S1
Bifunctional Rhodium Intercalator
Conjugates as Mismatch-directing DNA
Alkylating Agents
Ulrich Schatzschneider and Jacqueline K. Barton
*
Division of Chemistry and Chemical Engineering, Cal
ifornia Institute of Technology, Pasadena, CA 91125
jkbarton@caltech.edu
Supporting Information
Synthesis and characte
rization of conjugate
1
and intermediates
Scheme S1 - Synthesis of tris-h
eteroleptic rhodium complexes
Scheme S2 - Synthesis of an amino-functionalized bipyridine ligand
Scheme S3 - Introduction of the aminoalkyl-
substituted bipyridine to the rhodium-
chrysenequinone diimine complex
Scheme S4 - Synthesis of a carbox
y-functionalized aniline mustards
Literature
S2
Synthesis and characterization
of conjugate 1 and intermediates
Conjugate
1
of [Rh(phen)(chrysi)(bpy)]
3+
tethered to an aniline mustard was prepared in
a thirdteen step sequence as outlined in Schemes S1 to S4.
[Rh(phen)(chrysi)(NH
3
)
2
]
3+
2
was synthesized in four steps
from rhodium trichloride,
phenanthroline, and 5,6-chr
ysenequinone, and purif
ied as described by Mürner, Jack-
son, and Barton (Scheme S1).
[1]
The synthesis of the aminoal
kyl-functionalized bipyridine
6
is shown in Scheme S2. The
mono-anion of 4,4'-dimethylbipyrid
ine, obtained by deprotonation of
3
with one equiva-
lent of freshly prepared lithi
um diisopropylamine, was r
eacted with an excess of 1,6-
dibromohexane under carefully controlled conditions.
[2, 3]
After aqueous workup and col-
umn chromatography on silica with a gradient
of dichloromethane and diethylether, 4-(7-
bromoheptyl)-4'-methylbipyridine
4
was obtained in 58 % yield. This was converted to
phthalimide
5
in almost quantitative yield by r
eaction with potassium
phthalimide fol-
lowed by cleavage to 4-(7-a
minoheptyl)-4'-methylbipyridine
6
with hydrazine monohy-
drate in methanol.
[4]
Aminoalkyl-substituted bipyridine
6
was then reacted with [Rh(phen)(chrysi)(NH
3
)
2
]
3+
2
in
anhydrous acetonitrile at el
evated temperature to give [Rh(phen)(chrysi)(bipy
#
)]
3+
7
in 43
% yield after purification on
Sephadex SP C25 (Scheme S3).
The aniline mustard
12
was prepared in four
steps from 4-aminophenol
8
as outlined in
Scheme S4.
[5-7]
The starting material was reacted with bromoacetic acid ethyl ester and
separated from other alkylati
on products by column chromatography on silica to give
9
.
This compound was then reacted
with oxirane to give (4-(
N
,
N
-bis(2-hydroxy-ethyl)-
phenoxy)acetic acid ester
10
. The hydroxy groups were subs
tituted by chlorides upon
treatment with phosp
horoxytrichloride in benzene to give
11
and the ester protective
group then removed under acidic conditions to yield
12
.
Conjugate
1
was then prepared
by coupling of
7
with
12
in anhydrous dimethylforma-
mide in the presence of EDAC (1-ethyl-3
-(3-dimethylaminopropyl)carbodiimide) and
HOBt (1-hydroxybenzotriazol) as shown in
Scheme 1 and finally
purified by semi-
preparative HPLC.
S3
Scheme S1. Synthesis of tris-het
eroleptic rhodium complexes.
Rh
N
Cl
Cl
Cl
Cl
R
hCl
3
+
N
(H
3
O)
CF
3
SO
3
H
Rh
N
OTf
OTf
OTf
OTf
N
Rh
N
NH
3
NH
3
NH
3
NH
3
N
Rh
N
NH
NH
3
NH
NH
3
N
NaOH
NH
3
3
3
67 %
73 %
(two steps)
70 %
N
N
O
O
2
S4
Scheme S2. Synthesis of an aminoal
kyl-functionalized bipyridine ligand.
N
N
N
N
N
O
O
LDA
BrC
6
H
12
Br
N
2
H
4
N
O
O
K
Br
N
N
NH
2
N
N
50 %
98 %
76 %
34
5
6
S5
Scheme S3. Introduction of the aminoalkyl-s
ubstituted bipyridine
to the rhodium-
chrysenequinone diimine complex.
Rh
N
NH
NH
3
NH
NH
3
N
3
N
N
NH
2
+
Rh
N
HN
NH
N
N
N
NH
2
3
26
7
S6
Scheme S4. Synthesis of a carboxy-
functionalized aniline mustard.
NH
2
OH
Br
COOEt
NH
2
O
COOEt
O
N
O
COOEt
HO
OH
N
O
COOEt
Cl
Cl
POCl
3
N
O
COOH
Cl
Cl
conc. HCl
28 %
23 %
64 %
60 %
8910
11
12
S7
Synthetic procedures and characterization
4-(7-Bromoheptyl)-4'-methylbipyridine 4
Anhydrous tetrahydrofuran (25 ml) was plac
ed in a dry Schlenk bottle under argon and
cooled to -78 °C with an acetone/
dry ice mixture. Then diisopropylamine (4.6 ml, 32.8
mmol) was added via syringe followed by
n
-butyllithium (15 % so
lution in hexane, 17.2
ml, 27.4 mmol) to give a slightly yellow soluti
on which was kept at -78 °C. In a separate
Schleck bottle, 4,4'-dimethylbipyridine (5.0
g, 27.2 mmol) was dissolved in anhydrous
tetrahydrofuran (150 ml), prec
ooled to -78 °C and then added to the former solution. The
mixture, which turned deep brow
n, was stirred at that te
mperature for 1 h and then al-
lowed to warm to 0 °C by placing in an
ice bath. In another Schlenk bottle, 1,6-
dibromohexane (20 ml, 51.2 mmol) was dissolv
ed in anhydrous tetrahydrofuran (20 ml)
at room temperature
and then added to the former solution
at once(!). The mixture first
turned blue but the color changed to yellow wit
hin 1.5 h hours, after which the reaction
was quenched by addition of wa
ter (100 ml). After stirring
for 30 min, the pH was ad-
justed to pH = 5 and the mixture left to
stand overnight. The yellow organic phase was
separated, the aqueous phase extr
acted with ether (100 ml) and dichloromethan (2x 100
ml), and the combined organic phases washed
with 5 M sodium chloride, dried over
magnesium sulfate, and the solvent removed
in vacuo
at room temperature(!). The
slightly yellow oil obtained was purified by co
lumn chromatography on silica first with di-
chloromethane as the eluent to remove e
xcess 1,6-dibromohexane,
and then with a
gradient of dichloromethane/ether (100:0
→
0:100). After removal of the solvent, the
product was obtained as
a white solid (5.48 g,
15.78 mmol, 58.0 %).
1
H NMR (CDCl
3
,
300 MHz):
δ
= 8.55 (t,
J
= 4.8 Hz, 2H), 8.23 (m, 2H), 7.13 (d,
J
= 4.8 Hz, 2H), 3.40 (t,
J
=
6.7 Hz, 2H), 2.69 (t,
J
= 7.8 Hz, 2H), 2.44 (s, 3H), 1.84
(m, 2H), 1.70 (m, 2H), 1.37 (m,
6H); MS(ESIpos): 369 (M+Na), 347 (M+H), 267 (M-Br).
4-(7-phthalimidoheptyl)-4
'-methylbipyridine 5
4-(7-Bromoheptyl)-4'
-methylbipyridine
4
(0.70 g, 2.00 mmol) wa
s dissolved in anhydrous
dimethylformamide (125 ml) and solid potassi
um phthalimide (0.45
g, 2.40 mmol) was
added to the slightly yellow soluti
on. After stirring for 90.5 h,
the solution was diluted with
chloroform (80 ml) and then poured into wate
r (250 ml). The phases were separated and
the aqueous phase extracted with chloroform (2
x 100 ml). The combined organic phases
were washed with 0.2 M sodium hydroxide (
100 ml), water, and brine, dried over mag-
nesium sulfate, and the solvent removed to gi
ve the product as a colorless oil which so-
lidified to a white solid upon standi
ng (0.81 g, 1.96 mmol, 98.0 %).
1
H NMR (CDCl
3
, 300
MHz):
δ =
8.53 (m, 2H), 8.21 (m, 2H), 7.83 (m, 2H),
7.69 (m, 2H), 7.12 (m, 2H), 3.67 (t,
J
= 7.2 Hz, 2H), 2.67 (t,
J
= 7.8 Hz, 2H), 2.43 (s, 3H),
1.66 (m, 4H), 1.36 (m, 6H);
MS(ESIpos): 414 (M+H), 436 (M+Na).
4-(7-aminoheptyl)-4'-methylbipyridine 6
Phthalimide
5
(0.81 g, 1.96 mmol) was dissolved in
methanol (50 ml) by heating to 70 °C
to give a colorless solution. Then hydrazin
e monohydrate (0.2 ml, 4.1 mmol) was added
and the reaction mixture stirred at 70 °C for
19 h. The solvent was removed and 6 M hy-
drochloric acid (100 ml) added
to the white solid obtained. The suspension was ex-
S8
tracted with chloroform, the organic phase wa
shed with 6 M hydrochloric acid (60 ml),
and the combined aqueous phases then adjusted to
pH = 8 with 5 M sodium hydroxide.
The aqueous phase was extrac
ted with chloroform and ether, the combined organic
phases dried over magnesium su
lfate and evaporated to dryne
ss to give the product as
a white solid (420 mg, 1.48 mmol, 76 %).
1
H NMR (CDCl
3
, 300 MHz):
δ =
8.54 (t,
J
= 4.5
Hz, 2H), 8.21 (m, 2H), 7.13 (d,
J
= 4.8 Hz, 2H), 2.68 (m, 4H),
2.43 (s, 3H), 1.85 (s, 2H),
1.70 (m, 4H), 1.39 (m, 6H);
MS (ESIpos): 284 (M+H).
[Rh(phen)(chrysi)(bipy
#
)]Cl
3
7
4-(7-aminoheptyl)-4'-methylbipyridine
6
(160.0 mg, 0.56 mmol)
was dissolved in anhy-
drous acetonitrile (35 ml
) and then [Rh(phen)(chrysi)(NH
3
)
4
]Cl
3
2
(226.6 mg, 0.33 mmol),
dissolved in 25 ml of the same solvent with s
onication, was added to the former solution.
The mixture was heated to refl
ux under argon for 19.5 h, cool
ed to room temperature,
and the solvent removed to give a deep brown so
lid. The solid was dissolved in a mini-
mum amount of 0.05 M magnesium chloride
and methanol and then loaded onto a col-
umn of Sephadex SP C25 (20x
4 cm) pre-equilibrated with 0.
05 M magnesium chloride
and the product eluted with a gradient of 0.05
→
0.5 M magnesium chloride. All magne-
sium chloride solutions were acidified with a
drop of hydrochloric acid. The main fraction
was loaded on a C
18
Waters SepPak (10 g) which
was washed with cupious amount of
water, and the product then eluted with acetonitr
ile containing a drop
of trifluoroacetic
acid. In order to obtain a more pure product
(> 98 % from HPLC),
the purification proce-
dure was repeated once. After removal of the
solvent in a stream of air and drying
in
vacuo
, the product was obtained as
a red-brown solid (131.
4 mg, 0.13 mmol, 39.4 %).
MS (ESIpos): 820 (M
+
-3Cl-2H), 410 (M
2+
-3Cl-H); UV/Vis (water):
212, 264, 298 (sh), 312
(sh), 398 (6200) nm (l mol
-1
cm
-1
); HPLC (100:0
→
30:70 water/acetonitr
ile over 50 min)
> 98 %.
(4-Aminophenoxy)acetic acid ethyl ester 9
4-Aminophenol
8
(20.0 g, 183.3 mmol) was dissolved
in ice-cold degassed anhydrous
dimethylformamide (250 ml) under argon and then
solid sodium hydride (55 - 65 % in oil,
7.6 g, 190 mmol) was added in
small portions. The solution
was stirred at 0 °C for 30
min after which it had turned
deep red-brown. Then bromoacet
ic acid ethyl ester (20.2
ml, 182.2 mmol) was slowly added
at 0 °C via syringe and the re
sulting mixture stirred at
room temperature overnight. After removal of
the solvent, the resulting orange gum was
dissolved in a mixture of ethylacetate and wa
ter (250 ml, 1:1), the organic phase sepa-
rated, the aqueous phase extracted with ethy
l acetate (2x 100 ml),
the combined organic
phases washed with sodium hy
drogencarbonate, brin
e, and water, dr
ived over magne-
sium sulfate and the solvent removed. The re
sulting brown oil was
purified twice by col-
umn chromatography on silica wi
th chloroform/methanol (9:1) as
the eluent to give the
product as an orange oil which slowly
solidified (9.83 g, 50.4 mmol, 27.5 %).
1
H NMR
(CDCl
3
, 300 MHz):
δ
= 6.76 (d,
J
= 9.3 Hz, 2H), 6.62 (d,
J
= 9.0 Hz, 2H), 4.53 (s, 2H),
4.25 (q,
J
= 7.2 Hz, 2H), 1.29 (t,
J
= 7.2 Hz, 3H); MS (ESI
pos): 196 (M+H), 218 (M+Na).
S9
(4-Bis(2-hydroxyethyl)aminophenoxy)acetic acid ethyl ester 10
(4-Aminophenoxy)acetic acid ethyl ester
9
(7.89 g, 40.4 mmol) was dissolved in a de-
gassed mixture of acetic acid and water (
35 ml, 1:1) under argon to give a orange-to-red
solution which was cooled to 0 °C. Oxirane (9
.10 g, 206.6 mmol) wa
s condensed into a
separate Schlenk bottle immersed in aceton
e/dry ice and then added
with a syringe pre-
cooled to -20 °C to the former solution. Th
e mixture was allowed to warm to room tem-
perature while stirring under argon for 18.
5 h and the deep brown solution then concen-
trated in vacuo at < 40 °C. Wa
ter (25 ml) was added to the resu
lting oily material and the
product extracted with ether (4
x 100 ml). The combined orga
nic phases were carefully
washed neutral with 2 M sodium hydrogencar
bonate (100 ml) in a large separation fun-
nel (strong effervescence!) followed by water,
dried over magnesium
sulfate and the sol-
vent then removed at < 40 °C. The resulting ma
terial was twice recrystallized from tolu-
ene/hexanes (300 ml, 1:1) to remove some
brown oil and then purified by column chro-
matography on silica with chloroform/methanol
(9:1) as the eluent. The product was ob-
tained as a slightly pinkish oil which very sl
owly solidified to a white solid (2.58 g, 9.1
mmol, 22.5 %).
1
H NMR (CDCl
3
, 300 MHz):
δ
= 6.82 (d,
J
= 9.0 Hz Hz, 2H), 6.66 (d,
J
=
9.0 Hz, 2H), 4.53 (s, 2H), 4.24 (q,
J
= 7.1 Hz, 2H), 3.76 (t,
J
= 5.0 Hz, 4H), 3.45 (t,
J
=
4.8 Hz, 4H), 1.28 (t,
J
= 7.1 Hz, 3H); MS (ESI
pos): 284 (M+H), 306 (M+Na).
(4-Bis(2-chloroethyl)aminophenoxy)acetic acid ethyl ester 11
(4-Bis(2-hydroxyethyl)aminopheno
xy)acetic acid ethyl ester
10
(0.81 g, 2.86 mmol) was
dissolved in anhydrous benzene
(10 ml) and then phosphoroxy
trichloride (0.8 ml, 8.58
mmol) was added via syringe followed by heating
to 100-110 °C for 1 h. Afterwards, the
solution was poured on ice, the organic phase
separated, dried over
magnesium sulfate,
and shaken with activated alumina. After
removal of the solvent, the product was ob-
tained as a colorless oil which solidified to
a white solid (0.58 g,
1.81 mmol, 63.3 %).
1
H
NMR (CDCl
3
, 300 MHz):
δ
= 6.84 (d,
J
= 9.0 Hz, 2H), 6.62 (d,
J
= 9.3 Hz Hz, 2H), 4.52
(s, 2H), 4.24 (q,
J
= 7.2 Hz, 2H), 3.59 (m, 8H), 1.27 (t,
J
= 7.2 Hz, 3H); MS (ESIpos): 320
(M+H).
(4-Bis(2-chloroethyl)aminophenoxy)acetic acid 12
(4-Bis(2-chloroethyl)aminophenox
y)acetic acid ethyl ester
11
(0.62 g, 1.94 mmol) was
dissolved in concentrated hydrochloric acid (
10 ml), heated to refl
ux for 1 h and cooled
to room temperature. After addi
tion of water (50 ml), the pr
oduct was extracted into di-
ethylether (4x 50 ml), the combined organic
phases wahsed with wate
r, dried over mag-
nesium sulfate, and the solvent
removed to give a white soli
d (0.34 g, 1.16 mmol, 59.8
%).
1
H NMR (CDCl
3
, 300 MHz):
δ
= 6.87 (d,
J
= 9.0 Hz, 2H), 6.65 (d,
J
= 9.3 Hz, 2H),
4.60 (s, 2H), 3.62 (m, 8H);
MS (ESIpos): 292 (M+H).
S10
[Rh(phen)(chrysi)(bipy)]Cl
3
1
[Rh(phen)(chrysi)(bipy
#
)]Cl
3
7
(5.8 mg, 6.2 μmol) was di
ssolved in anhydrous dimethyl-
formamide under argon to give a bright orange solution. Then, (4-Bis(2-
chloroethyl)aminophe
noxy)acetic acid
12
(2.0 mg, 6.8 μmol) wa
s added as a solid fol-
lowed by HOBt (1-hydroxybenzotriazol, 2.1 mg
, 15.5 μmol). Finally, EDAC (1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide, 5 μl, 36.
7 μmol) was added and t
he solution stirred
at room temperature for 2.5
h. Then, water (5 ml) was added and the resulting solution
loaded on a C
18
Waters SepPak (5 g) which was wa
shed with cupious am
ount of water.
The product was eluted with acetonitrile containi
ng a drop of trifluoroacetic acid, the sol-
vent removed in a stream of air and the resu
lting red-brown solid stored at -80 °C. The
product was then dissolved in
600 μl of acetonitrile and pu
rified by semi-preparative
HPLC on a HP/Agilent 1100
system with a Dynamax 300
Ǻ
C
1
column using a gradient
of 0.5 % trifluoroacetic acid
in water and acetonitrile (100:0
→
30:70 over 60 min). The
main peak was collected, the solution froz
en at -80 °C, and the product lyophilized to
dryness. The resulting red-br
own solid was dissolved in wate
r (2 ml) and kept at -80 °C.
Aliquots were taken from the thawed solution
and diluted as needed. MS (ESIpos): 1093
(M
+
-3Cl-2H).
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Jackson, J.K. Barton,
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1998
,
37
, 3007-3012.
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y, B.L. Iverson,
T.E. Mallouk,
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116
, 4786-4795.
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