An Efficient Route to N 6 Deoxyadenosine Adducts of Diol Epoxides of Carcinogenic Polycyclic...

7/26/2019 An Efficient Route to N 6 Deoxyadenosine Adducts of Diol Epoxides of Carcinogenic Polycyclic Aromatic Hydrocarb

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P e r g a m o n

Bioorganic Medicinal Chemistry,

Vol. 3, No. 6, pp. 811-822, 1995

Copyright 1995 Elsevier Science Ltd

Printed in Great Britain. All rights reserved

0968-0896/95 9.50 + .00

0968-0896(95)00065-8

n E ffic ient R oute to

N

Deoxyadenos ine ddu cts o f Dio l

Ep oxides of Carcinogenic Polycycl ic rom atic H ydrocarbons

Seong Jin Kim,a Hemant K. J a j o o b Hye-You ng Kim,c Liang

Z h o u , a

Pamela Horton,c

Constance M. Harris, a and Thomas M. Harris a

Dep artme nts ofChem istry and bPharmacology and CCenter in M olecu lar Toxicology, Vanderb i l t Universi ty ,

Nashvi l le , TN37235, U.S.A.

Ab stra et--P oly cy clic aromatic hydrocarbons are metabolized to a wi de variety of oxidized derivatives, including highly

reactive diol epoxides which alkylate DN A. The reaction lacks regio- or stereospecificity but occurs primarily at the exo cyc lic

amino groups of deoxyguanosine and deoxyadenosine. An efficient ro ute to / adducts of deoxyadenosine is described using as

examples those arising from trans opening of the

anti-tetrahydro ol

epoxides of naphthalene, benzo[a]pyrene, and

benzo[c]phenanthrene. The adducts we re synthesized in 50-92 yields by reaction o f 6-fluoropurine 2'-deoxyriboside with

aminotriols formed by

t rans

opening of racemic dihydrod iol epoxides using liquid NH 3. The diastereomeric adducts we re

separated by H PLC and their absolute configurations were assigned by circular dichroism. ~H NM R studie s revealed significant

differences in conform ation of the tetrahydroarom atic ring between the sterically unrestricted naphthalene derivative an d the

sterically congested derivatives of benzo[a]pyrene an d benzo[c]phenanthrene. These differences m ay have a bearing on the

higher carcinogenicity shown by the latter hydrocarbons. Undecadeoxyoligonucleotides bearing regi o- and stereochemically

defined adenine

N6-anti-trans-benzo[a]pyrene

adducts have been prepared.

n t r o d u c t i o n

P o ly c y c l i c a r o ma t i c h y d r o c a r b o n s ( P A H s ) s u c h a s

n a p h th a l e n e , b e n z o [ a ] p y r e n e , a n d b e n z o [ c ] p h e n a n -

th r e n e a r e w id e s p r e a d i n t h e e n v i r o n me n t a s p r o d u c t s o f

c o mb u s t i o n . I n l i v in g c e l l s t h e y a r e c o n v e r t e d t o a

v a r i e ty o f o x y g e n a t e d d e r iv a t i v e s , I i n c lu d in g

d ih y d r o d io l e p o x id e s ( e . g. 1 - 3 i n S c h e m e 1 ). T h e

c o v a l e n t b i n d i n g o f P A H m e t a b o l it e s t o D N A i s

b e l i e v e d t o p l a y a n imp o r t a n t r o l e i n mu ta g e n e s i s a n d

in t h e i n i t ia t i o n o f t h e c a r c in o g e n i c p r o c es s . T h e ma jo r

D N A a d d u c t s r e s u l t f r o m a t t a c k o f t h e e x o e y c l i c a min o

g r o u ps o f d eo x y g u a n o s i n e a n d d e o x y a d e n o s i n e o n t h e

b e n z y l i c p o s i t i o n o f t h e e p o x id e s w i th b o th

t rans

a n d

cis

r ing o pening be ing o bse rved . 2

T h e r e i s c o n s id e r a b le i n t e r es t i n t h e p r e p a r a ti o n o f P A I l

a d d u c t s o f n u c l e o s id e s a n d o l i g o n u c l e o t i d e s f o r u s e i n

c h e m ic a l a n d b io lo g i c a l s t u d ie s . S e v e r a l g r o u p s h a v e

p r e p a r e d m o d e s t q u a n t i t i e s o f t h e n u c l e o s id e ad d u c t s b y

b io m ime t i c p r o c e d u r es , i .e . re a c t i o n o f d ih y d r o d io l

e p o x i d e s o f P A H s w i t h d e o x y g u a n o s i n e o r

d e o x y a d e n o s i n e o r w i t h D N A f o l l o w e d b y e n z y m a t i c

d e g r a d a t i o n ) 4 F r e q u e n t l y , t h e y i e ld s a r e l o w d u e t o

c o mp e t in g h y d r o ly s i s o f t h e e p o x id e s.

T h e p r e p a r a t i o n o f o l i g o n u c l e o t i d e s b e a t i n g s t r u c tu ra l l y

d e f in e d a d d u c t s p r e s e n t s f o r mid a b l e p r o b l e ms .

R e a c t i o n s o f P A I l d io l e p o x id e s w i th o l i g o n u c l e o t i d e s

f r e q u e n t l y g iv e c o mp le x mix tu r e s o f a d d u c t s . R e a c t i o n s

a t mu l t i p l e s i t e s a n d mix tu r e s o f

t rans and c i s

a d d u c t i o n p r o d u c t s a r e o b s e r v e d . E p o x id e s 2 o f

b e n z o [ a ] p y r e n e r e a c t p r ima r i l y a t t h e N 2 p o s i t i o n o f

g u a n in e . G e a c in to v h a s s t u d i ed e x t e n s iv e ly t h e

r e a c ti o n s o f t h e s e e p o x id e s w i th o l i g o n u c l e o t i d e s /

Y ie ld s c a n b e q u i t e h ig h i n c a s e s w h e r e t h e o l i g o m e r

c o n t a in s o n ly a s i n g l e g u a n in e b u t a r e s t r o n g ly

s e q u e n c e d e p e n d e n t .

Min o r a d d u c t s o f t h i s d io l e p o x id e a t t h e N s p o s i t io n o f

a d e n in e a p p e a r i n s o me c a s e s t o h a v e

dispropor t iona te ly impor tan t b io logica l impac t . An

imp o r t a n t e x a m p le i n v o lv e s mu ta t i o n s s e e n a t a n

a d e n in e re s id u e l o c a t e d a t t h e s e c o n d p o s i ti o n o f th e

6 1 s t c o d o n o f t h e r a s p r o to - o n c o g e n e b y V o u s d e n

et al,

i n t u mo r s r e s u l ti n g f r o m t r e a tme n t o f l a b o r ato r y a n im a l s

w i th ra c e mic e p o x id e 2 o f b e n z o [ a ] p y r en e .6 T h e s e

muta t ions a re ove r - represented re la t ive to the y ie ld of

t h i s min o r a d d u c t . T h e l imi t a t i o n s o f G e a c in to v ' s

me th o d a r e mo s t e v id e n t f o r s i t u at i o n s w h e r e mu l t i p l e

g u a n in e r e s id u e s a r e p r e s e n t o r w h e r e a d d u c t e d

o l i g o me r s c o n t a in in g min o r r e g io i s o me r s o r

s te reo isomers a re des i r ed . The reg ion of r a s codon 61

typi f ie s th is problem; n ot on ly i s the inh e ren t r ea c t iv i ty

o f t h e b e n z o [ a ] p y r e n e d io l e p o x id e s a t t h e a d e n in e N ~

p o s i t io n l o w b u t t h e s e q u e n c e s u r ro u n d in g t h e a d e n in e

in ques t ion , 5 ' -C-GGA-CAA-GAA-G-3 ' , conta ins four

a d e n in e a n d f o u r g u a n in e r e s id u e s .

A n a lt e r n a t i v e a p p r o a c h f o r sy n th e s i s o f a d d u c t e d

o l i g o n u c l e o t i d e s i n v o lv e s i n c o r p o r a t i o n o f a d d u c t e d

n u c l e o s id e s i n to o l i g o n u c l e o t i d e s . B o th c h e mic a l a n d

e n z y ma t i c s y n th e s e s h a v e b e e n e mp lo y e d w i th o th e r

c a r c in o g e n a d d u c t s . F o r P A H a d d u c t s , s i g n i f i c a n t

s u c c e s s h a s b e e n o b s e r v e d w i th t h e c h e mic a l a p p r o a c h ,

i n s p i t e o f t h e f a c t t h a t p r o t e c t i o n o f t h e P A H h y d r o x y

811


2/12

812 S J K~a ~

s O . ~ 4

H O ~ ~

H O 1

I NHs

HaN

H O % [ ~

. o ~ ~

H O 4

~o

H O

sOH

O H

N H

N N

N .N

7 dR

H O 2

NHa

NO

1tO

~ N H ~OH

~ O H

N N

N

8 dR

H O 3

NHa

HO.,~

H O O ~

H O $

0 b

H O

~ O H

%OH

>

d R

CI

N N

N s

d R

10a

J ~ N

4

N ~

N N

S S

dR

S c h e m e 1.

K F

F

d R

10b

groups i s requ i red dur ing DNA syn thesi s . ~ A ma jo r

shor t coming o f the p rocedure i s t ha t i t i s was te fu l o f the

expens ive d io l epox ides s ince they a re in t roduced in to

the syn the t i c pa thway a t an ear ly s t age and

o l igonucleo t ide syn theses a re normal ly car r i ed ou t wi th

the phosphoramid i t e reagen t s as the excess reagen t .

Here in we repor t a syn thes i s o f PAH adduct s a t t he N 6

pos i t ion o f deoxyad enos ine which p rov ides fu l l con t ro l

o f s t e reospec i f i c i ty and ov ercom es the p rob lem s o f

y ie lds . The method shou ld have a h igh degree o f

genera l i ty . In add i tion , an exam ple i s p resen ted o f an

equal ly e f f i c i en t app li ca t ion o f th i s s t ra t egy to the

syn thes i s o f the above men t ioned ras -61 o l igonucleo t ide

which p rov ides comple te con t ro l o f reg iospec i f i c i ty and

i n tr o d u c e s t h e PA H mo i e t y n e a r t h e e n d o f t h e

synthesis .

R e s u l t s a n d D i s c u ss i on

Thi s rou te fo r the syn thes i s o f adducted nuc leos ides

invo lves reversa l o f the nuc leoph i l e -e l ec t rop h i l e

re l a tionsh ip o f the nuc leos ide and PAH. s Thus, the

ha logen a tom of 6 -ha lopur ine 2 -deoxyr ibos ide (10) i s

d i sp laced v ia an a romat i c nuc leoph i l i c subs t i tu t ion

reac t ion by an amino t r io l der ived f rom the d io l epox ide .

The conf igura tions o f the subs t ituen t s in the am ino t rio l

determ ine the ~,3nfigurations of the subst i tuents in the

adduct .

A s imi l a r approach i s a l so be ing e mp loyed by o thers

invo lved in th i s research a rea , no tab ly by Lakshman

and by Se ide l . 7 How ever , ra ther than focus ing on the

syn thes i s o f p ro tec ted P AH ad duct s fo r use in

o l igonucleo t ide syn thes i s , we des i red a syn thes i s o f


3/12

Polyc yc l i c a rom a t ic hydroc a rbon a dduc t s 813

unprotected adducts which could be accomplished with

a minimum of steps (preferably no protection-

deprotection) and which would be very economical in

its use of valuable diol epoxides. As our targets we

chose adducts derived from t r a n s opening of diol

epoxides of an unhindered hydrocarbon (naphthalene),

a bay-region hydrocarbon (benzo[a]pyrene) and a fjord-

region PAH (benzo[c]phenanthrene).

The success of this strategy is, in no small part,

dependent on having an effective synthesis of the

aminotriols. Smith e t a l . described a two-step synthesis

of naphthalene aminotriol (4) from the corresponding

a n t i dihydrodiol epoxide by SN2 epoxide ring opening

with trimethylsilylcyanide to give an isonitrile,

hydrolysis of which gave the aminotriol. 9

Benzo[a]pyrene aminotriol (5) has been synthesized

from + ) - a n t i - d i h y d r o d i o l epoxide by Lehr and by

Meehan by two-step procedures using azide ring

opening followed by reduction, t

We have found that the conversion of a n t i - d i h y d r o d i o l

epoxides to the t r a n s - a m i n o t r i o l s can be achieved

efficiently in a single step by treatment o f the diol

epoxides with liquid ammonia for -24 h at 70-100 C

in a Parr high pressure reactor. Aminolysis has been

carried out with racemic a n t i - d i h y d r o d i o l epoxides of

naphthalene, benzo[a]pyrene, and benzo[c]phen-

anthrene to yield (+)-aminotriols 4--6. The reactions are

SN2 processes and give exclusively t r a n s opening

products in yields that are near quantitative. It is

noteworthy that even severely hindered bay-region

epoxides react readily with ammonia under these

conditions. Lakshman

e t a l . t l

in a similar study with

benzo[c]phenanthrene diol epoxide detected, in

addition to the major isomer 6, a minor isomer resulting

from t r a n s opening at the non-benzylic end of the

epoxide.

Racemic naphthalene aminotriol (4) reacted with 6-

chloropurine 2'-deoxyribonucleoside (10a) t2 to give the

diastereomeric N~ adducts Tab of deoxyadenosine in

-60% yield. Condensation with benzo[a]pyrene

aminotriol (5) under the same conditions gave only

-12% of adducts 8ab, reflecting steric retardation of

the condensation reaction. To improve the adduction

yield, the 6-chloropurine nucleoside was converted to

the fluoro analog 10b by displacement with

trimethylamine followed by fluoride ion. 'c't3 The more

reactive, but much more unstable, 6-fluoropurine

deoxyribonucleoside was treated with naphthalene

aminotriol (2.0 eq). The reaction, monitored by TLC

and HPLC, was complete within 1.5 days at 55 C with

no detectable side reactions. Preparative HPLC gave a

93% yield of 7ab. Under the same conditions the

reaction with benzo[a]pyrene aminotriol (5) required 5

days to reach completion (78% yield) and the reaction

with benzo[c]phenanthrene aminotriol 6 (50% yield)

was not finished after 5 days.

The diastereomeric products were separated by

reversed-phase HPLC. Each diastereomeric adduct was

analyzed by NMR and CD spectroscopy. Adducts of

(+)-2 at the N6 position of deoxyadenosine have

previously been prepared by Jeffrey e t a l . using

poly(deoxyadenosine) and Cheng e t a l . from calf

thymus DNA and deoxyadenosine 5'-phosphate by

reaction with diol epoxide followed by enzymatic

hydrolysis and HPLC isolation. 3 The adducts from (+)

and (-) forms of

s y n a n d a n t i - b e n z o [ c ] p h e n a n t h r e n e

diol

epoxides have been synthesized by Jerina e t a l . from 2'-

deoxyadenosine 5'-phosphate by reaction with the

enantiomeric diol epoxides followed by dephosph-

orylation and HPLC isolation. 4 The N~-adducts of a n t i -

benzo[c]phenanthrene diol epoxide have been prepared

in protected form by Lakshman

e t a l .

by a non-

biomimetic strategy similar to ours. tt

The CD spectrum of the more mobile diastereomer

prepared from benzo[a]pyrene aminotriol 5 was

identical to that reported by Cheng e t a l . for the t r a n s

adduct of (+)-2 (S configuration at the benzylic epoxide

carbon, i.e. 10S), while the slower isomer corresponded

to the t r a n s adduct of (-)-2 (10R). In the case of

benzo[c]phenanthrene, the more mobile diastereomer

(gb, 4S stereochemistry) is also the one derived from

the diol epoxide with R stereochemistry at the benzylic

epoxide carbon 4 [(-)-3]. 4 CD spectra for the adducts

derived from naphthalene aminotriol (4) (Fig. 1) have

not been reported previously but the stereochemical

assignments could be made by analogy with previously

analyzed spectra; 3'4 t4 i.e. the faster eluting isomer

exhibited a positive CD band in the 270--280 nm region

and was assigned the S configuration at the N-

substituted carbon.

l ~ f t ~ l ~ ~ l ~ ~ l

mdegl

4 I V

nml

Fig ure 1 . C i rc u la r d ic hro i sm spe c t r a of na phtha le n e d io l e poxide N 6-

de oxya de nos ine a ddue t s 7a 4S i som e r , uppe r spe c t rum ) a nd b 4R

isom e r, lowe r spe c t rum ) . Spe c t r a we re obta ine d in m e tha no l

The tH NMR spectra (Table 1) of the deoxyadenosine

adducts were assigned by a combination of COSY and

NOE difference spectroscopy. IH NMR spectra of

peracetylated derivatives of the deoxyadenosine

adducts of diol epoxides of benzo[a]pyrene and


4/12

8 1 4 S . J . K I M e t a / .

b e n z o [ c ] p h e n a n t h r e n e h a v e b e e n r e p o r t e d e a r l i e r; 3,4

h o w e v e r , i t w a s o f i n te r e s t t o e x a m i n e t h e n o n - a c y l a t e d

a d d u c t s t o d e t e r m i n e i f th e r e a r e c o n f o r m a t i o n a l

d i f f e re n c e s i n d u c e d b y a c e t y la t i o n . F o r b o t h P A H s t h e

c o u p l i n g c o n s t a n t s i n t h e t e t r a h y d r o a r o m a t i c r i n g w e r e

o n l y s l ig h t ly a f f e c t e d b y a c e t y l a t i o n ( f o r e x a m p l e , i n

a c e t y l a t e d 8 a J ~. s w a s 9 . 3 , J~ 9 w a s 2 . 3 a n d J g, o w a s - 3

H z ) , c o n f i r m i n g t h e c o n c l u s i o n r e a c h e d b y C h e n g

e t

a l . ~ ( b a s e d o n a c o m p a r i s o n o f a c e t y l a te d a n d

u n a c e t y l a t e d t e t r o l s ) t h a t n o d r a s t i c c h a n g e s a r e

i n d u c e d b y a c e t y l a ti o n . T h e d i a s t e r e o m e r i c p a i r s h a d

n e a r l y i d e n t i c a l c h e m i c a l s h i f ts , a l t h o u g h t h e r e i s a

s l i g h t u p f i e l d s h i f t o f t h e 2 ' a n d 3 ' p r o t o n s i n t h e i s o m e r

w i t h th e S c o n f i g u r a t i o n a t t h e l i n k a g e s i te . H o w e v e r , a

c o m p a r i s o n o f t h e t H N M R s p e c tr a o f t h e a d d u c t s

p r o v i d e s i n s i g h t i n t o t h e e f f e c t o f s t e r i c c o n s t r a i n t s

i m p o s e d b y b a y - r e g i o n h y d r o c a r b o n s a s c o m p a r e d w i t h

n o n - b a y - re g i o n . W i t h t h e n a p h t h a l e n e a d d u c t , t h e

c o u p l i n g c o n s t a n t b e t w e e n t h e N - s u b s t i t u t e d b e n z y l i c

C H a n d t h e v i e i n a l C H i s 7 . 9 H z . I n c o n t r a st , t h e

c o r r e s p o n d i n g c o u p l in g c o n s t a n ts i n t h e b e n z o [ a ] p y r e n e

a n d b e n z o [ c ] p h e n a n t h r e n e a d d u c t s a r e o n l y 3 . 3 a n d 4 . 2

H z , r e s p e c t i v e l y . I n a d d i t i o n , t h e r e i s a s i g n i f i c a n t

d o w n f i e i d s h i ft o f t h e a d e n i n e H - 2 p r o t o n i n t h e

b e n z o [ a ] p y r e n e a n d b e n z o [ c ] p h e n a n t h r e n e d e r i v a t iv e s .

T h e d i f f e r e n c e s i n c o u p l i n g c o n s t a n t s a n d c h e m i c a l

s h i f t s r e f l e c t t h e f a c t t h a t t h e a d e n i n e m o i e t y i s f o r c e d

o u t o f t h e p l a n e o f th e a r o m a t i c r in g m u c h m o r e

s e v e r e l y i n t h e c a s e o f th e b a y r e g i o n d e r i v a t iv e s . T h e

d i f f e re n c e i n n u c l e o s id e c o n f o r m a t i o n c a n b e e x p e c t e d

t o o c c u r i n P A H - a d d u c t ed D N A a s w e l l a n d m a y

c o n t r i b u t e t o t h e g r e a t e r c a r c i n o g e n i c i t y o f b a y r e g i o n

P A H d i o l e p o x id e s .

T h e a d d u c ts w e r e a l s o c h a r a c t e r i z e d b y m a s s

s p e c t r o m e t r y u s i n g t h e r m o s p r a y a n d f a s t a t o m

b o m b a r d m e n t . T h e r m o s p m y g a v e m u c h c l e a n e r s p e c t r a

w i t h w e a k b u t d e t e c t a b l e M H i o n s. I n a l l c a s e s

e x t e n s i v e f r a g m e n t a t i o n o c c u r r e d l e a d i n g t o f o r m a t i o n

o f i o n s c o r r e s p o n d i n g t o p r o to n a t e d a d e n i n e a n d

T a b l e 1 . H N M R d a ta f o r d e o x y a d e n o s i n e - / ~ - P A H a d d u c t s

P r oton 7a /b 8a 8b 9a 9b

Adenine

2 8.23 s br 8.53 s br 8.53 s br 83 4 s 8.54 s

8 8.24 s 8.18 s 8.18 8.18 s 8.18 s

Sugar

r 6.44 dd 6.44 ~t br 6. 44 ~t IX 6.44 ~t 6.44 - t

J - - 6 . 0 , 8.0

2' 2.82 ddd 2.85 ddd 2.83 ddd 2.83 ddd 2.81 ddd

2 2.42 ddd 2.42 ddd 2.43 ddd 2.42 ddd 2.42 ddd

3' 4.58 ddd 4.59 ddd 4.58 ddd 4.59 ddd 4.58 ddd

4' 4.08 -q 4.08 -.q 4.08 --q 4.07 &l 4.08 dd

5' 3.84 dd 3.85 dd 3.87 dd 3.84 dd 3.87 dd

5 3.74 dd 3.74 dd 3.76 dd 3.74 dd 3.75 dd

Hydro-

ca r bon

1 4.75 d 8.155 dd 8.15 dd 6.56 d 6.56 d

J,~5.6 Jl2 7.7 J,~ 7.7 Ji2 4.2 J~2 4.2

2 4.10 dd 7.97 t 7.97 t 4.67 dd 4.68 dd

Jz3 2.3 Jz3 7.7 Jz3 7.7 J23 2.5 Jz3 2.5

3 4.28 dd 8.19 dd 8.19 dd 4.24 dd 4.24 dd

J 3 . 7.9 J,3 12 J,~ 1.2 J3~ 7~ J3.4 7.8

4 5.71 Ix 8.04 d 8.04 d 4.96 d 4.99 d

J ~ s m a l l J . ~ 9.1 Jr5 9.1

5 -7.28 dd 8.12 d 8.12 d 8.01 dd 8.00 dd

. t ~ 7 . 9 j ~ 7 ~ J ~ 7 ~

Js.7 1.9

6 7.24 ddd 8.57 d 8.57 d 8.01 dd 8.00 dd

J~7 6.7 J~7 1.0 J ,, 1.0

Ju 1 4

7 7.31 ~ 5.24 dd 5.24 ~ 7.75 dd 7.75 dd

J,~ 7.6 J,s 9.0 J ~ 9.0 J,~ 88 J,~ 8,8

8 7.50 dd 4.25 dd 4.25 dd 7.75 dd 7.75 dd

JIs < I . 0 J s .o 2 . 2 J ~ 9 2 2

9 4.56 dd 4.56 dd 7.85 d 7.85 d

J9.to 3.3 Jg.,o 3.3 ,/9.1o 7.6 Jg.m 7.6

J~.11 1A J~ .. 1A

10 7.42 t 7.42 t

J i o . 6 . 1 J i o . H 6 . 1

11 7.10 t 7.11 t

Ju. n 8.7 Ju.12 8.7

8.65 d 8.65 d

2

--6.49 br -6.49 br

8.09 d br 8.09 d br

JtH2 9.4 Ju.n 9.4

8.03 d 8.03 d

Spectra were measured at 400 MHz in methan ol-d at 300 K with 0.1 Hz/pt resolution.


5/12

Polycyclic

rom tic

hydrocarbon adducts 8 5

aminotriol as well as ions arising from loss of water and

ammonia from the nucleoside adduct. Protonated

adenine (rrdz 136) was the base peak in the spectra of

8 and 9; in 7 the base peak was protonated aminotriol.

The aminotriol method should be fully applicable to

preparation of nucleosides of diol epoxides of other

PAHs and other stereoisomers of the diol epoxides.

With minor adaptation the synthetic strategy should

provide a convenient route to the adducted nucleotides

which are useful as standards for chromatographic

detection of PAH adducts in biological samples using

the very sensitive post-labeling procedures developed

by Randerath. 15 At present, PAH-deoxyadenylate

derivatives are not commercially available.

The application of this electrophile-nucleophile

reversal strategy to preparation of adducted

oligonucleotides offers the potential for substantial

benefits over the previously employed direct adduction

or the assembly of oligonucleotides from adducted

nucleosides, both in terms of structural specificity and

economics. The cost advantage lies in the fact that

introduction of the PAH residue would be delayed until

the end of the synthesis and excess aminotriol used to

promote reaction could be recovered for use in

subsequent syntheses. However, for the extension of this

strategy to oligonucleotides to be successful, several

formidable problems had to be overcome. Key amongst

them was management o f the somewhat labile 6-

fluoropurine residue during oligonucleotide synthesis.

This post-oligomerization or 'convertible nucleoside'

strategy, to use the term coined by Verdine, has been

employed by Verdine and others but for different

purposes. ~6 However, none of the previously reported

methods used 6-fluoropurine as the functionalized unit.

The fluoro substitutent is probably more active than the

ones used previously but is required with the hindered

and relatively non-nucleophilic PAH amino triols.

In conventional synthesis of oligonucleotides the

oligomer is assembled on a solid support. The 4,4'-

dimethoxytrityl group is released from the 5' position of

the terminal deoxyribose by brief treatment with an

acidic reagent and the protective groups, i.e. the acyl

groups on the exocyclic amino groups and cyanoethyl

on the phosphate, and the succinate linkage to the solid

support are cleaved with ammonia or other

nucleophiles. It was immediately apparent that

fluoropurine 10b would not be stable to ammonia

treatment, thus presenting a serious block to the

application of a post-oligomerization nucleophile-

electropbile reversal strategy to the synthesis of

oligonucleotides bearing adenine N6 PAH adducts.

By judicious choice of conditions it might be possible

to prepare oligonucleotides containing chloropurine

nucleoside 10a. However, the nucleoside studies had

shown that 10a was not sufficiently reactive to give

satisfactory yields with hindered amines. We

considered the possibility of preparing the

oligonucleotide containing lOa at the target site and

then converting it to the 6-fluoro analog immediately

before reaction with the amine. However, conditions

could not be found to achieve this transformation

efficiently. As a consequence, we chose to carry out the

aminotriol adduction reaction on immobilized

oligonucleotide while the protective groups were still in

place on exocyclic amino sites and phosphate groups.

This procedure offers the advantage that unreacted

aminotriol is readily recovered from reaction mixtures

but suffers from the inherent difficulties of solid phase

reactions: i.e. (1) the immobilized reagent cannot be

purified before the adduction reaction; (2) progress of

the adduction reaction is difficult to monitor; (3) the 6-

fluoropurine could have reduced reactivity in the solid

support.

For assembly of fluoro phosphoramidite 12, several

possibilities presented themselves as to the timing of

introduction of the fluoro substituent during assembly of

the trityl-protected phosphitylated nucleoside. After

some experimentation, the most dependable of these

appeared to be conversion of chloro nucleoside 10a to

4,4'-dimethoxytrityl derivative l l a 17 followed by

replacement of the chlorine by fluorine to give l l b

(Scheme 2). Phosphoramidite reagent 12 is then

prepared by treatment of l l b with 2-cyanoethyl-

N,N,N',N'-tetraisopropylphosphoradiamidite in the

presence of diisopropylammonium tetrazolide. I~

Purification was achieved by flash chromatography on

silica gel. A small amount of triethylamine was added

to the eluting solvent since gradual release of HF by

adventitious moisture causes precipitous destruction of

the reagent. The reagent when pure can be stored at low

temperature if protected from moisture.

Oligonucleotide synthesis was carried Out by standard

solid-phase procedures to prepare 5'-d(C-GGA-CXA-

GAA-G)-3' where X is fluoronucleoside 10b (Scheme

3). After the final detritylation, the immobilized

oligonucleotide was carefully washed with acetonitrile

and dried to remove occluded acid and moisture.

Treatment with racemic aminotriol 5 was carded out at

65 C for 5 days using approximately a 10-fold excess

of the aminotriol. Our earlier studies had employed

dimethylacetamide as the solvent but dry

dimethylsulfoxide containing diisopropylethylamine has

been found to give cleaner reactions and higher yields.

Subsequently, the cyanoethyl and acyl protecting

groups were removed by treatment with concentrated

NH4OH (60 C, 7.5 h) to give benzo[a]pyrene-adducted

oligonucleotides 13ab. The diastereomeric mixtures

resulting from use of racemic aminotdol were purified

by reversed-phase HPLC (Fig. 2). The lipophilicity of

the PAH moieties facilitated separation from

unadducted oligonucleotides. The diastereomers present

in 13 separated readily in spite of the fact that the

diastereoisomerism reflects only a small portion of the

oligonucleotide structures.

Oligonucleotides 13ab were obtained in a combined

yield of 13 . The oligonucleotides were further purified

by polyacrylamide gel electrophoresis for use in


6/12

816 S.J. KIMetal.

10a

4-CH30-CIsH4)2CsHsCCI

CI

D M T r - O - ~

1 1 a O H

1. Me3N

2 . K F

F

D M T r - O - ~

O , I P r

1 2 ~ ) ' ~N ' I P r

\ c

NCCH2CH 2OP N-IPra)2

Scheme

2.

F

D M T r - O - ~

O H

1 1 b

1.50

1.00

0.,qo

o.e0

0 . 0 1 U 2 0 . 0 ~ 4 O O 8 0 . 0

Figure 2. H PLC elution profile of oligonucleotides 13a and b

CGGAC.AN~ ~v~-~ ~-AGAAG). PRP-1 column, 55 C, linear

gradient, 10 mM ethylenediamine acetate, pH 7.45 , 0-20

acetonitrile ove r 20 rain.

mu ta g e n e s i s a n d / n

v i t r o

p o ly m e r a se s t u d ie s , n T h e

p u r i f i ed o l i g o n u c l e o t i d e s w e r e c h a r a c t e r i z e d b y c i r c u l a r

d ichro ism (F ig . 3 ) , cap i l la ry ge l e lec t rophores is (F ig .

4 ) , a n d e n z y m e d ig e s t i o n ( F ig . 5 ) . I n o u r p r e l imin a r y

r e p o r t w e w e r e u n a b l e t o o b t a in c o mp le t e h y d r o ly s i s

w i th sn a k e v e n o m p h o sp h o d ie s t e r a se a n d a lk a l i n e

p h o sp h a t a se ; s t h i s i s i n a c c o r d w i th r e su l t s o b t a in e d b y

o th e rs . 19 C o m p le t e d ig e s t i o n w a s a c c o m p l i sh e d w i th a

c o mb in a t i o n o f n u c l e a se P l h y d r o ly s i s f o l l o w e d b y

t r e a tme n t w i th sn a k e v e n o m p h o sp h o d ie s t e r a se a n d

a lk a li n e p h o sp h a t a se. T h e n u c l e o s id e r at i o s f o r b o th 1 3 a

and b were dC , 2 ; dG, 3 .9 ; dA, 3 .8 ; BP-nuc leos ides

8 a /b , 1 .1 ( u n mo d i f i e d o l i g o n u c l e o t i d e g a v e v a lu e s o f

d C , 2 ; d G , 4 . 0 ; d A , 4 .8 ) . S t e re o c h e m ic a l a s s ig n m e n t s

f o r th e B P m o ie ty i n 1 3 a a n d b w e r e ma d e f r o m th e C D

sp e c t ra b y c o m p a r i so n w i th C D sp e c t r a o f 8 a a n d b ; ~ '

c o n f i rm a t i o n w a s o b ta i n e d b y H P L C c o m p a r is o n o f t h e

a d d u c t e d n u c l e o s id e s fr o m th e e n z y m e d ig e s t i o n w i th

8a an d b (F ig . 6 ) ; the in-s t e lu ted o l igon uc leo t id e , 13a ,

b a d t h e s a me B P s t e r e o c h e mis t r y a s 8 b , i .e . 1 0 R .

D e ta i l e d h ig h f i e ld N MR s tu d i es o n t h e

o l ig o n u c l e o t i d e s d u p l e x e d w i th t h e i r c o mp le me n ta r y

s t rands a re in progress .

I ' ' ' I ' ' ' ' I '

.

1 0

oi@

I , , , , I , . , ,

I I I I O

Iml

Figu re 3. Circular dichroism

spectr of

oligonucleotides 13a (dash ed

line) and b (solid line) n 20 mM sodiumphosphate, pH 7.0. Spectra

are no rmalized o 1.0 absorbancc at 260 nm.

T h e sy n th e t i c me th o d p r o v id e s a v e r s a ti l e p r o c e d u r e f o r

th e p r e p a r a t i o n o f o l i g o n u c l e o t i d e s b e a r in g s t r u c tu ra l l y

d e f in e d a d d u c t s o f p o ly c y c l i c a r o ma t i c h y d r o c a r b o n s a t


7/12

Polyc yclic aromatic hydrocarbon adducts 8 7

12

I~A

~ n t l ~ d s

F

~ N I ~ N

) =

P r o t e c t a d

n u c l e o s i d e

, ) , 5

A r

N H

I N H 3

A r

N

C G G A C ] A G A A G

5 1 t I I I I I I I I

1 3 a A r :

3

O H O H

O H 1 3 b A r =

1 0 R

1 S

S c h e m e

3 .

th e/ ~ position of adenine. The success of the method is

independent of sequence; i.e. the sequence chosen for

the present study contained a large number of other

target sites which if the adducts had been prepared by

direct adduction would have led to an extremely

complex mixture of products. The strategy should be

equally applicable to other PAlls and other

stereochemical arrangements of functional substituents.

We have yet to demonstrate that the method will be as

useful for preparation of oligonucleotides bearing

adducts on the N 2 position of guanine. Halogen

substituents at the C-2 of purines are substantially less

reactive with nucleophiles than those at C-6. While

displacements occur readily with small sterically

unencumbered nucleophiles bay-region aminotriols of

PAHs may be too stefically hindered to give

satisfactory yields.

An inherent limitation in the post-oligomerization

strategy for preparation of PAH adducted oligo-

nucleotides is that clean separation of diastereomeric

adducts will not in all cases be feasible by HPLC.

Indeed in examples which will be reported elsewhere

we have seen separations which are inadequate to

completely resolve the diastereomers. The use of

individual enantiomers would circumvent this problem

but the high cost and poor availability of optically pure

species are problems which need to be overcome. We

are currently developing an enantioselective route to

PAH diol epoxides.

E x p e r i m e n t a l

CAUTION. The diol epoxides of polycyclic aromatic

hydrocarbons are potent carcinogens.

Spectroscopic ethods

NMR spectra were obtained at 300 and 400 MHz on

Bruker spectrometers. Mass spectral data were obtained


8/12

8 1 8 S . J . K I M e t a l .

0.20

0.10

J:l

An Efficient Route to N 6 Deoxyadenosine Adducts of Diol Epoxides of Carcinogenic Polycyclic...

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