Asymmetric trimethine 3H-indocyanine dyes: Efficient synthesis and protein labeling

Article abstract of DOI:10.1039/c0ob00227e

We present an efficient method to synthesize three new asymmetric trimethine cyanine dyes containing only one carboxylic acid group for bioconjugation. Two of them have better protein labeling performance than other conventional cyanine dyes due to their

Full text of DOI:10.1039/c0ob00227e

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/obc | Organic & Biomolecular Chemistry
Asymmetric trimethine 3H-indocyanine dyes: efficient synthesis and protein
labeling†
Fengling Song,a Li Wang,a Xiaoqiang Qiao,b Bingshuai Wang,a Shiguo Sun,a Jiangli Fan,a Lihua Zhangb and
Xiaojun Peng*a
Received 8th June 2010, Accepted 9th July 2010
DOI: 10.1039/c0ob00227e
We present an efficient method to synthesize three new
asymmetric trimethine cyanine dyes containing only one
carboxylic acid group for bioconjugation. Two of them have
better protein labeling performance than other conventional
cyanine dyes due to their particular structure design.
Here we developed a stepwise synthesis route for three new
asymmetric trimethine 3H-indocyanine (Cy3) dyes 1d–f with
multi-sulfo groups and one carboxylic acid group with much
better yields. As so far, no detailed investigation has been reported
about protein labeling efficiency of such asymmetric cyanine dyes.
This work highlights excellent protein labeling performance of two
asymmetric dyes 1e–f because of their particular structure.
These asymmetric dyes could be obtained by mixing two
different quaternary salts 2a–e and a coupling reagent N, N¢-
diphenylformamidine. But the purification is difficult and the yield
is horrible. In this study, dyes 1c–d were synthesized by a stepwise
route (Scheme 2). N, N¢-diphenylformamidine (1.2 eq.) reacted
with one quaternary salt 2a (1.0 eq.) to afford a hemicyanine inter-
mediate 3. Then another quaternary salt 2 was added to react with
3 in acetic anhydride to produce an asymmetric dye . The adding
sequence of different quaternary salts 2 has a great influence on
the total yield of dyes. We found that in the first step, adding
quaternary salt 2a–c instead of 2d–e can improve apparently the
yield of the desired asymmetric dyes, which is because the electron-
withdrawing group on the 3H-indolium ring of 2a–c can reduce
the reactivity of quaternary salt 2 with the formed hemicyanine
intermediate 3 to form undesired symmetric dyes.
As shown in Scheme 2, new asymmetric dyes 1e (35%) and 1f
(42%) were obtained by using the quaternary salt 2a (with low
reactivity) as the first quaternary salt and 2d or 2e (with relatively
high reactivity) as the second quaternary salt. Their yields are
greatly improved as compared with reported asymmetric Cy3 dyes
1c (5%) in the literature,8 and better than the yields of 1d (21%)
which was obtained from the reaction with quaternary salt 2b or
2c (with low reactivity) as the second quaternary salt.
After purified on C18-RP column using methanol–water mix-
ture as eluent, these asymmetric dyes were converted to their NHS
active ester for protein labeling (ESI Scheme S1†). The NHS
esters were used without further purification due to easy to be
contaminated and deactivated via hydrolysis.
Intensive research efforts have been devoted to the design and
synthesis of novel water-soluble fluorescent organic dyes for life
science applications especially protein research during the past
decade.1,2 Among them, 3H-indocyanine dyes with multi-sulfo
groups have received considerable attention and were widely
used as fluorescent labeling compounds for proteins. This is
because they have large molar extinction coefficients, moderate-
to-high fluorescence quantum yields, and a broad wavelength
tunability.3 Usually these cyanine dyes have two or four sulfo
groups symmetrically on the two ends of their molecular structure.
And two active functional groups (like carboxylic acid) are often
introduced for bioconjugation, as seen in a commercially available
dye 1a (Scheme 1). Its more stable alterative, 1b, was reported by
our group previously.4,5
As well known, single labeling site is more desirable for protein
labeling to obtain specific-position target and to avoid protein
crosslinking.1,6,7 However, in many cases it was found difficult to
synthesize and purify asymmetric cyanine dyes with multi-sulfo
groups and single labeling site, such as one carboxylic acid group.
For example, one asymmetric cyanine dye 1c (Scheme 2) with this
kind of structure was prepared in a very low yield (5%).8
aState Key Laboratory of Fine Chemicals, Dalian University of Tech-
nology, 158 Zhongshan Road, Dalian 116012, P.R. China. E-mail:
pengxj@dlut.edu.cn; Fax: +86-411-39893800; Tel: +86-411-39893899
bCAS 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
† Electronic supplementary information (ESI) available: Synthesis and
characterization of all the intermediate and Cy3 dyes, Spectra of free dyes,
Methods of determination of D/P, details of experiments of SDS-PAGE
and HPLC. See DOI: 10.1039/c0ob00227e
Bovine serum albumin (BSA) was chosen as the protein for
bioconjugation. After labeling reaction, the obtained dye-BSA
Scheme 1 Two symmetric trimethine cyanine dyes.
The Royal Society of Chemistry 2010 Org. Biomol. Chem., 2010, 8, 4249–4251 | 4249
This journal is
©

View Article Online
Table 1 Spectral properties of dye-BSA conjugates in water
Table 2 D/Ps and LODs of dyes for BSA by SDS-PAGE
Entry
l
abs /lem/nm
Stokes shifts/nm
Ua
Udye-BSA/Udye
Entry
1a
1.5
100
1b
1.4
100
1c
0.9
500
1d
0.8
500
1e
2.5
50
1f
2.2
50
dye-BSA
D/P
LOD/nga
1a-BSA 555/570
1b-BSA 556/572
1c-BSA 553/569
1d-BSA 551/570
1e-BSA 554/572
15
16
16
19
18
16
0.32
0.35
0.34
0.22
0.39
0.36
1.6
2.5
1.9
2.2
3.5
2.8
a BSA denaturation: pH = 8.7, SDS 2.5%, boiling for 10min; Optimal Cy3
labeling conditions were obtained based on experiments of dye 1b (see ESI
Figure S8 and Figure S9†): pH = 8.7, at 30 ◦C for 30 min.
1f-BSA
555/571
a The fluorescence quantum yields were determined in reference to
Rhodamine B in ethanol (U = 0.97)9. lem: 554 nm, error ca. 10%.
conjugates were separated by HPLC to remove the excess dyes,
and their spectral properties were tested (see Table 1).
All dye-BSA conjugates were found to have a slight red-shift (4–
7 nm) in their absorption and emission spectra when compared
with the free dyes (Table 1 and ESI Table S1†).10 Moreover
the fluorescence was significantly enhanced. The fluorescence
quantum yields of dye-BSA conjugates reach about 0.39 which are
1.6–3.5 folds of the free dyes’ in water. Fig. 1 shows the emission
spectra of 1f-BSA and 1b-BSA conjugates and their free form.
The fluorescence-enhancing effect might be resulted from the rigid
microenvironment when the dyes are conjugated to proteins.8 The
fluorescence quantum yields of dyes-BSA conjugates are similar
and around 0.35, expect that of 1d-BSA.
Dye/protein (D/P) ratios are usually used to evaluate labeling
efficiency of dyes. After these Cy3 dyes were labeled on BSA under
the same conditions, their D/P ratios were calculated based on the
absorbances of free dyes and dye-BSA bioconjugates. The results
show that the D/P of dye 1e (2.5) and 1f (2.2) were about 2–3 folds
of that of dye 1a–d (Table 2).
To further confirm the better protein labeling performance of
dye 1e–f, sodium dodecyl sulfate polyacrylamide gel electrophore-
sis (SDS-PAGE) was carried out for all these dye-BSA conjugates.
The LOD (limit of detection) value is used for quantification of
the minimum amount of protein for the specified method.11 As
expected, new dyes 1e–f do possess the best LOD, 1a–b the second,
but 1c–d are poor (Table 2). Fig. 2 shows the comparison of 1f-BSA
and 1b-BSA on gel photographs.
Fig. 1 Fluorescence enhancement after labeling BSA with dye 1f (red)
and 1b (black) in water (when the absorption maxima (Almax) of these
samples were about 0.095).
Because no significant difference can be found in the fluores-
cence quantum yield of all these dye-BSA conjugates (see Table 1),
we speculate that the molecular structure effect claims the better
protein labeling performance of dyes 1e–f than that of 1c and
1d. Both 1e-NHS and 1f-NHS have a hydrophobic end at the
NHS ester reaction center; whereas 1c-NHS and 1d-NHS have
a hydrophilic sulfo-group at the end with the NHS ester (Fig. 3).
The hydrophilic sulfo-group might have a repulsing effect from the
protein chain (with carboxylic groups), which retards the labeling
reaction between dyes and protein.
Scheme 2 Synthesis route of the asymmetric trimethine cyanine dyes.
4250 | Org. Biomol. Chem., 2010, 8, 4249–4251 This journal is The Royal Society of Chemistry 2010
©

View Article Online
with the decrease in BSA concentration from 60 nM to 20 nM,
the fluorescent signal of 1f-BSA decreased about 3 times. In
other words, the fluorescent signal was proportional to BSA
concentration in this range. With fluorescence detector and under
the optimized labeling conditions, the LOD of 1f could decrease
to 4.8 ¥ 10-10 M (or 0.6 ng), considering that the signal-to-noise
ratio of should be above 3 to check the LOD of HPLC.12 For
comparison, by the same method the LOD of 1b was detected and
found to be 8.7 ¥ 10-10 M (or 1.1 ng) (see ESI Figure S9†), about
2-fold higher than that of 1f.
Fig. 2 Gel photographs showed the LODs of dye 1f and 1a.
In conclusion, a series of asymmetric Cy3 dyes were synthesized.
The stepwise route affords an asymmetric dye with yield 42%.
SDS-PAGE and HPLC with fluorescence detector are applied to
evaluate the labeling performance of the Cy3 dyes on BSA. Under
the optimal conditions of SDS-PAGE, the LOD of 1f-BSA is as
low as 50 ng, which can be directly observed by naked eyes under
UV light. And in HPLC experiments, the LOD could decrease to
4.8 ¥ 10-10 M (or 0.6 ng). The excellent labeling performance of
1f is attributed to its particular structure with the hydrophobic
groups on one end and hydrophilic sulfo-groups on the other
end. We believe dyes 1e–f will be found beneficial in the bioassay
applications.
Acknowledgements
This work was supported financially by the NSF of China
(20706008, 20705621, 20876024 and 20923006), National Basic
Research Program of China (2009CB724700), the Fundamental
Research Funds for the Central Universities, Ministry of Edu-
cation of China (Program for Changjiang Scholars and Inno-
vative Research Team in University, IRT0711; and Cultivation
Fund of the Key Scientific and Technical Innovation Project,
707016), and Innovative Research Team of Liaoning Provence
(2006T026).
Fig. 3 Repulsion effect between the dye and the protein chain.
Both dye 1e and 1f exhibit excellent protein labeling perfor-
mance. But dye 1f should have better photostability since it
contains one N-p-carboxybenzyl (N-p-CH2C6H4COOH) on the
indole ring.4 So we choose to quantify the LOD of dye 1f in a
more accurate way by HPLC with a florescence detector excited
at labs-max 554 nm. As shown in Fig. 4, 1f-NHS could label BSA
with a concentration as low as 20 nM by HPLC. In addition,
Notes and references
1 K. Peneva, G. Mihov, F. Nolde, S. Rocha, J. Hotta, K. Braeckmans, J.
Hofkens, H. Uji-I, A. Herrmann and K. Mullen, Angew. Chem., Int.
Ed., 2008, 47, 3372–3375.
2 I. Miller, J. Crawford and E. Gianazza, Proteomics, 2006, 6, 5385–
5408.
3 G. Patonay, J. Salon, J. Sowell and L. Strekowski, Molecules, 2004, 9,
40–49.
4 L. Q. Wang, X. J. Peng, R. Zhang, J. N. Cui, G. Q. Xu and F. G. Wang,
Dyes Pigm., 2002, 54, 107–111.
5 X. Q. Qiao, L. Wang, J. F. Ma, Q. L. Deng, Z. Liang, L. H. Zhang,
X. J. Peng and Y. K. Zhang, Anal. Chim. Acta, 2009, 640, 114–
120.
6 O. Dilek and S. L. Bane, Bioorg. Med. Chem. Lett., 2009, 19, 6911–
6913.
7 C. Bouteiller, G. Clave, A. Bernardin, B. Chipon, M. Massonneau,
P. Y. Renard and A. Romieu, Bioconjugate Chem., 2007, 18, 1303–
1317.
8 R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, C. J. Lewis and A. S.
Waggoner, Bioconjugate Chem., 1993, 4, 105–111.
9 D. G. Taylor and J. N. Demas, Anal. Chem., 1979, 51, 712–717.
10 A. L. Tatarets, I. A. Fedyunyayeva, T. S. Dyubko, Y. A. Povrozin, A. O.
Doroshenko, E. A. Temetschnig and L. D. Patsenker, Anal. Chim. Acta,
2006, 570, 214–223.
11 E. A. Arriaga, Y. N. Zhang and N. J. Dovichi, Anal. Chim. Acta, 1995,
299, 319–326.
12 D. M. Pinto, E. A. Arriaga, D. Craig, J. Angelova, N. Sharma, H.
Ahmadzadeh, N. J. Dovichi and C. A. Boulet, Anal. Chem., 1997, 69,
3015–3021.
Fig. 4 Analysis of 1f-NHS labeled BSA with low concentration (a) 60 nM
BSA and (b) 20 nM BSA by HPLC with fluorescence detector (excited at
554 nm and detected at 572 nm). Experimental conditions were mentioned
in ESI.†
This journal is
The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 4249–4251 | 4251
©