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a nd
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2 9
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h
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atog
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s atef
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t
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exp
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toE
B3 0
F i
g
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.C h
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atog
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of
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s atef
rom
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m
u sV I
L
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Rfl
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L S I
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ary
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a s s sp
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S /
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ta nd
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sp
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N
M
R
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n etic r e s o n a n c e
N
T
H
n e utr al
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PE
sol i d
p
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o nT L
C
th i
nl
ay
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atog
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I
N
T
R
O D U
C T
I
O N
1
^
-B
u tad
i
e n e(
B D
)
, a c o
l
o rl
e s sg
a s,h
a sb
e e nm
ai
nly
u s ed
i
nt
h
em
a n uf
a ctu r e of
sy
n th
eti
c r ub b
er.E
m
i
s si
o n of
B D
m
ay
o ccu rd
uri
ng
i
tsm
an uf
a ctu re, storag
e, tr an sp
o rt, u s e o rd
i
sp
o s al
.B D
h
a s al
s ob
e e nf
o u nd
i
nauto
m
ob i l
e exh
au st[
1
]
and
ci g
a r et te sm
ok
e[
2]
.B D
i
sl i
sted
a s on e of 18 9
h
a z a rd
o u s ai
rp
ol l
u ta n tsi
n th
e19 9 0 C l
e a nA i
rA
ct A
m
e nd
m
e n ts[
3
]
.T h
e In te rna ti
on al A g
en cy f
o rR
ese a r ch
o nC
an c e rh
a s cl
a s si f i
ed B D
a s ag
r o up
2 A
c a r ci
n og
e n[
4
]
w
i
th s uf f i
ci
e n t e vi
d
e n c ef
o r c a r ci
n og
e ni
ci
ty
i
n exp
eri
m
e ntal
a ni
m
al
s,b
u tl i
m
i
ted
evi d
e n c ef
or c a r ci
n og
e ni
cit
y
of
B D i
nh
um
a n s.E
p
i
d
em
i
ol
og
i
c al
stud i
e s of
w
ork
e r s e xp
o s ed
toB D
p
ro vi
d
e c o ntr o v e r si
al
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d
e n c ew
i
th
the r e s ult
s sh
ow
i
ng
th
e a s s o ci
ati
o n of
B D
e xp
o s ur ew
i
th
l y
m
p
h
ati
c a nd
h
em
atop
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eti
c c a n c ersd
e sp
i
te a n o v er al l
d
ef i
cit
i
n c a n c e rm
o rtal i
ty
[
5
,6
]
.R
e c e ntf i
nd i
ng
si
n r od
en ti
nh
al
atio n c a rci
n og
e ni
ci
ty
stud i
e s of
B D
h
a v ed
em
o n str ated
t
h
e stri k i
ng
l
y
h i
g
h
s e n si
tivi
ty
of
m
i
c e toB D
-i
n
d
u c ed
tu
m
o r s c om p
ar ed
to r ats[
7
-9
]
.
L
ow
exp
o s u re of B D
, eve n at6
.
2 5
p p
m
,i
n c r e a s ed
i
n ci
d
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tum
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nf
em
al
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m
i
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n c o nt
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d
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r ag
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a
w
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r ats e xp
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to8 0 0 0
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m
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em
ark
ed
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n ca rci
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eni
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of B D
i
s n ea rl
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th
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ers of
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i
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s ap
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i
c e a nd
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c a r ci
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n th
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c co rd i
ng
to s e v e r al
stud
i
e s o n th
em
etab
ol i
sm
of
B D
,B D
i
sf i
r stm
etab
ol i
z ed
b y
cy
to ch
r om
eP 4 5 0
2 E 1
m
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e n a s e(
C
Y P 2 E 1
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to1
,2
-e
p
o xy b
ute n e(
E B
)
, a nd
th
e nE B
c a nb
e ei
the r o xi
d
i
z ed f
u rth
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od
i
ep
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uta n e(
D
E B)
o rh y d
r ol y
zed
vi
a ep
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ase to3
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u te n e- l ,2
-
d i
o
l
,w
h i
ch
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b
s eq
u e nt
ly m
et
ab
ol i
z ed
t
o3
,4
-ep
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-
b
u tP 4
5 0
[
10
- 13
]
. T
h
e el i
m
i
n ati
o n of
BD
m
ay
ei
th
e rp
r o c e ed
b
y
g
l
utath i
o n ec o n
j
ug
atio
n toE
B o rD E
B, o rb y h y d
rol y
si
s of E B
orD E B
[
13
]
.B D
m
utag
eni
ci
ty
i
n th
ep
re s e n c e of
m
etab
ol i
c a cti
vati
o nh
asb
e e nd
em
o n str ated i
n th
eS
. ty
p h i m
u ri
um
a s s ay
. Th
e r e a cti
v em
etab
ol i
te sE B
a nd D E
Ba r e str o n
g
l y
m
u tag
e ni
ci
n te st sy
stem
s su ch
a sS
alm
o n el l
a,S
a c ch
a r om
y
c e s, a nd
D
r o s op
h i l
a[
1 4,15
]
.T h
em
u tag
e nic
i
ty
of
al l
th
r eem
etab
ol i
tes, E B,E
BD
and D E B
,h
a sb
e e n sh
ow
n atb
oth
tk
a nd hp
rt l
o ci i
n c ul
tu r ed T K 6 h
um
a nl
y
m
p h
ob
l
a stoid
c e
l ls
[
16
]
.Si
m
i l
arl y
,
B D
,E
B a nd D E B
w
er em
u tag
e ni
ci
n sp
l
e ni
cT
c el l
sf
r om
ex
p
o s ed B 6 C 3 F 1
m
i
c e[
1 7
]
.I
n cre a s e si
n si
ster ch
r om
a ti d
ex ch
a ng
e, ch
rom
o s om
al
a
b
err a ti
o n s, a nd m
i
c r o n u cl
e ated
p
e ri
p
h
e r al b l
o od
ery
th
r o cy
te sw
er ef
o u nd
af
te ri
n vi
v o e xp
o s u r e toB D
[
18
,19
]
.E
xp
o s u r e of B D
al
s o c a u s ed
c o v al
e n tb i
nd
i
ng
toh
em
og l
ob i
n[
2 0
]
a nd
n ucl
e a rp
ro tei
n s[
2 1
,2 2
]
a nd p
r o tei
n-
D
N
A
a nd D
N
A
-D
N
A
cro s s
l i
nk
s[
2 3
]
i
nm
i
c e.A
s th
em
aj
o rm
etab
ol i
te of B D
m
etab
ol i
sm
,E B h
asb
e e n sh
ow
n to re a ctw
i
th d
e oxy g
u a n o si
n e a nd
i
n vi
tr oD
N
A
tof
o rm
tw
om
ai
n ad d
u ctsi d
e n ti f i
ed
a s7
-(
2
-h y d
rox
y
-3
-b
u te n-
l
-y
l
)
g
u ani
n e(
E
B-G
uaI
)
a nd
7
-(
l
-h
y
d
ro xy
-3
-b
ute n -2
-y
D
g
u a ni
n e(
E B
-G
u a
I I
) [
2 4
]
.D E B
h
a s al
s ob
e e n sh
ow
nt
o r e a ct
w
i t
h g
u a ni
n et
og
i
v e a n ad
d
u ct,7
-N
-(
2
,
3
,4
-tr
i h
y d
ro xy
b
u ty
l
)
g
u a ni
n e[
23
]
.E B
-G
u a
I I
a nd
7
-N
-(
2
,3
,4-tr
i h y d
r o xy b
u ty l
)
g
u ani
n ew
e red
ete cted
,b
u t n otq
u a nti
tated i
n th
el i
v erD
N
A h
y
d
r ol
y
s atef
r om
B 6 C 3 F 1
m
i
c e e xp
o s ed
to5 0 0
p p
m (
l
,4
- "
C
)
l
,3
-
b
u ta
d
i
e n e;h
ow
e v er, th
ey w
e r e n otd
ete cted i
nD
N
A f
r om
r ats af
te r e xp
o s u r e to ^''
C
-B D
[
2 3
]
.
T h
e r e s ul
ts of
th
em
u tati
o n al
sp
e ctrum
i
nd
u c ed by
E B i
n sp
l
e ni
cT
-c e
ll
s of
m
al
eB 6 C 3 F 1
m
i
c eh
a v e sh
ow
nf
o u rA
:T
- >G
:C tr a n si
ti
o n s a nd
o n eA
:T
- >C
:G
tr a n s v er s
i
o n am
o ng 18 b
as ep
ai
r s ub
sti
tu ti
o r \s[
1 7
]
.F
u rth
erm
o r e, n e ar
l y h
al f
of
t
h
ep
oi
n tm
u tati
o n si
nd
u c ed b y
BD
, E B, and D E B
o cc ur ed
atA
:T b
as ep
ai
rs[
1 7
]
.A d d i
ti
o nal l
y
, tw
o stud i
es o n th
ef
req
u e n cy
of
m
u tati
o n si
n th
eb
o n em
a r r ow
of
b
as ep
ai
rsf
ol l
ow
i
ng
B D in
h
al
ati
o n e xp
o sur e s[
25
,2 6
]
. Th
e sei
n vi
v od
ata s ugg
e stt
h
at ad
e ni
n eD
N
A
ad d
u ctsm
ay
b
ei
n v ol
v ed
i
nB D
m
u tag
e n e si
s a nd
c a r c
in o
g
e n e sis
.T h
ep
urp
o s e of
th
ep
r e s e n t stud y i
s to ch
ara cteri
z e tw
om
ai
na
d d
u cts th
at a r ef
o rm
ed
f
r om
th
e r e a cti
on of E B
w
i
th
ad
eni
n e, and
to u ses
j
m
th
e si
zed
E B
-g
u a ni
n e a nd E B
-a
d
e nin e
ad d
u cts a s ref
e r e n c e c om
p
o u nd
st
od
ete ct and
q
u anti
tate th
e cor r esp
o nd i
ng
ad d
u ct sp
e ci
e sf
orm
ed
i
nD
N
A
M
A
T E R I
A
L S
A N D
M
E T
H
O D
S
C h
em
i
c al
sE B
(
9 8 %
)
w
a s ob
tai
n ed f
r om
A l d
ri
ch
(M
i l
w
a uk
e e,W
I
)
.G l
a ci
al
a c eti
c a ci d
w
a sb
o ug h
tf
r om
E
M
S
ci
e n c e(
G i b b
stow
n,N
J)
.A
d
e ni
n e,g
uani
n e,g
u a n o si
n e,c a c o
d
y
l i
c a ci d
a nd
c al f
th y
m
u sD
N
A
w
er ep
ur ch
a s ed f
rom
Si
g
m
a(
S
t.L
o ui
s,M
O
)
.A
c eto n e,d
i
eth
y
l
eth
e r,m
eth
a n ol
, a nd
H
C l
w
e r ep
u r ch
a s ed
f
r om
M
al l i
n ck
r od
t(
P
a ri
s,K Y
)
.A
c eti
c a ci d
, am
m
o ni
um
f
o rm
ate,T
ri
s,p
o ta s si
um
d i h
y
d
rog
enp
h
o sp
h
ate, and
p
ota s si
um
h y d
r oxi d
ew
er eb
o ug
h
tf
r om
F i
sh
e rSc
i
e n ti f i
c(
P i
t tsb
u rg
,P A
)
.C
I8
re v er s ep
h
a s e r e si
n a nd
p
h
o sp
h
o r u sp
e n ta o xi d
ew
e rep
u rch
a s ed f
r om
B
ak
er(
P h i l l i
p
sb
u rg
,N
J)
.H
i
g
h
Pe rf
o rm
an c e Li q
ui d C h
rom
a tog
r ap h y
(
H
P L C
)
A C 1 8
re v e rs ep
h
a s e c ol
um
n(
d
es cri b
ed
b
el
ow
i
n e ach
s ecti
o n of
m
eth
od
s)
,a R
h
od y
nei
nj
ecto r(
Bax te r,C h
a rl
o t te,N
C
)
eq
ui p p
ed
w
i
th
a 2 -m
l i
nj
e cti
o nl
o op
,2
W
a te r s5 10
H
P L C
p
um p
s(M
i l l i
p
or e,M
i l f
o rd
,M
A
)
, a nd
o n e of
tw
od
ete cti
o ns
y
stem
sw
ere u s ed
f
or th
e a n al
y
si
s of
s om
e r e a cti
o np
rod
u cts:(
1
)
aH
ew
l
et tP
a ck
ard 1 04 0 A
d i
od
e-ar ray d
ete cto r(
D A D
)
eq
ui
p p
ed
w
i
th
aH
P L C
-d
ete cti
o nc o
m p
u ter sy
stem
,(
2
)
a nA
p p
l i
ed B i
o sy
stem
s,I
n c.(
R
am
s ey
,N
J)
75 7
ab
s o rb
a n c ed
ete cto r.T h
e el
u e nts u s ed i
n th
eH
P L C
s ep
a r ati
o n of
th
e r e a cti
o np
r od
u ctsi
n ar ea ct
i
o nm
i
xtur e ar ed
e s c ri b
ed b
el
ow
.M
a ssS
p
ectrom
etry
L i q
ui d
se co nd
a ry i
o niz a
ti
o nm
a ss sp
ectrom
etry
(
L S
IM
S
)
w
as c o nd
u cted
too
b
tai
nm
a ss sp
e c tr a of
an al y
te s usi
ng
a VG
7 0-25 0 S
EQ
h y b
ri d
m
a s s sp
ectr om
etere
q
ui
p p
ed
w
i
th
aC
s*g
u n op
e r ati
ng
at ap
ri
m
a ry
i
o nb
e am
of 3 0 k V
.T h
eC
s*
i
o n so
f
m
eth
a n ol
a nd
w
ate r .T
a nd
em
sp
e ctrom
etry
(M
S
/ M
S
)
stud i
e sw
er ep
erf
o rm
ed
b y
m
as s-sel
e c ti
ng p
o si
ti
vei
o n sf
r om
th
em
ag
n etic an al y
zer and
al l
ow
i
ng
th
em
toc o
l l
id
ew
i th n eu tr al
A
r atom
s(
p
r e s s ur e 2 x 10'^
to r r
)
p
r e s enti
n th
ef i
rstq
u ad
rup
ol
e at a c ol l i
si
o n e ne rg y
of 5 0
eV f
ol l
ow
ed b y
th
em
a s s a n al y
si
sw
i
th
th
es e con
d q
uad
rup
ol
e.R
e a cti
o n of E B
w
i
th G
u a n o si
n eT h
ep
r o c ed
u re u s ed
w
a sm
od i f i
ed f
r om
th
at of C i
t ti
et ah
[
2 4
]
.G
u a n o si
n e(
1 0 5
m g
,0
.3 7
m
m
ol
)
w
a s r e a cted
w
i
th E
B(
26 6
m g
,3
.7 2
m
m
ol
)
i
n1
.75
m
l
ac eti
ca c
i d
(
d i l
u ted f
r om
eq
u al
v ol
um
e,0
.8
75
m
l
, of d i
sti l l
ed
w
a te r)
.T h
e r e a cti
o nm
i
xtu r ew
a sh
e ated
at5 0
°C f
o r5 h
o u r s.T h
e r e a cti
o nw
a s stop p
ed
w
it
h 1
.7 5
m
l
acetone an
d
7.0
m
l d i
eth y l
eth
er . Th
ep
r eci
p
i
tate af
ter c e n tri f
ug
ati
oni
n aC
entr a-8 R T
ab l
etop
c e ntri f
ug
e(
I
n tern atio n al E
q
ui p
m
e ntC
om p
a ny
,N
e ed
h
am H
ei
g
h
ts,M
A
)
at4 0 0
rp m
(
2 0
xg
)
f
o r5
m
i
n u te sw
a sd i
s s ol
v ed
i
n2 0
m
M
p
ota s si
um
p
h
o sp
h
atep
H
5
.5
(
2
m
l
)
.T h i
sp
rod
u ct s ol
u ti
o nw
a sh
y d
r ol
y
z ed b
y
h
e ati
ng
at8 0
°C f
o r5 h
o u r si
n1
N H
C l
, a nd
n e utr al i
z ed w
i
th 1
N
K
O H
a nd
th
e nf
r o z e nu nt
i
l
l
aterH
P L C
a n al
y
si
s.T
h
eH P L C
c ol
um
n u s ed
i
n th
e s ep
a r ati
o n of
E B
-g
u a n o si
neh y d
rol
y
s atew
a s a Baxter(M
cG
aw
P
ark
,I L
)
B &
J
O
D 5
o ctad
e cy
l
a n a
l
y
ti
c c ol
um
n(
2 5
x0
.4 6
cm
,5
m m
)
.T h
e el
u e n ts u s ed i
nH
P L C
w
er e(
a)
7 0
%
a
q
u e o u sm
etha n ol
(
d
eg
a s s ed
)
a nd
(
b
)
2 0
m M
p
ota s si
um p
h
o sp
h
ateb
uf f
e r,p H
5
.5
(
d
eg
a s s ed
)
.T h
e c o nd
i
ti
o n s of f l
ow
r ate(
P R
)
a nd g
r ad i
e n ti
n th
eH
P L C
s e
p
a r ati
o nw
er e:(
1
)
P R 1
.5
m
l
/ m
i
n, a n
d
(
2
)
al i
n e a rg
r ad
i
e n tf
r om
1 5
%
(
a)
a nd
8 5 %
(
b
)
to5 0 %
of
e ach
o v e r3 0
m
i
n u te s.P
u ri f i
ed f
r a cti
o n s at th
e s am
e rete nti
o nti
m
ew
er ep
o ol
ed i
n to a s ol
uti
o n a nd
a sm
al
l p
o rti
o n of
th
ep
o ol
ed
s ol
uti
o nw
a su se
d f
o rH
P L C
a n al
y
si
sw
i
th D A D
to ob
tai
n ap
urit
y
i
nd
ex(
sp
e ctr am
atch
v al
u ew
h i
ch
r a ng
e sf
rom
0
to1 0 0 0
,i
nd i
c ati
ng
n o ch
a ng
ei
n ab
s orp
ti
o n sp
e ctr a at
i
n te r e stw
e r ef
r eed f
r om p
h
o sp
h
ate s al
tsb
y
ap p
l
y
i
ng
to th
eH
P L C
col
um
n a nd
w
a sh i
ng
w
i
th d
eg
a s s ed
d i
sti l l
ed
w
aterf
or th
ef i
r st3 0
m
i
n a nd
th
en al i
n e a rg
r ad i
e n tf
rom
1 0 0 %
d
eg
a s sed d i
sti l l
ed
w
ater to1 0 0 % d
eg
a s s ed
m
eth
a n ol i
n5
m
i
n .T h
ef
ra cti
o n s of i
n ter e stw
ere c ol l
e cted
a nd
th
e n e v ap
orated i
n v a c u o tod
ry
n e s s sb y
aS
a v a nt(
F
a rm
i
ng d
al
e,N
Y
)
s v cl OO
sp
e ed
-v a c a n
d
th
e nd
i
s s ol
v ed i
nd
ist i lle
d
w
ate r tom
e as ur e^
ax ^^
^ ^
m
in^
^^^
aSh
im
ad
z u(
C
ol
um
b
ia,M
D
)
U V
-16 0 U
sp
e ctr op
h
o tom
ete r.A f
terd
ry i
ng
ag
ai
n th
ro ug
h
th
e sp
e ed
-v a c
,
t
h
ef
r a cti
o n sw
e r e s ub
j
e cted
toL S I
M
S
a nd
M
S
/ M
S
.R
e a cti
o n of E B
w
i
th A
d
e ni
n eA
d
e ni
n e(
5 0
m
g
,0
.3 7
m m
ol
)
w
a s r e acted
w
i
th E B
(
2 6 6
m
g
,3
.7 2
m m
ol
)
i
n am
i
x tu re of 1 5
m
l
10
m
M
T
ri
s-H
C l
p H
7
.2w
i
th 15
m
l
m
eth
a n ol
.T h
e r e a cti
o nm
i
x tu r ew
a sh
e ated
at5 0
°C f
o r5 h
o urs.U
nr e acted E B
w
a s extr a cted f
r om
th
er e a ct
i
o nm
i
x tu r eb
y
ad
d i
ng
a nd d
e c a n ti
ng
1 2 0
m
l d i
eth
y
l
eth
er tw
i
c e.T h
er e
m
ai
ni
ng
r e a cti
o nm
i
x tu rew
asf
r oze n u nti l l
aterH
PL C
a nal y
si
s.P
ri
m
ary
se
p
a rati
o n of E B
-ad
e ni
n ep
rod
u ctsw
a s c o nd
u cted
w
i
th
aB
e ck
m
a n(
F
uU
e r to n,C A
)
o ctad
e cy l
s em
i
-p
r ep
ara ti
v e c ol
um
n(
2 5
x1
cm
,5
m m )
c o up
l
ed w i
th
D
A
D
.T h
e el
u e n ts u s ed i
np
ri
m
a ry H
P L C
s ep
a r ati
o nw
e r e(
a)
7 0 %
aq
u e o u sm
eth
a n ol
(
d
eg
a s s ed
)
a nd
(
b
)
2 0
m
M
p
ota s si
um
p
h
o sp
h
ateb
uf f
e r,p H
5
.5
(
d
eg
a s s ed
)
.T h
eo t
h
e r c o nd i
ti
o n s of
th
ep
ri
m
a ry H
P L C
s ep
ar ati
o nw
e re :(
1
)
P R 3
.0
m
l
/ m
i
n, a n
d
(
2
)
al i
n e arg
r ad i
e n tf
r om
1 5 %
(
a)
a nd 8 5 %
(
b
)
to5 0
%
of
e a ch
o v e r3 0
m
i
n ute s.P
u ri f i
ed f
r a cti
o n sf
r om
th
e s am
e r ete n ti
o n ti
m
ew
e r ep
o ol
ed
a nd
a sm
all p
o rti
o no
f
th
ep
o ol
ed
s ol
u ti
o nw
a s u s ed
f
o r th
e s e c o nd
ary
H
P L C
a n al
y
si
sw
i
th
aB
eck
m
a n o ctad
e cy l
a n al y
ti
c c ol
um
n(
2 5
x0
.4 6
cm
,5
m m
)
a nd D A D
to ob
tai
n ap
u ri
ty
i
nd
exf
o r th
ep
o ol
ed
f
r acti
o n s. Th
e se c o nd
a ry
H
P LC
a n al y
si
sw
a sc o n
d
u cted
u nd
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i ti
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ed
i
n th
ep
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ri
m
ary
f
r e ed f
rom p
h
o sp
h
ate sal
tsb
y w
a sh i
ng
i
n s ol i d
p
h
as e ex tr a ctio n(
SP E
)
c ol
um
n s(
c om
p
ri
s ed
of
th
em
atri
x of C 18
r e v ers ep
h
a s e re si
n)
w
i
th 6
-8
m
l
d
i
stil
l
ed w
ate rf
ol l
ow
ed b y 6
m
l
of 7 0
-8 0 %
aq
u e o u sm
eth
a n ol
.T h
ef
ra cti
o n s of i
n ter e stw
e r eeva
p
o r ated i
n va c u o tod
ry
ne ss usi
ng
a sp
e ed
-v ac.
E
ach f
r a cti
o nw
asd i
s s ol
v ed
i
ntod i
sti l l
ed
w
ater tom
e a s ur e\
na x'K
mw
^ ^^
^
^ ab
s orp
ti
o n v al
u e s atX
^
a xan
d X
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sp
e ctr op
h
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etry
.A f
te rd
ry i
ng
ag
ainb y
th
e sp
eed
-v a
c, s o
m
e of
t
h
ef
r a cti
o n sw
e r e s ub
j
e cted
toL S I
M
S
, a nd
M
S
-
M
S
.
S
ub
s eq
u e nt sy
nth
e s e s of E B
-a
d
e ni
n ep
rod
u ctsw
er e c a r ri
ed
o utf
oll
ow
i
ng
t
h
e s am
ep
r o c ed
ur e tof
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er ch
arateri
ze th
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sp
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etry
a n
d
N
M
R
a n al y
si
s.A f
ter th
ep
ri
m
ary
s ep
arati
o n a nd
s e c o nd
ary
v e ri fi
c atio n of
t
h
ep
u ri
ty
of
p
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ed f
r action s of i
nte r e st, thef
r a cti
o n sw
er ed
esal
ted
a nd d
ri
ed
t
w
i
c e a sd
e s c ri b
ed
ab
ove to c om
p l
etel y
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ts.I
n ord
er tom
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or ep
r e ci
s el
y
th
ew
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g
h
t of
th
eE B
-ad
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n ep
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e tub
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ai
ni
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t
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ed
ry p
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ed p
r od
ucts af
te r sp
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i
th p h
o sp h
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ei
n ad
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h i
ch
w
a s s ub
s eq
u e ntl y
seti
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or3 d
ay
sb
ef
or ew
ei
g
h i
ng
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terd
e si
c c atio n a nd
m
e a s uri
ng
th
ew
ei
g h
t of
t
h
eE B
-a
d
e ni
n ep
r od
u cts, th
ey
w
e r ed i
s s ol
v ed i
nd i
sti
l
l
ed
w
ater a nd
a sm
al l f
ra cti
o n of
e a ch
s o
l
u ti
o nw
as tak
e n tog
et th
e ab
so rp
tio n v al
u e s atA
^
g ^ ^ ^ i
d X
^
^ ^, a nd
th
ec o r r e s
p
o nd
i
ng
ab
so rp
ti
vi
ty
c oef f i
ci
e n ts(
or exti
n cti
o n c o ef f i
ci
e n ts, e) (
p H
7
)
of
e a c
h
E B
-a
d
e ni
n ep
r od
u ct of i
n te r e stw
e r ed
ete rm
i
n ed
.F
ol l
ow
i
ng
th
e s am
ep
r o c ed
ur e ab
o v e ex c ep
tf
or r ep
l
a ci
ng w
ate rw
i
th
H
C l
(
p H
1
)
o rN
aO H
(
p H
13
)
,t
h
e ab
s orp
ti
vi
ty
c o ef f i
ci
e nt of
e a ch E B
-a
d
e ni
n ep
r od
u ct, a nd
the a s s o ci
ated X
j ^^^ ^a n
d A
^j^n ^tp H
^
o r13
w
er ed
ete rm
i
n ed
.T h
e u n u s ed p
ar t of
e a ch E B
- ad
e ni
n ep
r od
u cti
nw
ate rw
a sd
e si
c c ated
ag
ai
n, a nd
s ub
j
e cted
toN
M
R
a n al y
si
s to'
H
-N
M
R
S
p
e ctr aD
ata'
H
-N
M
R
s
p
ectr a of b
o th E B
- ad
e ni
n ep
r od
u ctsw
a s ob
tai
n ed
o n a Bruk
erA
M
X
-5 0 0
sp
e ctr om
ete ri
nD
M
S
O
-d 6
. E
B
-
A d
e
I
:5
4.1 0
(
b
rd
d
,I
H
,J
=13
.4 1
H
z,J
=
8
.
3
2H
z,C
H
j-N H
)
, 4.4 1(
b
rd d
, IH
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= 1 3.4 1H
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.6
9H
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H
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)
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(
b
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)
,
5
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d
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=1 0
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H
z, ci
sC
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2=C
H
)
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d
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H
z , tr a nC
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2=C
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(
b
r s,I
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(
m
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2=C
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8
.
0 0
(m
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6-H
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)
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and
8
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p
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.(
tw
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)
.
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d
eI I
:5
3
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2(
b
rd
d
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=13
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H
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H
z,C
H
2-
O H
)
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(
b
rd
d
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H
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=13
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H
z ,J
=8
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H
z,C
H
2-
O
H
)
,5
.2 0
-
5
.
3 0
(
m
,4
H
,C
H
-
C
H
=C
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2, a n
d
C
H
j-O H
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(
m
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2=C
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1 0
(
m
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H
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^-
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d N
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)
,
8
.3 5
a nd
8
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p
.p
.m
,(
tw
o s, IH
,H
-2
)
.
R
e a cti
o n of E
Bw
i
th C
al f
Th y
m
u sD
N
A
T
h
ep
ro c ed
ur ed
esc ri b
ed b
el
ow w
a s al
s om
od i f i
ed f
r om
th
at of C i
t ti et al
.[
2 4
]
.C
al f
th
y
m
u sD
N
A
(
3 6
m g
)
w
asd i
s s ol
ved i
n1 0
m
M
T ri
s-
H
C l b
u
f f
e rp H
7
.2
(
7
m
l
)
b y
g
e ntl
y
m
ix
i
ng
and
th
e nE B
(
3 3 4
m g
,4
.7 6
m m
ol
)
a nd
m
eth
a n ol
(
7
m
l
)
w
e r e ad d
ed
to th
e s ol
uti
o n .T h i
s r e acti
o nm
i
xtu r ew
a sh
eated
a t3 7
°
C f
o r1 8
h
o urs.T h
e r e a cti
o nm
i
x tu rew
a s ex tra cted
tw
i
c ew
i
th 2 8
m
l d i
eth
y
l
eth
e r tor e
m
o v e u nr e a cted E B
.E i
ther n e utr al
th
erm
al h
y
d
r ol y
si
s o r a ci d h
y
d
r ol
y
si
sw
a sd
o n ew
i
th
s am p
l
e sf
rom
th
e r em
ai
ni
ng
E B
-D
N
A
a
q
u e o u s s ol
u ti
o n.I
n th
en e u tr a
l
th
erm
al
h y d
rol
y
si
s, s am p
l
e s ol
u ti
o ni
n10
m
M
c a c od y l
i
c a ci
d w
a sh y d
r ol y
z ed b y h
e ati
ng
at9 5
°C f
or3 0
m
i
n ,w
h i l
ei
n a ci
d
h
y
d
r ol
y
si
s, s am
p
l
e s ol
u ti
o ni
n0
.1
N H
C l
w
a sh
e ated
a t7 0
°
C f
o r3 0
m
i
n.A f
te r n e utr al
th
e rm
al
h y d
r ol y
si
s, th
e r es ul
ti
ng
h y d
r ol y
s ate of
e a ch
s am
p
l
ew
e n t th
r o ug
h
m
i
cro co nce ntr atio n . Th
e neu tr al
th
e rm
al h y d
r ol y
s a te(N
TH ) w
a s ch i l l
ed
to 4°
C
a n