An efficient approach to the synthesis of water-soluble cyanine dyes using poly(ethylene glycol) as a soluble support

Article abstract of DOI:10.1016/j.tetlet.2007.06.060

An efficient synthesis approach to unsymmetrical water-soluble cyanine dyes has been established. Loading and activation of sulfoindolenium to poly(ethylene glycol) (PEG) have been achieved via a simple strategy. Cyanine dyes are released by the attack of heterocyclic carbon nucleophile and the cleavage of PEG-bound hemicyanine. The efficient approach delivers cyanine dyes in high purity without the nontrivial chromatographic separation.

Full text of DOI:10.1016/j.tetlet.2007.06.060

Tetrahedron Letters 48 (2007) 5825–5829
An efficient approach to the synthesis of water-soluble cyanine
dyes using poly(ethylene glycol) as a soluble support
Lin-Ling Jiang, Li-Fang Dou and Bao-Lin Li*
School of Chemistry and Materials Science, The Key Laboratory of Medicinal Plant Resource and Natural
Pharmaceutical Chemistry of Ministry of Education, Shaanxi Normal University, Xi’an 710062, PR China
Received 21 March 2007; revised 12 June 2007; accepted 15 June 2007
Available online 17 June 2007
Abstract—An efficient synthesis approach to unsymmetrical water-soluble cyanine dyes has been established. Loading and activa-
tion of sulfoindolenium to poly(ethylene glycol) (PEG) have been achieved via a simple strategy. Cyanine dyes are released by the
attack of heterocyclic carbon nucleophile and the cleavage of PEG-bound hemicyanine. The efficient approach delivers cyanine dyes
in high purity without the nontrivial chromatographic separation.
Ó 2007 Elsevier Ltd. All rights reserved.
Unsymmetrical water-soluble cyanine dyes (Cy3 and
Cy5), originally described by Waggoner and co-work-
ers,1,2 have been proven valuable in numerous applica-
tions involving conjugation with proteins. However,
these dyes were prepared by traditional solution-phase
chemistry and must be purified by chromatography,
the tedious process that resulted in a high cost of using
these dyes. Traditional solution synthesis of unsymmet-
rical cyanine dyes is illustrated in Scheme 1. The reac-
tion of nucleophilic heterocycle such as 2 with a
polyene chain precursor such as amidine 1 gives a hemi-
cyanine intermediate 3. The hemicyanine is susceptible
to react with a second molecule of heterocycle such as
4 to form unsymmetrical dye 5, meanwhile it can also
react with 2 to form the symmetrical dye, which often
requires nontrivial chromatographic separation.3–5
Therefore purification of the unsymmetrical dye is a
particular problem in traditional solution synthesis.
Solid-phase synthesis is a powerful approach to the syn-
thesis of important heterocyclic compounds6–9 because
of the ease of workup and purification.10–13 From earlier
research, it is known that solid-supported substrate
attacked by heterocyclic nucleophile is an effective way
for the synthesis of dyes. Balasubramanian’s group
improved the solid-phase synthesis of cyanine dyes
employing sulfonyl chloride resin and polystyrene as
loading materials.14,15 But they also pointed out that
the sulfonated heterocycles did not react well in the
loading reaction, which led to only one sulfo-group
attached to the cyanine dyes. Therefore, cyanine dyes
synthesized by solid-phase method suffered poor
solubility for biomolecular labelling.14–18
N
I
2
NHPh
NPh
NHPh
N
I
1
3
N
Br
(CH ) COOH
2 5
4
N
N
I
(CH ) COOH
2 5
In recent years, soluble polymer-supported synthesis has
been studied intensely.19 In fact, the advantages of both
homogeneous reaction (high reactivity) and solid-phase
synthesis methods (easy isolation and purification of
products) coexist in this technique. Our own interest
grew out of a desire to develop a soluble polymer-
supported synthesis strategy to synthesize unsymmetric
cyanine dyes with high solubility. The structure of
5
Scheme 1. Solution synthesis of a cyanine dye.
Keywords: Polymer-supported synthesis; Water-soluble cyanine dyes;
PEG; Cleavage.
*
Corresponding author. Tel.: +86 29 85303858; e-mail: baolinli@
snnu.edu.cn
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.06.060

5826
L.-L. Jiang et al. / Tetrahedron Letters 48 (2007) 5825–5829
O
O3S
SO3H
CH3CCH(CH3)2
O3S
N
R
N
n
HO3S
NHNH2
N
R'
H
11
10
Figure 1. Structure of sulfoindocyanine dye. R = CH2CH3, R0 =
(CH2)5COOH, n = 1, 2.
KOH
KO3S
12
EtI
N
sulfoindocyanine dyes synthesized in our work is shown
in Figure 1. They consist of two nitrogen-containing het-
erocycles linked to each other by an odd-number poly-
ene chain. Structural diversity is possible by varying
the length of polyene chain, the nitrogen substituents
or the heterocyꢀcle themselves. The sulfoindocyanine
dyes have ꢀSO3 and –SO3H groups at 5 and 50 posi-
tions, respectively, so as to increase their water solubil-
ity. The R0 substituent is carboxyl alkyl group, which
can be activated to form succinimidyl ester for easy con-
jugation with biomolecule.
BrCH2(CH2)4COOH
O3S
O3S
N
N
(CH2)5COOH
14
13
Scheme 3. Synthesis of sulfonated heterocycles.
nium-5-sulfonate 13 and 1-(e-carboxypentynyl)-2,3,3-
trimethylindolenium-5-sulfonate 14, were prepared for
the synthesis of cyanine dyes. Indolenium-5-sulfonate
11 was prepared from p-hydrazinobenzenesulfonic acid
10 and 3-methyl-2-butanone by conventional Fisher
indole synthesis (Scheme 3).23 The reaction of 11 and
KOH gave 2,3,3-trimethylindolenium-5-sulfonic potas-
sium salt 12. And then 13 was obtained by refluxing
12 in large excess of ethyl iodide. The indolium salt 14
was prepared by the reaction of 12 with 6-bromohexa-
noic acid in dichlorobenzene at 110 °C for 48 h in 81%
yield.
In order to realize the soluble polymer-supported syn-
thesis, the preparation of aniline scaffold 9 was required
(see Scheme 2). We selected poly(ethylene glycol) (PEG,
MW2000) as a soluble support. The choice of using
PEG was mainly motivated by its good solubility in
dichloromethane (DCM) and its poor solubility in
diethyl ether, which made the reaction homogeneous
and the separation heterogeneous. Furthermore, the
low cost and recyclability of PEG and its derivatives
made them attractive candidates as the support for the
synthesis of cyanine dyes.
Our strategy for the polymer-supported synthesis of
unsymmetrical water-soluble cyanine dyes is described
in Scheme 4. The reaction of PEG-bound aniline 9 with
1,1,3,3-tetramethoxypropane or triethyl orthoformate in
glacial acetic acid gave product PEG-bound-4-(3-meth-
oxyallylideneamino)benzonic acid ester 1524 or PEG-
bound formamidine 18,25 respectively. The structural
difference between 15 and 18 was confirmed by 1H
The synthesis of PEG-bound aniline 9 began with 4-
aminobenzoic acid 6 and was accomplished in three
steps. The amino group of 6 was protected with di-
tert-butyl dicarbonate to afford 7,20 which was coupled
with PEG in the presence of DCC leading to the forma-
tion of PEG-bound BOC–aniline 8.21 Intermediate 8
was characterized by FT-IR and 1H NMR. The loading
level of the BOC–aniline group was 0.19–0.32 mmol/g
determined by 1H NMR integral method22 using ethanol
as an internal standard. It is stable to storage at ambient
conditions. Subsequent cleavage of the BOC group
under classical condition (TFA/CH2Cl2) afforded 9,
quantitatively. The removal of BOC group was
confirmed by the complete disappearance of the methyl
1
NMR spectra. In the H NMR spectrum of 15, the sig-
nals at d 8.06 (m, 3H), 7.85 (d, 1H), 7.11 (d, 2H), 6.65
(dd, 1H) indicated that a phenyl and three methine
groups exist in molecule 15. However, three main peaks
at d 8.53 (1H), 8.12 (4H) and 7.26 (4H) in the 1H NMR
spectrum of 18 showed that 18 has a symmetric structure
in which hydrogen atom connected with nitrogen atom
can move to another nitrogen atom by tautomerization.
Subsequent reaction of 15 with heterocyclic carbon
nucleophile 13 resulted in the formation of PEG-bound
tetramethine hemicyanine dye 16.26 However, it was not
successful in preparing similar PEG-bound dimethine
hemicyanine 19 using the same conditions. A variety
of solvents, temperatures, and reaction time for 19 were
investigated. It was found that the reaction in ethanol
with reflux gave by far the best result.27 In fact, 16 or
19 could also react with 13 to give the byproducts (sym-
metrical dyes). However, the byproducts could be mini-
mized by using equivalent 13 and appropriate reaction
time. And the small quantity of symmetrical dyes could
be precipitated with ethyl acetate, while the PEG-bound
hemicyanine could be dissolved completely in it at
25 °C. The removal of the byproducts was confirmed
by thin-layer chromatography (TLC).28 Although a
small quantity of unreacted 15 or 18 existed in the prod-
uct, which did not affect the following reaction and the
1
signal in the H NMR spectrum. The loading yield of
1
aniline group on PEG was also estimated by H NMR
integration method.
In addition to the scaffold of aniline 9, another two
important intermediates, 1-ethyl-2,3,3-trimethylindole-
di-tert-butyl dicarbonate,
THF,reflux, 24 h
HOOC
NHBOC
HOOC
NH2
6
7
PEG, DCC, DMAP,
CH2Cl2, rt, 12 h
O
O C
O
O C
NH2
NHBOC
TFA, CH2Cl2,
reflux, 5 h
8
9
Scheme 2. Preparation of supported aniline on PEG.

L.-L. Jiang et al. / Tetrahedron Letters 48 (2007) 5825–5829
5827
O
O C
NH2
9
triethyl
1,1,3,3-tetramethoxy-
propane, glacial acetic
acid, 55 oC, 5.5 h
orthoformate,
glacial acetic acid,
55 oC, 5.5 h
O
O C
O
C O
O
C
N CH NH
N
O
OMe
15
18
13, glacial acetic
13, triethyl orthoformate,
EtOH, reflux, 2.5 h
acid, 80 oC, 1 h
O
SO3
SO3
O
O C
N
H
O C
19
N
N
H
N
16
14, acetic anhydride,
14, acetic anhydride,
pyridine, 110 oC, 15 min
pyridine, 110 oC, 15 min
HO3S
SO3
HO3S
SO3
N
(CH2)5COOH
N
N
N
HOOC(H2C)5
20
17
Scheme 4. Polymer-supported synthesis of trimethine and pentamethine cyanine dyes.
purification of final products. The important step was to
form unsymmetrical cyanine dye 17 or 20. In this step,
immobilized activated PEG-bound tetramethine hemi-
cyanine 16 or PEG-bound dimethine hemicyanine 19
reacted with a substoichiometric quantity of the second
heterocycle nuleophile 14 in a mixed solution of acetic
anhydride and pyridine, so that all 14 is consumed and
the unreacted hemicyanine would remain on the poly-
mer. At the same time, the possibility of forming the
symmetrical dyes would be eliminated. Finally, 17 or
20 was ably released by the attack of heterocycle 14
and the cleavage of immobilized hemicyanine 16 or 19.
When the reaction finished, the reaction mixture was
cooled and the blue or red gummy product was precip-
itated with ethyl acetate. Because a small number of
PEG-bound materials were precipitated at the same
time, the gummy product was washed with DCM, which
dissolved all PEG-bound materials. After being washed
two or three times with DCM, pure product 1729 and
2030 were obtained in 23.7% and 21.6% yields for the fi-
nal reaction, respectively, which were extremely higher
than that of similar compounds synthesized by tradi-
tional solution method.1,3 And furthermore the purity
of these products were greater than 98% determined
by HPLC.
affords products in high purity without complicated
chromatographic separation, and it is suitable to synthe-
size unsymmetrical water-soluble cyanine dyes bearing
sulfo-groups. Further utilization of this procedure in
the synthesis of other types of cyanine dyes, including
heptamethine cyanine dyes and other hydrophilic het-
erocyclic compounds, is in progress in our laboratory.
Acknowledgments
The authors are thankful to financial support from
Science and Technology Key Project of Ministry of
Education, PR China (No. 105153), Natural Science
Foundation of Shaanxi Province, PR China (No.
2004B06).
Supplementary data
Full experimental details for 7–14, details for resins
loading and spectroscopic data for all compounds. Sup-
plementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.tetlet.2007.06.060.
A simple isolation and purification procedure for the
products was achieved, thanks to the loading of PEG,
thus eliminating time-consuming chromatographic sepa-
ration and the use of costly equipment. The products
have high purities analyzed by HPLC and TLC. These
dyes are well suited to protein lableing, microscopy
imaging of living cells, fluorescence detection, etc., and
their succinmidyl ester derivatives for protein labelling
can be readily made using published syntheses.2
References and notes
1. Mujumdar, S. R.; Mujumdar, R. B.; Grant, C. M.;
Waggoner, A. S. Bioconjugate Chem. 1996, 7, 356–362.
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C. J.; Waggoner, A. S. Bioconjugate Chem. 1993, 4, 105–
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Talanta 2005, 67, 947–954.
5. Toutchkine, A.; Nalbant, P.; Hahn, K. M. Bioconjugate
Chem. 2002, 13, 387–391.
In summary, we have developed an efficient approach to
the synthesis of unsymmetric water-soluble cyanine dyes
from inexpensive precursors in a few steps. This method

5828
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dissolved in methanol and precipitated again with diethyl
ether for further purification. At this stage, a little of
symmetrical dye was existed in the product. And then the
brown precipitate was dissolved in ethyl acetate. The
PEG-bound tetramethine hemicyanine could be dissolved
completely in ethyl acetate at 25 °C, while the symmetrical
dye could not be dissolved in it. After the symmetrical dye
was filtered, the ethyl acetate layer was concentrated under
reduced pressure and then 30 ml cold diethyl ether was
added to the solution with vigorous stirring. And then 16
was filtered and collected. Yield 0.812 g (83.9%). IR
(KBr): 3440, 2885, 1704, 1627, 1599, 1467, 1173, 1113,
´
7. Krchnˇak, V.; Holladay, M. W. Chem. Rev. 2002, 102, 61–
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Zechel, C. Angew. Chem., Int. Ed. 1996, 35, 2288–2337.
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nologies; John Wileys & Sons: New York, 2000.
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13. Li, W.; Chen, Y.; Lam, Y. Tetrahedron Lett. 2004, 45,
6545–6547.
14. Mason, S. J.; Balasubramanian, S. Org. Lett. 2002, 4,
4261–4264.
15. Mason, S. J.; Hake, J. L.; Nairne, J.; Cummins, W. J.;
Balasubramanian, S. J. Org. Chem. 2005, 70, 2939–
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16. Jung, M. E.; Kin, W.-J. Bioorg. Med. Chem. 2006, 14, 92–
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1061, 1027 cmꢀ1
.
27. Preparation of PEG-bound dimethine hemicyanine (19): A
mixture of 18 (2.35 g, 1.05 mmol), 13 (0.28 g, 1.05 mmol),
triethyl orthoformate (0.34 ml, 2.08 mmol) and ethanol
(5 ml) was stirred and heated to reflux under nitrogen
atmosphere for 2.5 h. And then 50 ml cold diethyl ether
was added to the mixture with vigorous stirring to produce
a yellow precipitate, which was filtered and collected. The
precipitate was dissolved in methanol and precipitated
again with diethyl ether for further purification. At this
stage, a little of symmetrical dye was existed in the
product.
And then the yellow precipitate was dissolved in ethyl
acetate. The PEG-bound dimethine hemicyanine could be
dissolved completely in ethyl acetate at 25 °C, while the
symmetrical dye could not be dissolved in it. After the
symmetrical dye was filtered, the ethyl acetate layer was
concentrated under reduced pressure and then 30 ml cold
diethyl ether was added to the solution with vigorous
stirring. And then 19 was filtered and collected. Yield
1.83 g (69.3%). IR (KBr): 3435, 2883, 1697, 1633, 1604,
1528, 1175, 1114 cmꢀ1
.
23. Illy, H.; Funderburk, L. J. Org. Chem. 1968, 33, 4283–
4285.
28. Thin layer chromatography (TLC) was performed on
silica gel plates (GF254), which was developed with a
mixture of n-butanol/acetic acid/water (v/v/v: 4/1/2).
29. Cleavage of PEG-bound hemicyanine and formation
of Cy5 (17): A mixture of PEG-bound tetramethine
hemicyanine 16 (0.501 g, 0.18 mmol), 1-(e-carboxypentyn-
yl)-2,3,3-trimethylindolenium-5-sulfonate 14 (0.033 g,
0.09 mmol), acetic anhydride (Ac2O) (2 ml) and pyridine
(1 ml) was stirred and heated at 110 °C under nitrogen
atmosphere for 15 min. The mixture was cooled and the
blue dye was precipitated with ethyl acetate. The gummy
product 17 was washed with DCM until the blue free
powder was obtained. Yield 0.014 g (23.7%). 1H NMR
(300 MHz, D2O): d 7.86–7.70 (m, 6H, 4-H, 40-H, 6-H, 60-
H, b, b0 protons of the bridge), 7.27–7.25 (m, 2H, 7-H, 70-
H), 6.38 (dd, 1H, c proton of the bridge, J = 12.6,
12.5 Hz), 6.11–6.00 (m, 2H, a, a0 proton of the bridge),
4.00–3.95 (m, 4H, a-, a0-CH2), 2.18 (t, 2H, –CH2COOH,
J = 7.0 Hz), 1.72–1.27 (m, 21H, 3CH2 groups, 1CH3 and 2
(CH3)2 groups). 13C NMR (300 MHz, D2O): d 183.1,
173.9, 173.7, 154.3, 144.2, 143.7, 141.9, 141.8, 139.4, 139.3,
126.6, 119.8, 111.1, 110.9, 104.0, 103.8, 49.1, 49.0, 44.1,
39.4, 37.1, 26.8, 26.7, 26.1, 25.6, 11.7. MALDI-TOF-MS:
m/z for C33H40S2O8N2 [M]+ calcd 656.81. Found: 656.86.
[M+Na]+ calcd 679.80. Found: 679.90. HPLC (methanol–
water) tR = 1.98 min, purity >98%.
24. Preparation
of
PEG-bound-4-(3-methoxyallylidene-
amino)benzoic acid ester (15): A mixture of 9 (2.02 g, 0.90
mmol), 1,1,3,3-tetramethoxypropane (3.7 ml, 22.2 mmol)
and glacial acetic acid (9 ml) was stirred and heated at
55 °C for 5.5 h. And then 30 ml cold diethyl ether was
added to the solution with vigorous stirring to produce a
dark yellow precipitate, which was filtered and collected.
The precipitate was dissolved in dichloromethane (DCM)
and precipitated with diethyl ether for further purification.
Yield 1.56 g (73.0%). IR (KBr): 3435, 2885, 1704, 1639,
.
1601, 1465, 1114, 1060 cmꢀ1 1H NMR (300 MHz,
CDCl3): d 8.10–8.02 (m, 3H), 7.85 (d, 1H, J = 8.3 Hz),
7.11 (d, 2H, J = 8.2 Hz), 6.65 (dd, 1H, J = 2.7, 8.3 Hz),
4.46–3.33 (m, PEG).
25. Preparation of PEG-bound formamidine (18): A mixture
of 9 (3.02 g, 1.35 mmol), triethyl orthoformate (10 ml,
60 mmol) and glacial acetic acid (27 ml) was stirred and
heated at 55 °C for 5.5 h. And then 50 ml cold diethyl
ether was added to the solution with vigorous stirring to
produce a yellow precipitate, which was filtered and
collected. The precipitate was dissolved in DCM and
precipitated with diethyl ether for further purification.
Yield 2.43 g (80.1%). IR (KBr): 3430, 2881, 1701, 1638,
1604, 1534, 1111 cmꢀ1 1H NMR (300 MHz, CDCl3): d
.
8.53 (s, 1H), 8.12 (d, 4H, J = 8.1 Hz), 7.26 (d, 4H,
J = 8.1 Hz), 4.50–3.39 (m, PEG).
30. Cleavage of PEG-bound hemicyanine and formation
of Cy3 (20): A mixture of PEG-bound dimethine hemi-
cyanine 19 (0.592 g, 0.24 mmol), 1-(e-carboxypentynyl)-
26. Preparation of PEG-bound tetramethine hemicyanine
(16): A mixture of 15 (0.810 g, 0.34 mmol), 13 (0.090 g,
0.34 mmol) and glacial acetic acid (7.5 ml) was stirred and
heated at 80 °C under nitrogen atmosphere for 1 h. And
then 30 ml cold diethyl ether was added to the solution
with vigorous stirring to produce a brown precipitate,
which was filtered and collected. The precipitate was
2,3,3-trimethylindoleninium-5-sulfonate
14
(0.038 g,
0.11 mmol), acetic anhydride (Ac2O) (2 ml) and pyridine
(1 ml) was stirred and heated at 110 °C under nitrogen
atmosphere for 15 min. The mixture was cooled and the
red dye was precipitated with ethyl acetate. The gummy
product 20 was washed with DCM until the red free

L.-L. Jiang et al. / Tetrahedron Letters 48 (2007) 5825–5829
5829
powder was obtained. Yield 0.015 g (21.6%). 1H NMR
(300 MHz, D2O): d 8.31 (t, 1H, b proton of the bridge,
J = 13.9 Hz), 7.73–7.64 (m, 4H, 4-H, 40-H, 6-H, 60-H),
7.18–7.15 (m, 2H, 7-H, 70-H), 6.22–6.17 (m, 2H, a, a0
proton of the bridge), 3.94–3.89 (m, 4H, a-, a0-CH2), 2.03
(t, 2H, –CH2COOH, J = 7.1 Hz), 1.64–1.15 (m, 21H,
3CH2 groups, 1CH3 and 2(CH3)2 groups). 13C NMR
(300 MHz, D2O): d 183.5, 175.8, 175.5, 151.9, 144.1, 143.7,
141.7, 141.5, 139.7, 139.60, 126.8, 119.8, 111.5, 111.3,
103.4, 49.3, 44.3, 39.6, 37.3, 27.1, 27.0, 26.6, 26.1, 25.6,
11.9. MALDI-TOF-MS: m/z for C31H38S2O8N2 [M+Na]+
calcd 653.20. Found: 653.09. HPLC (methanol)
tR = 2.19 min, purity >98%.