S
1
Supporting Information
for
A r
hodiu
m
-
cyanine fluorescent probe:
detection and
signaling
of
mismatches in DNA
Adela Nano,
Adam N.
Boynton,
Jacqueline K. Barton
*
Di
v
ision of Chemistry and Chemical Engineering,
California Institute of
Technology, Pasadena, California, 91125
S
2
Table of Contents
1. Materials
................................
................................
................................
................................
....
3
2. Synthesis
................................
................................
................................
................................
.....
5
2.1. Synthesis
of Cy3
-
COOH
................................
................................
................................
....
6
2.2. Synthesis of DPA
-
NH
2
................................
................................
................................
.......
9
2.3.
Synthesis of RhCy3 conjugate
................................
................................
........................
11
2.4. Synthesis of the model compounds
................................
................................
.................
13
3. Photocleavage experiments
................................
................................
................................
....
15
4. Photophysical measurements
................................
................................
................................
.
16
4.1 Steady
-
state fluorescence
................................
................................
................................
..
16
4.2. Fluorescence lifetimes
................................
................................
................................
......
17
5. Cell cultures
................................
................................
................................
.............................
17
6. Genomic DNA extraction and purification procedure
................................
........................
17
7. Figures
................................
................................
................................
................................
......
19
8. Quantification of mismatches in genomic DNA
................................
................................
...
29
9. References
................................
................................
................................
................................
31
S
3
Table of
Figure
s
Figure S1.
Absorption spectra of RhCy3 and its spectroscopic models.
................................
.....
19
Figure S2.
Steady
-
state emission spectra of Cy3
-
linker.
................................
..............................
20
Figure S3.
Full TOF
-
MS ES+ spectrum of RhCy3..
................................
................................
....
21
Figure S4.
HPLC trace of RhCy3.
................................
................................
...............................
22
Figure S5.
1
H NMR spectrum of 3b.
................................
................................
............................
23
Figure S6.
13
C NMR spectrum of 3b..
................................
................................
..........................
23
Figure S7.
1
H NMR spectrum of DPA
-
NH
2
................................
................................
................
24
Figure S8.
13
C NMR spectrum of DPA
-
NH
2
................................
................................
...............
24
Figure S9.
1
H NMR spectrum of DPA
-
Cy3
................................
................................
.................
25
Figure S10
.
13
C NMR spectrum of DPA
-
Cy3
................................
................................
..............
25
Figure S11.
1
H NMR spectrum of RhCy3.
................................
................................
..................
26
Figure S12.
1
H NMR spectrum of Cy3
-
linker.
................................
................................
............
27
Figure S13.
13
C NMR spectrum of Cy3
-
linker.
................................
................................
...........
27
Figure S14.
Fluorescence lifetime decay curve for RhCy3 free in solution.
...............................
28
Figure S15.
Fluorescence lifetime decay curve for RhCy3 with WM DNA.
..............................
28
Figure S16.
Fluorescence lifetime decay curve for RhCy3 with WM DNA
...............................
29
S
4
1.
M
aterials
All reactions except when mentioned were performed under a dry atmosphere of argon.
RhCl
3
was purchased from Pressure Chemical, Inc. 2,2’
-
dipyridylamine (HDPA),
1,2
-
bis(
2
-
iodo
ethoxy
)ethane,
anhydrous
i
Pr
2
NH, anhydrous DMF, HBTU
and Sephadex ion exchange
resin were obtained from Sigma
-
A
l
drich and used as purchased. Sep
-
Pak C
18
solid phase
extraction cartridges were purchased from Waters Chemical Co.
1
H,
13
C NMR spectra were
recorded o
n a Bru
ker Spectrometer
s (400
or 300
MHz).
1
H,
13
C chemical shifts were reported to
the delta scale in ppm relative to the residual peak of the deuterated used solvent as internal
standards: chloroform
-
d
1
(
1
H: δ = 7.26 ppm;
13
C: δ = 77.16
ppm
),
methanol
-
d
4
(
1
H: δ = 3.31
ppm;
13
C
: δ = 49.00
ppm
),
dichloromethane
-
d
2
(
1
H: δ = 5.32 ppm;
13
C: δ = 54.00
ppm
),
a
cetonitrile
-
d
3
(
1
H: δ = 1.94
ppm;
13
C: δ = 118.26
ppm
).
Chromatographic purifications were
perfo
rmed using 40
-
63 μm silica gel or Sep
-
Pak C
18
cartridges.
HPLC purifications were
performed on an HP1100 high
-
pressure liquid chromatography system equipped with a diode
array detector using a Varian DynaMax C18 semi
-
preparative column.
Mass spectrometry was
performed at Caltech mass spectrometry facility.
UV
-
vi
s
absorption spectra
were recorded on a
Cary 100 Bio spectrophotometer
.
S
5
2.
Synthesis
S
6
2.1.
Synthesis of Cy3
-
COOH
Scheme S1
.
Synthesis pathway for
Cy3
-
COOH
and its prec
ursors.
Compound 2
a
.
1
4
-
Hydrazinobenzoic acid (1.5 g, 9.85 mmol) and 3
-
methyl
-
2
-
butanone (1.27 g,
14.79 mmol) were refluxed in glacial acetic acid (20 ml) solution for about 16 h. Afterwards, the
reaction was cooled down to room temperature and quenched with water. The crude wa
s
extracted
with dichloromethane (2x
)
and washed with water (3
x
)
followed by drying the organic
phase with anhydrous Na
2
SO
4
. D
ichloromethane was evaporated under vacuum and the crude
was purified over a silica gel column chromatography, eluting with aceton
itrile/dichloromethane
(2/8). The product was obtained as yellowish solids in 51
% yield
(1.014 g).
1
H NMR
(400
MHz, Methanol
-
d
4
) δ 8.38 (d,
J
= 1.5 Hz, 1H), 8.32 (dd,
J
= 8.4, 1.6 Hz, 1H), 7.97 (d,
J
= 8.4 Hz,
1H), 4.12 (s, 3H), 1.68 (s, 6H).
13
C NMR
(101 MHz, MeOD) δ 199.26, 166.68, 145.28, 141.93,
132.28, 130.81, 124.10, 114.96, 54.65, 34.43, 21.01.
MW = 203.24
Da
, chemical formula:
C
12
H
13
NO
2
.
ESI
-
MS
(positive)
m/
z
(% intensity)
: calculated for [M]
+
203.09
, found
203.13
S
7
Compound
3a
:
2
A
solution
of
compound
2
a
(1, 014g, 4.99 mmol
) and
iodomethane
(4.247g,
29.93 mmol
)
in
acetonitrile (20.0 ml
)
was
stirred
at 90 °C
under argon
during
30 h
.
The reaction
was cooled
down to room temperature
and the precipitate
was
fil
tered off and washed with
acetonitr
ile and hexane
. The
indolium salt
3a
was obtained as white solids which turn
slowly
yellowish under normal atmosphere (1.440g, 84%).
MW = 345.18
Da
, chemical formula:
C
13
H
16
INO
2
.
ESI
-
MS
(positive)
m/
z
(% intensity)
: calculated for [M
-
I]
+
218.1
2
(100)
, found
218.14
(100)
.
Compound 5
a
:
Method I
.
3
DMF
(25 ml)
was introduced in a flame
-
dried Schlenk flask under
vacuum which was then filled with argon. POCl
3
(1
.2
eq
,
2.12g,
0.013
mol
)
was added dropwise
followed by the addition
of 2
-
methylene
-
1,3,3
-
trimethylindoline
4a
(2g,
0.011 mol
)
.
The final
mixture was stirred at
50
C
during 45 min
.
After cooling it down to room temperature, t
he
reaction was neutralized with a saturated solution
of sodium acetate
and extracted with
dichloromethane
. Organic phase
was
dried with anhydrous Na
2
SO
4
and
concentrated under
vacuum. The crude was purified using a silica gel column chromatography
eluted
with
DCM/MeOH 0.6%.
This method afforded
5a
as a yellowish oil
in
60%.
Method
I
I
.
4
(Chloromethylene)dimethylammonium chloride
(8.86
g, 0.069 mmol
)
was dissolved
in
anhydrous
dichloromethane
(40
.0 ml)
and stirred at room temperature during 30 minutes.
A
solution of the Fischer base,
2
-
methylene
-
1,3,3
-
trimethylindoline
(2.0 g,
0.011 moles)
,
in
dichloromethane
(15.0 ml)
was added dropwise and the mixture was s
tirred at room temperature
over
night. Afterwards, the solvent was evaporated under vacuum and the resulting crude was
dissolved in THF
(30
.0
ml)
and a saturated
aqueous
solution of K
2
CO
3
(30.0 ml)
which was
added solely. Following
the addition of K
2
CO
3
solution
,
the
color of the
mixture turns from deep
S
8
red to orange. After stirring at room temperature for about 12 h, THF was removed under
vacuum and the aque
ous
phase extr
acted with dichloromethane.
The organic phase was washed
2x
with water and dried with anhydrous Na
2
SO
4
, f
iltered and concentrated under vacuum
. The
crude was purified over a silica gel column chromatography, eluted with DCM/MeOH 0.6%. The
aldehyde
-
derivati
ve was collected
as yellowish oil (43%,
954 mg).
1
H NMR
(400 MHz,
Chloroform
-
d) δ 10.03 (d, J = 8.9 Hz, 1H), 7.34
–
7.22 (m, 2H), 7.08 (td, J = 7.5, 0.9 Hz, 1H),
6.87 (d, J = 7.9 Hz, 1H), 5.41 (d, J = 8.9 Hz, 1H), 3.26 (s, 3H), 1.68 (s, 6H).
13
C NMR
(101
MHz, CDCl3) δ 186.54, 173.64, 143.45, 139.39, 128.04, 122.45, 121.82, 107.98, 99.01, 47.43,
29.62, 29.58.
MW = 201.27
Da
,
chemical formula:
C
13
H
15
NO.
E
SI
-
MS
(positive)
m
/
z
(%
intensity):
calculated
for [M
+
H]
+
202.12
(100)
, found
202.15
(100)
.
Cy3
-
COOH
:
I
ndolium salt
3
a
(343
mg,
0.99 mmol)
and the aldehyde
-
Fischer base
5
b
(200 mg,
0.99 mmol
)
were combined together in a solution of glacial acetic anhydride
(10
ml)
. The
solution was stirred at room te
mperature for 2.5 h
. The cyanine dye precipitate
d
durin
g the
formation, therefore it was
filtered off and washed with diethyl ether. To obtain the
final product
as highly pure,
sil
ica gel column chromatography was
performed eluting with
dichloromethane/methanol 5% to 8%
. The cyanine dye was
obtained as d
ark green solids which
in solution
were
bordeaux red
in 61% yield after column chromatography.
MW = 528.43 Da,
chemical formula
:
C
26
H
29
IN
2
O
2.
1
H NMR
(400 MHz, Methanol
-
d4) δ 8.59 (t, J = 13.5 Hz, 1H), 8.16
–
8.08 (m, 2H), 7.60 (dd, J =
7.4, 1.5 Hz, 1H), 7.54
–
7.33 (m, 3H), 6.55 (d, J = 13.7 Hz, 1H), 6.46 (d, J = 13.2 Hz, 1H), 5.51
(s, 1H), 3.76 (s, 3H), 3.69 (s, 3H), 3.37 (s, 4H), 1.81 (d, J = 2.7 Hz
, 12H).
E
SI
-
MS
(positive)
m
/z
(% intensity): calculated
for [M
-
I
]
+
401.22
(100)
, found
401.42
(100)
.
S
9
2.2.
Synthesis of DPA
-
NH
2
Scheme
S
2
.
Synthesis pathway for
DPA
-
NH
2
.
Compound 3b
:
2,2’
-
dipyridylamine
1b
(1.0 g, 5.84 mmol)
was
placed in a flame
-
dried Schlenk
flask
under vacuum and then filled with argon. THF
(15 ml)
was added followed by the slow
addition of NaH
(182 mg, 7.93 mmol)
under argon. The solution was stirred at room temperature
during
1.5 h prior to the addition of
1,
2
-
bis(2
-
iodoethoxy)ethane
(5.0 g, 13.51 mmol)
. The final
mixture was protected from light and was stirred at 70
°C
during 20 h. After cooling down to
room temperature, the
reaction m
ixture was quenched with water and
extracted with
dichloromethane
. The org
anic phase was washed 2x with water,
dried with anhydrous Na
2
SO
4
and
concentrated
in vacuo
.
Most of t
he
starting material
1b
was precipitated (
or
recrystallized)
from a solution of the crude in dichloromethane by adding diethyl ether
. Afterwards, the
filtrate
was concentrated under vacuum
and
purified over a silica gel column chromatography. The
mobile phase was dichloromethane/ethyl acetate 5% to 20 %. The desired product was
obtained
as a yellowish oil
in
about
10
% yield
after
column chromatography
purification.
Many fractions
were collected as a mixture with the starting material
1b
.
1
H NMR
(400 MHz, Chloroform
-
d
) δ
8.34 (ddd,
J
= 4.9, 2.0, 0.9 Hz, 2H), 7.54 (ddd,
J
= 8.4, 7.2, 2.0 Hz, 2H), 7.19 (dt,
J
= 8.4, 0.9 Hz,
S
10
2H), 6.88 (ddd,
J
= 7.2, 5.0, 1.0 Hz, 2H), 4.42 (t,
J
= 6.1 Hz, 2H), 3.83 (t,
J
= 6.1 Hz, 2H), 3.69
(dd,
J
= 7.4
, 6.5 Hz, 2H), 3.64
–
3.54 (m, 4
H), 3.21 (dd,
J
= 7.4, 6.5 Hz, 2H).
13
C NMR
(101
MHz, CDCl
3
) δ 157.40, 148.12, 137.17, 117.05, 114.90, 77.34, 77.02,
76.70, 71.94
, 70.36,
70.18, 69.46, 47.95, 2.96 (
C
H
2
-
I). MW = 413.26 Da,
c
hemical formula: C
16
H
20
IN
3
O
2
.
ESI
-
MS
(positive)
m/z
(% intensity): calculated for [M
+
H]
+
414.07 (100), found 414.04 (100).
Compound DPA
-
NH
2
:
5
A solution of 7 M ammonia in methanol
(5 ml)
was added to
3b
(130
mg, 0.314 mmol)
in
a 10 ml microwave vial. The reaction was stirred at 100
°C
during 15
minutes in a
n
auto
-
sampling microwave.
The final product
(which is soluble preferably in water)
was extracted with water and washed with ethyl acet
ate
to eliminate any residual of starting
material
. The water was evaporated and the
product was obtained pure in 95
% yield
and used
as
obtained
in the next step
(90 mg)
.
1
H NMR
(400 MHz, Acetonitrile
-
d
3
) δ 8.34 (ddd,
J
= 5.0, 2.0,
0.9 Hz, 2H), 7.65 (ddd,
J
= 8.4, 7.2, 2.0 Hz, 2H), 7.17
–
7.10 (m, 2H), 6.98 (ddd,
J
= 7.3, 4.9, 0.9
Hz, 2H), 4.33 (t,
J
= 5.8 Hz, 2H), 3.74 (t,
J
= 5.8 Hz, 2H), 3.72
–
3.66 (m, 2H), 3.52 (S, 4H),
3.13
–
3.05 (m, 2H).
13
C NMR
(101 MHz, CD
3
CN)
δ 157.75, 148.38, 138.37, 118.01, 117.79,
115.58, 70.34, 70.08, 69.10, 66.20, 48.18, 39.93 (
C
H
2
-
NH
2
).
15
N{
1
H} NMR
(41 MHz, CD
3
CN) δ
244.55 (s).
MW = 302.38 Da
, c
hemical formula: C
16
H
22
N
4
O
2
.
ESI
-
MS
(positive)
m/z
(%
intensity): calculated for [M
+
H]
+
303.18 (100), found 303.19 (100).