1
A Mismatch
-
Selective Bifunction
al Rhod
ium
-
Oregon
Green Con
jugate:
A Fluorescent Prob
e for Mismatched DNA
Bri
an M.
Zeglis and Jacq
ueline K. Ba
rton*
Division of Ch
emi
stry and Ch
emi
cal
Engineeri
ng,
Cal
ifornia Institute of Tech
nology, Pas
adena, California,
91125
jkbart
on@caltech
.edu
Sup
po
rti
ng
Information
Gen
era
l Sy
nthes
is:
Rh
Cl
3
was purch
ased from Pressure Ch
emi
cals and used as rec
eived. Oreg
on
Green
514
TM
succinimi
dyl ester was purchased fro
m
Molecu
lar
Pro
bes
(Invitrogen), stored
at
-
20
C, and used as recei
ved. Unless otherwise noted, all non
-
aqueous solvents were
purch
ased from
Fluka and stored under arg
on and over mo
lecu
lar sieves. All water used was purified using the
Mi
lliQ water
purificat
ion system.
All o
ther start
ing mat
eri
als were purchased fro
m
Aldrich
Ch
emi
cal
Co
mp
any and used as rec
eived.
1
H NMR were perform
ed on a 300 MHz Varian
Sp
ect
romet
er at room
temp
erat
ure using solvent res
idual signal as a refer
ence to TMS
. ESI mas
s
spect
romet
ry was perfo
rmed
a
t the Pro
tein/Peptide Mi
croanalytical
Laboratory (Cal
ifornia
Institute of Tech
nology). UV
-
Vis spect
ra were
taken on a Beck
man
DU7400 spect
rophotometer,
and extinction coeffi
cients were determi
ned using IC
P
-
MS
.
Ligand
Sy
nthesis:
The peg
-
linker
mo
dified bip
yridine linker
was synthesized
in two steps
(Su
pplem
entary
Figure
1) fro
m
4
-
(4’
-
met
hyl
-
[2,2’]b
ipyridinyl
-
4
-
yl)
-
butyric aci
d prep
ared
acco
rding to the published proced
ure in Della Ciana, L.; Ham
ach
i, I. Meyer, T.J.
JOC
1989
, 54,
1731
-
1725.
Sup
plementa
ry F
igur
e 1
N
N
O
O
N
O
O
H
2
N
O
O
N
H
2
N
N
O
O
H
N
O
O
H
O
C
H
2
C
l
2
,
1
0
0
%
N
N
O
N
H
O
O
N
H
2
1
.
5
,
D
C
C
D
M
F
,
A
r
p
e
g
b
i
p
y
4
-
(4’
-
methy
l
-
[2,2’]bipy
ridiny
l
-
4
-
yl)
-
buty
ric acid suc
cinimidy
l ester:
27 mg
of DCC (.129
mmo
l) and 14 mg
N
-
hydroxysucci
nimi
de (.129 mmo
l) were
added to 30 mg
(.117 mmo
l) of 4
-
(4’
-
methyl
-
[2,2’]b
ipyridinyl
-
4
-
yl)
-
butyric acid (prep
are
d acco
rding
to the published proced
ure
in
Della Ciana, L.; Ham
ach
i, I. Meyer, T.J.
JOC
1989
, 54, 1731
-
1725.) in CH
2
Cl
2
and stirred at
room
temp
erature
for 2 hours. After 2 hours, the react
ion mi
xture
was filtered and concen
trated in
vacu
o. The pure final product was o
btained as a clear
oil aft
er
column
chromatograp
hy (SiO
2
,
EtOAc, Hex).
1
H NMR:
8.55 (dd, 4H), 8.26 (d, 2H), 7.21 (d, 2H), 2.86 (m, 6H), 2.68 (t, 2H), 2.46
(s, 3H), 2.171 (t, 2H). ESI
-
MS:
m/
z = 354.
2
N
-
(2
-
[2
-
(2
-
amino
etho
xy)
-
etho
xy]
-
ethy
l)
-
4
-
(4’
-
methy
l
-
[2,2
’]bipy
ridiny
l
-
4
-
yl)
-
buty
ramide
(peg
-
bpy
):
100 mg
4
-
(4’
-
met
hyl
-
[2,2’]b
ipyridinyl
-
4
-
yl)
-
butyric aci
d succi
nimi
dyl ester was
dissolved in 3 mL DMF
, and added to a solution of 2 mL (ex
cess) of
2,2’(et
hylenedioxy)bis(et
hylami
ne) in 1 mL DMF. After two hours, .0
5 mL DIEA was added to
ensure
deprotonation. The react
ion mi
xture
was stirred
for 16 hours at room
temp
erature.
After 16
hours, the react
ion mi
xture
was concentrat
ed in vacu
o, taken up in CH
2
Cl
2
, extract
ed 2X with a
saturat
ed sodium
bicarb
onate solution, d
ried over Mg
SO
4
, filtered
, and re
-
concen
trat
ed in vacuo.
The final product was obtained pure
as a clear
oil without column
chromat
ography.
1
H NMR: 8.5
(m,
2H), 8,26 (s, 2H), 7.14 (t, 2H), 3,5
-
3.3 (m,
10H), 3.9
-
3.6 (m,
4H), 2.43 (s, 3H), 2.25
-
2.15 (m,
2H),
2.15
-
2.05 (m
, 2H). ESI
-
MS: 387.
Conjug
ate Sy
nthes
is:
As stated earl
ier,
the met
allointercal
ator mo
iety was synthesized
via the
sequential addition of phenanthroline, chrysenequinone, and peg
-
mo
dified bypyridine to Rh
Cl
3
acco
rding to the proced
ure
publishe
d in Pet
itjean
, A.; Bart
on, J.K.
J. Am. Ch
em. Soc.
2004,
126,
14728
-
14729. However, the peg
-
linker
mo
dified bipyridine was substituted for the alklyami
no
-
bipyridine ligand fro
m
that publicat
ion.
Meta
llointer
calator/Or
egon Gree
n Coupl
ing
:
5 mg
Oregon Gre
en 514 succi
nimi
dyl ester was
dissolved in DMF
and added to a solution of 5 mg
of
2
. After two hours of stirring, .5 mL DIEA
were
added, and the res
ultant react
ion mi
xture
was allowed to stir under arg
on overn
ight. After
16 hours, 4 ml
H
2=
O were
added to
the react
ion mi
xture, and the resultant solution was loaded
onto a Waters Sep
-
Pak, washed with water, and eluted with 1:1:.001 (H
2
O:MeC
N:TFA). The
solution was then fro
zen
with liquid nitrogen and lyophilized
. The desired
final product,
1
, was
purified by
prep
arat
ive HPLC using a grad
ient of 99.9:.1 (water:
TFA) to 99.9:1
(acet
onitrile:TFA) over
the course of 80 mi
nutes. ESI
-
MS:
m/
z = 709, 1418. UV
-
Vis: 302 nm
(
ε
=54,800); 313 nm
(
ε
=44,600); 519 (
ε
=78,000).