Synthesis of novel fluorescent 1,3,5-trisubstituted triazine derivatives and photophysical property evaluation of fluorophores and their BSA conjugates

Article abstract of DOI:10.1515/hc-2012-0024

Cyanuric chloride was allowed to react with N,N - diethylaniline to obtain 4-(4,6-dichloro-1,3,5-triazin-2-yl)- N,N -diethylaniline, which was converted into six novel 1,3,5-trisubstituted triazine derivatives on reaction with different amino acids. These

Full text of DOI:10.1515/hc-2012-0024

DOI 10.1515/hc-2012-0024ꢀ
ꢀHeterocycl. Commun. 2012; 18(3): 127–134
Vikas S. Padalkar, Vikas S. Patil, Rahul D. Telore and Nagaiyan Sekar*
Synthesis of novel fluorescent 1,3,5-trisubstituted
triazine derivatives and photophysical property
evaluation of fluorophores and their BSA
conjugates
Abstract: Cyanuric chloride was allowed to react with N,N- Parul et al., 2000; Talavera et al., 2000; Timtcheva et
diethylaniline to obtain 4-(4,6-dichloro-1,3,5-triazin-2-yl)- al., 2000), detection of compounds by HPLC (Liebes
N,N-diethylaniline, which was converted into six novel et al., 2001), capillary electrophoresis (CE) (Flana-
1,3,5-trisubstituted triazine derivatives on reaction with gan et al., 1995; Hegaard et al., 1998; Krylov, 2000), as
different amino acids. These compounds had UV absorp- well as coupling CE with laser-induced fluorescence
tion in the range 352–379 nm, accompanied by intense detection (Chiu et al., 1998; Zhang et al., 2001). They
single emission in the range 420–497 nm with fairly good also find application for determination of non-protein
quantum yield (0.106–0.383). The new compounds were cysteine in human serum (Lu et al., 2011), monitoring
1
characterized by FT-IR, H NMR, 13C NMR, mass spec- of glycolipid uptake by cells (Cheng et al., 2011), spe-
tral, and elemental analyses. These fluorophores were cific protein labeling of living cells (Arai et al., 2011),
conjugated with protein bovine serum albumin through red-shifted voltage-sensitive fluorescence (Perron et
carbodiimide chemistry between the negatively charged al., 2009), and targeting β-amyloid plaques (Parhi
carboxylate groups (-COO-) of the fluorophore and the et al., 2008). Several fluorescence probes have been used
surface terminated positively charged amino groups (-NH3+) to investigate biological processes through fluorescence
of the protein. The interaction between functionalized measurements (Fuller et al., 1998; Birch, 2001; Okerberg
amino acids with protein molecules was investigated using and Shear, 2001; Duan et al., 2009; Han and Burgess,
fluorescence spectroscopy showing fluorescence enhance- 2010; Wang et al., 2011). These are coumarin deriva-
ment or quenching of the fluorophore after conjugation.
tives (Cao et al., 2011), fluorescein isothiocyanates (Sun
et al., 1998; Zhang et al., 2001), anthracene derivatives
Keywords: α-amino acid; bioconjugation; bovine serum (DiCesare and Lakowicz, 2001), and β-naphthol deriva-
albumin (BSA); fluorescence; 1,3,5-triazine.
tives (Sartor et al., 2001).
The spectral changes observed on binding of fluo-
rophores with proteins are an important tool for investi-
gations of the topology of binding sites, conformational
changes, and characterization of substrate to ligand
binding (Song et al., 1996; Moreno et al., 1999). In addi-
tion, protein quantification in biological liquids is of
great importance in biology and medicine (Kessler and
Wolfbeis, 1992), and fluorescent probes are successfully
applied for this approach (Haughland, 1996).
*Corresponding author: Nagaiyan Sekar, Institute of Chemical
Technology (Formerly UDCT), N.P. Marg, Matunga, Mumbai 400 019,
Maharashtra, India, e-mail: n.sekar@ictmumbai.edu.in
Vikas S. Padalkar: Institute of Chemical Technology (Formerly
UDCT), N.P. Marg, Matunga, Mumbai 400 019, Maharashtra, India
Vikas S. Patil: Institute of Chemical Technology (Formerly UDCT),
N.P. Marg, Matunga, Mumbai 400 019, Maharashtra, India
Rahul D. Telore: Institute of Chemical Technology (Formerly UDCT),
N.P. Marg, Matunga, Mumbai 400 019, Maharashtra, India
An effective fluorophore for biological studies has
to show a good luminescent intensity and emission
spectra free of interferences due to the emitting sub-
stances present in the analyzed matrix. A high Stoke’s
shift is a desired characteristic for a fluorescent probe
Introduction
Fluorescent probes are useful tools in analytical tech- that permits an improved separation of the light inher-
niques (Mason, 1993), such as immunofluorescence and ent to the matrix and the light dispersed by the sample
immunofluorometric assays (Gosling, 1990; Diamandis, (Haughland, 1992).
1993), protein conformation studies (Sytnik and Kasha,
2-Aryl-4,6-disubstituted triazine derivatives have been
1994; Sytnik et al., 1994; Sytnik and Litvinyuk, 1996; reported as fluorescent probes (Cowley et al., 1991) but

128ꢀ
ꢀV.S. Padalkar et al.: Fluorescent 1,3,5-trisubstituted triazine derivatives
they are not biocompatible. As a part of ongoing research solution at pH 7. Protein conjugates were analyzed by
to develop novel materials for high-tech applications using fluorescence analysis.
(Sekar et al., 2010, 2011; Gupta et al., 2011; Padalkar et
al., 2012; Patil et al., 2012), here we report the synthesis,
characterization, and photophysical properties of novel Photophysical properties
fluorescent biocompatible fluorophores and their binding
study with protein. The novel triazine derivatives were The quantum yields of compounds 5a–f were determined
prepared from 4-(4,6-dichloro-1,3,5-triazin-2-yl)-N,N-dieth- using anthracene as standard. Samples were prepared in
ylaniline and different α-amino acids.
1-cm path length quartz cells with absorbance <ꢀ0.1 at exci-
tation and all emission wavelengths to uniformly illumi-
nate across the sample, and to avoid the inner-filter effect.
Absorption and emission characteristics of the standard
as well as the new compounds were measured at differ-
ent concentrations (0.1–1.4 ppm). Absorbance intensity
values were plotted against emission intensity values, and
a linear plot was obtained. Gradients were calculated for
each compound. All measurements were done by keeping
the parameters such as solvent and slit width constant.
Relative quantum yield of all synthesized derivatives 5a–f
were calculated by using Eq. (1), and for emission mea-
surements compounds were excited at their correspond-
ing absorption maxima.
Results and discussion
Chemistry
The reaction of cyanuric chloride with N,N-diethylaniline
yielded dichloro-1,3,5-triazine 3 (Scheme 1). Then com-
pound 3 was allowed to react with different α-amino acids
in methanol at room temperature to give the desired fluo-
rophores 5a–f. A summary of the synthesized derivatives
5a–f is given in Table 1. All compounds were characterized
by FT-IR, 1H NMR, 13C NMR, mass spectral, and elemental
analyses. The synthesized novel triazine derivatives 5a–f
are fluorescent in solution when irradiated with UV light,
ΦX = ΦST (GradX/GradST) (η2X/η2ST
)
(1)
and these compounds contain biocompatible free carbox- where ΦX = quantum yield of synthesized sample, ΦST
=
ylic acid groups, which were further explored for labeling quantum yield of standard used, GradX = gradient of syn-
with protein bovine serum albumin (BSA) in the presence thesized sample, GradST = gradient of standard used, η2ST
of the coupling agent N-hydroxysuccinimide and 1-ethyl- refractive index of solvent for standard sample.
=
3-(3-dimethyllaminopropyl)carbodiimide hydrochloride.
Gradient of standard (Figure 1), gradient of com-
To warrant an efficacious label, the unbound fluorophore pound 5c (Figure 2), and quantum yield calculation of 5c
was efficiently removed by dialysis in phosphate buffer (Table 2) are shown for illustration.
Cl
Cl
70oC, 8h
N
N
N
N
Et2N
Cl
N
Cl
N
Cl
1
2
3
Et2N
H2N
H
R
2
H2N
MeOH
K2CO3
rt, 12h
MeOH
K2CO3
rt, 12h
COOH
R
4a-e
H
COOH
4f
COOH
R
H
Cl
N
HN
N
N
N
N
NH
N
NH
R
R
H
COOH
Et2N
Et2N
H
COOH
5a-e
5f
Scheme 1ꢁSynthesis of amino acid substituted 1,3,5-triazine derivatives.

V.S. Padalkar et al.: Fluorescent 1,3,5-trisubstituted triazine derivativesꢀ
ꢀ129
Compound
R
Absorption
λmax (nm)
Emission
λem (nm)
Stoke’s
shift (Δλ)
Quantum
yield
COOH
R
H
5a
5b
5c
5d
H-
376
376
379
352
440
497
487
464
64
121
108
112
0.106
0.376
0.346
0.383
HSCH2-
PhCH2-
p-OH-C6H4-
HN
N
N
N
NH
R
Et2N
H
COOH
5e
379
452
112
0.289
5a-e
N
H
Cl
N
N
H
N
N
NH
5f
352
420
68
0.170
HN
R
Et2N
COOH
5f
a
Table 1ꢁAbsorption, emission, Stoke’s shift, and quantum yield of compounds 5a–f .
aAbsorption and emission analyses were carried out at room temperature; solvent used was DMSO/H2O (1 mL:9 mL); concentration of
samples: 1 × 10-6 μL; DMSO: dimethyl sulfoxide.
By putting values of gradients in Eq. (1), quantum are given in Figures 3 and 4, respectively. Absorption and
yield was calculated for each compound by taking anthra- emission wavelength range of all compounds are similar,
cene as standard (quantum yield of anthracene in ethanol which means that only the triazine and N,N-diethyl aniline
solution is 0.27).
groups are responsible for the delocalization of electrons
The fluorescence quantum yields of 5a–f were mea- from the electron donating terminus (N,N-diethyl) to the
sured in ethanol at room temperature. The quantum electron acceptor unit (1,3,5-triazine). Absorption, emis-
yields of 5b and 5d are much higher than those for 5a, 5c, sion maxima, and Stoke’s shift value are summarized in
5e, and 5f (Table 1). The higher quantum yields of 5b and Table 1.
5d are presumably due to presence of -OH and -SH groups
in fluorophore. Furthermore, it was observed that except
for fluorophore 5a all compounds show similar fluores- Protein labeling with fluorophores 5a–f
cence behavior with respect to absorption, emission, and
Stoke’s shift. The UV-Vis absorption and emission spectra Synthesized fluorophores have good quantum yields, high
of triazine derivatives 5a–f were recorded in DMSO/H2O fluorescence intensity, and contain biocompatible car-
(1:9) at room temperature using concentration 1 × 10-6 μL. boxylic acid groups which can be easily conjugated, so we
The absorption and emission spectra of compounds 5a–f explored fluorescence properties of new compounds after
100
90
80
70
60
50
40
30
20
10
0
60
50
40
30
20
10
0
0.10
0
0.02
0.04
0.06
0.08
0
0.05
0.10
Absorbance (a.u.)
Absorbance (a.u.)
Figure 1ꢁGraph of emission intensity vs. absorbance of standard
anthracene for gradient calculation.
Figure 2ꢁGraph of emission intensity vs. absorbance of compound
5c for gradient calculation.

130ꢀ
ꢀV.S. Padalkar et al.: Fluorescent 1,3,5-trisubstituted triazine derivatives
Gradient of anthracene
Gradient of compound 5c
GdST ꢂ=ꢂ Y2 - Y1/X2 - X1
Gd5c ꢂ=ꢂ Y2 - Y1/X2 - X1
GdST ꢂ=ꢂ (60.56–20.12)/(0.09–0.007)
GdST ꢂ=ꢂ 40.446/0.083
GdST ꢂ=ꢂ 487.30
Gd5c ꢂ=ꢂ (79.12–46.10)/(0.09–0.037)
Gd5c ꢂ=ꢂ 33.1/0.053
Gd5c ꢂ=ꢂ 624.52
Φ5c ꢂ=ꢂ (624.52/487.30) ΦST
Φ5c ꢂ=ꢂ (1.2815) (0.27)
Φ5c ꢂ=ꢂ 0.346
Table 2ꢁQuantum yield calculation of compound 5c.
conjugation with BSA. The extent of conjugation of BSA becomes steady at particular concentration (Figure 6).
with fluorophores 5a–f at various fluorophore/protein After conjugation fluorescence decreases gradually and
ratios was studied.
becomes steady at particular fluorophore concentration.
For protein labeling, the stock solution of protein A steady point in a graph indicates the amount of fluoro-
(1 mg/1 mL) was made in water and stock solutions of phore required for complete binding of fluorophore with a
fluorophores (different concentration: 0.1–1.4 ppm) were certain amount of protein. The decrease in fluorescence is
made in a DMSO/water mixture (0.5 mL DMSO/3.5 mL due to a change in microenvironment of the fluorophore.
water). The stock solution of fluorophore was treated with Only one carboxylic group in 5a–e is likely to bind with
2 molar equivalents of 1-ethyl-3-(3-dimethylaminopropyl) the protein. Comparison of results of bioconjugation of
carbodiimide hydrochloride and N-hydroxysuccinimide mono-carboxylic 5f and di-carboxylic compounds shows
and the mixture was stirred for 12 h at room temperature. no difference in their trends in photophysical properties.
After activation of fluorophore by carbodiimide chemistry,
corresponding protein stock solution was added and the
Conclusion
mixture was stirred for 12 h for conjugation with fluoro-
phore. After 12 h of stirring the mixture was dialyzed in
phosphate buffer (pH 7) to remove unconjugated fluoro- Six novel triazine-based fluorophores 5a– f were synthe-
1
phore from the conjugated derivative. The amount of fluo- sized, purified, and characterized by H NMR, 13C NMR,
rophores 5a–f needed for complete binding with available IR, UV-VIS, mass spectral, and elemental analyses.
amino groups of BSA was optimized by varying concen- Fluorescence emission properties were studied. These
tration of fluorophore and BSA. At higher concentrations fluorescent compounds are extremely fluorescent when
of fluorophore, complete binding takes place and graph irradiated under UV light and have potential use as
of intensity against concentration of fluorophore becomes fluorescent probes for protein labeling. Binding of the
steady. This was observed before conjugation as well as fluorophores with BSA is reported here.
after conjugation with BSA (Figure 5).
Before conjugation with BSA the fluorescence inten-
sity increases as fluorophore concentration increases and
Experimental
0.6
Biological and chemical materials
All commercial reagents and solvents were used without purifica-
tion. BSA was purchased from Sigma Aldrich. Column chromatogra-
phy was performed using silica gel 60–120 mm mesh size. Synthetic
reactions were monitored by TLC on 0.25-mm silica gel 60 F254 pre-
coated plates (Merck), which were visualized with UV light.
5c
5a
5b
5d
5e
5f
0.5
0.4
0.3
0.2
0.1
0
Instruments
The FT-IR spectra were recorded on a Perkin-Elmer 257 spectrometer
using KBr discs. 1H NMR and 13C NMR spectra were recorded on a Varian
VXR 400-MHz and a 100-MHz instrument, respectively, using TMS
as internal standard. Mass spectra (EI) were recorded on a Finnigan
320
340
360
380
400
420
Wavelength (nm)
Figure 3ꢁUV-Visible absorption spectra of compounds 5a–f.

V.S. Padalkar et al.: Fluorescent 1,3,5-trisubstituted triazine derivativesꢀ
ꢀ131
absorption maximum with an excitation slit width of 5 nm and emis-
sion slit width of 5 nm. The spectra were corrected. All concentrations
of the fluorophores were between 0.1 ppm and 1.4 ppm. Elemental
analysis was carried out by using a FLASH EA 1112 series instrument of
Thermo Finnigan make.
400
350
300
250
200
150
100
5c
5a
5b
5d
5e
5f
Synthesis of 4-(4,6-dichloro-1,3,5-
triazin-2-yl)-N,N-diethylaniline (3)
50
0
A mixture of N,N-diethylaniline (2) (27.0 g, 0.2 mol) and cyanuric chlo-
ride (1) (18.4 g, 0.1 mol) was heated at 70°C for 8 h under a slow stream
of dry nitrogen. After completion of the reaction monitored by TLC, the
mixture was extracted with hot chloroform (200 mL) and the white crys-
talline N,N-diethylaniline hydrochloride was removed by filtration. Slow
cooling and evaporation of the chloroform extract to a volume of 50 mL
yielded crystals of 3. The product was crystallized twice from acetone
400
450
500
550
600
650
700
Wavelength (nm)
Figure 4ꢁFluorescence emission spectra of compounds 5a–f.
instrument. Absorption spectra of the compounds were recorded on a (Padalkar, 2011) to give yellow crystalline solid; yield 11.7 g (40%); mp
Spectronic Genesys 2 UV-Visible spectrometer. Emission spectra were 156°C; IR:ν 3411, 2967, 1610, 1515, 1232, 1164, 839, 824, 715, 567 cm-1.
measured at room temperature on a Varian Cary Eclipse spectrofluo-
1H NMR (CDCl3): δ 8.29–8.33 (dd, 2H, J = 9.2, 2.8 Hz), 6.65–6.69
rometer in a standard 1-cm quartz cell. All dyes were excited at the (dd, 2H, J = 9.2, 2.8 Hz), 3.44–3.46 (m, 4H), 1.22–1.25 (m, 6H); 13C NMR
0.10
0.08
0.10
0.08
0.06
0.04
0.06
0.04
0.02
0
0.02
0
0
0.3
0.6
0.9
1.2
0
0.3
0.6
0.9
1.2
Conc. of compound 5b (ppm)
Conc. of compound 5a (ppm)
0.10
0.08
0.06
0.04
0.02
0
0.10
0.08
0.06
0.04
0.02
0
0
0.3
0.6
0.9
1.2
0
0.3
0.6
0.9
1.2
Conc. of compound 5d (ppm)
Conc. of compound 5c (ppm)
0.10
0.08
0.06
0.04
0.02
0
0.10
0.08
0.06
0.04
0.02
0
0
0.3
0.6
0.9
1.2
0
0.3
0.6
0.9
1.2
Conc. of compound 5e (ppm)
Conc. of compound 5f (ppm)
Before conjugation
After conjugation
Figure 5ꢁAbsorption intensity graph of compounds 5a–f before and after conjugation.

132ꢀ
ꢀV.S. Padalkar et al.: Fluorescent 1,3,5-trisubstituted triazine derivatives
45
40
35
30
25
20
15
10
5
70
60
50
40
30
20
10
0
0
0
0.3
0.6
0.9
1.2
0
0.3
0.6
0.9
1.2
Conc. of compound 5a (ppm)
Conc. of compound 5b (ppm)
240
220
200
180
160
140
120
100
80
140
120
100
80
60
40
0
0.3
0.6
0.9
1.2
0
0.3
0.6
0.9
1.2
Conc. of compound 5c (ppm)
Conc. of compound 5d (ppm)
180
160
140
120
100
140
120
100
80
60
40
20
0
0.3
0.6
0.9
1.2
0
0
0.3
0.6
0.9
1.2
Conc. of compound 5e (ppm)
Conc. of compound 5f (ppm)
Before conjugation
After conjugation
Figure 6ꢁFluorescence emission of compounds 5a–f before and after conjugation.
(CDCl3): δ 179.5, 172.3, 154.2, 130.7, 125.6, 114.6, 49.0, 15.6; MS: m/z (%) (49), 321 (23), 256 (43). Anal. Calcd for C17H22N6O4: C, 54.54; H, 5.92; N,
22.45. Found: C, 54.59; H, 5.89; N, 22.42.
298 (34), 299 (100), 161 (13), 105 (11).
Dye 5bꢁYield 1.147 g (67%); pale yellow solid; mp 220°C (dec.); IR: ν
3286, 1645, 1515, 1337, 1267, 1186, 786 cm-1; 1H NMR (DMSO-d6): δ 12.12
(bs, 2H), 8.10 (dd, 2H, J = 12.4, 8.8 Hz), 6.68 (dd, 2H, J = 12.4, 8.8 Hz),
3.40 (q, 4H), 2.03 (m, 4H), 1.99 (m, 2H), 1.78 (bs, 2H), 1.86 (bs, 2H), 1.10
(m, 6H); 13C NMR (DMSO-d6): δ 175.5, 170.3, 161.9, 149.3, 129.0, 120.3,
112.4, 74.9, 49.0, 28.3, 15.6; MS: m/z = 467 (M+1, 38), 466 (100), 412
(48), 388 (72), 258 (38). Anal. Calcd for C19H26N6O4S2: C, 48.91; H, 5.62;
N, 18.01; S, 13.74. Found: C, 48.92; H, 5.67; N, 18.02; S, 13.75.
General procedure for synthesis
of fluorophores 5a–f
A solution of α-amino acid 4a–f (20 mmol) in methanol (30 mL) was
treated with K2CO3 (20 mmol) and the mixture stirred at room temper-
ature for 30 min. A solution of 3 (10 mmol) in methanol (10 mL) was
added dropwise over the period of 15 min and the mixture was stirred
at room temperature until TLC analysis indicated disappearance of 3.
Then the mixture was concentrated and the residue was purified by
column chromatography eluting with MeOH/EtOAc, 1:9 to afford the
product 5a–f.
Dye 5cꢁYield 1.16 g (57%); yellow solid; mp 226–228°C; IR: ν 3286,
1
2928, 1650, 1518, 1337, 1267, 1191, 1148, 788, 698 cm-1; H NMR (DMSO-
d6): δ 12.13 (bs, 2H), 8.05 (dd, 2H, J = 9.2, 8.8 Hz), 7.20–7.35 (m, 10H), 6.68
(dd, 2H, J = 9.2, 8.8 Hz), 4.60 (bs, 2H), 3.12 (m, 4H), 2.67 (m, 2H), 2.48 (m,
4H), 1.05 (m, 6H); 13C NMR (DMSO-d6): δ 174.6, 164.8, 161.2, 149.6, 140.3,
136.9, 128.7, 127.8, 125.9, 124.6, 114.6, 72.6, 48.0, 35.7, 15.6; MS: m/z (%)
556 (M+1, 25), 557 (20), 278 (18), 256 (100), 225.3 (30). Anal. Calcd for
C31H34N6O4: C, 67.13; H, 6.18; N, 12.12. Found: C, 67.20; H, 6.19; N, 12.08.
Dye 5aꢁYield 0.85 g (62%); dark yellow crystalline solid; mp 234°C;
IR: ν 3398, 3305, 1733, 1644, 1570, 1509, 1415, 1212, 1149, 1078, 828, 783
cm-1. 1H NMR (DMSO-d6): δ 12.59 (bs, 2H, -OH), 8.15 (bs, 2H, -NH), 8.07
(dd, 2H, J = 7.7, 1.5 Hz), 6.72 (dd, 2H, J = 7.7, 1.5 Hz), 3.66 (s, 4H), 3.46 (m,
4H), 1.15 (m, 6H); 13C NMR (DMSO-d6): δ 172.7, 165.0, 161.2, 148.9. 128.7,
Dye 5dꢁYield 1.29 g (60%); yellow amorphous powder; mp 270°C
1
125.2, 115.0, 49.3, 46.4, 15.6; MS: m/z (%) 375 (M+1, 67), 374 (100), 389 (dec.); IR: ν 3277, 1684, 1340, 1269, 1194, 1076, 787, 740 cm-1; H NMR

V.S. Padalkar et al.: Fluorescent 1,3,5-trisubstituted triazine derivativesꢀ
ꢀ133
248 (45), 198.3 (32). Anal. Calcd for C19H22ClN7O2: C, 54.87; H, 5.33; N,
23.58. Found: C, 54.92; H, 5.37; N, 23.52.
(DMSO-d6): δ 12.13 (bs, 2H), 9.90 (bs, 2H), 8.05 (dd, 2H, J = 7.4, 1.8 Hz),
7.12 (dd, 2H, J = 7.4, 2.4 Hz), 7.03 (m, 4H), 6.69 (m, 4H), 6.64 (dd, 2H,
J = 9.2, 8.8 Hz), 4.72 (bs, 2H), 3.40 (m, 4H), 3.34 (m, 4H), 1.12 (m, 6H);
13C NMR (DMSO-d6): δ 171.6, 165.4, 162.2, 152.3, 148.7, 138.7, 130.0, 128.9,
124.6, 117.0, 113.1, 72.8, 64.3, 47.5, 15.1. MS: m/z (%) 587 (M+1, 56), 586
(100), 448 (34), 321 (23). Anal. Calcd for C31H34N6O6: C, 63.47; H, 5.84;
N, 14.33. Found: C, 63.52; H, 5.87; N, 14.32.
General procedure for activation
of fluorophore and labeling of protein
Dye 5eꢁYield 1.94 g (84%); yellow solid; mp 263°C (dec.); IR: ν 3728,
Protein labeling was carried out using N-hydroxysuccin-
imide and 1-ethyl-3-(3-dimethyllaminopropyl)carbodii-
mide hydrochloride. Stock solutions of fluorophores
(different concentrations) were prepared in 0.5 mL DMF
and 4.5 mL water. A mixture of 5a–f, 2 molar equivalents of
N-hydroxysuccinimide and 2 molar equivalents of 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide hydrochloride
were stirred at room temperature for 12 h. Then, BSA
(1 mg) was added to corresponding activated dye solution,
and the mixture was stirred for an additional 12 h, after
which time the labeled protein was separated from free
fluorophore by dialysis in phosphate buffer of pH 7 (Singh
et al., 2004).
1
2968, 1652, 1540, 1520, 1339, 1269, 1194, 1009, 784, 740, 699 cm-1; H
NMR (DMSO-d6): δ 12.42 (bs, 2H), 10.82 (m, 2H), 8.03 (dd, 2H, J = 8.0,
2.4 Hz), 7.30 (m, 2H), 7.18 (dd, 2H, J = 8.0, 2.4 Hz), 7.03 (m, 4H), 6.96
(dd, 2H, J = 7.4, 3.4 Hz), 6.63 (dd, 2H, J = 9.0, 2.2 Hz), 4.65 (s, 2H), 3.38
(d, 4H), 3.36 (m, 4H), 3.12 (m, 2H), 1.09 (m, 6H); 13C NMR (DMSO-d6):
δ 175.3, 165.0, 160.1, 150.6, 143.4, 139.0, 137.1, 128.9, 125.6, 122.2, 120.6,
118.6, 112.9, 111.3, 99.7, 73.0, 48.8, 30.1, 14.9; MS: m/z (%) 633 (M+1, 40),
633 (100%), 451 (30). Anal. Calcd for C35H36N8O4: C, 64.44; H, 5.73; N,
17.71. Found: C, 64.80; H, 5.77; N, 17.69.
Dye 5fꢁYield 1.43 g (73%); brown-yellow solid; mp 208–210°C (dec.);
1
IR: ν 3380, 2965, 1566, 1395, 1350, 1268, 1186, 1078, 923, 808 cm-1; H
NMR (DMSO-d6): δ 12.21 (s, 1H), 8.06 (s, 1H), 7.89 (s, 1H), 7.31 (s, 1H),
7.03 (dd, 2H, J = 7.3, 2.4 Hz), 6.63 (dd, 2H, J = 7.3, 2.4 Hz), 4.06 (s, 1H), 3.97
(m, 1H), 3.46 (m, 4H), 3.34 (m, 2H), 1.09 (m, 6H); 13C NMR (DMSO-d6):
δ 177.5, 174.3, 169.0, 162.0, 150.2, 138.0, 132.4, 130.7, 125.6, 118.5, 113.8,
74.0, 48.0, 30.1, 15.4; MS: m/z (%) 414 (M+1, 43), 413 (78), 357 (100),
Received February 3, 2012; accepted April 2, 2012
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