Novel asymmetric Cy5 dyes: Synthesis, photostabilities and high sensitivity in protein fluorescence labeling

Article abstract of DOI:10.1016/j.jphotochem.2010.01.002

Several novel water-soluble asymmetric pentamethine cyanine dyes were synthesized. The maximum absorption and emission wavelengths of the dyes in different solvents were in the range from 647 to 665 nm and exhibited negative solvatochromism with increasing solvent polarity. The fluorescence quantum yields of the dyes were about 0.1 in water, and were obviously higher than those of hydrophobic cyanine dyes. Dyes with N-benzyl groups and N-sulfo-groups displayed greater photostability than dyes with N-carboxypentyl groups in water. The limit of detection of dye 5a for BSA was 1.2 × 10^-8 mol L^-1 by high performance liquid chromatography with fluorescent director about 100-fold lower than that by UV detection (1.0 × 10^-6 mol L^-1). Therefore, Dye 5a could be used to improve photostability and detection sensitivity in protein analysis.

Full text of DOI:10.1016/j.jphotochem.2010.01.002

Journal of Photochemistry and Photobiology A: Chemistry 210 (2010) 168–172
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Novel asymmetric Cy5 dyes: Synthesis, photostabilities and
high sensitivity in protein fluorescence labeling
Li Wanga, Jiangli Fana, Xiaoqiang Qiaob, Xiaojun Penga,∗, Bin Daia,
Bingshuai Wanga, Shiguo Suna, Lihua Zhangb, Yukui Zhangb
a State Key Laboratory of Fine Chemicals, Dalian University of Technology, 158 Zhongshan Road, Dalian 116012, China
b CAS Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic Research & Analysis Center, Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Several novel water-soluble asymmetric pentamethine cyanine dyes were synthesized. The maximum
absorption and emission wavelengths of the dyes in different solvents were in the range from 647 to
665 nm and exhibited negative solvatochromism with increasing solvent polarity. The fluorescence quan-
tum yields of the dyes were about 0.1 in water, and were obviously higher than those of hydrophobic
cyanine dyes. Dyes with N-benzyl groups and N-sulfo-groups displayed greater photostability than dyes
with N-carboxypentyl groups in water. The limit of detection of dye 5a for BSA was 1.2 × 10−8 mol L−1 by
high performance liquid chromatography with fluorescent director about 100-fold lower than that by UV
detection (1.0 × 10−6 mol L−1). Therefore, Dye 5a could be used to improve photostability and detection
sensitivity in protein analysis.
Received 30 October 2009
Received in revised form
13 December 2009
Accepted 4 January 2010
Available online 6 January 2010
Keywords:
Cy5
Cyanine dye
Photophysical property
Photostability
© 2010 Elsevier B.V. All rights reserved.
Fluorescent labeling
High performance liquid chromatography
1. Introduction
In order to effectively reduce the background signal arising
from auto-fluorescence of the biological matrix and light scatter-
In the post-genome sequencing era, there has been more and
more attention paid to protein analysis. Until now, fluorescence
detection has proved to be one of the most sensitive methods
for protein analysis [1,2]. Fluorescence detection combined with
electrophoresis [3,4] and high performance liquid chromatography
(HPLC) techniques enable qualitative and quantitative analysis of
proteins. However, most proteins with important biological func-
tions, such as the drug targets and biomarkers, are of an extremely
low concentration [5]. Therefore, the study of more suitable fluo-
rophores for the improvement on detection sensitivity for proteins
is an imperative task.
Cyanine dyes play an indispensable role in biomedical appli-
cations [6,7], particularly in fluorescence detection of antibodies
and DNA [8], the imaging of biological targets in vivo [9], and flu-
orescent labeling compounds for proteins [10,11]. This is due to
their excellent spectral properties, including large molar extinction
coefficients and broad wavelength tunabilities.
ing, longwavelength cyanine dyes (>600 nm or within the near infra
red region) have been developed. Increasing the length of the con-
jugated chain of the cyanine dyes is the main approach to impart
the desired red shift; however, this reduces the photostability [12].
In our previous work, we firstly employed rigid N-
p-carboxybenzyl (N-p-CH2C6H4COOH) as substituents in
3H-indolenine trimethine cyanine dyes (Cy3) [13], and 3H-
indolenine heptamethine cyanine dyes (Cy7) [14], that not only
improved the photostability, but also increased the yields of inter-
mediates and products. The properties of pentamethine cyanine
dyes (Cy5) of 3H-indolenines, however, remain unreported. The
bio-labeling of the dyes to proteins via covalent conjugations
include the activation of N-p-carboxybenzyl in the dyes and the
reaction of the activated dyes to reactive groups (such as –NH2) on
proteins in aqueous buffer solutions under mild conditions. Sulfo-
groups are important for this kind of application because they
prevent protein precipitation during the derivatization procedures
[15,16].
In this paper, we describe the synthesis of the water-soluble
Cy5 dyes, the relationship between the molecular structure and
photostability and labeling performance on BAS. The structures of
water-soluble Cy5 dyes were shown in Fig. 1. Compared with well-
∗
Corresponding author. Tel.: +86 411 39893899; fax: +86 411 39893800.
E-mail address: pengxj@dlut.edu.cn (X. Peng).
1010-6030/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jphotochem.2010.01.002

L. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 210 (2010) 168–172
169
Dye 6b: Yield 69%. 1H NMR (400 MHz, D2O): ı 1.54 (s, 12H,
C(CH3)2), 5.19 (s, 4H, N–CH2–Ar), 6.02 (d, 2H, J = 13.6 Hz, CH CH),
6.19 (m, 1H, CH CH), 7.04–7.76 (m, 14H, Ar–H), 7.82 (t, 2H,
J = 13.6 Hz, CH CH). API-ES-MS, m/z: 390.6 [M−2H]2−
.
Dye 6c: Yield 60%. 1H NMR (400 MHz, D2O): ı 1.28–1.66 (m, 12H,
6CH2), 1.74 (s, 12H, C(CH3)2), 2.79 (m, 4H, CH2COOH), 3.93 (m, 4H,
N–CH2), 6.02 (m, 2H, CH CH), 6.44 (d, 1H, J = 13.2 Hz, CH CH), 7.49
(m, 2H, Ar–H), 7.64 (m, 2H, Ar–H), 7.76 (s, 2H, Ar–H), 7.94 (t, 2H,
J = 13.2 Hz, CH CH). API-ES-MS, m/z: 740.6 [M−2H]2−
.
2.2.1. Hemicyanine intermediate 4
The quaternary salt 3a (375 mg, 1 mmol) and malonaldehyde
dianil hydrochloride (310 mg, 1.2 mmol) were dissolved in a mix-
ture of acetic acid (5 mL) and acetic anhydride (5 mL), and then
heated to reflux. The reaction was monitored by thin-layer chro-
matography (TLC). Extended heating produced some symmetrical
dye (<5%). After 50 min, the mixture was cooled to room temper-
ature and diluted with diethyl ether. The supernatant fluid was
removed by decantation. The brown powder thus obtained was
simply separated by flash-column.
Fig. 1. Structures of water-soluble Cy5 dyes.
known symmetric cyanine dye 6c, the asymmetric dyes (5a and
5b) contain only one active group on their molecules, and should
benefit from quantitative protein labeling and purifying. All results
demonstrated that asymmetric dye 5a possesses better photosta-
bility, which can be a good fluorescent labeling reagent for protein
labeling.
2.2.2. Asymmetrical dyes
A solution of crude hemicyanine intermediate 4 (585 mg,
1 mmol) and quaternary salt 3b, 3c or 3d (1 mmol) in acetic anhy-
dride (5 mL) was heated to 120 ◦C for 40 min. The reaction was
monitored by TLC. The mixture was cooled to room temperature
and diluted with diethyl ether. After filtration, the crude dye was
chromatographied in a C18-RP column using a methanol–water
mixture as the eluent.
2. Experiments
Dye 5a: Yield 56%. 1H NMR (400 MHz, D2O): ı 1.53 (m, 4H, 2CH2),
1.74 (s, 12H, C(CH3)2), 2.74 (m, 2H, CH2SO3−), 3.92 (s, 2H, N–CH2),
5.18 (s, 2H, N–CH2–Ar), 6.02 (m, 2H, CH CH), 6.26 (t, 1H, J = 13.2 Hz,
CH CH), 7.02–8.42 (m, 12H, Ar–H), 7.76 (m, 2H, CH CH). API-ES-
2.1. Instruments and materials
Mass spectral determinations were taken on HP1100 API-
ES mass spectrometer. NMR spectra were recorded on a Varian
400 MHz NMR spectrometer (USA). Chemical shifts are expressed
in parts per million from D2O (ıH = 4.79) [17]. Fluorescence mea-
surements were performed on a PTI-C-700 Felix and Time-Master
system. Visible spectra were measured on a HP-8453 spec-
trophotometer. HPLC experiments were performed on the Waters
2695-2996-2475. Purification of dyes was performed by conven-
tional column chromatography with C18-RP absorbent (Sinochrom
C18, 40–75 mesh, 10 nm, 280 m2 g−1, Dalian Elite Company, China).
Deionized water was redistilled before use, and acetonitrile was of
chromatographic grade. Other chemicals used for the experiments
were of analytical grade.
MS, m/z: 391.1 [M−2H]2−
.
Dye 5b: Yield 30%. 1H NMR (400 MHz, D2O): ı 1.27–1.67 (m,
10H, 5CH2), 1.68 (s, 12H, C(CH3)2), 2.08 (m, 2H, CH2COOH), 2.81
(m, 2H, CH2SO3−), 3.94 (m, 4H, N–CH2), 6.14 (m, 2H, CH CH), 6.46
(t, 1H, J = 12.8 Hz, CH CH), 7.19 (m, 2H, Ar–H), 7.62 (m, 2H, Ar–H),
7.69 (d, 2H, J = 5.2 Hz, Ar–H), 7.84 (m, 2H, CH CH). API-ES-MS, m/z:
381.0 [M−2H]2−
.
Dye 5c: Yield 64%. 1H NMR (400 MHz, D2O): ı 1.71 (m, 4H, 2CH2),
1.73 (s, 12H, C(CH3)2), 3.17 (m, 2H, CH2SO3−), 4.15 (m, 2H, N–CH2),
5.36 (s, 2H, N–CH2–Ar), 6.37 (m, 2H, CH CH), 6.42 (m, 1H, CH CH),
7.13–7.78 (m, 11H, Ar–H), 8.34 (t, 2H, J = 12.8 Hz, CH CH). API-ES-
MS, m/z: 703.2 [M−H]−.
2.2. Synthesis
2.2.3. Synthesis of the succinimidyl esters 7 of Cy5 dyes
The synthetic routes of Cy5 dyes were shown in Fig. 2.
2,3,3-Trimethyl-3H-indolenine and 2,3,3-trimethylindolenine-5-
sulfonate (2) were obtained as starting materials by conventional
Fisher 3H-indole synthesis [18].
The dye with carboxyl group was dissolved in dry N,N-
dimethylformamide (DMF, 2 mL/100 mg of the dye). N,Nꢀ-
dicyclohexyl carbodiimide (DCC, 5 eq./carboxyl group) and N-
hydroxysuccinimide (NHS, 10 eq./carboxyl group) was added. The
mixture was left at room temperature for 10 h. After diluting the
mixture with dry ethyl acetate, the supernatant was centrifuged
and collected. By TLC, a nearly 100% yield of the active succin-
imidyl esters of Cy5 dyes (Cy5-NHS) was obtained. Because active
esters easily become deactivated via hydrolysis, the products were
prepared on the spot and used immediately without further purifi-
cation.
Intermediates of 3H-indolium quaternary salt 3 were syn-
thesized from the quaternization of 2 with 1,4-butane sultone,
6-bromohexanoic acid or p-(chloromethyl) benzoic acid, respec-
tively, and were used in further experiments without additional
purification. The yields of intermediates 3a, 3b, 3c and 3d were
78%, 58%, 39% and 80%, respectively. Symmetric Cy5 dyes, as refer-
ence dyes, were synthesized according to the previous procedure
[16].
Dye 6a: Yield 64%. 1H NMR (400 MHz, D2O): ı 1.48–1.69 (m, 8H,
4CH2), 1.78 (s, 12H, C(CH3)2), 2.79 (m, 4H, CH2SO3−), 3.94 (m, 4H,
N–CH2), 6.12 (m, 2H, CH CH), 6.39 (d, 1H, J = 13.6 Hz, CH CH), 7.19
(m, 2H, Ar–H), 7.59 (m, 2H, Ar–H), 7.64 (s, 2H, Ar–H), 7.84 (t, 2H,
2.3. Determination of quantum yield
The corresponding fluorescence quantum yields were calcu-
lated relative to a standard solution of Rhodamine B in ethanol
J = 13.2 Hz, CH CH). API-ES-MS, m/z: 261.2 [M−3H]3−
.

170
L. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 210 (2010) 168–172
Fig. 2. Synthetic routes of Cy5 dyes: (a) intermediates, (b) symmetric dyes, (c) asymmetric dyes, and (d) succinimidyl esters of Cy5 dyes.
(˚ = 0.56) [19] and were determined using the formula (1) [20]:
2.4. Photostability
ꢀ
ꢁꢀ ꢁꢀ
ꢁꢀ
ꢁ
2
Fx
Fs
Ax
ꢀex s
ꢀex x
nx
ns
The photostability tests were carried out in quartz cells (10 mm
in width) where sample solutions were irradiated with a 500 W
Iodine–tungsten (I/W) lamp at room temperature. The distance
between the cells and the lamp was 250 mm. The solutions of the
dyes (1 × 10−5 mol L−1) in water were radiated and the irreversible
bleaching of the dyes at the absorption peak was monitored as a
function of time.
˚
x
= ˚s
(1)
As
where ˚ is quantum yield, F is integrated area under the corrected
emission spectrum, A is absorbance at the excitation wavelength,
ꢀex is the excitation wavelength, n is the refractive index of the solu-
tion used in measurement [21] and the subscripts x and s denote
test and the standard, respectively.
Table 1
Spectra properties of the Cy5 dyes in the different solvents.
Dye
Solvent
ꢀab/ꢀem (nm)
˚
ε(×105 mol−1 cm−1 L)
Stocks shifts (nm)
DMF
Methanol
Water
660/678
650/670
647/664
0.15
0.13
0.10
0.95
1.14
1.12
18
20
17
5a
DMF
Methanol
Water
660/679
650/668
648/664
0.16
0.13
0.10
0.97
1.07
1.14
19
18
16
5b
5c
6a
6b
6c
DMF
Methanol
Water
656/674
649/667
645/662
0.14
0.11
0.09
0.95
1.12
1.28
18
18
17
DMF
Methanol
Water
660/678
649/670
647/663
0.14
0.13
0.09
1.03
1.16
1.15
18
21
16
DMF
Methanol
Water
662/680
653/672
648/663
0.16
0.12
0.11
1.02
1.13
1.58
18
19
15
DMF
Methanol
Water
660/677
651/668
648/665
0.14
0.11
0.10
1.12
1.15
1.23
17
17
17

L. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 210 (2010) 168–172
171
Fig. 3. Absorption and fluorescence emission spectra of dye 5a in water.
Fig. 4. Photofading behavior of the Cy5 dyes in water.
2.5. General protein labeling procedures
to find the relationship between structure and photostability of
cyanine dyes, the photostabilities of the dyes with different sub-
stituents on N-position of 3H-indolenine were studied in water.
From Fig. 4, it could be shown that after 7 h of irradiation, dye 6c
showed 74% photofading, and dyes 5b, 5c and 6a faded 64–68%.
However, dyes 5a and 6b showed about only 39–44% decrease in
maximal absorbance. The photostabilities of the dyes can be placed
in the order: 6b > 5a > 6a > 5b > 5c > 6c. The photostability of asym-
metric dye 5c was relatively lower because it contained only one
sulfo-group on its 3H-indolium ring, and sulfo-groups are strong
electron-withdrawing groups and can reduce the electron density
of the dye’s pentamethine chain.
An aqueous solution of BSA (14 ␮mol L−1, 60 ␮L) was mixed
with 2.5% (w/v) SDS aqueous solution (60 ␮L) and borate buffer
(50 mmol L−1, 30 ␮L, pH 9.0). After the mixture was stirred at 50 ◦C
for 30 min, Cy5-NHS in dry DMF (8.5 mmol L−1, 10 ␮L) was added,
and then stirred at 50 ◦C for 30 min in the dark. The sample was fur-
ther diluted with borate buffer (pH 9.0) and then cooled to 25 ◦C.
Subsequently, 10 ␮L aliquot of the derived protein mixture was
injected, and further analyzed by HPLC.
2.6. HPLC
The rate constants of photofading (k) for the Cy5 dyes could be
calculated according to the formula (2) [14]:
Column 250 × 4.6 mm (C8, 5 ␮m and 30 nm); eluent A, 100%
water with 0.1% TFA; eluent B, 95% acetonitrile with 0.1% TFA; gra-
dient conditions: 0–27 min, 25–80% B, 27–35 min: 80–80% B; flow
rate: 1.0 mL min−1; the column temperature: ambient tempera-
ture. Detector: fluorescence detection: ꢀex 648 nm, ꢀem 664 nm;
UV detection: ꢀabs 214 nm.
ꢀ
ꢁ
A0
At
ln
= kt
(2)
where A0 is the absorbance in maximal wavelength before the irra-
diation and At is the absorbance in maximal wavelength after the
irradiation. The rate constants of the photoreaction based on the
experimental data are shown in Table 2. The data showed that
except for dye 5c, the dyes with benzyl ring in the N-substituents
(5a and 6b) were more stable than those with N-alkyl substitution
(5b, 6a and 6c). In addition, for the dyes with N-alkyl substituents,
N-sulfonatobutyl groups were better than N-carboxypentyl groups
for stability. The photostability of commercial dye 6c with two N-
carboxypentyl groups was worst among these dyes. For example,
the photofading constant k of N-carboxybenzyl dye (5a) was less
than 60% of the k of N-sulfonatobutyl dye (6a) and less than half of
the k of N-carboxypentanyl dye (6c).
3. Results and discussion
3.1. Spectral properties
The absorbance and fluorescent properties of water-soluble
Cy5 dyes in different solvents are summarized in Table 1. Typical
absorption and emission spectra of Cy5 dye were shown in Fig. 3.
The maximum absorption and emission wavelengths of dyes in dif-
ferent solvents were in the range from 647 to 680 nm. The dyes
exhibited a strong absorption in red visible region with high molar
extinction coefficients (ε) in solutions (105 mol−1 cm−1 L).
As shown in Table 1, all these dyes exhibited negative sol-
vatochromism, with a blue shift of the absorption and emission
maximum with increasing solvent polarity (e.g. 5a: ꢀabs in DMF is
660 nm and 647 nm in water; ꢀem in DMF is 678 nm and 664 nm
in water). The effect of the solvent polarity on ꢀmax could be illus-
trated by interactions between the dye molecules and the solvents,
as the interactions make the ground state of dye more stable by
forming hydrogen bonds [22].
Therefore, the effect of different N-substituents on the photo-
stability of the dyes can be placed in the order: carboxylbenzyl
group > sulfonatobutyl group > carboxylpentanyl group. The main
reason for this may be steric hindrance of benzyl and sulfo-groups
preventing the attack of singlet oxygen, thus improving the photo-
stability of Cy5 dyes.
3.3. Fluorescent labeling on BSA
Although the fluorescence quantum yields (˚) of these water-
soluble dyes were lower in polar solvents, those were also moderate
(0.09–0.11) in water, obviously higher than those of hydrophobic
dyes (ca. 0.01 in water). The introduction of multi-sulfo-groups
could significantly enhance water-solubility, reduce aggregation
and fluorescence self-quenching in aqueous media.
Considering its high yield, good photostability and water-
solubility, novel dye 5a was converted to its NHS active ester, and
then used to label the standard protein BSA (Fig. 5).
Table 2
Rate constants of photofading.
3.2. Photostability
Molecule
5a
5b
5c
6a
6b
6c
Rate constants k
1.41
2.61
2.68
2.41
1.17
3.14
Good photostability is an important requirement for the appli-
cation of fluorescent dye in bio-labeling and bio-imaging. In order
(×10−3 mol min−1
)

172
L. Wang et al. / Journal of Photochemistry and Photobiology A: Chemistry 210 (2010) 168–172
Acknowledgements
This work was supported by NSF of China (20725621
and 20706008), National Basic Research Program of China
(2009CB724706), Ministry of Education of China (Program for
Changjiang Scholars and Innovative Research Team in University,
IRT0711, and Cultivation Fund of the Key Scientific and Technical
Innovation Project, 707016).
Fig. 5. Protein derivatization with Cy5-NHS.
References
[1] N.G. Giepmans Ben, R.A. Stephen, M.H. Ellisman, Y.T. Roger, The fluores-
cent toolbox for assessing protein location and function, Science 312 (2006)
217–223.
[2] M.J. Eggertson, D.B. Craig, Laser-induced fluorescence detector for liquid chro-
matography: applications to protein analysis, Biomedical Chromatography 14
(2000) 156–159.
[3] P.G. Righetti, A. Castagna, F. Antonucci, C. Piubelli, D. Cecconi, N. Campostrini,
P. Antonioli, H. Astner, M. Hamdan, Critical survey of quantitative proteomics
in two-dimensional electrophoretic approaches, Journal of Chromatography A
1051 (2004) 3–17.
[4] M. Unlu, M.E. Morgan, J.S. Minden, Difference gel electrophoresis: a single gel
method for detecting changes in protein extracts, Electrophoresis 18 (1997)
2071–2077.
[5] T. Liu, W.J. Qian, M.A. Gritsenko, D.G. Camp II, M.E. Monroe, R.J. Moore,
R.D. Smith, Human plasma N-glycoproteome analysis by immunoaffinity sub-
traction, hydrazide chemistry, and mass spectrometry, Journal of Proteome
Research 4 (2005) 2070–2080.
[6] H.J. Gruber, C.D. Hahn, G. Kada, C.K. Riener, G.S. Harms, W. Ahrer, T.G. Dax,
H.G. Knaus, Anomalous fluorescence enhancement of Cy3 and Cy3.5 ver-
sus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to
IgG and noncovalent binding to avidin, Bioconjugate Chemistry 11 (2000)
696–704.
Fig. 6. Analysis of 5a-NHS labeled BSA by HPLC (a) with fluorescent detector (ꢀex
648 nm, ꢀem 664 nm), (b) with UV detector (ꢀabs 214 nm). Experimental conditions
were mentioned in Experimental Procedures.
[7] K.D. Volkova, V.B. Kovalska, A.L. Tatarets, L.D. Patsenker, D.V. Kryvorotenko, S.M.
Yarmoluk, Spectroscopic study of squaraines as protein-sensitive fluorescent
dyes, Dyes and Pigments 72 (2007) 285–292.
[8] M.F. Kircher, R. Weissleder, L. Josephson, A dual fluorochrome probe for imaging
proteases, Bioconjugate Chemistry 15 (2004) 242–248.
[9] R. Weissleder, V. Ntziachristos, Shedding light onto live molecular targets,
Nature Medicine 9 (2003) 123–128.
[10] G. Patonay, J. Salon, J. Sowell, L. Strekowski, Noncovalent labeling of
biomolecules with red and near-infrared dyes, Molecules 9 (2004) 40–49.
[11] C.H. Tung, Fluorescent peptide probes for in vivo diagnostic imaging, Biopoly-
mers 76 (2004) 391–403.
[12] W. Pham, W.F. Lai, R. Weissleder, C.H. Tung, High efficiency synthesis of a bio-
conjugatable near-infrared fluorochrome, Bioconjugate Chemistry 14 (2003)
1048–1051.
[13] L.Q. Wang, X.J. Peng, R. Zhang, J.N. Cui, G.Q. Xu, F.G. Wang, Syntheses and spec-
tral properties of fluorescent trimethine sulfo-3H-indocyanine dyes, Dyes and
Pigments 54 (2002) 107–111.
[14] X.Y. Chen, X.J. Peng, A.J. Cui, B.S. Wang, L. Wang, R. Zhang, Journal of Photo-
chemistry and Photobiology A: Chemistry 181 (2006) 79–85.
[15] B. Schuler, L.K. Pannell, Specific labeling of polypeptides at amino-terminal cys-
teine residues using Cy5-benzyl thioester, Bioconjugate Chemistry 13 (2002)
1039–1043.
[16] R.B. Mujumdar, L.A. Ernst, S.R. Mujumdar, C.J. Lewis, A.S. Waggoner, Cya-
nine dye labeling reagents: sulfoindocyanine succinimidyl esters, Bioconjugate
Chemistry 4 (1993) 105–111.
It should be noted that the limit of detection (LOD) here refers
to the minimum amount of protein that could be derivatized
with a detection signal-to-noise ratio of 3 [23]. In this paper, the
LODs were evaluated by HPLC with fluorescent detector and UV
detector. In our previous work [24], the LOD of commercial flu-
orescein isothiocyanate (FITC) for BSA was 0.4 × 10−6 mol L−1. In
comparison with isothiocyanate-containing dyes (e.g. FITC), NHS-
carboxyl-containing Cy3 dyes had relatively high reaction activity
and selectivity in protein labeling under mild conditions.
As shown in Fig. 6, the fluorescent signal of 5a-BSA could
be clearly detected when the UV signal of BSA was quite weak.
Therefore, compared to results using UV detection, more proteins
with low concentrations could be labeled by 5a-NHS, result-
ing in improved detection sensitivity for protein analysis. For
BSA, the LOD of dye 5a as measured by fluorescent detec-
tion was 1.2 × 10−8 mol L−1, about 100 times lower than that
by UV detection (1.0 × 10−6 mol L−1). These results further sug-
gested that a high sensitivity detection of proteins could be easily
achieved after 5a-NHS derivatization and detection by HPLC with
a fluorescence detector, especially for those at a low concentra-
tion.
[17] H.E. Gottlieb, V. Kotlyar, A. Nudelman, NMR chemical shifts of common lab-
oratory solvents as trace impurities, Journal of Organic Chemistry 62 (1997)
7512–7515.
[18] H. Illy, L. Funderburk, Fisher indolesynthesie, direction of cyclization of iso-
propylmethyl ketone phenylhydrazone, Journal of Organic Chemistry 33 (1968)
4283–4285.
[19] T. Karstens, K. Kobs, Rhodamine B and rhodamine 101 as reference substances
for fluorescence quantum yield measurements, The Journal of Physical Chem-
istry 84 (1980) 1871–1872.
4. Conclusion
[20] R.A. Velapoldi, H.H. Tonnesen, Corrected emission spectra and quantum yields
for a series of fluorescent compounds in the visible spectral region, Journal of
Fluorescence 14 (2004) 465–472.
[21] S. Fery Forgues, D.J. Lavabre, Are fluorescence quantum yields so tricky to mea-
sure? A demonstration using familiar stationery products, Journal of Chemical
Education 76 (1999) 1260–1264.
[22] A. Mishra, R.K. Behera, P.K. Behera, B.K. Mishra, G.B. Behera, Cyanines during
the 1990s: a review, Chemical Reviews 100 (2000) 1973–2011.
[23] D.M. Pinto, E.A. Arriaga, D. Craig, J. Angelova, N. Sharma, H. Ahmadzadeh,
N.J. Dovichi, C.A. Boulet, Picomolar assay of native proteins by capillary elec-
trophoresis precolumn labeling, submicellar separation, and laser-induced
fluorescence detection, Analytical Chemistry 69 (1997) 3015–3021.
[24] X.Q. Qiao, L. Wang, J.F. Ma, Q.L. Deng, Liang Zhen, L.H. Zhang, X.J. Peng, Y.K.
Zhang, High sensitivity analysis of water-soluble, cyanine dye labeled pro-
teins by high-performance liquid chromatography with fluorescence detection,
Analytica Chimica Acta 640 (2009) 114–120.
In this paper, novel water-soluble asymmetric Cy5 dyes were
synthesized and their photostability in water were investigated to
ensure the good reproducibility of protein fluorescent labeling. It
is demonstrated that the benzyl group and sulfo-group on the N-
position of 3H-indolium pentamethine cyanine dyes improved the
photostability in aqueous solutions. Furthermore, the LOD of dye
5a-NHS for BSA was 1.2 × 10−8 mol L−1 as measured by HPLC using
a fluorescent detector. Compared with UV detection, the sensitivity
of detection was increased about 100 times by fluorescent labeling
of dye 5a. All results demonstrated that dye 5a was a good fluo-
rescent labeling reagent to achieve high sensitive detection for low
concentration proteins.