S
1
Supporting information
1.
Synthesis of conjugate
1
.
2. O
bserved and simulated
isotope patterns
of conjugate 1 (ESMS)
.
3
. Methods used to study the extent of
oligonucleotide
platination
.
4
.
Figure S
1
: Autoradiogram of the denaturing gel revealing the photo
cleavage pattern
within the DNA adducts after cyanide reversal.
S
2
1.
Synthesis of conjugate 1
.
Synthetic scheme:
1) Synthesis of the bipyridine ligand precursor:
2) Synthesis of the rhodium complex core:
S
3
3) Synthesis of the platinum unit and assembl
y of the full conjugate:
Scheme S1
: Synthetic steps to complexes
1
(conjugate) and
2
(rhodium recognition
unit control).
Materials and Methods
: Commercially available chemicals
and dry
solvents
were
purchased from Aldrich, Lancaster, Fluka, Acros and Pr
essure Chemical Company and
used as received.
[Rh(chrysi)(phen)(NH
3
)
2
]Cl
3
was prepared as described in Mürner, H;
Jackson, B.A.; Barton, J.K.
Inorg. Chem
.
1998
,
37
, 3007
-
3012.
Dichloro[(d,l)
-
diaminopropionicacid]platinum
II
was prepared according to a publi
shed procedure
(Altman, J.; Wilchek, M.
Inorg. Chim.
Acta
1985
,
101
, 171
-
173).
1
H
(
13
C NMR) were
run on a 300 MHz
(75 MHz)
Varian spectrometer in CDCl
3
at room temperature using
S
4
the solvent residual signal as a reference relative to TMS. ESI mass spectrome
try was
performed at the Protein/peptide Micro Analytical Laboratory (California Institute of
Tech
nology)
while elemental analysis was performed by Desert Analytics Laboratories
(Tucson, AZ).
S
ynthesized
DN
A and
DNA adducts were characterized by MALDI
spec
trometry
on a Voyager DE
-
PRO MALDI
-
TOF mass spectrometer
with a 337 nm
nitrogen laser source
from Applied Biosystems using
a
hydropicolinic acid
/picolinic acid/
ammonium citrate matrix.
Infrared spectra were recorded on a Biorad Win
-
IR pro
spectrometer as
KBr pellets.
4’
-
(7
-
Bromoheptyl)
-
4
-
methyl
-
[2,2’]bipyridine
: 16 mL of 2.5M
n
BuLi in hexanes
(40 mmol; 1.05
equiv.
) were added dropwise to a mixture of N,N
-
diisopropylamine (6.4
mL; 46 mmol; 1.2
equiv.
) and dry THF (25 mL) at
-
78°C under argon. After stirring
at
that temperature for 15 minutes, 7.05
g of 4,4’
-
dimethyl
-
[2,2’]bipyridine (38 mmol)
solubilized in 150 mL of dry THF were
quickly
cannulated into the reaction
mixture
at
-
78°C under argon. The chocolate brown solution was stirred at
-
78°C for 1h under
argon
and warmed up in an ice bath for 5 minutes before 30 mL of 1,6
-
dibromohexane (0.19
mol; 5.1
equiv.
) in 30 mL of dry THF were added all at once. The orange
-
yellow solution
was stirred at 0°C for 1.5h before water (100 mL) was added and the pH adjusted
to 7.0
with 2 M HCl. 100 mL of diethyl
-
ether were added and the aqueous layer extracted with
dichloromethane (3x50 mL). The combined organic layers were dried (Na
2
SO
4
), filtered
and concentrated in vacuo, and the pure
product
obtained
as a colorless oil
i
n 80% yield
after column chromatography (SiO
2
; CH
2
Cl
2
, Et
2
O).
1
H NMR:
8.55 (d,
3
J
=5.1 Hz, 1H),
8.53 (d,
3
J
=4.2 Hz, 1H), 8.21 (m, 2H), 7.10
-
7.15 (m, 2H), 3.39 (t,
3
J
=7 Hz, 2H), 2.69 (t,
3
J
=8 Hz, 2H), 2.44 (s
, 3H), 1.84 (quint.,
3
J
=6.6 Hz,
2H), 1.70 (quint.,
3
J
=7.3 Hz, 2
H),
S
5
1.3
-
1.5 (m, 6H).
13
C NMR:
155.8, 155.7,
152.7, 148.8, 148.7, 148.1, 124.5, 123.8, 121.9,
121.2,
35.3, 33.8, 32.6, 30.1 28.9, 28.4, 27.7, 21.1.
R
f
(Al
2
O
3
, CH
2
Cl
2
)
= 0.68
.
ESMS:
349.2
(MH
+
)
, 369.0 (MNa
+
)
.
N
-
[
7
-
(4’
-
Methyl
-
[2,2’]bipyridin
yl
-
4
-
yl)
-
h
eptyl]
-
phthalimide
:
8.69 g of
4’
-
(7
-
b
romoheptyl)
-
4
-
methyl
-
[2,2’]bipyridine
(25 mmol) and 5.13 g of
potassium phthalimide (27.7 mmol, 1.11
equiv.
) were suspended in 60 mL of dry DMF
and heated at 50°C for 16h. T
he mixture was poured o
nto ice and dich
loromethane (200
mL) added. After separation, the aqueous layer was extracted with dichloromethane (3x
40 mL) and the combined organic layers dried (Na
2
SO
4
), filtered and concentrated in
vacuo.
The pure product was obtained
as a white solid
in
80
% yield af
ter digestion in
ethanol.
1
H NMR: 8.53
(d,
3
J
=
5.1 Hz, 1H), 8.5
2
(d,
3
J
=5.4 Hz, 1H), 8.20 (
m
, 2H),
7.80
-
7.84 (m, 2H), 7.67
-
7.70 (m, 2H), 7.10
-
7.15 (br d, 2H), 3.66
(t,
3
J
=7
.3 Hz, 2H), 2.67
(t,
3
J
=8 Hz, 2H), 2.42 (s, 3H), 1.62
-
1.72
(
m
,
4
H),
1.3
-
1.4
(m, 6H
).
13
C NMR: 168.4, 156.1,
156.0, 152.7, 148.9, 148.8, 148.0, 133.8, 132.0, 124.6, 123.9, 123.1, 122.0, 121.2, 37.9,
35.4, 30.3, 29.1, 28.9, 28.5, 26.7, 21.1.
R
f
(Al
2
O
3
, CH
2
Cl
2
)
= 0.5
1.
ESMS:
414.2
(MH
+
)
,
436.2 (MNa
+
)
.
EA:
C
26
H
27
N
3
O
2
calc
. %C
75.52
, %H
6.58
, %
N
10.16
,
obs
. %C
75.24
,
%H
6.67
, %N
9.85
.
7
-
(
4’
-
Methyl
-
[2,2’]bipyridinyl
-
4
-
yl)
-
heptylamine
(bpy
-
NH
2
)
:
1.3 mL of
hydrazine hydrate (27 mmol, 5.2
equiv.
) were added to a suspension of
N
-
[7
-
(4’
-
m
ethyl
-
[2,2’]bipyridinyl
-
4
-
yl)
-
heptyl]
-
phthalimide
(2.1 g, 5.08
mmol) in ethanol (40 mL), and
the mixture heated at 40°C under argon for 12h. The white precipitate was filtered
,
washed with ethanol (60 mL) and the filtrate concentrated in vacuo. The residue was
taken up in chloroform (50 mL), filtered once
more and ext
racted with 1 M
HCl (4x30
S
6
mL). After washing with chloroform (3x30 mL), the combined aqueous layers were
basified to pH~10 with NaHCO
3
(s) and 4 M aq
.
KOH. The mixture was extracted with
5x50 mL CHCl
3
and the combined organic layers dried (Na
2
SO
4
), filtere
d and
concentrated in vacuo to yield the desired product as a white solid in 98%.
1
H NMR: 8.55
(d,
3
J
=4.5 Hz, 1H), 8.53
(d,
3
J
=4.2 Hz, 1H), 8.2s (s
, 2H),
7.1
2 (
d,
3
J
=5.2 Hz,
2H),
2.6
-
2.75
(
m
,
4H), 2.43
(s, 3H), 1.6
9
(
quint.,
3
J
=7 Hz
,
2
H),
1.3
-
1.5 (m, 8
H).
1
3
C NMR:
156.1,
156.0, 152.7, 148.9, 148.8, 148.1, 124.6, 123.9, 122.0, 121.2, 41.4, 35.4, 32.0, 30.3, 29.1,
29.0, 26.6, 21.1.
ESMS:
284.2
(MH
+
).
EA: C
18
H
25
N
3
.0.19 CHCl
3
calc
. %C 71.38, %H
8.29, %N 13.73,
obs
. %C 71.40, %H 8.07, %N 13.70.
[Rh(chrysi)(phen)
(bpyNH
2
)]
Cl
3
(
2
)
:
A solution of
[Rh(chrysi)(phen)
(NH
3
)
2
]Cl
3
(103 mg) and
7
-
(4’
-
m
ethyl
-
[2,2’
]bipyridinyl
-
4
-
yl)
-
heptylamine (55 mg, 1.94 mmol, 1.5
equiv.
) in a mixture of acetonitrile (25 mL) and ethanol (1 mL) was
refluxed for 16h.
After evaporation of the
solvents, the brown red residue was taken up in water and loaded
on a Sephadex SPC25 cation exchange column (chloride form) and eluted with a gradient
of MgCl
2
in water (0
-
0.5 M). The brown band was desalted by
ad
sorption onto a SepPak
C1
8 Cartridge (Water
s), washed with copious water and eluted
with a mixture of
H
2
O/CH
3
CN/TFA
(0.5:0.5:0.001). 67 mg of a dark red
hygroscopic
solid were obtained
after lyophilization (
60
%).
ESMS: 410.2 (
2
-
3Cl
-
H)
2+
, 431.2 [
2
-
3Cl
-
H
+
CH
3
CN]
2+
, 820.2
(
2
-
3Cl
-
2H)
+
.
UV
-
vis (H
2
O)
λ
ma
x
(nm)
(log
10
ε
):
207 (4.9), 267 (4.7),
288 (sh),
300 (
4.4),
312 (
sh
), 396 (3.8), 459 (3.6).
IR (KBr, cm
-
1
):
3460 (br), 3090, 2941, 2860, 1677, 1620
(sh)
, 1516, 1432, 1202, 1136, 834, 718
. EA:
C
48
H
45
N
7
RhCl
3
.
CF
3
CO
2
H
.2.7 H
2
O
calc. %C
54.99
, %H
4.75
, %N
8.98
,
obs. %C
54.99
, %H
4.75
, %N
8.77
.
S
7
1
: To a solution of
2
(19 mg, 2.0 10
-
5
mol) and dichloro[(d,l)
-
diaminopropionic
-
acid]platinum
II
(18 mg, 4.9 10
-
5
mol, 2.4
equiv.
) in 1.0 m
L of d
ry DMF were added 20
mg of EDCI (5
equiv.
) and two drops of dry triethylamine u
nder argon. The brown
mixture was stirred
at room temperature for 20h,
adsorbed onto a SepPak C18 Cartridge,
washed thoroughly with water, dilute sodium hydroxide and water again, before elution
with a mixture of H
2
O/CH
3
CN/TFA (0.5:0.5:0.001).
The
desired
complex was obtained
quantitatively as a
hygroscopic
brown solid after lyophilization. ESMS:
586.9
(
1
-
3Cl
-
H)
2+
,
1172.1
(
1
-
3Cl
-
2H)
+
.
ESI MS MS on peak 1172.1: 1136.0 [(
1
-
3Cl
-
2H)
-
Cl]
+
, 1099.1
[(
1
-
3Cl
-
2H)
-
2Cl]
+
.
UV
-
vis (H
2
O)
λ
max
(nm) (log
10
ε
): 207 (4.9
), 227
(sh
),
270 (4.7), 290
(sh),
300 (sh),
310 (sh), 395 (3.8), 459 (3.7
)
[NB: The spectra of
1
and
2
are very similar
and show superimposed signals
]
.
IR (KBr, cm
-
1
): 3440 (br), 3180, 3093, 2936, 2862,
1677, 1625
(sh), 1566, 1523, 1431, 1202, 1133, 833
,
801, 71
9
.
EA: C
51
H
51
N
9
ORhPtCl
5
.
6
CF
3
CO
2
H.
14.5
H
2
O
calc
. %C 34.08, %H 3.63, %N 5.68,
obs
. %C 34.01, %H 3.46, %N
5.95.
2. O
bserved and simulated
isotope patterns of conjugate 1 (ESMS)
.
O
bserved
(2+)
S
imulated
(2+)
S
8
Observed (1+)
Simulated (1+)
3
. Methods used to study the extent of platination
10
μ
M of labeled duplex
[1]
CXn (X= C, G; n = 0, 3, 9) were prepared by heating a
mixture of each strand (same concentration
, estimated by UV
-
vis spectroscopy
; one of
which contained radiolabeled strands) solubilized in a 20 mM NaCl, 20 mM Na
phosphate pH 7 b
uffer at 90° C for 10 minutes and letting th
e samples slowly cool down
to room temperature
(~3h)
. Duplex CXn (10
μ
M in buffer) and complex 1 or 2 (10
μ
M in
water) were then mixed and incubated in the absence of light for up to 24h. 20
μ
M
aliquots were take
n out at determined times, diluted to 70
μ
M by addition of 0.5 M NaCl
(to stop the platination reaction
[2]
), filtered through molecular sieves (to remove free
platinum reactant
[3]
) and dried in vacuo. After quanti
fication of their activity, the dry
sampl
es
were dissolved in a appropriate volume of loading dye (
mixture of
formamide,
bromophenol and xyleneblue [1]
; typically 10
μ
M
)
and analyzed by gel electrophoresis
under denaturing
conditions (0.3 mm thick, 18%
acrylamide,
TBE buffer, 90W, 2000 V
for 90 m
in). The PtenCl
2
controls were run for about twice as much time, until the fast
moving
dye
was eluted out of the gel, conditions under which the major adducts separates
from the parent band well enough to be quantified. The
gel was then exposed
to phosphor
S
9
screens
at room temperature for an appropriate amount of time (typically 4
-
10h) in order
to collect a quantifiable signal without saturating the screen (less than 300 000 counts
total). Molecular Dynamics phosphorimager was used to collect data from the s
torage
screen and the gel image analyzed using
the ImageQ
uant
program
.
References:
[1] Sambrook, J.; Frisch, E.F.; Maniatis, T.;
Molecular Cloning: A Laboratory
Manual
2
nd
ed.; Cold Spring Harbor Laboratory; New York,
1989
.
[2] Gonnet, F.; Kozelka, J.; Ch
ottard, J.
-
C.;
Angew. Chem. Int. Ed. Engl
.
1992
,
31
,
1483
-
1485.
[3] Micro Bio
-
spin Chromatography Columns, Bio
-
Gel P
-
6 gel, Bio
-
Rad; 6,000
daltons exclusion limit.
S
10
3. Autoradiogram of the denaturing gel revealing the photocleavage pattern within
the DNA a
dducts after cyanide reversal.
3’
C
G
C
G
G
T
A
T
C
A
C
G
T
A
C
A
C
5’
3’
C
G
C
G
G
T
A
T
C
A
C
G
T
A
C
A
C
5’
A+G
C+T
A+G
C+T
LC
DC
2
DC
1
h
ν
1
h
ν
2
h
ν
1
F
LC
DC
2
DC
1
h
ν
1
h
ν
2
h
ν
1
F
PtenCl
2
PtenCl
2
CG3
CC3
S
11
Figure S1
: autoradiogram of the gel
electrophoresis of products after
the following
treatments: 1) incubation of duplexes
CG3
and
CC3
(5
μ
M) with complexes
1
or
2
(5
μ
M) in sodium phosphate buffer pH 7 (10 mM) in the presen
ce of sodium chloride (10
mM) at 37°C for 12h
, protected from light
; 2) irradiation of the mixture for 15 min. at
442 nm wavelength (
HeCd
laser; 12.5 mW power); 3) treatment with 0.2 M NaCN
(basic) at 60°C for 24h
, protected from light
(all samples except
Maxam Gilbert)
.
DC n:
duplex incubated
with complex
n
in the absence of light
, h
ν
n: irradiation after incubation
with complex
n
.
h
ν
n F: irradiation after molecular sieve purification of the incubated
duplexes.
PtenCl
2
:
duplex incubated
with
PtenCl
2
.