Practical syntheses of dyes for difference gel electrophoresis

Article abstract of DOI:10.1016/j.bmc.2005.07.068

The three dyes (two indocyanine and one benzoxazolium) useful in difference gel electrophoresis-methyl Cy5 1, propyl Cy3 2, and the benzoxazolium dye Cy2 3-and their NHS esters have been prepared by efficient routes in good overall yield from inexpensive

Full text of DOI:10.1016/j.bmc.2005.07.068

Bioorganic & Medicinal Chemistry 14 (2006) 92–97
Practical syntheses of dyes for difference gel electrophoresis
Michael E. Jung* and Wan-Joong Kim
Department of Chemistry and Biochemistry, University of California, Los Angeles, 405 Hilgard Ave, Los Angeles, CA 90095, USA
Received 25 June 2005; revised 28 July 2005; accepted 28 July 2005
Available online 23 September 2005
Abstract—The three dyes (two indocyanine and one benzoxazolium) useful in difference gel electrophoresis—methyl Cy5 1, propyl
Cy3 2, and the benzoxazolium dye Cy2 3—and their NHS esters have been prepared by efficient routes in good overall yield from
inexpensive precursors.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Me Me
N
Me
Me Me
N
Me
Me
N
Me
N
The use of fluorescent dyes in biology is ever-increasing
and serves as the basis for many advancements in
science;1 for example, the sequencing of the human gen-
ome. Fluorescent two-dimensional difference gel electro-
phoresis is a relatively new technique for the multiplex
quantitative analysis of the component proteins of relat-
ed, but different, protein samples.2 This technique has
been quite useful in solving problems related to compar-
ing different protein samples by two-dimensional gel
electrophoresis. The application of this technique re-
quires a supply of fluorescent dyes with different absorp-
tion characteristics activated for attachment to the
proteins of interest. Usually, this signifies a group of sev-
eral indocyanine dyes, although recently other dyes have
also been used, in particular, the three dyes methyl Cy5
1, propyl Cy3 2, and the benzoxazolium dye Cy2 3
(Scheme 1).3 The dyes are generally converted into their
N-hydroxysuccinimide esters 4–6 for attachment to the
proteins. Although these dyes are well-known and even
commercially available,4 there is no detailed experimen-
tal procedure for their efficient synthesis. We report here
detailed procedures for the synthesis of these three dif-
ference gel electrophoresis (DIGE) dyes from relatively
inexpensive precursors.
Me
Pr
O
O
R
R
O
O
1 R = H
4 R = NHS
2 R = H
5 R = NHS
O
O
N
N
+
Et
O
O
R
3 R = H
6 R = NHS
Scheme 1. Dyes methyl Cy5, propyl Cy3, and Cy2.
prepared. Condensation of commercially available mal-
ondialdehyde bis(dimethyl acetal) with aniline under
acidic conditions afforded the anilino anilinium salt 7
in 85% yield.5 The reaction of 7 with the commercially
2. Results and discussion
available 1,3,3-trimethyl-2-methyleneindoline
8
in
refluxing acetic acid afforded the anilinobutadienyl salt
9 in 66% yield.6 The second component of the dye, the
indolium salt 11, was prepared by alkylation of commer-
cially available 2,3,3-trimethyl-3H-indole 10 with 6-
bromohexanoic acid in dichlorobenzene at 110 °C for
12 h to give 67% yield of 11.7 Finally, the reaction of
For the synthesis of the methyl Cy5 dye 1 and its NHS
ester 4 (Scheme 2), the three-carbon spacer 7 had to be
*
Corresponding author. Tel.: +1 310 825 7954; fax: +1 310 206
3722; e-mail: jung@chem.ucla.edu
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.07.068

M. E. Jung, W.-J. Kim / Bioorg. Med. Chem. 14 (2006) 92–97
93
Me
Me
O
Me
O
Br
Me
COOH
1,2-Cl2C6H4
CH(OMe)2
CH(OMe)2
PhNH2
AcOH
66%
+
+
N
PhNH
Me
NHPh
N
_
aq HCl
85%
110 ˚C/24h
80%
+
N
Cl
Me
14
7
8
CH3CH2Br
Me Me
Me
1,2-Cl2C6H4
110 ˚C/24h
22%
Me
Br(CH2)5COOH
1,2-Cl2C6H4
110 ˚C/12h
67%
PhNH
N
N
COOH
Ac
N
+
Me
O
Me
16
O
PhNH
Ac2O/
93%
NPh
9
10
Ph
N
+
N
+
Me Me
Et
Me
Me
Me Me
1) NaOAc
EtOH
9
Et
Me
15
17
+
N
N
+
N
/4h
69%
O
O
Me
2) DSC
pyr
92%
1)Et3N
EtOH
N
+
N
Et
16
+ 17
/30 min
75%
2) DSC
HOOC
1 R = H
4 R = NHS
O
R
O
pyr
11
86%
O
Scheme 2. Synthesis of dye methyl Cy5 and its NHS ester.
O
R
3 R = H
6 R = NHS
the activated indolium salt 9 with the methylindolium
salt 11 in ethanol in the presence of sodium acetate afforded
the desired dye 1 in 69% yield as a blue powder. The dye
was easily converted into the N-hydroxysuccinimide
ester (NHS) by treatment with commercially available
N,N0-disuccinimidyl carbonate (DSC) in the presence
of pyridine to give the activated dye 4 in 92% yield.
All spectroscopic data, especially high field NMR and
mass spectrometry, were in agreement with the
structures assigned.8
Scheme 4. Synthesis of the benzoxazolium dye Cy2 and its NHS ester.
with N,N0-disuccinimidyl carbonate (DSC) in the pres-
ence of pyridine to give the activated dye 5 in 96% yield.
Again, all the pertinent spectroscopic data, especially
high-field NMR and mass spectrometry, were in agree-
ment with the structures assigned.8
Finally, the synthesis of the Cy2 dye, the benzoxazolium
dye 3, and its NHS ester 6 (Scheme 4) required
completely different coupling components. Thus, the
commercially available 2-methylbenzoxazole 14 was
alkylated with two different alkyl halides: alkylation
with ethyl bromide gave the ethyl salt 1511 in 22% yield
(this compound is also commercially available), while
alkylation with the commercially available 4-bromo-
methylphenylacetic acid gave an 80% yield of the salt
16.12 Condensation of the ethyl salt with diphenylform-
amidine in the presence of excess acetic anhydride as sol-
vent afforded the acetanilidylvinyl indolium salt 17 in
93% yield.13 This compound was then reacted with the
other benzoxazolium salt 16 in ethanol in the presence
of triethylamine to give the desired dye 3 in 75% yield
as a deep yellow powder. The dye was easily converted
into the N-hydroxysuccinimide ester (NHS) by treat-
ment with N,N0-disuccinimidyl carbonate (DSC) in the
presence of pyridine to give the activated dye 6 in 86%
yield. The spectroscopic data were in agreement with
the structures assigned.8
The synthesis of the propyl Cy3 dye 2 and its NHS ester
5 (Scheme 3) began with the alkylation of the trimethyl-
indole 10 with propyl bromide to give the 1-propyl-2-
methyleneindoline 129 in an unoptimized yield of 44%.
Condensation with commercially available N,N0-diphen-
ylformamidine in the presence of excess acetic anhydride
as solvent afforded the acetanilidylvinyl indolium salt 13
in 87% yield. This compound was then reacted with the
same methylindolium salt 11 in ethanol in the presence
of triethylamine to give the desired dye 2 in 85% yield
as a red powder.10 The dye was easily converted into
the N-hydroxysuccinimide ester (NHS) by treatment
Me Me
Me Me
Me
PhNH
Ac2O/∆
NPh
CH3CH2CH2Br
1,2-Cl2C6H4
110 ˚C/24h
44%
N
N
Pr
87%
10
12
Ac
N
Me
Me
N
Me Me
Me
Me
N
1) 11
Et3N
EtOH
30 min
85%
Ph
3. Conclusion
N
+
Pr
+
Pr
We have thus developed simple practical methods for
the synthesis of the three dyes—methyl Cy5 1, propyl
Cy3 2, and Cy2 3—from inexpensive precursors in only
a few steps and reasonable overall yields. The synthesis
of each dye requires only 4 steps from commercially
available materials and generally the yields are quite
high, especially for the coupling of the components.
2) DSC
pyr
96%
13
O
2 R = H
5 R = NHS
R
O
Scheme 3. Synthesis of dye propyl Cy3 and its NHS ester.

94
M. E. Jung, W.-J. Kim / Bioorg. Med. Chem. 14 (2006) 92–97
The utility of these dyes for DIGE is currently under
investigation and will be reported shortly.14
1.80 mmol) in 1,2-dichlorobenzene was heated at
110 °C for 12 h. The solution was cooled to room tem-
perature, and the residue obtained was filtered and
washed with a mixture of acetonitrile/diethyl ether (1/
1). The solid obtained was dried under vacuum to give
the product 11 as a light red powder (2.3 g, 67%).7 IR
(neat): 3405, 2936, 1724, 1624, 1460, 1392, 1168,
4. Experimental
4.1. General information
767 cmꢀ1 1H NMR (400 MHz, DMSO-d6): d 7.93–
.
All reactions were carried out under an atmosphere of
nitrogen and all commercial reagents were used direct-
7.97 (1H, m), 7.79–7.87 (1H, m), 7.56–7.64 (2H, m),
4.43 (2H, t, J = 7.7 Hz), 2.82 (3H, s), 2.19 (2H, t,
J = 7.2 Hz), 1.81 (2H, m), 1.52 (2H, m), 1.50 (6H, s),
1.35 (2H, m). 13C NMR (100 MHz, DMSO d6): d
196.98, 174.77, 142.33, 141.51, 129.85, 129.40, 123.99,
115.97, 54.62, 47.90, 33.83, 27.41, 25.87, 24.48, 22.47,
14.51.
1
ly. H and 13C NMR NMR data were obtained on a
Bruker 400 MHz spectrometer. 1H NMR and 13C
NMR data are reported in parts per million (d) down-
field from tetramethylsilane. The following abbrevia-
tions are used: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), and br (broad). Infrared spec-
tra were recorded on a Thermo Nicolet Avatar 370
FT infrared spectrophotometer as a liquid film or as
a thin crystalline film. All IR data are reported in
wavenumbers (cmꢀ1). Thin-layer chromatography
(TLC) was performed using Merck silica gel 60 F254
0.2 mm alumina-backed plates. Visualization was
accomplished using ultraviolet light or one of the fol-
lowing stains: anisaldehyde, phosphomolybdic acid,
and potassium permanganate. Flash chromatography
was carried out using ICN Biomedicals silica gel 60
(230–400 mesh).
4.1.4. 2-[5-[1-(5-Carboxypentyl)-1,3-dihydro-3,3-dimeth-
yl-2H-indol-2-ylidene]-1,3-pentadien-yl]-1,3,3-trimethyl-
3H-indolium, Methyl Cy5, 1. A solution of the anil 9
(0.20 g, 0.65 mmol), the acid 11 (0.18 g, 0.65 mmol),
and anhydrous sodium acetate (0.11 g, 0.79 mmol) in
absolute ethanol (50 mL) under nitrogen was refluxed
for 4 h. The solid was purified by flash chromatogra-
phy on silica gel (dichloromethane/methanol = 5:1) to
give the methyl Cy5 dye 1 (0.22 g, 69%) as a blue
powder.4 IR (neat): 3406, 2925, 1716, 1575, 1470,
1425, 1371, 1335, 1217, 1146, 1016, 1040, 923, 796,
750, 708 cmꢀ1 1H NMR (400 MHz, DMSO-d6): d
.
4.1.1. N-((1E)-3-(Phenylimino)prop-1-enyl)benzenamine
hydrochloride 7. A solution of distilled water (140 mL),
HCl (10 mL), and aniline (7.4 mL, 0.08 mol) was added
dropwise to a solution of distilled water (171 mL),
HCl (8.5 mL), and malondialdehyde bis(dimethyl
acetal) (10.5 mL, 0.06 mol) with stirring at 50 °C. The
precipitate was isolated by filtration to give malondial-
dehyde dianil hydrochloride 7 (12 g, 85%) as an orange
powder. 5 IR (neat): 3425, 1642, 1620, 1580, 1492, 1343,
8.30 (2H, t, J = 13.1 Hz), 7.58 (2H, d, J = 7.4 Hz),
7.34 (4H, m), 7.21 (2H, m), 6.53 (1H, t,
J = 12.3 Hz), 6.25 (2H, dd, J = 13.8, 13.9 Hz), 4.06
(2H, t, J = 7.2 Hz), 3.56 (3H, s), 2.15 (2H, t,
J = 7.2 Hz), 1.69 (2H, m), 1.64 (12H, s), 1.52 (2H,
m), 1.32 (2H, m). 13C NMR (100 MHz DMSO-d6): d
175.03, 173.69, 172.95, 154.47, 143.21, 142.47, 141.54,
141.47, 128.80, 125.83, 125.16, 125.05, 122.89, 122.76,
111.49, 103.77, 103.51, 55.39, 49.30, 43.67, 34.16,
31.56, 27.61, 27.44, 27.15, 26.12, 24.73.
1273, 1194, 749, 683 cmꢀ1
.
1H NMR (400 MHz,
DMSO-d6): d 12.72 (2H, d, J = 13.2 Hz), 8.92 (2H, t,
J = 12.4 Hz), 7.40 (8H, m), 7.20 (2H, m), 6.50 (1H, t,
J = 11.5 Hz). 13C NMR (100 MHz, DMSO-d6): d
158.89, 139.18, 130.33, 126.31, 117.86, 99.09.
4.1.5. 2-[5-(1,3-Dihydro-1,3,3-trimethyl-2H-indol-2-yli-
dene)-1,3-pentadienyl]-1-[6-[(2,5-dioxo-1-pyrrolidi-nyl)oxy]-6-
oxohexyl]-3,3-dimethyl-3H-indolium, Methyl Cy5-NHS
ester, 4. Anhydrous pyridine (0.1 mL) and N,N0-disucc-
inimidyl carbonate (DSC, 23.6 mg, 0.09 mmol) were
added to a stirred solution of the acid 1 (0.36 g,
0.06 mmol) in dry DMF (2 mL) under nitrogen. The
reaction mixture was stirred at 60 °C for 1.5 h. After
evaporation of the solvent, the deep blue residue was
purified by column chromatography on silica gel
(dichloromethane/hexane/methanol = 5:1:1) to give the
pure NHS ester of the dye 4 (0.33 g, 92%) as a blue pow-
der.3,4b IR (neat): 2926, 1733, 1496, 1481, 1456, 1372,
1336, 1217, 1182, 1149, 1097, 1040, 1016, 924, 796,
4.1.2. 2-(4-Phenylamino-1E,3E-butadien-1-yl)-1,3,3-trim-
ethylindolium chloride 9. A mixture of malondialdehyde
dianil hydrochloride 7 (1 g, 4.49 mmol) and 1,3,3-tri-
methyl-2-methyleneindoline 8 (0.93 mL, 4.49 mmol) in
glacial acetic acid (10 mL) was refluxed for 4 h. The
solution was cooled to room temperature, the acetic acid
was removed under reduced pressure, and the residue
was purified by flash chromatography on silica gel
(dichloromethane/hexane/methanol = 5:1:1) to give the
product 9 as a red powder (0.9 g, 66%).6 1H NMR
(400 MHz, DMSO-d6): d 8.18 (1H, d,J = 9.8 Hz), 7.52
(1H, t, J = 13.3 Hz), 6.74–7.36 (9H, m), 6.48 (1H, dd,
J = 14.1, 9.9 Hz), 5.55 (1H, d, J = 12.5 Hz), 3.24 (3H,
s), 1.55 (6H, s). 13C NMR (100 MHz, DMSO-d6): d
129.41, 129.17, 128.04, 125.24, 121.71, 121.36, 120.41,
118.18, 107.27, 46.68, 28.38.
1
756, 708, 668 cmꢀ1. H NMR (400 MHz, DMSO-d6): d
8.30 (2H, t, J = 13.1 Hz), 7.58 (2H, d, J = 7.4 Hz), 7.36
(4H, m), 7.20 (2H, m), 6.53 (1H, t, J = 12.4 Hz), 6.25
(2H, t, J = 14.2 Hz), 4.05 (2H, t, J = 7.2 Hz), 3.56 (3H,
s), 2.77 (4H, s), 2.64 (2H, t, J = 7.2 Hz), 1.68 (2H, m),
1.65 (2H, m), 1.64 (12H, s), 1.44 (2H, m). 13C NMR
(100 MHz, DMSO-d6):
d 173.74, 173.26, 172.93,
4.1.3. 1-(5-Carboxypentyl)-2,3,3-trimethyl-3H-indolium
bromide 11. A mixture of 2,3,3-trimethyl-3H-indole 10
(0.2 g, 1.25 mmol) and 6-bromohexanoic acid (0.36 g,
170.71, 169.35, 154.49, 151.10, 143.21, 142.44, 141.54,
141.47, 129.11, 128.81, 125.83, 125.19, 125.05, 122.87,
122.76, 111.50, 103.44, 49.31, 49.03, 43.63, 31.54,

M. E. Jung, W.-J. Kim / Bioorg. Med. Chem. 14 (2006) 92–97
95
30.44, 27.61, 27.43, 26.84, 25.90, 25.66, 24.37. MS (EI):
m/z (%) = 580 (100).
4.1.9. 2-[3-(1,3-Dihydro-3,3-dimethyl-1-propyl-2H-indol-
2-ylidene)-1-propenyl]-1-[6-[(2,5-di-oxo-1-pyrro-lid-
inyl)oxy]-6-oxohexyl]-3,3-dimethyl-3H-indolium, Propyl
Cy3-NHS ester, 5. Anhydrous pyridine (0.1 mL) and
4.1.6. 3,3-Dimethyl-2-methylene-1-propylindoline 12. A
mixture of 2,3,3-trimethyl-3H-indole 10 (0.2 g,
0.01 mol) and 1-bromopropane (2.28 mL, 0.02 mol) in
1,2-dichlorobenzene was heated at 110 °C for 24 h.
The solution was cooled to room temperature, and the
residue obtained was filtered and washed with a mixture
of acetonitrile/diethyl ether (1/1). The solid obtained was
dried under vacuum to give the 3,3-dimethyl-2-methy-
lene-1-propylindoline 12 as a light red powder (0.11 g,
44%).8 IR (neat): 2966, 2925, 1617, 1601, 1474, 1454,
N,N0-disuccinimidyl
carbonate
(DSC,
21 mg,
0.08 mmol) were added to a stirred solution of the
dye 2 (27 mg, 0.05 mmol) in dry DMF (2 mL) under
nitrogen. The reaction mixture was stirred at 60 °C
for 1.5 h. After evaporation of the solvent, the deep
red residue was purified by column chromatography
on silica gel (dichloromethane/hexane/methanol =
5:1:1) to give the pure propyl Cy3-NHS ester 5
(31 mg, 96%) as a red powder.3,4b IR (neat): 2924,
2853, 1737, 1555, 1456, 1428, 1371, 1248, 1196,
1
1356, 1290, 1119, 931, 767 cmꢀ1. H NMR (400 MHz,
1
D2O): d 7.66 (1H, m), 7.60 (1H, m), 7.45–7.50 (2H,
m), 4.65 (2H, s), 4.30 (2H, t, J = 7.4 Hz), 1.86 (2H, m),
1.42 (6H, s), 0.87 (3H, t, J = 7.4 Hz). 13C NMR
(100 MHz, D2O): d 141.77, 140.92, 129.75, 128.99,
123.26, 115.15, 54.33, 49.20, 21.72, 20.85, 10.18.
1158, 1116, 1019, 930, 796, 579, 680 cmꢀ1. H NMR
(400 MHz, DMSO-d6): d 8.32 (1H, dd, J = 13.4,
13.4 Hz), 7.61 (2H, d, J = 7.6 Hz), 7.38–7.47 (4H,
m), 7.26 (2H, t, J = 7.4 Hz), 6.48 (2H, d,
J = 13.5 Hz), 4.09 (4H, m), 2.74 (4H, s), 2.66 (2H, t,
J = 7.2 Hz), 1.74 (2H, m), 1.69 (2H, m), 1.66 (12H,
s), 1.48 (2H, m), 1.19 (2H, m), 0.95 (3H, t,
4.1.7. 2-(2-Phenylacetamido-E-1-ethenyl)-3,3-dimethyl-
1-propylindolium salt 13. A mixture of N,N0-diphenyl-
formamidine (0.35 g, 1.78 mmol) and 3,3-dimethyl-
2-methylene-1-propylindoline 12 (0.30 g, 1.48 mmol) in
acetic anhydride (10 mL) was refluxed for 30 min. The
solution was cooled to room temperature, the solvent
was removed under reduced pressure, and the residue
was purified by flash chromatography on silica gel
(dichloromethane/hexane/methanol = 5:1:1) to give the
salt 13 as a light yellow powder (0.45 g, 87%). IR (neat):
2965, 2926, 1680, 1638, 1603, 1580, 1553, 1492, 1369,
J = 7.4 Hz). 13C NMR (100 MHz, DMSO-d6):
d
174.20, 173.40, 169.17, 168.61, 151.50, 142.30,
140.52, 129.11, 128.86, 125.04, 122.03, 111.14,
110.96, 105.17, 48.90, 46.40, 44.20, 30.70, 29.71,
28.20, 27.21, 25.82, 25.62, 24.53, 21.17, 11.30. MS
(EI): m/z (%) = 582 (100), 485 (22).
4.1.10. N-Ethyl-2-methylbenzoxazolium bromide, 15. A
mixture of 2-methylbenzoxazole 14 (2.24 g, 0.01 mol)
and bromoethane (2.5 mL, 0.03 mol) in 1,2-dichloroben-
zene was heated at 110 °C for 24 h. The solution was
cooled to room temperature, and the residue obtained
was filtered and washed with diethyl ether. The solid
obtained was dried under vacuum to give the salt 15
as a white powder (0.60 g, 22%).10 This compound is
also commercially available from various suppliers. IR
(neat): 3084, 3047, 2974, 2929, 2859, 2729, 1593, 1462,
1
1311, 1200, 1130, 996, 757 cmꢀ1. H NMR (400 MHz,
DMSO-d6): d 9.10 (1H, d, J = 14.2 Hz), 7.05–7.70 (9H,
m), 5.34 (1H, d, J = 14.2 Hz), 4.06 (2H, t, J = 7.1 Hz),
2.05 (3H, s), 1.70 (6H, s), 1.60 (2H, m), 0.67 (3H, t,
J = 7.4 Hz). 13C NMR (100 MHz CDCl3): d 162.80,
154.24, 142.85, 139.56, 129.48, 128.41, 125.42, 123.32,
121.96, 119.42, 109.13, 94.35, 47.84, 45.19, 29.26,
27.86, 20.15, 11.45.
1388, 1188, 1147, 1025, 759 cmꢀ1
.
1H NMR
(400 MHz, DMSO-d6): d 8.09–8.23 (2H, m), 7.71–7.80
(2H, m), 4.60 (2H, q, J = 7.3 Hz), 3.02 (3H, s), 1.43
(2H, t, J = 7.3 Hz). 13C NMR (100 MHz, DMSO-d6):
d 169.03, 147.91, 129.73, 129.04, 128.13, 115.00,
113.42, 42.38, 13.84, 13.40.
4.1.8. 2-[3-[1-(5-Carboxypentyl)-1,3-dihydro-3,3-dime-
thyl-2H-indol-2-ylidene]-1-propenyl]-3,3-dimethyl-1-propyl-3H-
indolium, Propyl Cy3, 2. A mixture of the salt 11 (0.08 g,
0.28 mmol), enamide 13 (0.10 g, 0.28 mmol), and dry tri-
ethylamine (0.1 mL) in absolute ethanol (10 mL) was re-
fluxed for 30 min. The solvent was removed under
reduced pressure and the crude residue was purified by
flash chromatography on silica gel (dichloromethane/
hexane/methanol = 5:1:1) to give the dye propyl Cy3 2
as a deep red powder (0.12 g, 85%).9 IR (neat): 3407,
2969, 2934, 2876, 2734, 2673, 1557, 1453, 1427, 1242,
4.1.11. N-[(4-Carboxymethyl)phenylmethyl]-2-methyl-
benzoxazolium bromide, 16. A mixture of 2-methylbenz-
oxazole 14 (2.24 g, 0.01 mol) and 4-(bromomethyl)phen-
ylacetic acid (3.22 g, 0.01 mol) in 1,2-dichlorobenzene
was heated at 110 °C for 12 h. The solution was cooled
to room temperature, and the residue obtained was fil-
tered and washed with acetonitrile. The solid obtained
was dried under vacuum to give the salt 16 as a light yel-
low powder (3.20 g, 80%).11 IR (neat): 3014, 1733, 1579,
1
1192, 1130, 1030, 930, 754 cmꢀ1. H NMR (400 MHz,
DMSO-d6): d 8.31 (1H, dd, J = 13.4, 13.5 Hz), 7.61
(2H, d, J = 7.4 Hz), 7.36–7.47 (4H, m), 7.26 (2H, t,
J = 7.1 Hz), 6.54 (1H, d, J = 13.5 Hz), 6.53 (1H, d,
J = 13.4 Hz), 4.08 (2H, t, J = 7.2 Hz), 3.43 (2H, t,
J = 7.2 Hz), 2.14 (2H, t, J = 7.2 Hz), 1.75 (2H, m),
1.66 (12H, s), 1.52 (2H, m), 1.40 (2H, m), 1.19 (2H,
m), 0.84 (3H, t, J = 7.4 Hz). 13C NMR (100 MHz
DMSO-d6): d 174.20, 161.01, 146.45, 143.11, 141.37,
137.32, 129.11, 127.92, 126.13, 122.98, 122.21, 118.45,
105.73, 102.72, 74.11, 49.36, 44.06, 43.30, 30.26, 27.94,
27.90, 26.26, 20.89, 19.44, 11.79, 11.46.
1
1456, 1360, 1226, 1164, 753 cmꢀ1. H NMR (400 MHz,
DMSO-d6): d 7.13 (4H, s), 7.01 (1H, br t, J = 7.8 Hz),
6.95 (1H, br d, J = 8.1 Hz), 6.85 (1H, br d,
J = 7.8 Hz), 6.68 (1H, br t, J = 7.6 Hz), 5.20 (1H, d,
J = 15.0 Hz), 4.19 (1H, d, J = 15.0 Hz), 3.48 (2H, s),
3.17 (3H, s). 13C NMR (100 MHz DMSO-d6): d
173.18, 170.49, 153.38, 136.75, 133.98, 130.15, 130.04,
129.54, 129.46, 128.38, 119.71, 117.15, 50.79, 40.84,
22.21.

96
M. E. Jung, W.-J. Kim / Bioorg. Med. Chem. 14 (2006) 92–97
4.1.12.
2-(2-Phenylacetamido-E-1-ethenyl)-N-ethyl-
Acknowledgment
benzoxazolium salt, 17. A mixture of N,N0-diphenyl-
formamidine (0.38 g, 1.98 mmol) and the salt 15
(0.26 g, 1.65 mmol) in acetic anhydride (10 mL) was re-
fluxed for 30 min. The solution was cooled to room tem-
perature, the solvent was removed under reduced
pressure, and the residue was purified by flash chroma-
tography on silica gel (dichloromethane/hexane/metha-
nol = 5:1:1) to give the salt 17 as a light yellow powder
(0.41 g, 93%).12 IR (neat): 3084, 3064, 2978, 1719,
1646, 1613, 1589, 1491, 1466, 1413, 1372, 1319, 1252,
We thank Senior Associate Dean Leonard Rome of the
David Geffen School of Medicine at UCLA for generous
support of this work.
References and notes
1. For the chemistry involved in the fluorescent labeling of
DNA for genetic analysis, see: (a) Menchen, S.; Benson,
S.; Upadhya, K.; Theisen, P.; Hauser, J.; Kenney, P. Proc.
SPIE Int. Soc. Opt. Eng. 2000, 3926, 88–94; (b) , For other
chemicals uses of various cyanine dyes, see: Kondo, T.;
Seike, M.; Mori, Y.; Fujii, K.; Yamada, T.; Hirohashi, S.
Proteomics 2003, 3, 1758–1766; (c) Owen, D. J.; VanDer-
veer, D.; Schuster, G. B. J. Am. Chem. Soc. 1998, 120,
1705–1717; (d) Guenther, R.; Reddington, M. V. Tetra-
hedron Lett. 1997, 38, 6167–6170; (e) Grahn, W.; Johan-
nes, H. H.; Rheinheimer, J.; Knieriem, B.; Wuerthwein, E.
U. Liebigs Annalen 1995, 1003–1009; (f) Mujumdar, R. B.;
Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.; Waggoner, A.
S. Bioconjugate Chem. 1993, 4, 105–111; (g) Ernst, L. A.;
Gupta, R. K.; Mujumdar, R. B.; Waggoner, A. S.
Cytometry 1989, 10, 3–10.
1151, 1004, 755, 702 cmꢀ1
.
1H NMR (400 MHz,
CDCl3): d 9.19 (1H, d, J = 13.8 Hz), 7.26–7.83 (9H,
m), 5.33 (1H, d, J = 13.8 Hz), 4.44 (2H, q, J = 7.4 Hz),
2.02 (3H, s), 1.31 (3H, t, J = 7.4 Hz).13C NMR
(100 MHz, CDCl3): d 173.25, 169.92, 162.86, 147.68,
146.88, 138.12, 131.30, 130.86, 129.62, 128.57, 127.85,
113.93, 112.21, 87.14, 42.36, 23.45, 13.79.
4.1.13. 2-[3-[3-[4-[2-carboxyethyl]phenylmethyl]-2(3H)-
benzoxazolylidene]-1-propenyl]-3-ethylbenzoxazolium, Cy2,
3. A mixture of the enamide 17 (0.10 g, 0.32 mmol), the
salt 16 (0.92 g, 0.32 mmol), and dry triethylamine
(0.1 mL) in absolute ethanol (10 mL) was refluxed for
30 min. The solvent was removed under reduced pres-
sure and the crude residue was purified by flash chroma-
tography on silica gel (dichloromethane/hexane/
methanol = 5:1:1) to give the dye 3 as a deep yellow
powder (0.11 g, 75%). IR (neat): 3408, 2927, 1710,
1609, 1565, 1508, 1461, 1394, 1347, 1280, 1201, 1154,
2. (a)Luppens,S. B. I.;tenCate, J. M. J. ProteomeRes. 2005, 4,
232–237; (b) Sitek, B.; Apostolov, O.; Stuehler, K.; Pfeiffer,
K.; Meyer, H. E.; Eggert, A.; Schramm, A. Mol. Cell.
Proteomics 2005, 4, 291–299; (c) Lilley, K. S.; Friedman, D.
B. Expert Rev. Proteomics 2004, 1, 401–409; (d) Chakrav-
arti, D. N.; Gallagher, S.; Chakravarti, B. Curr. Proteomics
2004, 1, 261–271; (e) Prime, J.; Alban, A.; Hawkins, E.;
Hughes, B. Methods Proteome Protein Anal. 2004, 231–250;
(f) Van den Bergh, G.; Arckens, L. Curr. Opin. Biotechnol.
2004, 15, 38–43; (g) Jones, A.; Wastling, J.; Hunt, E. Comp.
Funct. Genomics 2003, 4, 492–501; (h) Shaw, J.; Rowlinson,
R.;Nickson,J.;Stone,T.;Sweet,A.;Williams,K.;Tonge,R.
Proteomics 2003, 3, 1181–1195; (i) Gade, D.; Thiermann, J.;
Markowsky, D.; Rabus, R. J. Mol. Microbiol. Biotechnol.
1
1116, 1083, 978, 906, 748 cmꢀ1. H NMR (400 MHz,
CDCl3): d 8.38 (1H, dd, J = 13.2, 13.3 Hz), 7.16–7.48
(12H, m), 5.90 (1H, d, J = 13.3 Hz), 5.88 (1H, d,
J = 13.2 Hz), 5.22 (2H, s), 4.12 (2H, br q, J = 7.2 Hz),
3.51 (2H, s), 1.30 (3H, t, J = 7.2 Hz). 13C NMR
(100 MHz CDCl3): d 174.31, 166.37, 162.25, 161.74,
148.00, 146.88, 146.73, 131.23, 130.93, 130.58, 130.38,
127.17, 126.33, 126.23, 125.59, 125.38, 111.01, 110.86,
110.76, 110.53, 85.95, 85.26, 47.41, 45.52, 39.60, 12.98.
¨
2003, 5, 240–251; (j) Unlu, M.; Minden, J. Mod. Protein
¨
¨
Chem. 2002, 227–244; (k) Unlu, M.; Minden, J. Prot.
¨
Protocols Handbook 2002, 2nd ed., pp 185–196.
3. Tonge, R.; Shaw, J.; Middleton, B.; Rowlinson, R.;
Rayner, S.; Young, J.; Pognan, F.; Hawkins, E.; Currie,
I.; Davison, M. Proteomics 2001, 1, 377–396.
4. Commercially available from Amersham Biosciences, GE
Healthcare. (a) Mader, O.; Reiner, K.; Egelhaaf, H.-J.;
Fischer, R.; Brock, R. Bioconjugate Chem. 2004, 15, 70–
4.1.14.
2-[3-[3-[[4-[2-[(2,5-Dioxo-1-pyrrolidinyl)oxy]-
2-oxoethyl]phenyl]methyl]-2(3H)-benzo-xazolylidene]-1-pro-
penyl]-3-ethyl-benzoxazolium, Cy2-NHS ester, 6. Anhy-
drous pyridine (0.1 mL) and N,N0-disuccinimidyl
carbonate (DSC, 21 mg, 0.08 mmol) were added to a
stirred solution of the dye 3 (25 mg, 0.05 mmol) in dry
DMF (2 mL) under nitrogen. The reaction mixture
was stirred at 60 °C for 1.5 h. After evaporation of the
solvent, the deep yellow residue was purified by column
chromatography on silica gel (dichloromethane/hexane/
methanol = 5:1:1) to give the pure Cy2-NHS ester 6
(26 mg, 86%) as an orange powder. IR (neat): 2924,
2851, 1736, 1565, 1507, 1461, 1395, 1348, 1280, 1201,
¨
78; (b) Unlu, M.; Morgan, M. E.; Minden, J. S. Electro-
¨
phoresis 1997, 18, 2071–2077; (c) Southwick, P. L.; Ernst,
L. A.; Tauriello, E. W.; Parker, S. R.; Mujumdar, R. B.;
Mujumdar, S. R.; Clever, H. A.; Waggoner, A. S.
Cytometry 1990, 11, 418–430; (d) Guether, R.; Redding-
ton, M. V. Tetrahedron Lett. 1997, 38, 6167–6170; (e)
Schafer, J.; Hamon, C.; Schwarz, J.; Pearce, C. PCT Int.
Appl. 2005, WO2005012914; (f) Bourget, C.; Kotera, M.;
Lhomme, J.; Trevisiol, E.; Laayoun, A.; Tora, C.; Sothier,
I. PCT Int. Appl. 2002, WO2002090584.
1115, 1082, 747 cmꢀ1. H NMR (400 MHz, CDCl3): d
1
5. (a) Shiobara, Y.; Ishida, S. 1960, JP35017020.; (b) Ref. 4b;
(c) Compound 7 is also commercially available.
6. (a) Kiprianov, A. I.; Buryak, V. Yu. Zh. Org. Khim. 1972,
8, 1707–1712; (b) Ref. 4b.
7. (a) Lee, L. G.; Woo, S. L.; Head, D. F.; Dubrow, R. S.;
Baer, T. M. Cytometry 1995, 21, 120–128; (b) Ref. 4a,b.(c)
Reichardt, C.; Engel, H. D. Chem. Ber. 1988, 121, 1009–
1011.
8. We have no information on the counterions of these final
quaternary ammonium salt dyes but they are almost
8.40 (1H, t, J = 13.2 Hz), 7.22–7.49 (12H, m), 6.52
(1H, d, J = 13.2 Hz), 6.40 (1H, d, J = 13.2 Hz), 5.49
(2H, s), 4.31 (2H, br q, J = 7.1 Hz), 3.85 (2H, s), 2.78
(4H, s), 1.46 (3H, t, J = 7.1 Hz). 13C NMR (100 MHz,
CDCl3): d 168.99, 166.51, 162.18, 161.73, 146.95,
146.82, 132.87, 132.02, 131.37, 130.82, 130.13, 128.36,
126.09, 125.28, 125.08, 110.98, 110.90, 110.76, 110.66,
87.15, 86.49, 37.19, 25.60, 13.34. MS (EI): m/z
(%) = 550 (100), 453 (6), 304 (6).

M. E. Jung, W.-J. Kim / Bioorg. Med. Chem. 14 (2006) 92–97
97
certainly not acetate since no peak for an acetate group
appears in the proton NMR of the final compounds.
9. (a) Wurthner, F. Synthesis 1999, 2103–2113; (b) Durr, H.;
Ma, Y.; Cortellaro, G. Synthesis 1995, 294–298.
10. (a) Cummins, W. J.; West, R. M.; Smith, J. A. PCT Int.
Appl. 1999 WO 9905221; (b) Jackson, P.; Cummins, W. J.;
West, R.; Smith, J. A.; Briggs, M. S. J. PCT Int. Appl.
1998 WO 9815829.
12. Hamilton, A. L.; Birch, M. N.; Hatcher, M. J.; Bosworth,
N.; Scott, B. PCT Int. Appl. 1999, WO9964519.
13. (a) Schouten, J. A.; Ladame, S.; Mason, S. J.; Cooper, M.
A.; Balasubramanian, S. J. Am. Chem. Soc. 2003, 125,
5594–5595; (b) Gorb, L. T.; Romanov, N. N.; Fedotov, K.
V.; Tolmachev, A. I. Khim. Geterotsikl. Soedin. 1981, 481–
484; (c) Abramenko, P. I.; Zhiryakov, V. G. Khim.
Geterotsikl. Soedin. 1975, 475–479; (d) Bailey, J.; Elvidge,
J. A. J. Chem. Soc., Perkin Trans. 1973, 1, 823–828.
14. Haykinson, M. J.; UCLA, private communication.
11. Jedrzejewska, B.; Kabatc, J.; Pietrzak, M.; Paczkowski, J.
Dyes Pigments 2003, 58, 47–58.