Water soluble indodicarbocyanine dyes based on 2,3-dimethyl-3-(4- sulfobutyl)-3H-indole-5-sulfonic acid

Article abstract of DOI:10.1016/j.dyepig.2012.09.007

A series of mono-reactive, water-soluble, indodicarbocyanine dyes based on 2,3-dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid was synthesized. These dyes contain two aromatic sulfo groups and in addition to (up to 3) sulfoalkyl groups in the indolenine end groups. The impact of the number and position of sulfobutyl groups on the spectral properties and photostabilities of the dyes free in solutions and after binding to an antibody (IgG) was investigated. These dyes have absorption maxima between 649 and 653 nm, molar absorptivities between 240,000 and 256,000 M^-1 cm^-1, emission maxima from 668 to 672 nm, and fluorescence quantum yields between 28 and 32% in free form and up to 43% when bound to IgG. Independent of the number of sulfo groups, these dyes exhibit no aggregation in water at concentrations up to 5 × 10^-5 M. The number of sulfoalkyl groups was found to directly correlate with the fluorescence quantum yields and photostabilities of the IgG conjugated dyes.

Full text of DOI:10.1016/j.dyepig.2012.09.007

Dyes and Pigments 96 (2013) 535e546
Contents lists available at SciVerse ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Water soluble indodicarbocyanine dyes based on 2,3-dimethyl-3-(4-sulfobutyl)-
3H-indole-5-sulfonic acid
Larysa I. Markova a, Iryna A. Fedyunyayeva a, Yevgen A. Povrozin a, Olga M. Semenova a,
,b,
Sania U. Khabuseva a, Ewald A. Terpetschnig b, Leonid D. Patsenker a
*
a SSI “Institute for Single Crystals” of the National Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001, Ukraine
b SETA BioMedicals, LLC, 2014 Silver Court East, Urbana, IL 61802, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 July 2012
Received in revised form
10 September 2012
Accepted 13 September 2012
Available online 1 October 2012
A
series of mono-reactive, water-soluble, indodicarbocyanine dyes based on 2,3-dimethyl-3-(4-
sulfobutyl)-3H-indole-5-sulfonic acid was synthesized. These dyes contain two aromatic sulfo groups
and in addition to (up to 3) sulfoalkyl groups in the indolenine end groups. The impact of the number and
position of sulfobutyl groups on the spectral properties and photostabilities of the dyes free in solutions
and after binding to an antibody (IgG) was investigated. These dyes have absorption maxima between
649 and 653 nm, molar absorptivities between 240,000 and 256,000 Mꢀ1 cmꢀ1, emission maxima from
668 to 672 nm, and fluorescence quantum yields between 28 and 32% in free form and up to 43% when
bound to IgG. Independent of the number of sulfo groups, these dyes exhibit no aggregation in water at
concentrations up to 5 ꢁ 10ꢀ5 M. The number of sulfoalkyl groups was found to directly correlate with
the fluorescence quantum yields and photostabilities of the IgG conjugated dyes.
Keywords:
Cyanine dyes
Protein conjugates
Spectral properties
Fluorescence
Ó 2012 Elsevier Ltd. All rights reserved.
Photostability
Aggregation
1. Introduction
was reported to have a positive effect on the quantum yields [11],
fluorescence lifetimes [12], and photostabilities [13] mostly attrib-
Water soluble indodicarbocyanine dyes containing reactive
groups are widely used in biomedical applications as fluorescent
labels [1,2]. The carboxylic groups in these dyes can be converted to
N-hydroxysuccinimide ester (NHS) or maleimide for covalent
binding under mild conditions to amino or thiol groups of
biomolecules [3]. To avoid cross-linking, labels containing only one
reactive group are preferred [4,5]. The widely used, commercially
available indodicarbocyanine labels such as Cy5 (GE Healthcare) [6]
or Alexa 647 (Life Technologies) [3] contain only one reactive
carboxylic group and, respectively, two and four sulfo groups
rendering the dyes soluble in aqueous media.
uted to a reduced dye aggregation tendency in aqueous media [14].
Thequantumyield of Cy5inphosphate buffer wasreported tobe 27%
[6], while for Alexa 647 it is 33% [3,15] and the difference is even
more pronounced when the dyes are bound to proteins. Further the
photostability of Alexa 647 is also increased compared to Cy5 [16].
There are several positions in the indocyanine dye molecules
which are available for introduction of sulfo, sulfoalkyl and/or
carboxyalkyl groups. Most commonly the sulfo groups were
introduced in the position 5 of indolenine moiety, while sulfoalkyl
and carboxyalkyl groups are in the positions 1 and/or 3. Although
selected indodicarbocyanine dyes containing carboxyalkyl and
sulfoalkyl groups in different positions of the indolenine moiety
have been reported in several papers [6] and patents [17,18],
a systematic study of such compounds has not been done.
In this work we synthesized a series of unsymmetrical mono-
reactive indodicarbocyanine dyes based on quaternized 2,3-
dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid, investigate
the spectral properties, quantum yields, fluorescence lifetimes, and
photostabilities of these dyes free in aqueous solutions and after
binding to IgG.
There are many reports on improving the quantum yields [7],
fluorescence lifetimes [8], and photostabilities [9,10] of long-
wavelength cyanine labels. An increased number of sulfo groups
* Corresponding author. SSI “Institute for Single Crystals” of the National
Academy of Sciences of Ukraine, 60 Lenin Ave., Kharkov 61001, Ukraine. Tel.: þ38
057 3410102; fax: þ38 057 3409343.
E-mail address: patsenker@isc.kharkov.com (L.D. Patsenker).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.dyepig.2012.09.007

536
L.I. Markova et al. / Dyes and Pigments 96 (2013) 535e546
2. Experimental
photostabilities were calculated as the ratio between (i) the
measured absorbencies at the long-wavelength maximum before
and after exposure (A/A0) and (ii) the relative fluorescence inten-
sities before and after exposure (I/I0), and the corresponding plots
were generated. The photostability was estimated via half-life (s1/2),
which is the period of time it takes for the absorption and
2.1. General
1H NMR spectra were measured on a Varian Mercury-VX-200
(200 MHz) spectrometer in DMSO-d6 using TMS as an internal
standard. Chemical reactions were monitored by TLC (Silica gel 60
RP-18, Merck) and spectrophotometrically. The purities of the dyes
were determined by HPLC (column: DiscoveryÒ Bio Wide Pore C18,
250 mm ꢁ 4.6 mm; mobile phase A: water þ 0.05% TFA; mobile
phase B: acetonitrileewater (50:50) þ 0.05% TFA; gradient: 0% Be
80% B in 30 min). The dyes purities were 85e98%.
emission intensity to decrease by half.
The formation energy (DHf) and electronic charge distribution
on dye molecules were calculated by the PM3 method using stan-
dard parameterization [21,22]. Full geometry optimization was
undertaken.
Cy5 was from GE Healthcare. Immunoglobulin G (IgG) from
bovine serum (reagent grade ꢃ95%) and Sephadex G50 were
purchased from Sigma. All other reagents, materials and solvents
were from Aldrich, Merck, and Acros and were used as is.
Absorption spectra for the dye concentrations (c) ranging
between 1 ꢁ10ꢀ8 M and 5 ꢁ 10ꢀ5 M in water and phosphate buffer
(PB, pH 7.4) were recorded at room temperature on a PerkinElmer
Lambda 35 UV/Vis spectrophotometer. The molar absorptivities ( )
3
were measured in water for the same dye concentrations. Each dye
(2e4 mg) was dissolved in 50 mL water and the absorbance (A) was
measured. Then the solution was diluted (1:2) and the absorbance
was measured again. The dilution followed by the absorbance
measurement was repeated 11e13 times. The measurements were
done using standard 10, 5, 1, and 0.1-cm quartz cells. The molar
absorptivities were calculated according to the LamberteBeer’s
2.2. Synthesis
2.2.1. 5-(Ethoxycarbonyl)-5-methyl-6-oxoheptane-1-sulfonic acid
(1)
To a solution of potassium tert-butoxide (9.5 g, 0.085 mol) in
tert-butanol (140 mL), ethyl 2-methyl-3-oxobutanoate (10 mL,
0.071 mol) and then 1,4-butane sultone (7.2 mL, 0.071 mol) were
added dropwise. The mixture was refluxed for 2 h. After cooling,
the solvent was removed under reduced pressure using a rotary
evaporator. The residue was dissolved in 50 mL water, impurities
and co-products were extracted with hexane on a separatory fun-
nel, the aqueous layer was collected and acidified using 1 M
hydrochloric acid until pH ¼ 1. The solvent was removed under
reduced pressure and residue was dried. The product was dissolved
in methanol (170 mL) and the insoluble precipitate was filtered and
the obtained methanolic solution was used in the next step without
purification. A sample for analysis was obtained after removal of
law. The molar absorptivities (
3 ) of each dye were independently
measured 3e4 times, corrected by the dye purity, and the average
value was taken. The reproducibility of the values was no less than
3
ꢂ2000 Mꢀ1 cmꢀ1
.
Emission spectra and quantum yields were measured in water
or phosphate buffer (PB, pH 7.4) at room temperature on a Varian
Cary Eclipse spectrofluorometer. The dye concentrations measured
were in the range of 1 ꢁ10ꢀ8 M to 5 ꢁ 10ꢀ5 M. The emission spectra
were corrected. To reduce the inner filter effect, the quantum yields
were measured at c w 1e3 ꢁ 10ꢀ7 M in 0.1-cm quartz cells.
Absorption and emission maxima were determined at c w 1e
3 ꢁ 10ꢀ7 M with accuracy ꢂ0.3 nm and ꢂ0.5 nm, respectively,
and rounded off.
methanol. 1H NMR (200 MHz, DMSO-d6, ppm):
d 4.12 (2H, q,
J ¼ 7.1 Hz, OCH2), 2.38 (2H, t, J ¼ 7.7 Hz, CH2SO3H), 2.10 (3H, s,
COCH3), 1.78e1.44 (4H, m, CH2), 1.27e1.01 (2H, m, CH2), 1.22 (3H, s,
CCH3), 1.17 (3H, t, J ¼ 7.3 Hz, CH3).
For the determination of the quantum yields (FF), the integrated
relative intensities of the dyes were measured against Cy5
(
FF ¼ 27% [6] at c w 1e3 ꢁ 10ꢀ7 M) as the reference. Optical density
of the dye solutions at the excitation wavelength (610 nm) was
between 0.04 and 0.11 measured in a 5-cm cell. The emission
spectra of the solutions were recorded and the absolute quantum
yields of the dyes were determined as described in [19,20]. The
quantum yield of each sample was independently measured 3e5
times and the average value was calculated. The reproducibility
was no less than 7%.
2.2.2. 5-Methyl-6-oxoheptane-1-sulfonic acid (2)
A solution of sodium hydroxide (9.5 g) in water (95 mL) was
added to the methanolic solution of 5-(ethoxycarbonyl)-5-methyl-
6-oxoheptane-1-sulfonic acid (1). The mixture was heated at 50 ꢄC
for 10 h. Methanol was removed under reduced pressure and
residue was acidified using 1 M hydrochloric acid until pH ¼ 1. The
solvent was removed under reduced pressure and the residue was
dried. The product was dissolved in acetone, insoluble precipitate
was filtered, and acetone was removed. Yield: 13.2 g (90% for the
two stages starting from ethyl 2-methyl-3-oxobutanoate). 1H NMR
Fluorescence lifetime measurements were acquired using
a ChronosFD (ISS, Champaign, IL) frequency domain fluorometer
with a 635-nm laser diode as the excitation light source. In all
lifetime measurements the dye concentrations in PB pH 7.4 were in
the range of 10ꢀ6e10ꢀ7 M. Instrument control, data acquisition and
data analysis were performed using Vinci-Multidimensional Fluo-
rescence Spectroscopy software (ISS, Champaign, IL). The analysis
of the time-resolved fluorescence data was carried out using the
traditional non-linear least squares method. The Marquardte
Levenberg algorithm was utilized for the minimization routine of
(200 MHz, DMSO-d6, ppm):
2.47e2.34 (1H, m, CH), 2.08 (3H, s, COCH3), 1.64e1.42 (4H, m, CH2),
1.32e1.14 (2H, m, CH2), 0.97 (3H, d, J ¼ 7.1 Hz, CH3).
d
2.40 (2H, t, J ¼ 7.8 Hz, CH2SO3H),
2.2.3. 2,3-Dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid (3)
A mixture of 5-methyl-6-oxoheptane-1-sulfonic acid (1 g,
4.8 mmol), 4-hydrazinobenzenesulfonic acid (0.71 g, 3.8 mmol),
and acetic acid (10 mL) was refluxed for 6 h. The solvent was
removed under reduced pressure using a rotary evaporator and the
residue was dried. The product was triturated with hot iso-prop-
anol and the obtained precipitate was filtered and dried. Yield:
the
Photostability measurements were performed in water solu-
c
2-function.
tions at c w 5 ꢁ 10ꢀ7 M. The optical density of the long-wavelength
maximum was controlled to be between 0.10 and 0.20, and all
measurements were carried out in 1-cm cells. These solutions were
placed at the distance of approximately 30 cm from a 500 W
halogen lamp with heat filter and irradiated with occasional stir-
ring. The absorption and emission spectra of the solutions were
taken before irradiation and during light exposure. The relative
1.17 g (85%). labs 260 nm. 1H NMR (200 MHz, DMSO-d6, ppm):
d 7.83
(1H, s, arom H), 7.53 (1H, d, J ¼ 7.8 Hz, arom H), 7.54 (1H, d,
J ¼ 7.9 Hz, arom H), 2.58 (3H, s, 2-CH3), 2.36 (2H, t, J ¼ 7.5 Hz,
CH2SO3H), 2.09e1.86 (2H, m, CH2), 1.53e1.36 (2H, m, CH2), 1.43 (3H,
s, 3-CH3), 0.88e0.47 (2H, m, CH2).

L.I. Markova et al. / Dyes and Pigments 96 (2013) 535e546
537
2.2.4. Dipotassium 2,3-dimethyl-3-(4-sulfonatobutyl)-3H-indole-5-
sulfonate (4)
To a solution of 2,3-dimethyl-3-(4-sulfobutyl)-3H-indole-5-
sulfonic acid (1.17 g, 3.24 mmol) in methanol (10 mL), a solution
of potassium hydroxide in iso-propanol was added dropwise until
pH ¼ 8. The mixture was stirred for 10 min and the precipitate was
filtered and dried. Yield: 1.1 g (77%). 1H NMR (200 MHz, DMSO-d6,
2.2.9. Dipotassium 2,3-dimethyl-1-(4-sulfonatopropyl)-3-(5-
carboxypentyl)-3H-5-indoliumsulfonate (5e) was synthesized
according to Ref. [18]
Yield: 53%. labs 274 nm. 1H NMR (200 MHz, DMSO-d6, ppm):
7.99 (1H, d, J ¼ 6.8 Hz, arom H), 7.97 (1H, s, arom H), 7.82 (1H, d,
d
J ¼ 8.2 Hz, arom H), 4.50 (2H, t, J ¼ 6.8 Hz, NCH2), 2.87 (3H, s, 2-CH3),
2.44e2.34 (2H, m, CH2SO3K), 2.08 (2H, t, J ¼ 7.2 Hz, CH2COOK),
2.02e1.69 (4H, m, CH2), 1.53 (3H, s, 3-CH3), 1.21e1.01 (2H, m, CH2),
0.82e0.37 (2H, m, CH2).
ppm):
d
7.55 (1H, s, arom H), 7.53 (1H, d, J ¼ 9.1 Hz, arom H), 7.31
(1H, d, J ¼ 8.0 Hz, arom H), 2.23 (2H, t, J ¼ 8.2 Hz, CH2SO3K), 2.17
(3H, s, 2-CH3), 1.91e1.70 (2H, m, CH2), 1.50e1.31 (2H, m, CH2), 1.21
(3H, s, 3-CH3), 0.74e0.37 (2H, m, CH2).
2.2.10. Potassium 2,3,3-trimethyl-1-(4-sulfonatobutyl)-3H-
indolium-5-sulfonate (5f) was synthesized according to Ref. [6] with
almost quantitative yield
2.2.5. Dipotassium 2,3-dimethyl-1,3-di(4-sulfonatobutyl)-3H-5-
indoliumsulfonate (5a)
labs 273 nm. 1H NMR (200 MHz, DMSO-d6, ppm):
d 8.01 (1H, s,
A mixture of dipotassium 2,3-dimethyl-3-(4-sulfonatobutyl)-
3H-indole-5-sulfonate (1 g, 2.28 mmol), 1,4-butane sultone
(930 mg, 6.85 mmol) and 1,2-dichlorbenzene (10 mL) was stirred at
150 ꢄC under argon atmosphere for 8 h, cooled down to room
temperature, and 40 mL ether was added. The raw product was
filtered, washed with ether, dried, and column purified (RP-18,
H2O). Yield: 660 mg (50%). labs 275 nm. 1H NMR (200 MHz, DMSO-
arom H), 7.96 (1H, d, J ¼ 8.4 Hz, arom H), 7.81 (1H, d, J ¼ 8.0 Hz,
arom H), 4.46 (2H, t, J ¼ 6.9 Hz, NCH2), 2.84 (3H, s, 2-CH3), 2.53e
2.38 (2H, m, CH2SO3K), 2.10e1.84 (2H, m, CH2), 1.84e1.65 (2H, m,
CH2), 1.54 (6H, s, 3-(CH3)2).
2.2.11. Potassium 2,3,3-trimethyl-1-(3-sulfonatopropyl)-3H-
indolium-5-sulfonate (5g) was synthesized according to Ref. [18]
with almost quantitative yield
d6, ppm):
d
8.00 (1H, d, J ¼ 6.6 Hz, arom H), 7.98 (1H, s, arom H), 7.82
(1H, d, J ¼ 8.0 Hz, arom H), 4.49 (2H, t, J ¼ 6.8 Hz, NCH2), 2.87 (3H, s,
2-CH3), 2.44e2.22 (4H, m, CH2SO3K), 2.01e1.69 (4H, m, CH2), 1.65e
1.35 (4H, m, CH2), 1.53 (3H, s, 3-CH3), 0.90e0.41 (2H, m, CH2).
labs 273 nm. 1H NMR (200 MHz, DMSO-d6, ppm):
d 7.98 (1H, s,
arom H), 7.93 (1H, d, J ¼ 8.4 Hz, arom H), 7.81 (1H, d, J ¼ 8.0 Hz,
arom H), 4.60 (2H, t, J ¼ 6.9 Hz, NCH2), 2.80 (3H, s, 2-CH3), 2.61 (2H,
m, CH2SO3K), 1.84e1.65 (2H, m, CH2), 1.52 (6H, s, 3-(CH3)2).
2.2.6. Potassium 1-(5-carboxypentyl)-2,3-dimethyl-3-(4-
sulfonatobutyl)-3H-5-indoliumsulfonate (5b)
2.2.12. 1-(5-Carboxypentyl)-2,3,3-trimethyl-3H-indolium-5-
sulfonate (5h) was synthesized according to Ref. [6]
A mixture of dipotassium 2,3-dimethyl-3-(4-sulfonatobutyl)-
3H-indole-5-sulfonate (2.4 g, 5.48 mmol), 6-bromohexanoic acid
(3.38 g, 17.32 mmol) and 1,2-dichlorbenzene (18 mL) was stirred at
150 ꢄC under argon atmosphere for 6 h, cooled down to room
temperature, and 50 mL ether was added. The raw product was
filtered, washed with ether, dried, and column purified (RP-18,
H2O). Yield: 1.24 g (44%). labs 276 nm. 1H NMR (200 MHz, DMSO-d6,
Yield: 77%. labs 270 nm. 1H NMR (200 MHz, DMSO-d6, ppm):
d
8.02 (1H, s, arom H), 7.93 (1H, d, J ¼ 8.4 Hz, arom H), 7.81 (1H, d,
J ¼ 7.8 Hz, arom H), 4.44 (2H, t, J ¼ 6.3 Hz, NCH2), 2.84 (3H, s, 2-CH3),
2.22 (2H, t, J ¼ 7.1 Hz, CH2COOH), 1.95e1.73 (2H, m, CH2), 1.69e1.26
(4H, m, CH2), 1.54 (6H, s, 3-(CH3)2).
ppm):
d
7.99 (1H, s, arom H), 7.94 (1H, d, J ¼ 8.2 Hz, arom H), 7.83
2.2.13. General procedure for synthesis of 1,3-butadienyl-N-
phenylacetoamides (6b, 6d, 6fe6h)
(1H, d, J ¼ 8.2 Hz, arom H), 4.47 (2H, t, J ¼ 7.1 Hz, NCH2), 2.87 (3H, s,
2-CH3), 2.38e2.15 (2H, m, CH2SO3K), 2.23 (2H, t, J ¼ 7.1 Hz,
CH2COOH), 1.92e1.74 (2H, m, CH2),1.64e1.32 (8H, m, CH2),1.53 (3H,
s, 3-CH3), 0.86e0.42 (2H, m, CH2).
A mixture of 2.5 mmol of indolenine quaternary salt, 3.75 mmol
N,N0-prop-1-en-1-yl-3-ylidenedianiline hydrochloride,15 mL of acetic
anhydride and 15 mL of acetic acid was refluxed for 4.5 h. The solvent
was removed under reduced pressure and the residue was triturated
with ethyl acetate. The obtained precipitate was filtered, washed with
ethyl acetate until colorless filtrate, and dried to yield product as
orange powder. The products obtained with almost quantitative yield
were used for further syntheses without purification.
2.2.7. Potassium 1-ethyl-2,3-dimethyl-3-(4-sulfonatobutyl)-3H-5-
indoliumsulfonate (5c)
A mixture of dipotassium 2,3-dimethyl-3-(4-sulfonatobutyl)-
3H-indole-5-sulfonate (1 g, 2.28 mmol), iodoethane (2 mL) and
acetonitrile (20 mL) was stirred at 90 ꢄC for 40 h. Additional
portions of iodoethane (0.3 mL) were added every 10 h. The solvent
was removed under reduced pressure and the residue was column
purified (RP-18, H2O). Yield: 310 mg (32%). labs 275 nm. 1H NMR
2.2.13.1. Potassium 1-(5-carboxypentyl)-3-methyl-2-[4-
methyl(phenyl)carboxamido-1,3-butadienyl]-3-(4-sulfonatobutyl)-
3H-5-indoliumsulfonate (6b). labs 453 nm. 1H NMR (200 MHz,
(200 MHz, DMSO-d6, ppm):
d
7.99 (1H, s, arom H), 7.93 (1H, d,
DMSO-d6, ppm):
J ¼ 13.2 Hz, CH), 7.79 (1H, s, arom H), 7.61e7.16 (7H, m, arom H),
7.03 (1H, d, J ¼ 7.9 Hz, CH), 4.27e
d
8.82 (1H, d, J ¼ 13.1 Hz,
d CH), 8.33 (1H, t,
J ¼ 8.2 Hz, arom H), 7.84 (1H, d, J ¼ 8.3 Hz, arom H), 4.50 (2H, q,
J ¼ 7.1 Hz, NCH2), 2.86 (3H, s, 2-CH3), 2.27 (2H, t, J ¼ 6.3 Hz,
CH2SO3K), 2.41e2.03 (2H, m, CH2), 1.53 (3H, s, 3-CH3), 1.42 (3H, t,
J ¼ 6.9 Hz, CH3), 1.57e1.35 (2H, m, CH2), 0.89e0.41 (2H, m, CH2).
b
a
CH), 5.86 (1H, t, J ¼ 12.1 Hz,
g
4.03 (2H, m, NCH2), 2.34e2.12 (4H, m, CH2SO3K, CH2COOH), 1.91
(3H, s, COCH3),1.79e1.20 (10H, m, CH2),1.65 (3H, s, 3-(CH3)2), 0.97e
0.42 (2H, m, CH2).
2.2.8. Dipotassium 2,3-dimethyl-1-(4-sulfonatobutyl)-3-(5-
carboxypentyl)-3H-5-indoliumsulfonate (5d) was synthesized
according to Ref. [23]
2.2.13.2. Dipotassium
6-[3-methyl-2-[4-methyl(phenyl)carbox-
amido-1,3-butadienyl]-5-sulfonato-1-(4-sulfonatobutyl)-3H-3-
Yield: 49%. labs 274 nm. 1H NMR (200 MHz, DMSO-d6, ppm):
indoliumyl]hexanoate (6d). labs 455 nm. 1H NMR (200 MHz, DMSO-
d
7.99 (1H, d, J ¼ 6.8 Hz, arom H), 7.97 (1H, s, arom H), 7.82 (1H, d,
d6, ppm):
7.73 (1H, s, arom H), 7.66e7.17 (7H, m, arom H), 7.03 (1H, d, J ¼ 7.7 Hz,
CH), 4.39e4.22 (2H, m, NCH2), 2.22e
1.95 (4H, m, CH2SO3K, CH2COOK), 1.90 (3H, s, COCH3), 1.83e1.48
(10H, m, CH2), 1.68 (3H, s, 3-(CH3)2), 0.80e0.29 (2H, m, CH2).
d
8.94 (1H, d, J ¼12.5Hz,
d
CH), 8.59(1H, t, J ¼13.7Hz,
b CH),
J ¼ 8.2 Hz, arom H), 4.50 (2H, t, J ¼ 6.8 Hz, NCH2), 2.87 (3H, s, 2-CH3),
2.44e2.34 (2H, m, CH2SO3K), 2.08 (2H, t, J ¼ 7.2 Hz, CH2COOK),
2.02e1.69 (4H, m, CH2), 1.67e1.28 (4H, m, CH2), 1.53 (3H, s, 3-CH3),
1.21e1.01 (2H, m, CH2), 0.82e0.37 (2H, m, CH2).
a
CH), 5.57 (1H, t, J ¼ 12.2 Hz,
g

538
L.I. Markova et al. / Dyes and Pigments 96 (2013) 535e546
2.2.13.3. 1-(4-Sulfonatobutyl)-3,3-dimethyl-2-[4-methyl(phenyl)car-
synthesized starting from 1,3-butadienyl-N-phenylacetoamide 6b
and quaternary salt 5f. Yield: 42%. 1H NMR (200 MHz, DMSO-d6,
boxamido-1,3-butadienyl]-3H-5-indoliumsulfonate (6f). labs 445 nm.
1H NMR (200 MHz, DMSO-d6, ppm):
d
8.94 (1H, d, J ¼ 12.5 Hz,
CH), 7.97 (1H, s, arom H), 7.84e7.33 (7H, m,
CH),
d
CH),
ppm):
arom H), 7.62 (2H, d, J ¼ 8.4 Hz, arom H), 7.36 (1H, d, J ¼ 7.9 Hz, arom
H), 7.29 (1H, d, J ¼ 8.4 Hz, arom H), 6.59 (1H, t, J ¼ 12.9 Hz, H), 6.38
H), 4.18e3.91 (4H,
d
8.35 (2H, t, J ¼ 13.5 Hz,
b H), 7.80 (1H, s, arom H), 7.76 (1H, s,
8.58 (1H, t, J ¼ 12.0 Hz,
b
arom H), 6.89 (1H, d, J ¼ 14.5 Hz,
a
CH), 5.54 (1H, t, J ¼ 12.2 Hz,
g
g
4.40e4.17 (2H, m, NCH2), 2.28e2.10 (2H, m, CH2SO3H), 1.91 (3H, s,
COCH3), 1.77e1.24 (6H, m, CH2), 1.70 (6H, s, 3-(CH3)2).
(1H, d, J ¼ 12.9 Hz,
a
H), 6.34 (1H, d, J ¼ 12.9 Hz,
a
m, NCH2), 2.45e2.25 (4H, m, CH2SO3H), 2.20 (2H, t, J ¼ 7.0 Hz,
CH2COOH), 1.85e1.30 (14H, m, CH2), 1.68 (6H, s, 3-(CH3)2), 1.66 (3H,
s, 3-CH3), 0.93e0.37 (2H, m, CH2).
2.2.13.4. 1-(4-Sulfonatopropyl)-3,3-dimethyl-2-[3-methyl(phenyl)car-
boxamido-1,3-butadienyl]-3H-5-indoliumsulfonate (6g). labs 450 nm.
1H NMR (200 MHz, DMSO-d6, ppm):
d
8.94 (1H, d, J ¼ 12.5 Hz,
CH), 7.97 (1H, s, arom H), 7.84e7.33 (7H, m,
CH),
d
CH),
2.2.14.4. 1-(5-Carboxypentyl)-3,3-dimethyl-2-5-[3-methyl-5-sulfo-
1,3-di(4-sulfobutyl)-2,3-dihydro-1H-2-indolyliden]-1,3-pentadienyl-
3H-5-indoliumsulfonate (10). The dye was synthesized starting
from 1,3-butadienyl-N-phenylacetoamide 6h and quaternary salt
8.58 (1H, t, J ¼ 12.0 Hz,
b
arom H), 6.89 (1H, d, J ¼ 14.5 Hz,
a
CH), 5.54 (1H, t, J ¼ 12.2 Hz,
g
4.40e4.17 (2H, m, NCH2), 2.28e2.10 (2H, m, CH2SO3H), 1.91 (3H, s,
COCH3), 1.77e1.24 (4H, m, CH2), 1.70 (6H, s, 3-(CH3)2).
5a. Yield: 31%. 1H NMR (200 MHz, DMSO-d6, ppm):
d
8.36 (2H, t,
H), 7.80 (1H, s, arom H), 7.77 (1H, s, arom H), 7.61 (2H,
d, J ¼ 7.6 Hz, arom H), 7.36 (1H, d, J ¼ 8.2 Hz, arom H), 7.31 (1H, d,
J ¼ 8.4 Hz, arom H), 6.61 (1H, t, J ¼ 12.4 Hz, H), 6.41 (1H, d,
H), 4.17e4.02 (4H, m,
J ¼ 12.9 Hz,
b
2.2.13.5. 1-(5-Carboxypentyl)-3,3-dimethyl-2-[4-methyl(phenyl)car-
boxamido-1,3-butadienyl]-3H-5-indoliumsulfonate (6h). labs 452 nm.
g
1H NMR (200 MHz, DMSO-d6, ppm):
d
8.94 (1H, d, J ¼ 12.5 Hz,
CH), 7.97 (1H, s, arom H), 7.84e7.33 (7H, m,
CH),
d
CH),
J ¼ 14.0 Hz,
a
H), 6.31 (1H, d, J ¼ 13.7 Hz,
a
8.58 (1H, t, J ¼ 12.0 Hz,
b
NCH2), 2.44e2.34 (4H, m, CH2SO3H), 2.20 (2H, t, J ¼ 7.0 Hz,
CH2COOH), 1.69 (6H, s, 3-(CH3)2), 1.66 (3H, s, 3-CH3), 1.87e1.27
(14H, m, CH2), 0.94e0.39 (2H, m, CH2).
arom H), 6.89 (1H, d, J ¼ 14.5 Hz,
a
CH), 5.54 (1H, t, J ¼ 12.2 Hz,
g
4.40e4.17 (2H, m, NCH2), 2.28e2.10 (2H, m, CH2COOH), 1.91 (3H, s,
COCH3), 1.77e1.24 (6H, m, CH2), 1.70 (6H, s, 3-(CH3)2).
2.2.14.5. 3-(5-Carboxypentyl)-3-methyl-2-5-[3-methyl-5-sulfo-1,3-
di(4-sulfobutyl)-2,3-dihydro-1H-2-indolyliden]-1,3-pentadienyl-1-
(4-sulfobutyl)-3H-5-indoliumsulfonate (11). The dye was synthe-
sized starting from 1,3-butadienyl-N-phenylacetoamide 6d and
quaternary salt 5a. Yield: 19%. 1H NMR (200 MHz, DMSO-d6, ppm):
2.2.14. General procedure for synthesis of cyanine dyes 7e16
A solution of 0.3 mmol of 1,3-butadienyl-N-phenylacetoamide in
7 mL of acetic anhydride was stirred at 50 ꢄC for 30 min. Then
0.365 mmol of indolenine quaternary salt and 7 mL of dry pyridine
were added. In case of synthesis of cyanines 10e12, 7 mL of acetic
acid were added. The mixture was stirred at 110 ꢄC for 30 min. After
cooling, dryether was added and the mixturewas kept at 0 ꢄC for 2 h.
The obtained precipitate was filtered and washed with ether. Then
the ion exchange resin Dowex 50WX8-100 was prepared as
described in Ref. [6]. The raw product was dissolved in water, passed
through column with Dowex 50WX8-100, and column purified (RP-
18, watereacetonitrile) to give the title dye as blue powder.
d
8.34 (2H, t, J ¼ 12.8 Hz,
J ¼ 8.2 Hz, arom H), 7.35 (2H, d, J ¼ 8.5 Hz, arom H), 6.60 (1H, t,
H), 4.21e4.00 (4H, m, NCH2),
b H), 7.75 (2H, s, arom H), 7.62 (2H, d,
J¼ 12.2 Hz,
g
H), 6.41 (2H, d, J¼ 13.4 Hz,
a
2.51e2.32 (6H, m, CH2SO3H), 2.06 (2H, t, J ¼ 6.6 Hz, CH2COOH), 1.66
(6H, s, 3-CH3), 1.93e1.02 (18H, m, CH2), 0.96e0.33 (4H, m, CH2).
2.2.14.6. 1-(5-Carboxypentyl)-3-methyl-2-5-[3-methyl-5-sulfo-1,3-
di(4-sulfobutyl)-2,3-dihydro-1H-2-indolyliden]-1,3-pentadienyl-3-
(4-sulfobutyl)-3H-5-indoliumsulfonate (12). The dye was synthe-
sized starting from 1,3-butadienyl-N-phenylacetoamide 6b and
quaternary salt 5a Yield: 11%. 1H NMR (200 MHz, DMSO-d6, ppm):
2.2.14.1. 3-(5-Carboxypentyl)-2-5-[1-ethyl-3-methyl-5-sulfo-3-(4-
sulfobutyl)-2,3-dihydro-1H-2-indolyliden]-1,3-pentadienyl-3-
methyl-1-(4-sulfobutyl)-3H-5-indoliumsulfonate (7). The dye was
synthesized starting from 1,3-butadienyl-N-phenylacetoamide 6d
and quaternary salt 5c. Yield: 16%. 1H NMR (200 MHz, DMSO-d6,
d
8.36(2H, t, J ¼ 12.7Hz,
arom H), 7.35 (1H, d, J ¼ 8.6 Hz, arom H), 7.30 (1H, d, J ¼ 8.6 Hz, arom
H), 6.60(1H, t, J¼ 12.1Hz, H),6.40(1H, d,J¼ 13.7Hz, H), 6.33 (1H, d,
H), 4.21e4.00 (4H, m, NCH2), 2.45e2.31 (6H, m,
b
H), 7.76 (2H, s,aromH), 7.63(2H, d,J¼ 8.0Hz
g
a
ppm):
d, J ¼ 8.5 Hz arom H), 7.35 (1H, d, J ¼ 8.6 Hz, arom H), 7.30 (1H, d,
J ¼ 8.2 Hz, arom H), 6.55 (1H, t, J ¼ 12.4 Hz, H), 6.42 (1H, d,
H), 4.21e4.03 (4H, m,
d
8.34 (2H, t, J ¼ 13.3 Hz,
b
H), 7.75 (2H, s, arom H), 7.63 (2H,
J ¼ 13.4 Hz,
a
CH2SO3H), 2.20 (2H, t, J ¼ 7.4 Hz, CH2COOH),1.67 (6H, s, 3-CH3),1.83e
1.31 (18H, m, CH2), 0.94e0.40 (4H, m, CH2).
g
J ¼ 13.8 Hz,
a
H), 6.34 (1H, d, J ¼ 13.7 Hz,
a
NCH2), 2.43e2.28 (4H, m, CH2SO3H), 2.06 (2H, t, J ¼ 7.1 Hz,
CH2COOH), 1.66 (6H, s, 3-CH3), 1.87e1.04 (14H, m, CH2), 1.24 (3H, t,
J ¼ 7.2 Hz, CH3), 0.95e0.36 (4H, m, CH2).
2.2.14.7. 1-(5-Carboxypentyl)-2-5-[3,3-dimethyl-5-sulfo-1-(4-
sulfobutyl)-2,3-dihydro-1H-2-indolyliden]-1,3-pentadienyl-3,3-
dimethyl-3H-5-indoliumsulfonate (13). The dye was synthesized
starting from 1,3-butadienyl-N-phenylacetoamide 6h and quater-
2.2.14.2. 1-(5-Carboxypentyl)-2-5-[1-ethyl-3-methyl-5-sulfo-3-(4-
sulfobutyl)-2,3-dihydro-1H-2-indolyliden]-1,3-pentadienyl-3-
methyl-3-(4-sulfobutyl)-3H-5-indoliumsulfonate (8). The dye was
synthesized starting from 1,3-butadienyl-N-phenylacetoamide 6b
and quaternary salt 5c. Yield: 7%. 1H NMR (200 MHz, DMSO-d6,
nary salt 5f. Yield: 5%. 1H NMR (200 MHz, DMSO-d6, ppm):
d
8.35
H), 7.80 (2H, s, arom H), 7.63 (2H, d, J ¼ 8.4 Hz,
arom H), 7.36 (1H, d, J ¼ 8.4 Hz, arom H), 7.31 (1H, d, J ¼ 8.3 Hz,
arom H), 6.60 (1H, t, J ¼ 12.7 Hz, H), 6.39 (1H, d, J ¼ 14.4 Hz, H),
H), 4.18e3.97 (4H, m, NCH2), 2.63e2.49
(2H, t, J ¼ 13.2 Hz,
b
g
a
6.31 (1H, d, J ¼ 13.8 Hz,
a
ppm):
d, J ¼ 8.1 Hz, arom H), 7.31 (2H, d, J ¼ 8.2 Hz, arom H), 6.58 (1H, t,
H), 4.21e4.02 (4H, m,
d
8.38 (2H, t, J ¼ 12.9 Hz,
b
H), 7.77 (2H, s, arom H), 7.63 (2H,
(2H, m, CH2SO3H), 2.20 (2H, t, J ¼ 7.0 Hz, CH2COOH), 1.85e1.46
(8H, m, CH2), 1.69 (12H, s, 2 ꢁ [3-(CH3)2]), 1.46e1.29 (2H, m, CH2).
J ¼ 12.2 Hz, H), 6.33 (2H, d, J ¼ 12.4 Hz,
g
a
NCH2), 2.46e2.34 (4H, m, CH2SO3H), 2.20 (2H, t, J ¼ 7.3 Hz,
CH2COOH), 1.65 (6H, s, 3-CH3), 1.80e1.32 (14H, m, CH2), 1.25 (3H, t,
J ¼ 7.1 Hz, CH3), 0.94e0.38 (4H, m, CH2).
2.2.14.8. 2-5-[3-(5-Carboxypentyl)-3-methyl-5-sulfo-1-(3-
sulfopropyl)-2,3-dihydro-1H-2-indolyliden]-1,3-pentadienyl-3,3-
dimethyl-1-(3-sulfopropyl)-3H-5-indoliumsulfonate (14). The dye
was synthesized starting from 1,3-butadienyl-N-phenylacetoamide
6g and quaternary salt 5e. Yield: 14%. 1H NMR (200 MHz, DMSO-d6,
2.2.14.3. 2-5-[1-(5-Carboxypentyl)-3-methyl-5-sulfo-3-(4-
sulfobutyl)-2,3-dihydro-1H-2-indolyliden]-1,3-pentadienyl-3,3-
dimethyl-1-(4-sulfobutyl)-3H-5-indoliumsulfonate (9). The dye was
ppm):
d
8.36 (2H, t, J ¼ 13.2 Hz,
b H), 7.80 (1H, s, arom H), 7.75 (1H, s,
arom H) 7.61 (2H, d, J ¼ 8.4 Hz, arom H), 7.39 (2H, d, J ¼ 8.3 Hz, arom

L.I. Markova et al. / Dyes and Pigments 96 (2013) 535e546
539
H), 6.58 (1H, t, J ¼ 12.7 Hz,
g
H), 6.43 (2H, d, J ¼ 14.4 Hz,
a
H), 4.26
2.5. Purification of the dyeeIgG conjugates
(2H, t, NCH2), 4.09 (2H, t, NCH2), 2.64 (4H, t, J ¼ 7.0 Hz, CH2SO3H),
2.04 (2H, t, J ¼ 7.0 Hz, CH2COOH),1.85e1.46 (8H, m, CH2),1.68 (9H, s,
3-CH3, 3-(CH3)2), 1.31e0.46 (4H, m, CH2).
Separation of the dyeeIgG conjugate from unconjugated dye
was performed using gel permeation chromatography on
a 1.5 cm ꢁ 25 cm column (Sephadex G50, 67 mM PB, pH 7.4). The
fraction with the shortest retention time containing the blue dyee
IgG conjugate was collected.
2.2.14.9. 2-5-[3-(5-Carboxypentyl)-3-methyl-5-sulfo-1-(3-
sulfopropyl)-2,3-dihydro-1H-2-indolyliden]-1,3-pentadienyl-3,3-
dimethyl-1-(4-sulfobutyl)-3H-5-indoliumsulfonate (15). The dye
was synthesized starting from 1,3-butadienyl-N-phenylacetoamide
6f and quaternary salt 5e. Yield: 5%. 1H NMR (200 MHz, DMSO-d6,
2.6. Determination of the dye-to-protein ratios (D/P)
ppm):
arom H) 7.60 (2H, d, J ¼ 8.4 Hz, arom H), 7.37 (2H, d, J ¼ 8.3 Hz, arom
H), 4.26
(4H, m, NCH2), 2.60 (4H, m, J ¼ 7.0 Hz, CH2SO3H), 2.04 (2H, t,
J ¼ 7.0 Hz, CH2COOH), 1.85e1.46 (8H, m, CH2), 1.68 (9H, s, 3-CH3, 3-
(CH3)2), 1.32e0.48 (6H, m, CH2).
d
8.38 (2H, t, J ¼ 13.2 Hz,
b H), 7.80 (1H, s, arom H), 7.75 (1H, s,
A series of labeling reactions were performed and the corre-
sponding conjugates were purified and isolated as described above.
The absorption spectra of the conjugates were measured and the
dye-to-protein ratios (D/P) were calculated using the following
equation with the assumption that the molar absorptivities for the
free dye and the dyeeIgG conjugate are the same [24,25]:
H), 6.58 (1H, t, J ¼ 12.7 Hz,
g
H), 6.43 (2H, d, J ¼ 14.4 Hz,
a
2.2.14.10. 2-5-[3-(5-Carboxypentyl)-3-methyl-5-sulfo-1-(4-
sulfobutyl)-2,3-dihydro-1H-2-indolyliden]-1,3-pentadienyl-3,3-
dimethyl-1-(4-sulfobutyl)-3H-5-indoliumsulfonate (16). The dye
was synthesized starting from 1,3-butadienyl-N-phenylacetoamide
6f and quaternary salt 5d. Yield: 15%. 1H NMR (200 MHz, DMSO-d6,
AConjð
3
IgG
lmax
Þ
ꢀ
ꢁ
D=P ¼
;
A
Conjð278Þ ꢀ xA
3
Dye
Conjð maxÞ
l
where
3
Dye
is the molar absorptivity of the dye at the long-
wavelength maximum,
3
¼ 201,700 Mꢀ1 cmꢀ1 is the molar
IgG
ppm):
arom H) 7.60 (2H, d, J ¼ 8.4 Hz, arom H), 7.37 (2H, d, J ¼ 8.3 Hz, arom
H), 4.10
d
8.35 (2H, t, J ¼ 13.2 Hz,
b H), 7.80 (1H, s, arom H), 7.74 (1H, s,
absorptivity of IgG at 278 nm, x ¼ ADyeð278Þ=A
Þ; AConjð
;
Dyeðlmax
lmax
Þ
A
Conjð278Þ; A
and ADyeð278Þ are the absorbances of the
H), 6.59 (1H, t, J ¼ 12.7 Hz,
g
H), 6.40 (2H, d, J ¼ 14.4 Hz,
a
Dyeð maxÞ
l
(4H, m, NCH2), 2.60 (4H, m, J ¼ 7.0 Hz, CH2SO3H), 2.03 (2H, t,
J ¼ 7.0 Hz, CH2COOH), 1.85e1.46 (8H, m, CH2), 1.68 (9H, s, 3-CH3, 3-
(CH3)2), 1.31e0.43 (8H, m, CH2).
dyeeIgG conjugate and free dye at the long-wavelength
maximum and at 278 nm, respectively.
3. Results and discussion
2.3. General procedure for synthesis of cyanine dyes NHS-esters
3.1. Synthesis
1 mg of the dye was dissolved in anhydrous DMF (100
mL). Then
A synthetic pathway to indole sulfonic acid 3, a key intermediate
for these cyanine dyes, is shown on Scheme 1. In accordance with
Ref. [17], alkylation of ethyl 2-methyl-3-oxobutanoate by 1,4-
butane sultone with sodium hydride in DMF followed by hydro-
lysis and decarboxylation of the intermediate sulfoalkyl derivative
1 under reflux in concentrated hydrochloric acid gives ketone 2,
which requires purification by column chromatography (Method
A). By this way ketone 2 could be synthesized with a 79% yield
starting from ethyl 2-methyl-3-oxobutanoate.
0.5 mg of TSTU and 4 L of DIPEA were added and the mixture was
m
stirred at room temperature for 1 h. The reaction was monitored by
TLC (RP-18, watereacetonitrile). The solution without purification
was used for preparation of dyeeIgG conjugates.
2.4. General protein labeling procedures
A stock solution of 1 mg of the NHS-activated dye in 100 mL of
anhydrous DMF was prepared as described above. A series of
solutions containing 1.5 mg of IgG dissolved in 0.5 mL of 50 mM
bicarbonate buffer pH 9.0 were prepared. Then appropriate aliquot
In our attempt to synthesize intermediate 1, potassium tert-but-
oxidewasused in the above alkylation step (Method B). Asa result the
reaction time was reduced from 16 to 2 h. The hydrolysis of 1 with
alkaline followed by decarboxylation in acidic medium gives 5-
methyl-6-oxoheptane-1-sulfonic acid 2 with 90% yield. Importantly,
the obtained ketone 2 can be introduced in the next step without
of the dye stock solution (in the range of 5e50
mL) was added
slowly to each IgG solution. The mixtures were allowed to stir for
2 h at 25 ꢄC to form the dyeeIgG conjugates.
Scheme 1. Synthesis of 2,3-dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid.

540
L.I. Markova et al. / Dyes and Pigments 96 (2013) 535e546
purification and reacts with 4-hydrazinobenzenesulfonic acid in
acetic acid according to Fisher’s method [17,26] in high yield.
Importantly the solubility of the starting materials can be
increased also by protonation of the sulfonic acid group using ion
exchange chromatography. Indolenine 5a with protonated sulfo
groups showed much higher solubility in the reaction mixture
(acetic anhydride/pyridine) compared to the dipotassium salt of 5a
and cyanines 10e12 were obtained with satisfactory yields even in
absence of acetic acid.
The indole sulfonic acid 3 was transformed to the dipotassium
salt 4 and quaternized by an excess of 1,4-butane sultone and
6-bromohexanoic acid in 1,2-dichlorbenzene to quaternary salts 5a
and 5b, respectively, while the same salt 4 being refluxed with
iodoethane in acetonitrile yielded quaternary salt 5c (Scheme 2).
The products 5ae5c were obtained from dipotassium salt 4 with
50%, 44%, and 32% yields, respectively. These compounds can be
synthesized also by melting of reagents without solvent (5a) or
using tetramethylene sulphone as the solvent (5b and 5c) [17]. The
quaternary salts 5de5h were obtained according to Refs. [6,18,23].
The synthesis of the mono-reactive unsymmetrical cyanine dyes
is presented in Scheme 3. In the first step the quaternized indolenine
is reacted with N,N0-prop-1-en-1-yl-3-ylidenedianiline hydrochlo-
ride and the obtained 1,3-butadienyl-N-phenylacetoamide is
condensed with a 2nd quaternized indolenine. We introduced the
sulfoindolenines 5ae5h in the reaction in such order to obtain the
widest range of dyes by using the smallest number of 1,3-
butadienyl-N-phenylacetoamide precursors. The condensation of
indolenines 5b, 5d and 5fe5h with N,N0-prop-1-en-1-yl-3-
ylidenedianiline hydrochloride in a mixture of acetic acid with
acetic anhydride yielded the corresponding 1,3-butadienyl-N-phe-
nylacetoamides 6b, 6d and 6fe6h (Scheme 3, Table 1). Synthesis of
compounds 6d and 6fe6h was described also in Ref. [18].
The substituents in both the 1,3-butadienyl-N-phenyl-
acetoamides 6b, 6d and 6fe6h and indolenines 5a and 5ce5f were
found to have a strong influence on the reaction conditions to the
final cyanine dye: 1,3-butadienyl-N-phenylacetoamides were
reacted with indolenines 5ce5f containing two sulfo groups at
reflux in acetic anhydride with pyridine (1:1) as described in Ref.
[6] and yielded cyanines 7e9 and 13e16 with satisfactory yields.
However, our attempts to synthesize cyanines 10e12 via reaction of
1,3-butadienyl-N-phenylacetoamides 6b, 6d and 6h with indole-
nine 5a containing three sulfo groups failed under these conditions.
Moreover, addition of DMF, which was supposed to help in certain
cases [18] had no effect on the reaction. No detectable amounts of
the aimed products were obtained under both conditions.
Thus in this paper we synthesized new cyanine dyes 7, 9e11, 15
and 16. The structures of the cyanines were confirmed using 1H
NMR (DMSO-d6). The pentamethine chain is characterized by the
triplet signal of two
of -hydrogen at 6.55e6.61 ppm, and two doublet signals of
hydrogens at 6.33e6.43 ppm. The triplet signals of two -methy-
b
,b
0-hydrogens at 8.34e8.38 ppm, triplet signal
a0
g
a, -
a
lene hydrogens nearby to carboxylic groups are at 2.20 ppm for
cyanines 8e10, 12, and 13 and 2.06 ppm for 7, 11 and 14e16.
3.2. Spectral properties
All investigated cyanine dyes 7e16 contain the same
5-sulfoindolenine moieties as the end groups but differ by the
number and position of sulfoalkyl groups and also in regards to the
position of carboxypentyl substitution. The majority of cyanines
(dyes 7e13 and 16) contain sulfobutyl groups while cyanines 14
and 15 differ by the length of the sulfoalkyl chains: 14 contains two
sulfopropyl and 15 one sulfopropyl and one sulfobutyl group.
The spectral properties of the cyanine dyes such as the
absorption and emission maxima (lmax), molar absorptivities ( ),
3
and quantum yields (FF) measured free in aqueous phosphate
buffer (pH 7.4) and after binding to IgG at different dye-to-protein
ratios (D/P ¼ 1 and 4) are given in Table 2. The dyes in the table are
listed by order of increasing numbers of sulfoalkyl groups, which
was found to correlate well with the long-wavelength shift of the
absorption and emission maxima.
3.2.1. Free dyes
The concentration dependent spectral characteristics of the free
dyes were investigated in the concentration range between
1 ꢁ 10ꢀ8 M and 5 ꢁ 10ꢀ5 M. Within the measured concentration
range a linear correlation of the absorbances vs. concentrations was
found, which evidenced no aggregation tendency within the
investigated concentration range for the investigated cyanines. As
a result the data in the Table 2 measured for the free dyes at c w 1e
3 ꢁ 10ꢀ7 M can be attributed to non-aggregated cyanines.
The absorption and emission maxima ranged between 647e
653 nm and 665e672 nm, respectively, are slightly dependent on
the dyes structure: a tendency of the red-shift of the absorption and
emission maxima in the order of Cy5 < 13 < 16 < 9 z 10
< 7 z 8 < 11 z12 was found to correlate well with the increase of
the number of sulfobutyl groups. The most pronounced red-shift
(6e7 nm) was noted for cyanines 11 and 12 containing the three
sulfobutyl groups. The position of sulfobutyl and carboxypentyl
The fact that the indolenine 5a did not react under these
conditions is most likely due to its low solubility in the reaction
mixture. In contrast to the potassium disulfoindolenines 5ce5f and
sulfoindolenine 5h, the dipotassium salt 5a is totally insoluble in
a mixture of acetic anhydridedpyridine even upon addition of
DMF. At the same time indolenine 5a is highly soluble in acetic acid.
Indeed, adding acetic acid to the reaction mixture facilitated the
reaction of 1,3-butadienyl-N-phenylacetoamides 6b, 6d and 6h
with the indolenine 5a and cyanines 10e12 were synthesized with
satisfactory yields. The acetic acid concentration also was found to
have a strong impact on the reaction rate: cyanine products were
only isolated when the reaction mixture contained at least equal
amounts of acetic acid compared to acetic anhydride.
Scheme 2. Quaternization of 2,3-dimethyl-3-(4-sulfobutyl)-3H-indole-5-sulfonic acid.

L.I. Markova et al. / Dyes and Pigments 96 (2013) 535e546
541
Scheme 3. Synthesis of cyanine dyes.
groups in the dyes pairs (9 and 10), (7 and 8), and (11 and 12) as
well as the shortening of the sulfoalkyl chain in the order of
16 > 15 > 14 have almost no effect on the spectral maxima.
quantum yields from 27% to 32%, while the reduction of the sul-
foalkyl chain length in the order of 16 > 15 > 14 did not have
a noticeable impact on the quantum yield.
The molar absorptivities (
3
) are in the range of 240,000e
Due to the minimal aggregation tendency of the investigated
cyanine dyes in aqueous solutions at dye concentrations below
5 ꢁ 10ꢀ5 M, the increase in quantum yields with increasing number
of sulfo groups cannot be explained by the increased solubility of
the dye molecules but is more likely due to other reasons. The
electron withdrawing effect of sulfo groups could be considered as
one of these reasons: simulations of the electron density in a model
diethyl (C1), dibutyl (C2), and di-(sulfobutyl) (C3a) cyanines,
carried out by the PM3 method (Fig. 2), demonstrate that substi-
tution of the ethyl and butyl groups with sulfobutyl groups results
in a rearrangement of the electron density in such a way that the
positive charge at indolenine nitrogen increases from þ0.300 (C1)
and þ0.303 (C2) up to þ0.351 (C3a). The simulations indicate two
possible reasons for this effect: protonation of aromatic sulfo
groups and a direct electron withdrawing effect of the aliphatic
sulfo-group on the indolenine nitrogen by inductive (eI) mecha-
nism, which is further enhanced by the field effect through space.
While the aromatic sulfo group of the diethyl and dibutyl
derivatives C1 and C2 more likely exists in the deprotonated state,
the protonated form C3b seems to be the dominant form for di-
sulfobutyl-cyanines. According to our calculations form C3a with
deprotonated aliphatic sulfo groups is substantially more stable
(42 kcal/mol) compared to that of C3b (Fig. 2). The positive charge
on indolenine nitrogen in C3a (þ0.321) increases to þ0.351 in C3b.
Furthermore the aliphatic sulfo group has a direct electron with-
drawing effect on the indolenine nitrogen. The value of this effect
can be estimated via the positive charge on the indolenine nitrogen,
which increases from þ0.300 to þ0.303 in the diethyl (C1) and
dibutyl (C2) derivatives, respectively, to þ0.321 in the di-(sulfo-
butyl) cyanine C3b. It seems that the above electron withdrawing
effect propagates not only inductively (eI), evidenced by the elec-
tronic charge redistribution on the carbon atoms within the alkyl
chain (Fig. 2), but also due to the electrostatic interaction (attrac-
tion) between negatively charged sulfonyl oxygen and positively
charged indolenine nitrogen. The “field” interaction is supported by
the fact that the straight conformation of sulfobutyl chain with
s-trans allocation of all alkyl chain bonds in C3a and C3b (just this
conformation realizes in dibutyl derivative C2) becomes energeti-
cally unstable. At the same time C3a and C3b were found to exist in
the folded conformation, where the sulfonyl oxygen is approaching
260,000 Mꢀ1 cmꢀ1 and constant for each dye within the concen-
tration at least between 1 ꢁ 10ꢀ8 M and 5 ꢁ 10ꢀ5 M. The molar
absorptivities have demonstrated no clear correlation on the
cyanine dye structures.
The absorption and emission spectra exhibit a well-defined
shoulder (Fig. 1), which is known to be attributed to vibrational
transitions [27,28]. The shapes of the absorption and emission
spectra of the investigated dyes measured in a 0.1 cm cell are
unchanged within the concentrations between 1 ꢁ 10ꢀ8 M and
5 ꢁ 10ꢀ5 M regardless of the number and position of sulfoalkyl
groups, which indicates no aggregation in this concentration range.
Fig. 1 shows that the shapes of the spectra of cyanine 12 with three
sulfobutyl substituents are about the same as for Cy5 containing no
sulfoalkyl groups. Independent of the number and position of sul-
foalkyl groups, the shape and position of the absorption and
emission spectra of all the investigated cyanines measured at
c w 1 ꢁ10ꢀ8 M e 5 ꢁ 10ꢀ5 M is very similar (Fig. 1). This is evidence
that the dye molecules do not aggregate but also exhibit no
noticeable conformational changes and solvation effects in the
excited state. The Stokes’ shifts are between 18 and 19 nm
(w400 cmꢀ1) and independent on the positions of carboxypentyl
and sulfobutyl substituents on the indolenine ring and the sul-
foalkyl chain length.
Due to the observed high inner filter effects in both the excita-
tion and the emission paths at concentrations above w3 ꢁ 10ꢀ7 M
that can be attributed to the small Stokes shift of the dyes, and the
pronounced overlap between the absorption and emission bands
the emission spectra and quantum yields (FF) were measured using
0.1 cm cuvettes and at low dye concentrations.
Increasing the number of sulfo groups from two (Cy5) to five
(cyanine 12) (Table 2) in general leads to an increase of the
Table 1
The substituents in the dyes 7e16.
Dye
R1
R2
R3
R4
7
8
9
(CH2)4SO3H
(CH2)5COOH
(CH2)5COOH
(CH2)5COOH
(CH2)4SO3H
(CH2)5COOH
(CH2)5COOH
(CH2)3SO3H
(CH2)4SO3H
(CH2)4SO3H
(CH2)5COOH
(CH2)4SO3H
(CH2)4SO3H
Me
(CH2)5COOH
(CH2)4SO3H
Me
Me
Me
Me
Et
Et
(CH2)4SO3H
(CH2)4SO3H
Me
(CH2)4SO3H
(CH2)4SO3H
(CH2)4SO3H
Me
(CH2)5COOH
(CH2)5COOH
(CH2)5COOH
(CH2)4SO3H
(CH2)4SO3H
(CH2)4SO3H
(CH2)4SO3H
(CH2)4SO3H
(CH2)3SO3H
(CH2)3SO3H
(CH2)4SO3H
10
11
12
13
14
15
16
ꢀ
ꢀ
the indolenine nitrogen at a distance of about 3.8 A in C3a and 4.0 A
in C3b (Fig. 2).
Aliphatic sulfo groups are considered electron withdrawing
groups and expected to reduce the electronic charge on indolenine
nitrogen. In general electron withdrawing substituents at the

542
L.I. Markova et al. / Dyes and Pigments 96 (2013) 535e546
Table 2
Spectral characteristics of cyanine dyes (c w 1e3 ꢁ 10ꢀ7 M) and dyeeIgG conjugates in phosphate buffer pH 7.4.
Dye
Free dye
Dye IgG conjugate
lmax
3
ꢁ 10ꢀ3
,
lmax
FF, %
D/P ¼ 1
D/P ¼ 4
FF, %
(Abs), nm
Mꢀ1 cmꢀ1
(Em), nm
lmax
lmax
FF, %
(Abs), nm
(Em), nm
O3S
SO3H
Cy5
647
648
250
665
667
27
651
651
670
28
9
N
Et
N
(CH2)5
COOH
O3S
SO3H
13
260
240
28
32
670
670
33
35
15
N
N
(CH2)5
COOH
(CH2)4
SO3H
COOH
(CH2)5
O3S
SO3H
14
649
668
652
22
N
N
(CH2)3
SO3H
(CH2)3
SO3H
COOH
(CH2)5
O3S
SO3H
15
16
649
649
240
240
668
668
32
32
652
653
670
671
40
38
25
25
N
N
(CH2)4
SO3H
(CH2)3
SO3H
COOH
(CH2)5
O3S
SO3H
N
N
(CH2)4
SO3H
(CH2)4
SO3H
SO3H
(CH2
)
4
O
3S
SO3H
10
650
650
250
256
669
669
30
32
654
654
672
672
38
39
25
25
N
(CH2
COOH
N
(CH2
SO3
)
)
4
H
5
SO3H
(H2C)4
O
3S
SO3H
9
N
N
(CH2
COOH
)
(CH2
SO3
)
4
H
5
COOH
(H2C)5
SO3H
(CH2
)
4
O3S
SO3H
7
652
252
670
28
653
672
35
25
N
N
(CH2
)
Et
4
SO3H
SO3H
(H2C)4
SO3H
(CH2)4
O3S
SO3H
8
652
242
670
28
655
673
35
28
N
N
(CH2)5
COOH
Et

L.I. Markova et al. / Dyes and Pigments 96 (2013) 535e546
543
Table 2 (continued )
Dye
Free dye
Dye IgG conjugate
lmax
3
ꢁ 10ꢀ3
,
lmax
FF, %
D/P ¼ 1
D/P ¼ 4
(Abs), nm
Mꢀ1 cmꢀ1
(Em), nm
lmax
lmax
FF, %
FF, %
(Abs), nm
(Em), nm
673
COOH
(H2C)5
SO3H
(CH2
)
4
O3S
SO3H
11
653
250
672
31
655
38
32
N
N
(CH2
SO3
)
H
(CH2
SO3
)
4
H
4
SO3
(H2C)4
H
SO3
(CH2
H
)
4
O3S
SO3H
12
653
248
672
32
655
674
43
36
N
N
(CH2
)
(CH2
)
4
5
COOH
SO3
H
heterocyclic end groups are known to increase the quantum yields
of cyanine dyes [29,30], which is in good agreement with our
observations (Table 2). The reduced quantum yields of cyanines 7
and 8 could be explained by electron-donating effect of ethyl group
on indolenine nitrogen.
length on the photostability can be explained by different electron
withdrawing influence of sulfobutyl and sulfopropyl groups on the
electron distribution on cyanine dye molecules. The photostability
of cyanine dyes is known to increase upon introduction of electron
withdrawing substituents into the heterocyclic end groups [9,31].
Indeed quantum chemical simulations show that the sulfopropyl
group in the model cyanine C4 has a stronger electron withdrawing
effect in particular on indolenine nitrogen and polymethine chain
The fluorescence lifetimes (s) of cyanines 7e16 were measured to
be in the range between 1.00 and 1.24 ns (Table 3). There is a trend
of increased lifetimes with an increasing number of sulfobutyl
groups in the molecules in the order of Cy5 < 13 < 16 z
10 z 9 < 11 < 12. The sulfoalkyl chain length in cyanines 14e16
does not have a noticeable effect on the fluorescence lifetime.
The photostabilities of cyanine dyes were estimated via their
a-carbon atom as compared to sulfobutyl group in C3a (Fig. 2).
The electron withdrawing effect and the different impact of
sulfopropyl and sulfobutyl substituents is revealed also from the 1H
NMR data (200 MHz, DMSO-d6): The hydrogen signals of
ylene NCH2 group at indolenine nitrogen exhibit different chemical
shifts ( ) depending on how far they are from the electron-
a-meth-
photobleaching half-life (s1/2), which is the time to decrease the
absorption and emission intensity by half upon irradiation.
d
Increasing the number of sulfobutyl groups noticeably increases
withdrawing sulfo group: 4.25 ppm for sulfopropyl cyanine 14
and 4.10 ppm for sulfobutyl cyanine 16, while cyanine 15 has the
two signals: 4.26 ppm and 4.10 ppm. The same effect of the sul-
foalkyl chain length on the hydrogen chemical shifts of the indo-
lenines precursors 5g/5f: NCH2, 4.60/4.46 ppm, triplet; 2-CH3, 2.80/
2.84 ppm, singlet; 3-(CH3)2, 1.52/1.54 ppm, singlet.
photostability in the order: Cy5 < 13 ꢅ 10
ꢅ
9
< 16
ꢅ
8 z 7 < 12 z 11. The photostability increases by factor 2.2e2.3
(Table 4), when the total number of sulfo groups increases from
two (Cy5) to five (cyanines 11 and 12). The position of sulfobutyl
and carboxypentyl groups on the cyanine molecule seems to have
less of an effect on the photostability.
Importantly a shortening of the sulfoalkyl chain from sulfobutyl
in cyanine 16 to sulfopropyl (14) remarkably increases photo-
stability by about 30% (Table 4, Fig. 3). The effect of the alkyl chain
3.3. Dye IgG conjugates
Upon conjugation of cyanine dyes to antibodies (IgGs) the
absorption and emission maxima are typically shifted by up to
5 nm. This trend is more pronounced for less hydrophilic cyanines
such as Cy5 and 13 containing lower number of sulfoalkyl groups
(Table 2). Similar to free dyes, an increase in the number of sulfo-
butyl groups results in a red-shift of up to 4 nm. The dye-to-protein
(D/P) ration seems to have almost no effect on the spectral maxima.
The absorption spectra of cyanines show absorption of the dye
aggregates which are typically blue-shifted compared to the
absorption maximum (Fig. 1) and its intensity increases with
increasing D/P ratios. These aggregation bands with maxima
around 605 nm are well-defined for Cy5eIgG conjugates at D/P ¼ 4,
but are strongly reduced for more hydrophilic cyanines such as 12.
The quantum yields (FF) of dye-conjugates are known to be
strongly dependent on the dye-to-protein ratio (D/P): in general an
increased D/P is accompanied with a decrease in FF (Fig. 4) but the
decrease in quantum yields becomes less pronounced for dyes with
an increased number of sulfobutyl groups (Table 2). The decrease in
FF is most likely due to the interaction of dye molecules on the
protein, which can lead to the formation of non-fluorescent or low-
fluorescent aggregates and or homo-FRET (fluorescence resonance
Fig. 1. Absorption and emission spectra of Cy5 and cyanine 12 free in solution at
concentrations 1 ꢁ 10ꢀ8 M and 5 ꢁ 10ꢀ5 M (solid line) and bound to IgG at D/P ¼ 4
(dashed line).

544
L.I. Markova et al. / Dyes and Pigments 96 (2013) 535e546
Table 3
Fluorescence lifetimes (s) of cyanine dyes in phosphate buffer (pH 7.4).
Dye
s, ns
c2
Cy5
13
14
15
16
10
9
7
8
11
12
1.00
1.04
1.11
1.10
1.11
1.11
1.12
1.05
1.06
1.18
1.24
1.20
1.07
1.33
1.38
0.91
1.48
1.17
1.62
0.92
1.22
1.13
Table 4
Photostability (
s
1/2) of cyanine dyes (c w 10ꢀ7 M) in aqueous solutions.
Dye
(
s1/2), min
Absorption
Emission
Cy5
13
14
15
16
10
9
7
8
11
12
103
132
247
270
185
144
144
190
185
237
230
114
152
235
237
176
152
170
210
204
263
258
Fig. 3. Decrease of the long-wavelength absorption (a) and emission (b) band of
cyanine dyes and their IgG conjugates under light exposure vs. the sulfoalkyl chain
length.
Fig. 2. The PM3 calculated conformational structures, electron charges, and enthalpies
of formation (DHf) of the model cyanine molecules.

L.I. Markova et al. / Dyes and Pigments 96 (2013) 535e546
545
sulfobutyl groups has a strong influence on the aggregation
tendency of the dyes upon covalent labeling to IgG resulting in
increased quantum yields and photostabilities of the IgG-
conjugates.
Acknowledgment
This work was supported by the Science and Technology Centre
in Ukraine (project No. P313) and the National Academy of Sciences
of Ukraine (project No. 0801051004).
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