Supplementary material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2003
Supporting information for
Development of Novel Cell-permeable DNA Sensitive Dyes Using Combinatorial
Synthesis and Cell-based Screening
Jae Wook Lee, Michelle Jung, Gustavo R. Rosania, Young-Tae Chang*
Experimental Section
General: Unless otherwise noted, starting materials and solvents were purchased from
Aldrich and Acros organics, and used without purification. Ethanol (EtOH) and ethyl
acetate (EA) from Acros organics were used as the reaction solvents without any prior
purification. Salmon testes solid dsDNA, and phosphate buffered saline (PBS) (NaCl 120
mM, KCl 2.7 mM, and phosphate buffer 10 mM, pH=7.4 at 24 oC) were purchased from
Sigma-Aldrich and needed no further purification.
Fluorescence intensities were
measured by a Jobin Yvon Horiba Fluoro MAX-3 fluorimeter with a quartz cuvette cell
(10 mm10 mm4.5 cm; Starna (Atascadero, CA)).
1
H-NMR (200 MHz) spectra were
determined on Varian Genini 200 spectrometer. Chemical shifts were reported in parts
per million (ppm) relative to the line of a singlet at 2.50 ppm for DMSO-d6 and coupling
constants (J) are in Hertz (Hz).
The following abbreviations are used for spin
multiplicity: s=singlet, d=doublet, t=triplet, m=multiplet, and b=broad. All dye products
were identified by LC-MS from Agilent Technology, using a C18 column (204.0 mm),
with 4 minutes elution using a gradient of 5-95% CH3CN (containing 1% acetic acid)H2O (containing 1% acetic acid), with UV detector at =400 nm and an electrospray
ionization source.
Preparation of 2-[4-[5-(trimethyoxy)phenyl]-ethenyl-1-adamantyl]-4methylpyridinium bromide (1):
1-(1-adamantyl)-4-methylpyridinium bromide (20 mg, 0.06 mmol) and 2,4,5trimethoxybenzaldehyde (30mg, 0.15 mmol) were dissolved in ethanol (4 mL).
piperidine (0.4 mL) was added to the reaction mixture.
refluxed for 3 hours.
overnight.
The reaction mixture was
After the reaction was completed, the mixture stood at 0oC
Orange solid was filtered and washed with EtOAc (10 mL) yielding 1
(23.4mg, 80%) as yellow solid. LC-MS: RT=1.87 m/z: 406.2 [M]+ ; 1H-NMR (DMSO-
1
Supplementary material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2003
d6):  1.75 (s, 6H), 2.24 (s 9H), 3.80 (s, 3H, OCH3), 3.89 (s, 3HOCH3), 3.9 (s, 3H, OCH3)
6.78 (s, 1H), 7.39 (s, 1H), 7.44 (d, 1H, J=12 Hz), 8.1 (m, 3H), 9.1 (d, 2H, J=6Hz)
Preparation of 3-[4-[5-(trimethyoxy)phenyl]-ethenyl-1-adamantyl]-4methylpyridinium bromide (2):
1-(1-adamantyl)-4-methylpyridinium
bromide
(30
mg,
0.1
mmol)
and
3,4,5-
trimethyoxybenzaldehyde (57.3 mg, 0.3 mmol) were dissolved in ethanol (5 mL).
piperidine (0.4 mL) was added to the reaction mixture.
refluxed for 2 hours.
The reaction mixture was
After the reaction was completed, the mixture stood at 0oC
overnight. The yellow solid was filtered and washed with EtOAc (10mL) yielding 2
(11.2 mg, 23%) as yellow solid. LC-MS: RT=1.809 m/z: 406.2 [M]+; 1H-NMR (DMSOd6):  1.75 (s, 6H), 2.30 (s 9H), 3.74 (s, 3H, OCH3), 3.89 (s, 6H, OCH3), 7.15 (s, 2H),
7.60 (d, 1H, J=14 Hz), 8.07 (d, 1H, J=14 Hz), 8.2 (d, 3H, J=6Hz), 9.2 (d, 2H, J=6Hz)
Preparation of 2-[3-[4-(trimethyoxy)phenyl]-ethenyl-1-adamantyl]-4methylpyridinium bromide (3):
1-(1-adamantyl)-4-methylpyridinium
bromide
(30
mg,
0.1
mmol)
and
2,3,4-
trimethyoxybenzaldehyde (57.3 mg, 0.3 mmol) were dissolved in absolute ethanol (5
mL). piperidine (0.2 mL) was added to the reaction mixture. The reaction mixture was
refluxed for 2 hours.
After the reaction was completed, the mixture stood at 0oC
overnight. Solid was filtered and washed with EtOAc (10 mL) yielding 3 as a yellow
solid (14.9 mg, 30%). LC-MS: RT=1.833 m/z: 406.2 [M]+; 1H-NMR (DMSO-d6):  1.75
(s, 6H), 2.28 (s 9H), 3.80 (s, 3H, OCH3), 3.89 (s, 6H, OCH3), 6.96 (d, 1H, J=6Hz), 7.49
(d, 1H, J=14 Hz), 8.55 (d, 1H, J=6 Hz), 8.0 (d, 1H, J=14Hz), 8.23 (d, 2H, J=6Hz), 9.15
(d, 2H, J=6Hz)
Preparation of 1-Methyl-4[2-(2,4,5-trimethoxy-phenyl)-vinyl]pyridinium iodide (4)
1,4-dimethyl pyridinium iodide (20 mg, 0.085 mmol) and 2,4,5-trimethoxybenzaldehyde
(49 mg, 0.25mmol) were dissolved in absolute ethanol (5 mL). piperidine (0.13 mL) was
added to the reaction mixture. The reaction mixture was refluxed for 3 hours. After the
reaction was completed, the mixture stood at 0 oC overnight. The solid was filtered and
2
Supplementary material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2003
washed with EtOAc (10 mL) yielding 4 as a light yellow solid (6.2 mg, 17.7 %). LCMS: RT=1.56 m/z: 286.1 [M]+; 1H-NMR (DMSO-d6):  3.80 (s, 3H, OCH3), 3.89 (s, 3H,
OCH3), 3.89 (s, 3H, OCH3), 4.20 (s, 3H, CH3), 6.79 (s, 1H), 7.34 (s, 1H), 7.41 (d, 1H,
J=14 Hz), 8.04 (d, 1H, J=14 Hz), 8.11 (d, 2H, J=6Hz), 8.75 (d, 2H, J=6Hz).
3
Supplementary material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2003
Figure S1. LC-MS diagram of compound 1
LC data (400 nm)
1.857
DAD1 A, Sig=400,16 Ref =of f (E:\LJW\LC-MS\TLC00360.D)
mAU
800
700
600
500
400
300
200
0.307
100
0
0
0.5
1
1.5
2
2.5
3
3.5
min
MS data
*MSD1 SPC, time=1.915:1.933 of E:\LJW\LC-MS\TLC00360.D
API-ES, Pos, Scan, 70
100
API-ES, Pos, Scan, Frag: 70
406.1
406.1
*MSD1 SPC, time=1.968 of D:\GROUP\LJW\TLC00057.D
100
2.44685e+006
Max:Max:
1.31226e+006
OCH3
OCH3
H3CO
80
80
N
6060
Exact Mass: 406.24
407.1
40
407.1
40
20
20
0
100
200
300
400
500
600
700
800
900
m/z
0
200
400
600
800
4
1000
1200
m/z
Supplementary material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2003
Figure S2. LC-MS diagram of compound 2
LC data (400 nm)
1.835
DAD1 A, Sig=400,16 Ref =of f (E:\LJW\LC-MS\TLC00357.D)
mAU
250
200
150
100
1.531
0.306
50
0
0
0.5
1
1.5
2
2.5
3
3.5
min
MS data
API-ES, Pos, Scan, 70
406.1
*MSD1 SPC, time=1.879:1.897 of E:\LJW\LC-MS\TLC00357.D
100
H3CO
Max: 1.77152e+006
OCH3
OCH3
80
N
60
Exact Mass: 406.24
407.1
40
20
0
100
200
300
400
500
5
600
700
800
900
m/z
Supplementary material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2003
Figure S3. LC-MS diagram of compound 3
LC data (400 nm)
1.872
DAD1 A, Sig=400,16 Ref =of f (E:\LJW\LC-MS\TLC00359.D)
mAU
2000
1750
1500
1250
1000
750
500
1.079
0.313
250
0
0
0.5
1
1.5
2
2.5
3
3.5
min
MS data
API-ES, Pos, Scan, 70
406.1
*MSD1 SPC, time=1.915:1.951 of E:\LJW\LC-MS\TLC00359.D
100
Max: 2.23966e+006
OCH3
H3CO
H3CO
80
N
60
Exact Mass: 406.24
407.1
40
20
0
100
200
300
400
500
6
600
700
800
m/z
Supplementary material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2003
Figure S4. LC-MS diagram of compound 4
LC data (400 nm)
MS data
API-ES, Pos, Scan, 70
286.1
*MSD1 SPC, time=1.592:1.664 of E:\LJW\LC-MS\TLC00375.D
100
Max: 1.75626e+006
OCH3
OCH3
80
H3CO
60
N
+
Exact Mass: 286.14
287.1
40
20
0
260
280
300
320
7
340
360
m/z
Supplementary material (ESI) for Chemical Communications
This journal is © The Royal Society of Chemistry 2003
Scheme S1. General procedure of the synthesis and screening of the
styryl dyes in 96-well format
R
Diversity 2
Diversity 1
H3C
+
R CHO
cat. pyrrolidine
DMSO-EtOH
microwave
N+ XR'
A
N+ XR'
B
.
DNA binding tested by fluorescence
intensity changes.
Building blocks A
CH3
Cl
Br
Br
CHO
Br
CHO
A1
A3
CHO
A4
CHO
CHO
A6
A7
A5
OCH3
H3CO
H3CO
CHO
CHO
CHO
A2
OCH3
H3C
CHO
CHO
CHO
A9
A8
CHO
A11
A10
A12
CHO
OCH2 H CO
3
OCH3
OCH3
CHO
CHO
OMe
OH
OCH3
CHO
HO
OH
O
CHO
CHO
CHO
A16
A17
NH
A15
A14
CHO
A18
CHO
H
N
OMe
A20
A19
CHO
A27
A26
CHO
CHO
CHO
A37
OCH3
OCH3
A33
A34
CHO
A35
COOHCHO
CHO
N
NO2
A36
F
F
CHO
CHO
CHO
CHO
F
F
A32
A31
A30
A29
F
CHO
CHO
N
A28
A24
F
F
HOOC
CHO
CHO
A23
CHO
CHO
CHO
CHO
A22
A21
CHO
A25
NO2
NO2
CHO
CHO
A13
NO 2
CHO
N
CHO
H2CO
A38
H3CO
O
A40
A39
N
CHO
O
CHO
A44
A43
A42
A41
A45
Building blocks B
O
N
N
N
N
N
B1
B2
B3
N
N
N
B4
B5
B6
Br
N
N
B7
B8
B10
B9
N
O2N
OH
N
OMe
N
N
N
B11
B12
N
N
B16
B17
N
N
N
B13
B14
B15
8
B18
N
N
B19