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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
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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
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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
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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.
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7/26/2019 An Efficient Route to N 6 Deoxyadenosine Adducts of Diol Epoxides of Carcinogenic Polycyclic Aromatic Hydrocarb
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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
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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
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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
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7/26/2019 An Efficient Route to N 6 Deoxyadenosine Adducts of Diol Epoxides of Carcinogenic Polycyclic Aromatic Hydrocarb
8/12
8 1 8 S . J . K I M e t a l .
0.20
0.10
J:l