a r c i n o g e n s , i . e . , compounds t h a t a r e c h e m i c a l l y or b i o l o g i c a l l y a c t i v e by v i r t u e of t h e i r s p e c i f i c s t r u c t u r e and c o n f o r m a t i o n . Secondary o r p r o c a r c i n o g e n s , i . e . , compounds w h i c h a r e c h e m i c a l l y o r b i o l o g i c a l l y i n e r t , but undergo c o n v e r s i o n s w h i c h t r a n s f o r m them t o u l t i m a t e c a r c i n o g e n s . In a d d i t i o n , t h e PAHs o r t h e i r m e t a b o l i t e s may be promoting-agents o r c o - c a r c i n o g e n s , i . e . , non-carcinogens w h i c h s e r v e t o p o t e n t i a t e t h e e f f e c t o f e i t h e r a p r i m a r y o r secondary c a r c i n o g e n . Research i n t h i s a r e a has p r o ceeded a l o n g s e v e r a l p a t h s : ( i ) The i d e n t i f i c a t i o n o f m e t a b o l i t e s o f PAHs i n b i o l o g i c a l systems, e x p e c i a l l y t h e arene o x i d e s , d i h y d r o d i o l s and t e t r a h y d r o d i o l - e p o x i d e s . ( i i ) The d e t e r m i n a t i o n o f t h e c a r c i n o g e n i c i t y and m u t a g e n i c i t y o f t h e s e compounds. ( i i i ) The i d e n t i f i c a t i o n o f adducts formed w i t h DNA and t h e d e t a i l s o f t h e s t r u c t u r e s and c o n f o r m a t i o n s o f the complexes. I t appears t h a t co v a l e n t bond f o r m a t i o n between an a c t i v e compound and c e l l u l a r macrom o l e c u l e s may be an i m p o r t a n t s t e p i n t h e c a r c i n o g e n i c event (2,23-

26). The importance o f t h e r o l e o f DNA i n t h e g e n e t i c code has f o cused r e s e a r c h on t h e s t r u c t u r e o f t h e a c t i v e compound and t h e o v e r a l l c o n f o r m a t i o n o f DNA as a r e c e p t o r . In a d d i t i o n t h e i d e n t i f i c a t i o n o f benzo[a]pyrene a s an i m p o r t a n t c a r c i n o g e n (27) has s t i m u l a t e d e x t e n s i v e r e s e a r c h t o determine t h e o r i g i n and i t s activity.



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T h i s p r o c a r c i n o g e n undergoes m e t a b o l i c c o n v e r s i o n t o benzo[a]pyrene d i o l e p o x i d e s , BPDEs (5,28-31), w h i c h have been t h e focus o f s t r u c t u r a l and c o n f o r m a t i o n a l s t u d i e s by t h e o r e t i c a l and e x p e r i m e n t a l methods. These c h e m i c a l l y r e a c t i v e BPDEs a r e i n v o l v e d i n c o v a l e n t b i n d i n g t o DNA (13-22). I n a d d i t i o n , c o v a l e n t bond f o r m a t i o n t o t h e NT r i n g n i t r o g e n i n guanine w i t h r i n g opening and s t r a n d s c i s s i o n , and p h o s p h o r y l a t i o n , w h i c h i s a minor r e a c t i o n pathway, a r e two o t h e r r e a c t i o n p r o c e s s e s w h i c h have been i d e n t i f i e d (8-11,32,33). However, t h e a n a l y s i s o f a d d u c t s t o DNA w i t h HPLC t e c h n i q u e s e n t a i l s h y d r o l y s i s w i t h a sub sequent l o s s o f a l l but t h e BPDE bonded t o t h e amino n i t r o g e n o f t h e b a s e s , t h e r e b y f o c u s i n g a t t e n t i o n on these amino groups a s a c t i v e s i t e s (13-22). B i n d i n g t o N2 on guanine i s most p r e v a l e n t , and t h i s has r e c e i v e d much a t t e n t i o n (13-20). Of p a r t i c u l a r i n t e r e s t i s t h e s t r u c t u r e o f BPDE l ( + ) bound t o N2 on g u a n i n e . To d a t e , no c r y s t a l s t r u c t u r e has been r e p o r t e d . The p r o d u c t s r e s u l t i n g from t h e BPDE l(+)-N2(G) r e a c t i o n w i t h n u c l e i c a c i d s are m o s t l y t r a n s w i t h a minor component o f c i s a d d i t i o n (13-1T). In r e a c t i o n s o f racemic BPDE Il(±) w i t h p o l y ( G ) the t r a n s adduct c o n s t i t u t e s t h e major component (22)• I t i s b e l i e v e d t h a t t h e s t r u c t u r e and c o n f o r m a t i o n o f DNA and t h e adduct may e x p l a i n t h e t r a n s a d d i t i o n and s t e r e o s e l e c t i v i t y of t h e BPDE l ( + ) t o t h e DNA. Meehan and Straub (3h) and P u l k r a b e k e t a l . (35) have examined t h e s t e r e o s e l e c t i v i t y o f BPDE l ( + ) and i ( - ) by DNA. BPDE l ( + ) b i n d s most e x t e n s i v e l y t o guanine w i t h minor com ponents i n v o l v i n g adenine and c y t o s i n e . BPDE i ( - ) b i n d s most e x t e n s i v e l y t o adenine w i t h minor components i n v o l v i n g guanine. They (3*0 f i n d t h a t the l e v e l o f c o v a l e n t b i n d i n g t o N2 on guanine i s o f t h e same o r d e r as t h e mutagenic a c t i v i t y between the (+) and (-) a n t i d i o l epoxides i n mammalian c e l l s (36). This c o r r e l a t e s with t h e s t e r e o s e l e c t i v i t y o f BPDE l ( + ) f o r N2TG) (3T). I n another study Brookes and Osborne (3^) f i n d t h a t the i n d u c t i o n o f 8-azaguanine r e s i s t a n t mutants i n Chinese hamster VT9 c e l l s by BPDE l ( + ) and i ( - ) depends on the e x t e n t o f r e a c t i o n w i t h DNA and t o the r e a c t i o n products. For e q u a l e x t e n t s o f r e a c t i o n b o t h were e q u a l l y c y t o t o x i c but BPDE l ( + ) was more mutagenic. The e x t e n t o f b i n d i n g o f the l ( + ) isomer i s 9k% a t N2(G), k% t o u n i d e n t i f i e d p o s i t i o n s on G and 2% t o N6(A), whereas t h e e x t e n t o f b i n d i n g o f t h e I ( - ) isomer i s 59% t o N2(G), 21% t o 06(G), 18% t o N6(A) and 2% w h i c h may be 06(G) (8). However, t h e r e l a t i v e b i n d i n g t o DNA o f l ( + ) and i ( - ) i s 10:1 (3*0. They p o s t u l a t e t h a t t h e d i f f e r e n c e between t h e l ( + ) and I ( - ) isomers r e s u l t s from t h e d i f f e r e n c e s i n s p a t i a l orientation. We show t h a t s t e r e o s e l e c t i v e b i n d i n g t o N2(G) (3T), N6(A) and 06(G) can o c c u r f o r t r a n s a d d i t i o n d u r i n g t h e i n t e r c a l a t i v e c o v a l e n t b i n d i n g mode, and t h a t a minor component from c i s ad dition y i e l d s a complementary s t e r e o s e l e c t i v e component. We p o s t u l a t e that e x t e r n a l b i n d i n g r e s u l t s through r e l a x a t i o n (de n a t u r a t i o n and r e n a t u r a t i o n ) o f the DNA w h i c h a l l o w s the pyrene m o i e t y t o a d j u s t i t s e l f t o one o f the g r o o v e s , but a combination o f t h e s e p r o c e s s e s may o c c u r . G r e a t e r i n s i g h t i n t o t h e c o n f o r m a t i o n a l s t r u c t u r e o f the r e c e p t o r s i t e ( s ) i s gained by a s e r i e s o f p h y s i c a l measurements. L e f k o w i t z et a l . (39) performed an o p t i c a l l y d e t e c t e d magnetic r e s o -



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nance s t u d y t o show t h a t t h e BPDE l(±) c o v a l e n t adduct t o DNA l i e s o u t s i d e the h e l i x , i . e . , that i t i s not i n t e r c a l a t e d . P r u s i k and coworkers (UP,Ul) f i n d w i t h f l u o r e s c e n c e measurements t h a t t h e BPDEs are bound e x t e r n a l l y . D r i n k w a t e r e t a l . (1*2) demonstrate t h e u n w i n d i n g o f s u p e r c o i l e d SVl+0 DNA by c o v a l e n t b i n d i n g o f a s e r i e s o f hydrocarbon epoxides. A l t h o u g h not n e c e s s a r y f o r t h e i r work, t h e y i m p l i e d use o f a c l a s s i c a l i n t e r c a l a t i o n h y p o t h e s i s by comparison w i t h t h e e f f e c t b y e t h i d i u m bromide. Namely, p a r a l l e l base p a i r s s e p a r a t e d by 6.8 A w i t h t h e BPDE p a r a l l e l t o t h e base p a i r s and s t a c k e d a t 3.1+ A. C o n s e q u e n t l y , r e a c t i o n w i t h t h e e x o c y c l i c amino groups (13-22) o f G, A, and C p r e s e n t s a problem w i t h t h e i n t e r p r e t a t i o n o f a r e a s o n a b l e C l O - n i t r o g e n bond l e n g t h and bond a n g l e s f o r p r o p e r h y b r i d i z a t i o n about CIO and t h e amino group (j*JjjM0 . Subse quent measurements o f t h e o r i e n t a t i o n o f t h e pyrene m o i e t y and o f the base p a i r s demonstrated t h a t a c l a s s i c a l i n t e r c a l a t i o n site i s not i n v o l v e d . I n f a c t , f o r t h e c o v a l e n t l y bound adduct D r i n k w a t e r et a l . (1*2) and Gamper e t a l . (1*5) o b t a i n u n w i n d i n g a n g l e s o f 22°±3° and 30°-35°, r e s p e c t i v e l y , f o r r e l a x e d DNA and Meehan e t a l . (1*6) i n t e r p r e t t h e i r u n w i n d i n g a n g l e o f 13° f o r t h e p h y s i c a l l y bound BPDE l ( + ) ( r e v e r s i b l y bound i n a c l a s s i c a l i n t e r c a l a t i o n site). Thus BPDE unwinds DNA i n two ways: by c l a s s i c a l i n t e r c a l a t i o n and b y i n t e r c a l a t i v e covalent binding. In o r d e r t o determine t h e o r i e n t a t i o n o f t h e pyrene m o i e t y and t h e base p a i r s r e l a t i v e t o t h e average h e l i x a x i s s e v e r a l approaches have been t a k e n . G e a c i n t o v and coworkers 0*7) have found w i t h e l e c t r i c l i n e a r d i c h r o i s m s p e c t r a an upper l i m i t o f 35° f o r t h e o r i e n t a t i o n between t h e pyrene chromophore and t h e e l e c t r i c f i e l d v e c t o r . The a n g l e s o b t a i n e d r e f l e c t average o r i e n t a t i o n s o f t h e l o n g a x i s o f the pyrene m o i e t y r e l a t i v e t o average o r i e n t a t i o n s o f a l l o f t h e DNA b a s e s . The DNA t e n d s t o a l i g n i t s e l f a l o n g t h e e l e c t r i c f i e l d . In o r d e r t o i n t e r p r e t t h e e x p e r i m e n t a l r e s u l t s w i t h a p h y s i c a l model, i t has been assumed t h a t , on t h e a v e r a g e , t h e h e l i c a l a x i s i s a l i g n e d a l o n g t h e e l e c t r i c f i e l d v e c t o r . W i t h an upper l i m i t o f 35° t h e y proposed a model i n w h i c h t h e pyrene chromophore i s o r i e n t e d on the o u t s i d e o f t h e DNA. To e x p l a i n t h i s o r i e n t a t i o n s e v e r a l t h e o r e t i c a l models were proposed i n i t i a l l y . B e l a n d (1*8) and l a t e r J e f f r e y e t a l . (1*9) a d j u s t e d t h e BPDE l ( + ) adduct t o N2TG) t o f i t i n the minor groove o f B-DNA. A l t h o u g h t h e l o n g a x i s o f t h e pyrene m o i e t y y i e l d s an o r i e n t a t i o n r e l a t i v e t o t h e h e l i x a x i s in quali t a t i v e agreement ( c a . 50°-60°) w i t h t h e e x p e r i m e n t a l v a l u e o f 35°, the s t e r i c contacts are extremely poor. To r e s o l v e t h i s p r o b l e m , e i t h e r t h e DNA must be a d j u s t e d , i . e . , unwound (1*3) o r bent (1*1*) t o p e r m i t a b e t t e r f i t , o r a new r e c e p t o r s i t e must be p r o p o s e d . In a n o t h e r r e c e n t s t u d y on t h e o r i e n t a t i o n o f base p a i r s i n c a l f thymus DNA m o d i f i e d by c o v a l e n t l y bound BPDE l ( + ) Hogan, e t a l . (50) have shown t h a t t h e h e l i c a l axes o f DNA segments a r e n o t o r i e n t e d along the e l e c t r i c f i e l d . The o r i e n t a t i o n o f t h e h e l i x a x i s o f DNA segments i n t h e e l e c t r i c f i e l d y i e l d s y(DNA) = 29° a t 0.0*4 a d d u c t s p e r base p a i r , and t h e l o n g a x i s o f t h e pyrene i n t h e BPDE a d d u c t i s o r i e n t e d a t ot(BPDE) =1+3° from t h e h e l i x a x i s o f m o d i f i e d DNA regions. T h i s v a l u e a g r e e s w i t h t h a t o f 35° measured b y G e a c i n t o v e t a l . (kj) f o r an " a v e r a g e " h e l i x a x i s o f DNA a l i g n e d



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w i t h t h e e l e c t r i c f i e l d . The problem o f i n t e r p r e t a t i o n i s d i s c u s s e d by T a y l o r e t a l • (kk). Thus, Hogan e t a l • (j>0) p o s t u l a t e d a k i n k e d r e c e p t o r s i t e w i t h an i n t e r c a l a t i v e c o v a l e n t l y bound BPDE l ( + ) i n c o n t r a s t t o G e a c i n t o v and coworkers (kj) who p o s t u l a t e e x t e r n a l binding. A l t e r n a t e i n t e r p r e t a t i o n s o f t h i s d a t a may i n c l u d e l o c a l d e n a t u r a t i o n o f t h e duplex o r some form o f DNA damage. However, i f t h e DNA remains double s t r a n d e d a t t h e s i t e o f c o v a l e n t adduct f o r m a t i o n , then a model f o r t h e s t e r e o s e l e c t i v i t y o f t h e BPDE can be presented. T h e o r e t i c a l l y determined r e c e p t o r s i t e s f o r k i n k e d DNA were generated by T a y l o r e t a l . (kk) t o model t h i s t y p e o f b i n d i n g . The k i n k e d DNA accommodates t h e c o v a l e n t b o n d i n g r e g i o n w i t h t e t r a h e d r a l and t r i g o n a l h y b r i d c o n f i g u r a t i o n s about t h e CIO o f BPDE l ( + ) and N2(G), r e s p e c t i v e l y . However, t h e l o n g a x i s o f t h e pyrene 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 h e l i c a l a x i s , i . e . , ot(BPDE) > 80° i n a l l s t r u c t u r e s examined. An a l t e r n a t i v e model p r e s e n t e d i n t h i s paper shows t h a t t h e d a t a o f Hogan e t a l . (50) and G e a c i n t o v e t a l . (kj) a r e c o n s i s t e n t w i t h an e x t e r n a l l y bound adduct t o a deformed DNA k i n k e d t o accommodate t h e pyrene i n t h e minor groove. None t h e l e s s t h e r e c e p t o r s i t e k i n k e d t o accommodate an i n t e r c a l a t i v e c o v a l e n t bound adduct y i e l d s s t e r e o s e l e c t i v i t y f o r t h e l ( + ) d i a s t e r e o i s o m e r ( o f a l l f o u r s t u d i e d ) (37) i n an i n t e r m e d i a t e s t e p which has n o t been i d e n t i f i e d e x p e r i m e n t a l l y . Because t h e l i n e a r dichroism r e s u l t s f o r t h e o r i e n t a t i o n o f t h e pyrene m o i e t y i n t h e BPDE l ( + ) DNA adduct a r e v e r y s i m i l a r i n e x p e r i m e n t s performed by t h e e l e c t r i c f i e l d and f l o w t e c h n i q u e s (51) i t i s t e m p t i n g t o assume t h a t t h e angles r e f l e c t measurements r e l a t i v e t o t h e "average" DNA a x i s . Thus, t h e r e a r e two i n t e r p r e t a t i o n s t o t h e o r i e n t a t i o n o f 35°-^3° for t h e pyrene moiety: a p u r e l y e x t e r n a l l y bound adduct and an i n t e r c a l a t i v e c o v a l e n t l y bound a d d u c t . I n o u r proposed mechanism f o r l(+)-N2(G) a d d u c t s , t h e i n t e r c a l a t i v e c o v a l e n t l y bound form i n a k i n k e d r e c e p t o r s i t e has an o r i e n t a t i o n a(BPDE) > 80°, whereas t h e e x t e r n a l l y bound forms i n a r e l a x e d B-DNA t y p e s t r u c t u r e have v a l u e s o f ot(BPDE) « 15° f o r t h e G base i n t h e a n t i c o n f o r m a t i o n and a(BPDE) « 50° f o r G i n t h e s y n c o n f o r m a t i o n . The n o n - c o v a l e n t l y bound BPDEs t o DNA formed i n i t i a l l y appear t o be i n t e r c a l a t i o n complexes (U6,52-55). Meehan e t a l . (k§) r e p o r t t h a t t h e BPDE i n t e r c a l a t e s i n t o DNA on a m i l l i s e c o n d time s c a l e w h i l e t h e BPDE a l k y l a t e s DNA on a time, s c a l e o f m i n u t e s . Most o f t h e BPDE i s h y d r o l y z e d t o t e t r o l s (53-56). G e a c i n t o v e t a l . (5k) have shown w i t h l i n e a r d i c h r o i s m s p e c t r a l measurements t h a t t h e d i s appearance o f i n t e r c a l a t e d BPDE l ( + ) i s d i r e c t l y p r o p o r t i o n a l t o t h e r a t e o f appearance o f c o v a l e n t a d d u c t s . These r e s u l t s suggest t h a t e i t h e r t h e r e may be a c o m p e t i t i o n between t h e p h y s i c a l l y nonc o v a l e n t l y bound BPDE l ( + ) and an e x t e r n a l l y bound adduct o r as sug g e s t e d by t h e mechanism i n t h e p r e s e n t p a p e r , an i n t e r c a l a t i v e c o v a l e n t s t e p f o l l o w e d by a r e l a x a t i o n o f t h e DNA t o y i e l d an e x t e r n a l l y bound a d d u c t . T h e i r r e s u l t s f o r t h e BPDE i ( - ) e x h i b i t b o t h i n t e r c a l a t i v e and e x t e r n a l l y bound a d d u c t s . The l i n e a r dichroism measurements do n o t d i s t i n g u i s h between p h y s i c a l l y bound and c o v a l e n t bound forms w h i c h a r e i n t e r c a l a t i v e i n n a t u r e . Hence t h e a s sumption t h a t a s u p e r p o s i t i o n o f i n t e r n a l and e x t e r n a l s i t e s o c c u r s f o r t h i s isomer.



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The b i n d i n g o f i ( - ) t o base atoms a s s o c i a t e d w i t h t h e minor g r o o v e , s p e c i f i c a l l y N6(A) (38), 06(G) and N*+(C), have been observed (13-22,3*4,38). We propose t h a t t h e DNA can be k i n k e d t o a l l o w i n t e r c a l a t i v e c o v a l e n t b i n d i n g t o t h e s e atoms, b u t t h a t t h e f i n a l o r i e n t a t i o n may be i n such a s i t e o r e x t e r n a l l y bound t o t h e DNA and t h a t t h i s f i n a l s i t e may a l s o be k i n k e d . G e a c i n t o v and coworkers (51,57) have s t u d i e d t h e o r i e n t a t i o n o f t h e pyrene moiety i n BPDE l ( + ) and i ( - ) adducts w i t h DNA and Undeman e t a l . (58) have s t u d i e d t h e o r i e n t a t i o n o f BPDE l(±) and Il(±) t o DNA. They i n t e r p r e t t h e i r e l e c t r i c l i n e a r d i c h o r i s m measurements i n terms o f a s u p e r p o s i t i o n o f two t y p e s o f b i n d i n g s i t e s which t h e y l a b e l s i t e I and s i t e I I . To a v o i d c o n f u s i o n w i t h t h e n o t a t i o n f o r our i n t e r c a l a t i o n s i t e s , t h e i r s i t e s w i l l be r e f e r r e d t o as IQ and IIX. I n s i t e IQ t h e complex has t h e pyrene m o i e t y o r i e n t e d w i t h i n 30° o f t h e DNA b a s e s , i . e . , 60°-90° r e l a t i v e t o t h e h e l i x a x i s . They c a l l t h i s q u a s i i n t e r c a l a t i o n . We r e f e r t o i t as i n t e r c a l a t i v e c o v a l e n t b i n d i n g . I t r e s u l t s a f t e r i n t e r c a l a t i o n and k i n k i n g o f t h e DNA and c o v a l e n t bond f o r m a t i o n . I n s i t e I I X t h e complex has t h e l o n g a x i s o f t h e t h e pyrene moiety o r i e n t e d 15°-27° f o r BPDE l ( + ) and 37°-*45° f o r BPDE i ( - ) r e l a t i v e t o t h e h e l i x a x i s . They i n t e r p r e t t h i s as e x t e r n a l b i n d i n g . They a t t r i b u t e s i t e I I X b i n d i n g t o N2(G) and S i t e IQ b i n d i n g t o 06(G) and N6(A) i n agreement w i t h t h e i n t e r p r e t a t i o n o f Brookes e t a l . (38), t h a t t h e d i f f e r e n c e i n mu t a g e n i c i t y between t h e l ( + ) and i ( - ) isomers r e s u l t s from d i f f e r ences i n s p a t i a l o r i e n t a t i o n . S p e c i f i c a l l y , t h e l ( + ) isomer ex h i b i t s s i t e I I X b i n d i n g w i t h 88-9*+% bound. The i ( - ) isomer e x h i b i t s s i t e I I X b i n d i n g w i t h 50-60% bound, t h e r e m a i n i n g being a t t r i b u t e d t o s i t e IQ. The i n t e r p r e t a t i o n o f e x p e r i m e n t a l d a t a and p r e s e n t a t i o n o f a d e t a i l e d m o l e c u l a r model can be a c c o m p l i s h e d o n l y a f t e r c e r t a i n a s sumptions have been made about t h e a l i g n m e n t o f t h e DNA by an e l e c t r i c f i e l d and i n f l o w t e c h n i q u e s , ( l ) The h e l i x a x i s o f t h e DNA i s o r i e n t e d a l o n g t h e e l e c t r i c f i e l d and f l o w a x i s i n e x a c t l y t h e same manner. F o r DNA w i t h s u p e r t u r n s t h i s assumption i m p l i e s o n l y t h e "average" h e l i x a x i s . (2) The c o n f o r m a t i o n a l change t h r o u g h t h e r e c e p t o r s i t e i s smooth. That i s , t h e r e a r e no sharp bends which may o r i e n t t h e pyrene m o i e t y a l o n g t h e e l e c t r i c f i e l d o r f l o w a x i s w h i l e i t i s q u a s i i n t e r c a l a t e d , i m p l y i n g t h a t i t i s e x t e r n a l l y bound. (3) The average h e l i c a l a x i s t h r o u g h t h e r e c e p t o r s i t e l i e s a l o n g t h e e l e c t r i c f i e l d o r f l o w a x i s so t h a t t h e b e n d i n g i s s y m m e t r i c a l about the kink. I n F i g u r e 2, an i d e a l i z e d o r i e n t a t i o n o f t h e BPDEs i n b o t h bound forms i s i l l u s t r a t e d w i t h one s p e c i f i c example. F o r t r i g o n a l h y b r i d i z a t i o n about N2(G) and t e t r a h e d r a l h y b r i d i z a t i o n about CIO(BPDE), v a l u e s o f t h e k i n k , a , y(DNA) and a(BPDE) a r e i n dicated. However, i n p r a c t i c e t h e pyrene m o i e t y i s n o t p a r a l l e l t o t h e a d j a c e n t base p a i r , and i t s l o n g a x i s does n o t l i e i n t h e plane o f t h e l o c a l h e l i c a l a x e s , t and X', and t h e average h e l i c a l axis, Thus, a(BPDE) > 6*5°, t h e i d e a l i z e d v a l u e . Similarly for t h e o u t s i d e form, a(BPDE) >20°. I n t h i s p a p e r , a m o l e c u l a r model i s p r e s e n t e d i n which a(BPDE) « 80° f o r t h e i n t e r c a l a t i v e c o v a l e n t l y bound form, and a(BPDE) « 15° and y(DNA) * 30° f o r t h e e x t e r n a l l y bound form. Both forms a r e bound t o DNA w i t h d i f f e r e n t k i n k c o n f o r m a t i o n s . I n summary, a k i n k i n t h e DNA need not i m p l y i n t e r c a l a t i v e x



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F i g u r e 2. Geometry f o r i d e a l i z e d o r i e n t a t i o n s o f t h e pyrene m o i e t y ( ) i n (a) an i n t e r c a l a t i v e c o v a l e n t l y bound form and i n (b) an e x t e r n a l l y bound form. The average h e l i x a x i s i s assumed t o l i e along The l o c a l segments o f DNA l i e a l o n g t and X'. They a r e o r i e n t e d by y(DNA) r e l a t i v e t o £ and t h e l o n g a x i s o f t h e pyrene m o i e t y i s o r i e n t e d by 6'0). (b) L e a s t squares t e c h n i q u e s a r e used t o r e l a x a DNA fragment u n t i l c o n s t r a i n t s f o r t h e g e o m e t r i c a l c o n d i t i o n s o f p r o p e r bond l e n g t h s , bond a n g l e s w i t h a s p e c i f i c placement o f bases a r e s a t i s f i e d (6l,62). (c) S y s t e m a t i c adjustment o f bases


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t h r o u g h p a r a l l e l , k i n k e d o r any o t h e r o r i e n t a t i o n o f i n t e r e s t t o s e a r c h f o r backbone c o m p l e t i o n (63-66). A l a r g e number o f duplex DNA r e c e p t o r s i t e s have been o b t a i n e d f o r t h e i n t e r c a l a t i o n p r o c e s s , s e v e r a l d i n u c l e o s i d e monophosphate r e c e p t o r s i t e s have been used f o r modeling BPDE-DNA i n t e r a c t i o n s , and o n l y one s e t o f k i n k e d DNA s i t e s a r e a v a i l a b l e (36,66). The q u e s t i o n a r i s e s : which receptor s i t e s s h o u l d be used i n the modeling o f t h e b i n d i n g o f molecules w i t h DNA? In p r i n c i p a l , i t cannot be answered w i t h o u t t r y i n g a l l o f t h e them. In p r a c t i c e , c a l c u l a t i o n s w i t h t h e o r e t i c a l l y determined i n t e r c a l a t i o n s i t e s (67-71), c a l c u l a t i o n s u s i n g e x p e r i m e n t a l d a t a (72,73), d i n u c l e o s i d e t r i p h o s p h a t e u n i t s (7**, 75) t e t r a m e r duplexes w h i c h f i t i n t o B-DNA (61,62,66-68), and t e t r a m e r - d u p l e x e s (76) w i t h the same and w i t h mixed sugar puckers y i e l d r e s u l t s w h i c h demonstrate f o r s i m p l e systems t h a t the optimum b i n d i n g o r i e n t a t i o n s a r e i n agree ment w i t h e x p e r i m e n t a l d a t a and t h a t the t r e n d s i n b i n d i n g e n e r g i e s f o r a l l p o s s i b l e sequences remain a p p r o x i m a t e l y the same. The t e t r a m e r duplex r e c e p t o r s i t e s used i n t h i s study have t h e f o l l o w i n g characteristics: ( l ) t h e y f i t i n t o B-DNA, (2) t h e y r e p r e s e n t con f o r m a t i o n s which possess s p e c i f i c f e a t u r e s , i . e . , i n t e r c a l a t i o n , k i n k s or DNA d i s t o r t e d from the B-DNA form. T h e r e f o r e , we proceed w i t h the assumption t h a t the i n t e r c a l a t i o n p r o c e s s w i l l r e v e a l the manner i n which the r e a c t i v e atoms approach each o t h e r t o p e r m i t i n t e r c a l a t i v e c o v a l e n t b i n d i n g as a f i r s t s t e p f o l l o w e d by an ad j u s t m e n t o f the base p a i r s t o non-planar o r i e n t a t i o n s ( k i n k ) t o a c commodate the p r o p e r h y b r i d c o n f i g u r a t i o n s about CIO on BPDE and N2(G), N6(A), 06(G) and NU(C) f o r c o v a l e n t bond f o r m a t i o n . For b i n d i n g , two o r i e n t a t i o n s a l o n g the DNA must be c o n s i d e r e d : 3' and 5" b i n d i n g . They a r e i l l u s t r a t e d w i t h the BPDEs bound t o a p u r i n e (pu) and t o a p y r i m i d i n e ( p y ) .


5

The arrow denotes the 5" 3' d i r e c t i o n a l o n g t h e backbone, i . e . , 5'(sugar)3' o r e q u i v a l e n t l y 3'(p)5'. The diagram corresponds t o v i e w i n g the DNA i n t o the minor groove as seen i n most f i g u r e s i n t h i s paper. T h e r e f o r e , t h e energy o f opening t h e DNA to sites f a v o r i n g 3' o r 5" b i n d i n g p r o v i d e s t h e c o n t r i b u t i o n o f t h e DNA t o the process. Once the s i t e i s c r e a t e d , the a b i l i t y o f each BPDE t o f i t i n t o t h e r e c e p t o r i s examined. Two p r o c e s s e s a r e c o n s i d e r e d : intercala t i o n and c o v a l e n t i n t e r c a l a t i v e b i n d i n g r e p r e s e n t e d by

A BP? BPl

_R

+ BPDE

AE

B P D E

^

A B PBPo I A 1

I

2

BPDE BP!

I


250


The energy

AE

J

B P D E - BPDE

+

AC

BPDE

r e p r e s e n t s t h e energy f o r i n s e r t i o n o f BPDE i n t o s i t e R. c a l c u l a t e d w i t h a Coulomb p o t e n t i a l

( 2 )

It is


(3)

a 6-lU p o t e n t i a l

and a t o r s i o n a l p o t e n t i a l

T = J ( V / 2 ) [ 1 + c o s i^] b b

(5)

f o r i n t e r a c t i n g atoms i and j and f o r r o t a t i o n s about each bond b . A s s o c i a t e d w i t h t h e atoms a r e t h e n e t a t o m i c charges q^ and q j , and t h e van d e r Waals r a d i i and p j . The d i s t a n c e between atoms i and j i s r-j^ , t h e sum o f van d e r Waals r a d i i i s p i j = P i + P i > t h e r e duced d i s t a n c e i s s-y = r i j / p i j , A±j = 1 . 5 a i c y I j l j / C l i + I j ; where a i , a j and I i , I j a r e t h e a t o m i c p o l a r i z a b i l i t i e s and i o n i z a t i o n p o t e n t i a l s respectively, i s t h e b a r r i e r t o i n t e r n a l r o t a t i o n and l\> i s t h e p e r i o d i c i t y f o r a complete r o t a t i o n about bond b . The i n t e r m o l e c u l a r energy Igp^g c o n s i s t s o f a sum Q+U. The summation i n t h e two-body i n t e r a c t i o n s proceeds o v e r a l l atoms i i n t h e BPDE and j i n t h e DNA. The i n t r a m o l e c u l a r i n t e r a c t i o n s between non-bonded atoms i n BPDE as w e l l as t h e t o r s i o n a l energy a r e g i v e n by t h e change i n c o n f o r m a t i o n a l energy, A C ^ ^ g , measured relative t o t h e g l o b a l minimum f o r t h e f r e e m o l e c u l e . I t c o n s i s t s o f a sum o f terms Q + U + T. I n t h i s case t h e summation proceeds o v e r nonbonded atoms i and J i n d i f f e r e n t fragments o f BPDE, i . e . , fragments s e p a r a t e d by r o t a t a b l e bonds. The c o n f o r m a t i o n a l energy o f t h e benzo r i n g i s a l s o i n c l u d e d . For t h i s contribution the r e s t r i c t i o n s a r e made t h a t i > j and t h a t atoms i and j a r e non-bonded. The d e f i n i t i o n s and t h e d e t a i l s o f t h e energy terms and t h e parameters a r e p r e s e n t e d elsewhere (69)• The t o t a l energy change o f t h e complex i s g i v e n by

A

W

AE

- DNA

+

A

4>DE


( 6 )

10.

MILLER ET AL.

For c o n v e n i e n c e , minimum as

t h e energy w i l l be r e p o r t e d r e l a t i v e t o

0G

BPDE

=

e

BPDE "

U e

the g l o b a l

;

BPDE min

F o r t h e c o v a l e n t l y bound adduct t h e b i n d i n g energy must be added t o t o AEgp^g. ^ For tj^e DNA, o n l y a c o n f o r m a t i o n a l change occurs and ^t)NA ^DNA ^ g l ° l minimum i s assumed t o be f o r B-DNA. By c a l c u l a t i n g t h e energy changes f o r t h e i n t e r c a l a t i o n p r o c e s s , t h e p r e f e r e n c e f o r o r i e n t a t i o n o f t h e epoxide i n t h e major o r minor groove can be determined. This step provides a r a t i o n a l e f o r t h e f i r s t s t e p i n c o v a l e n t b i n d i n g by examining whether t h e r e a c t i n g atoms a r e i n p r o x i m i t y t o each o t h e r . The i m p o r t a n t s t e p i n s t e r e o s e l e c t i v i t y i s t h e a b i l i t y o f t h e BPDEs t o f i t i n t o t h e s i t e once a c o v a l e n t bond has formed. The c o n f o r m a t i o n s o f t h e benzo r i n g a r e i m p o r t a n t i n t h e f i t o f each o f t h e d i a s t e r e o i s o m e r s t o t h e k i n k s i t e d u r i n g adduct f o r m a t i o n . They m o d i f y t h e o r i e n t a t i o n o f t h e d o u b l e bond o f t h e benzo r i n g and hence t h e e n t i r e pyrene m o i e t y . A


251


=

A(

w t i e r e

Mechanism f o r BPDE-DNA Adduct

e

Da

Formation

F o r m a t i o n o f t h e BPDE-DNA adduct r e q u i r e s a study o f ( l ) Benzo r i n g c o n f o r m a t i o n s o f BPDEs and a d d u c t s ; (2) The r e h y b r i d i z a t i o n o f amino groups on the benzo f o r t h e C10-N bond f o r m a t i o n ; (3) The r e c e p t o r s i t e s r e s u l t i n g from a c o n f o r m a t i o n a l adjustment o f DNA t o accommodate an i n t e r c a l a t e d and f i n a l l y an i n t e r c a l a t i v e c o v a l e n t l y bound BPDE, and t h e base sequence s p e c i f i c i t y i n t h e f o r m a t i o n o f t h e r e c e p t o r s i t e ; (k) C l a s s i c a l i n t e r c a l a t i o n and the o r i e n t a t i o n o f CIO o f the BPDEs toward t h e r e a c t i v e N2(G), N6(A), 06(G) and Nl+(C) base atoms; (5) The s t e r e o s e l e c t i v i t y o f t h e BPDEs d u r i n g i n t e r c a l a t i v e c o v a l e n t b i n d i n g i n k i n k e d DNA; and (6) The p o s s i b l e r e o r i e n t a t i o n o f t h e complex t o y i e l d an e x t e r n a l l y bound a d d u c t . The e n e r g e t i c s f o r each o f t h e s e p r o c e s s e s w i l l be p r e s e n t e d t o i d e n t i f y the important steps t h a t i n f l u e n c e the b i n d i n g of s p e c i f i c isomers. I t w i l l be shown t h a t t h e o r i e n t a t i o n o f each d i a s t e r e o i s o m e r o f BPDE about s p e c i f i c base atoms i n k i n k e d r e c e p t o r s i t e s i n t h e du p l e x DNA d u r i n g c o v a l e n t bond f o r m a t i o n i s t h e d e t e r m i n i n g f a c t o r i n stereoselectivity• The parameters which d e f i n e t h e o r i e n t a t i o n o f t h e BPDE adduct t o N2 on guanine a r e g i v e n i n F i g u r e 3 i n terms o f t h e r e a c t i o n c o o r d i n a t e s R, a , $, Y> 5l) t o DNA. The major p r o d u c t i n v o l v i n g t h e t r a n s l(+)-N2(G) adduct suggests t h a t a(BPDE) and y ( ) r e l a t e d t o the t h e o r e t i c a l s t r u c t u r e s . The measured unwinding a n g l e s (1+2,1+5) i n v o l v e o t h e r a d d u c t s and i n t e r c a l a t i v e c o v a l e n t l y bound forms. B o t h o f t h e s e s t r u c t u r e s can a r i s e a f t e r a d e n a u t r a t i o n and r e n a t u r a t i o n o f t h e DNA. There i s e x p e r i m e n t a l e v i d e n c e f o r t h e dynamic p r o c e s s o f opening and c l o s i n g o f t h e DNA. I n hydrogen ex change s t u d i e s o f the amino and, i n Watson-Crick base pairing, b u r i e d imide g r o u p s , T e i t e l b a u m and Englander (105,106) c o n c l u d e t h a t t h e G»C and A»T base p a i r s a r e open about 1% o f t h e t i m e , and t h a t t h e opening r a t e c o n s t a n t i s about 0.01+ t o 0.06 s e c " i n both c a s e s . A study o f i n t e r c a l a t i o n by Gabbay e t a l . (107) o f two mole c u l e s , a 1,8-naphthylimide w i t h one b u l k y s u b s t i t u e n t and a 1,8,1+, 5n a p h t h y l i m i d e w i t h b u l k y s u b s t i t u e n t s on each end o f t h e m o l e c u l e t o d n a

x

z

D N A

a

r

e

1


278


Table

K+:), K->, I I ( +)

XIII.

E x t e r n a l l y Bound Adducts t o N2(G) and N6(A)

Adduct

base

anti l ( + )-N2(G)*> l(+)-N2(G)


Il(-)-N6(A)

c

sugars

syn

a

o r i e n t a t i o n of

and I I ( -)

trans

a

z

y (DNA)

a(BPDE)

5e

BPDE

a f f e c t e d base

6.6 9.6

syn syn

s_ s^

0 +10

36.0 36.1

0 5

51 h9

syn

s_

0

36.0

0

51

10

10 11+1.7 60.8

3

a n t i o r i e n t a t i o n o f a l l bases l(+)-N2(G) l(+)-N2(G) l(+)-N2(G)

anti anti anti

s_ s^ s^

0 -20 -1+0

36.0 22.5 22.0

0 10 20

50 33

l(+)-N2(G) l(+)-N2(G) l(+)-N2(G) l(+)-N2(G)

anti anti anti

a a a

-30

26.0 19.2 7.3 1-11+

15 26 35 29

29 16 15 15-^3

63.7 5.7 -10.1

36.1 38.0 38.6

5 10 15

1+2 38

377. k 1+1+.6 7.5

l(-)-N6(A) l(-)-N6(A) K-)-N6(A) l(-)-N6(A)

b

d

anti anti anti

a

b

c

d

s_ s_ js

10 20 30

e

Il(+)-N2(G) Il(+)-N2(G) Il(+)-N2(G) Il(±)-N2(G) Il(-)-N6(A) II(-)-N6(A) II(-)-N6(A)

-50 -70

anti anti anti

a a a

-30

-50 -70

26.0 19.2 7.3

15 25 35

f

C

anti anti anti

s_ s_ s^

0 20 30

36.0 38.0 38.6

0 10 15

30

31

37-^5

5

exp

exp

5 7 15 >65

215.7 -5.3 -13.8

1+6 39

3xl0 67.2 12.7

31

f

3

The s u g a r s a r e C(2')-endo t o C(2')-endo (s_) and C(2')-endo t o C(3^)-endo (a) i n t h e 5 " ( p ) 3 ' o r 3 ' ( s u g a r ) 5 ' d i r e c t i o n . Base sequences: +G-C,OG,BPT,G«C,OG+ and +G«C,T«A,BPT,A«T,C«G+. E n e r g i e s f o r t h e BPDE-N2(G) and -N6(A) a d d u c t s a r e measured r e l a t i v e t o t h e 5 ' - o r i e n t a t i o n o f t h e c o r r e s p o n d i n g isomers i n T a b l e s V I I I and I X , r e s p e c t i v e l y . See F i g u r e 11. See F i g u r e 13. E x p e r i m e n t a l r e s u l t s (50,51,58). a = 36° - u n w i n d i n g angle f r o m (1+2,1+5). E x p e r i m e n t a l r e s u l t s (51.). N6(A) adduct f o r m a t i o n i s assumed. E x p e r i m e n t a l r e s u l t s a r e f o r t h e r a c e m i c m i x t u r e (j>8_). z

e

exp



10.

MILLER ET AL.


279

prevent d i r e c t i n s e r t i o n into an i n t e r c a l a t i o n s i t e , also supports a dynamical structure of DNA. Their k i n e t i c studies show that DNA complexes form i n ca. 100 msec for these two compounds, respectively. The f i r s t compound can be inserted into DNA d i r e c t l y from one groove; however, f o r the second naphthylimide to be i n t e r calated, the substituents must l i e i n opposite grooves. The slower time needed for complex formation suggests a denaturation and renaturation of the DNA t o accommodate each substituent i n each groove. Their r e s u l t s suggest that there are two modes a v a i l a b l e for i n t e r c a l a t i o n : rapid (involving opening, RO) and slow ( i n v o l ving denaturation, SD) equilibrium processes, and conversely, that these two modes are a v a i l a b l e for the reverse process. For a co v a l e n t l y bound adduct which r e s u l t s during i n t e r c a l a t i o n , the RO process w i l l not permit the pyrene moiety to be dislodged from the i n t e r c a l a t i o n s i t e , whereas the SD process w i l l . Therefore, the f i n a l state w i l l depend on the d i r e c t i o n of the equilibrium pro cess. The a n t i ->» syn r e o r i e n t a t i o n of G, and A i s i l l u s t r a t e d i n Figure 12. This transformation takes N2(G) from i t s p o s i t i o n i n the minor groove and places i t i n the major groove and quite f a r outside the h e l i x . Watson-Crick p a i r i n g i s l o s t . There are no poor s t e r i c contacts i n t h i s model, and energy can be recovered by hydrogen bonding with water. An a n t i syn r o t a t i o n of A displaces N6(A) only s l i g h t l y farther into the major groove and s i m i l a r l y for 06(G). As already shown, the a n t i o r i e n t a t i o n of G y i e l d s the most favor able f i t and the pyrene orientations are i n agreement with experi mental r e s u l t s f o r the l(+)-N2(G) adducts. Similar calculations were performed f o r the BPDE l ( - ) - and Il(-)-N6(A) adducts. The stereoselected Cda conformation of the BPDE i ( - ) and I l ( - ) adducts to N6(A) were chosen f o r study i n a reoriented complex with an e x t e r n a l l y bound pyrene moiety. In Figure 13, the adduct i s shown i n i t s optimum o r i e n t a t i o n i n B-DNA with adenine a f t e r an anti syn transformation for which the non-bonded contacts are poor, and with the normal a n t i base o r i e n t a t i o n with favorable con t a c t s . The f i t improves f o r the a n t i base as ct 30°. The o r i e n t a t i o n of the pyrene moiety i s a(BPDE) =31° and the l o c a l h e l i c a l a x i s of the DNA i s oriented at y(DNA) = 15°. Calculations were not performed with e x t e r n a l l y bound BPDE-DNA adducts to 06(G) and NU(C). Calculations of e x t e r n a l l y bound BPDE l(-)-N6(A) adducts with kinked DNA with a 30° y i e l d s an o r i e n t a t i o n a(BPDE) = 31° i n good agree ment with experimental r e s u l t s for the e x t e r n a l l y bound component (51). The energies reported i n Table XIII f o r the e x t e r n a l l y bound forms are measured r e l a t i v e to that f o r the i n t e r c a l a t i v e covalently bound form. Thus, the trans BPDE l(+)-N2(G) adduct i s 10.1 k c a l / mole more stable and the trans BPDE Il(-)-N6(A) adduct i s 12.7 kcal/mole l e s s stable i n the e x t e r n a l l y bound form. S i m i l a r i l y , the trans BPDE Il(+)-N2(G) adduct i s -13.8 kcal/mole more stable and the trans BPDE l(-)-N6(A) adduct i s 7*5 kcal/mole l e s s stable. There f o r e , s i t e IQ ( i n t e r c a l a t i v e covalent) which i s favored by the i ( - ) isomer (5l) may be due to N6(A) and NMc) adduct formation, s p e c i f i c a l l y trans a d d i t i o n . The d i s t r i b u t i o n of BPDE i ( - ) adducts observed by Brookes et a l . (38) as 59% N2(G), 21% 06(G), lQ% N6(A) and 2% other must be ad dressed. Although the y i e l d of N2(G) adduct i s 59% compared t o x

x


280



t h a t w i t h o t h e r base atoms, i t i s a c t u a l l y 0.1 o f t h e l(+)-N2(G) a d duct. The b i n d i n g t o N2(G) and t h e r e s u l t i n g form ( e x t e r n a l l y o r i n t e r n a l l y bound) w i l l predominant. I n t r a n s a d d i t i o n t h e i ( - ) i s o mer i s u n f a v o r e d i n t h e i n t e r c a l a t i v e c o v a l e n t s t e p ; however, c i s a d d i t i o n i s p o s s i b l e as d i s c u s s e d i n t h e p r e v i o u s s e c t i o n . The c i s adduct s h o u l d y i e l d a s t a b l e complex analogous t o t h e r e s u l t s f o r

F i g u r e 12. A n t i ( - ) and s y n ( ) r e o r i e n t a t i o n o f G and A about t h e i r g l y c o s i d i c bonds (Cl'-O) i n B-DNA.

the l ( + ) adduct i n T a b l e X I I I . The c o n t r i b u t i o n t o b o t h IQ ( i n t e r n a l ) and I I X ( e x t e r n a l ) b i n d i n g s i t e s by t h e i ( - ) isomer may be due t o t h e s m a l l amount o f c i s adduct which b i n d s t o N2(G) ( f o r IQ) and l o w r e a c t i v i t y toward N6(A) ( f o r s i t e I Q ) . Two o t h e r models which o r i e n t t h e pyrene m o i e t y e x t e r n a l l y have been proposed. Aggarwal e t a l . (108) s u c c e s s f u l l y f i t b o t h t h e l ( + ) and i ( - ) i n t o A-DNA w h i c h c a n a r i s e from a l o c a l d i s t o r t i o n o f BDNA. I n t h e l ( + ) a d d u c t , t h e chromophore i s d i r e c t e d out o f t h e minor g r o o v e , whereas f o r t h e i ( - ) i t f i t s s n u g l y i n t o t h e g r o o v e . The a n g l e subtended by t h e l o n g a x i s o f BP w i t h r e s p e c t t o t h e h e l i x a x i s i s 67° f o r l ( + ) and 63° f o r i ( - ) . H i n g e r t y and Broyde (109) have o p t i m i z e d t h e c o n f o r m a t i o n o f t h e dCpdG-BPDE l ( + ) a d d u c t . They f i n d a pyrene base s t a c k e d c o n f o r m a t i o n w i t h C and o r i e n t e d by a(BPDE) = 25°-30°. I f t h e i r complex can be i n c o r p o r a t e d i n t o a d o u b l e h e l i x , i t may r e p r e s e n t a l o c a l d e f o r m a t i o n o f an e x t e r n a l l y bound form w i t h t h e pyrene o r i e n t e d i n t h e minor g r o o v e . Thus, a l l t h r e e models f o r t h e " f i n a l " e x t e r n a l l y bound BPDE l ( + ) t o N2(G) i n the minor groove e x h i b i t s i m i l a r p h y s i c a l c h a r a c t e r i s t i c s . At the t i m e o f w r i t i n g t h i s m a n u s c r i p t , s t e r e o s e l e c t i v i t y has not been demonstrated w i t h t h e s e l a t t e r two models (108,109).



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F i g u r e 13. An e x t e r n a l l y bound BPDE I l ( - ) - N 6 ( A ) adduct w i t h t h e pyrene m o i e t y p l a c e d i n t h e major g r o o v e . The r e c e p t o r s i t e s a r e (upper) B-DNA except f o r an a n t i s y n r o t a t i o n b y 180° about t h e g l y c o s i d i c bond o f A, and ( l o w e r ) DNA w i t h an a = 30° k i n k . x

D i s c u s s i o n and C o n c l u s i o n T h e o r e t i c a l r e s u l t s have been p r e s e n t e d w i t h a n a l y t i c a l d a t a and s t e r e o g r a p h i c p r o j e c t i o n s t o support a mechanism g i v e n i n F i g u r e ll+ i n which s t e r e o s e l e c t i v i t y occurs during i n t e r c a l a t i v e covalent binding. We propose a m o l e c u l a r model and demonstrate t h a t each step plays the f o l l o w i n g roles: The i n t e r c a l a t i o n s t e p o r i e n t s t h e e p o x i d e toward t h e major o r minor groove w i t h t h e r e a c t i v e CIO atom i n t h e groove a d j a c e n t t o t h e a p p r o p r i a t e base atoms: N 2 ( G ) , N 6 ( A ) , 06(G) and NU(c). A proton catalyzed nucleophilic SJJ2 r e a c t i o n i s favored d u r i n g i n t e r c a l a t i o n because p o s i t i v e i o n s , e s p e c i a l l y H , r e s i d e i n t h e grooves and t h e i r p r e s e n c e a s s i s t s i n t h e a c t i v a t i o n o f t h e BPDEs. However, d u r i n g c o v a l e n t bond f o r m a t i o n , t h e DNA must k i n k t o o r i e n t t h e pyrene m o i e t y w i t h i n t h e k i n k e d s i t e f o r proper b o n d i n g between CIO o f t h e BPDEs and t h e r e a c t i v e base atoms, i . e . , t o a c h i e v e a bond l e n g t h o f a p p r o x i m a t e l y 1.5 A and t e t r a h e d r a l h y b r i d i z a t i o n on CIO o f t h e BPDE and t r i g o n a l h y b r i d i z a t i o n on t h e r e a c t i v e base atoms. In t h i s s t e p t h e base p a i r s a r e deformed from the p a r a l l e l o r i e n t a t i o n o f a c l a s s i c a l i n t e r c a l a t i o n s i t e while the BPDE remains i n s e r t e d . The DNA k i n k s o r bends w i t h a wedge o p e n i n g i n t o t h e major o r minor g r o o v e , r e s p e c t i v e l y , w h i l e t h e a d j a c e n t base p a i r s remain s e p a r a t e d t o accommodate t h e q u a s i i n t e r c a l a t e d BPDE as i t undergoes i n t e r c a l a t i v e c o v a l e n t b i n d i n g t o DNA. I t was +


282


shown t h a t s t e r e o s e l e c t i v i t y o c c u r s d u r i n g t h i s s t e p . Specifically, BPDE l ( + ) and I l ( + ) a r e t h e o n l y isomers w h i c h f i t i n a k i n k e d s i t e (+39 ) when bound t o N2 on g u a n i n e . I n c o n t r a s t , BPDE i ( - ) and Il(-) a r e t h e o n l y isomers w h i c h f i t i n a k i n k e d s i t e (-30°) when bound t o atoms N 6 ( A ) , 06(G) and i t appears t h a t b i n d i n g t o Hk(c) may not be s t e r e o s e l e c t i v e . The l e s s f a v o r e d p r o t o n a d d i t i o n v i a an Sjji r e a c t i o n forms t h e carbonium i o n o f BPDE. This permits both trans and c i s a d d i t i o n , and minor p r o d u c t s . For c i s a d d i t i o n , the mirror images a r e s t e r e o s e l e c t e d , namely, i ( - ) and I I ( - ) by N2(G), l ( + ) and I l ( + ) by N6(A) and 06(G) and I l ( - ) and l ( + ) by N U ( C ) . I f both trans and c i s a d d i t i o n occurred t o an e q u a l extent, s t e r e o s e l e c t i v i t y would n o t be o b s e r v e d . T h e r e f o r e , t h e f a v o r e d t r a n s a d d i t i o n as w e l l as t h e s t e r i c f i t o f s p e c i f i c s t e r e o i s o m e r s during i n t e r c a l a t i v e covalent binding contribute t o s t e r e o s e l e c t i v i t y . Possible r e arrangements o f t h e DNA t o y i e l d o u t s i d e b i n d i n g c a n o c c u r i n two ways: F i r s t , an a n t i s y n r o t a t i o n about t h e g l y c o s i d i c bond o f t h e a f f e c t e d bases a l l o w s t h e r e m a i n i n g p o r t i o n o f t h e DNA t o resume i t s normal B-DNA c o n f o r m a t i o n w i t h an e x t e r n a l l y bound adduct t h a t f i t s w e l l i n t h e c a s e o f BPDE l ( + ) and I l ( + ) bound t o N2(G), b u t n o t w e l l f o r BPDE i ( - ) and I l ( - ) bound t o N 6 ( A ) . Second, a denatura t i o n , rearrangement o f t h e adduct and r e n a t u r a t i o n o f t h e DNA a l l o w s t h e adduct t o l i e i n a groove i n a s l i g h t l y k i n k e d DNA.


P

H+ BPT+

BPDE

BPDE

B-DNA

Activation

Intercalation

H+ Relaxation

I n t e r c a l a t i v e covalent binding; stereoselectivity; final predominant s t r u c t u r e f o r t r a n s a d d i t i o n t o N 6 ( A ) and 06(A1 o f BPDE I ( - ) and I l ( - ) .

Figure lU.

Outside b i n d i n g ; f i n a l predominant structure for trans a d d i t i o n t o N2(G) o f BPDE l ( + ) and I l ( + ) .

Mechanism f o r BPDE-DNA

adducts.



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The main f e a t u r e s o f t h i s proposed mechanism a r e ( l ) t h e s t e r e o s e l e c t i v i t y o f t h e BPDEs by t h e DNA d u r i n g i n t e r c a l a t i v e c o v a l e n t b i n d i n g and (2) t h e f i n a l o r i e n t a t i o n o f t h e bound pyrene w h i c h may be o r i e n t e d i n t e r n a l l y ( i n t e r c a l a t i v e c o v a l e n t ) o r ex t e r n a l l y (outside the h e l i x ) . The s t e r e o s e l e c t i v i t y o c c u r s d u r i n g c o v a l e n t bond f o r m a t i o n and a f t e r i n t e r c a l a t i o n . Relaxation of the DNA a l l o w s t h e adduct t o a d j u s t t o i t s f i n a l o r i e n t a t i o n . I f the e x p e r i m e n t a l measurements a r e assumed t o be made on t h e DNA-adducts a f t e r t h e f i n a l o r i e n t a t i o n has been a c h i e v e d , t h e n t h e f o l l o w i n g i n t e r p r e t a t i o n s can be made. The l ( + ) and I l ( + ) isomers a r e s t e r e o s e l e c t e d by N2(G), whereas t h e i ( - ) and I l ( - ) isomers a r e s t e r e o s e l e c t e d by t h e N6(A) and 06(G) during i n t e r c a l a t i v e covalent steps w i t h t r a n s a d d i t i o n . The l ( + ) and I l ( + ) - N 2 ( G ) adducts a r e r e a r r a n g e d t o an e x t e r n a l l y bound form w i t h t h e pyrene i n t h e minor g r o o v e , b u t t h e l ( - ) - N 6 ( A ) and I l ( - ) - 0 6 ( G ) adducts remain q u a s i i n t e r c a l a t e d . T h i s i s determined by t h e r e l a t i v e energy change between t h e two forms as we see from Table X I I I . However, t h e r e i s a s u p e r p o s i t i o n o f t h e two t y p e s o f s i t e s , IQ and I I X (51,57,58), and BPDE i ( - ) DNA adducts e x h i b i t b o t h types o f binding. By symmetry, t h e c i s BPDE l ( - ) - N 2 ( G ) adduct i s p r e d i c t e d t o behave s i m i l a r i l y t o t h e t r a n s l(+)-N2(G) adduct. I t s h o u l d be e x t e r n a l l y bound. The N2(G) adducts a r e more s t a b l e t h a n t h e N6(A) and 06(G) and Nl*(c) a d d u c t s . Because c i s a d d i t i o n p r o d u c t s a r e p r e s e n t , minor amounts o f t h e o t h e r adducts a r e found. I f o n l y c i s a d d i t i o n o c c u r r e d , t h e n t h e i ( - ) and I l ( - ) isomers would be s t e r e o s e l e c t e d by N2(G), and t h e l ( + ) and I l ( + ) isomers would be s t e r e o s e l e c t e d by N6(A) and 06(G). A l t h o u g h we d i d n o t p e r f o r m c a l c u l a t i o n s on t h e c i s a d d u c t s , i t can be seen from t h e s t e r e o g r a p h i c p r o j e c t i o n s t h a t t h e change accompanied b y a r e f l e c t i o n o f o n l y t h e BPDE atoms t h r o u g h t h e p l a n e o f t h e pyrene changes t h e ( + ) i n t o (-) i s o m e r s . Thus, t h e r u l e s o f s t e r e o s e l e c t i v i t y a r e r e v e r s e d . However, t h e s m a l l amount o f c i s adduct y i e l d s t h e s e minor components; t h e l ( - ) - N 2 ( G ) adduct i s most p r e v a l e n t (38) f o r r e a c t i o n s o f BPDE i ( - ) w i t h DNA and we assume t h a t t h i s a r i s e s from t h e c i s a d d i t i o n . I f b o t h t r a n s and c i s a d d i t i o n o c c u r r e d e q u a l l y , we p r e d i c t t h a t s t e r e o s e l e c t i v i t y would not be o b s e r v e d . Based on t h e r e s u l t s i n t h i s p a p e r , t h e f o l l o w i n g e x p e r i m e n t a l d a t a s h o u l d be o b t a i n e d f o r each o f t h e d i a s t e r e o i s o m e r s . ( l ) The r e l a t i v e y i e l d s o f t r a n s and c i s a d d i t i o n p r o d u c t s s h o u l d be d e t e r mined f o r adduct f o r m a t i o n t o each base atom. (2) A l t e r n a t i n g and n o n - a l t e r n a t i n g homopolymers s h o u l d be used t o e v a l u a t e t h e base s e quence s p e c i f i c i t y . (3) B i n d i n g t o s i t e s IQ and I I X s h o u l d be c o r r e l a t e d t o t r a n s and c i s adducts and t o s t e r e o s e l e c t i v i t y .

Acknowledgments The a u t h o r s acknowledge t h e g r a n t o f computer t i m e from R e n s s e l a e r P o l y t e c h n i c I n s t i t u t e and support by 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 under Grant CA-28921+. A l s o , t h e a u t h o r s w i s h t o thank C h r i s Bonesteel f o r her t e c h n i c a l assistance with the preparation of t h i s manuscript•



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pyrene Diol Epoxides to DNA - American Chemical Society

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