Promising near-infrared non-targeted probes: Benzothiazole heptamethine cyanine dyes

Article abstract of DOI:10.1080/17415993.2013.778258

A series of benzothiazole heptamethine cyanine dyes have been synthesized and their photophysical properties evaluated in relation to their structural features. These have been compared against two classical probes of this type: Indocyanine Green (IGC) and New Indocyanine Green (IR-820). Growth inhibitory studies were also performed using a eukaryotic, unicellular organism, fission yeast Schizosaccharomyces pombe. Herein we highlight some potentially interesting candidates with improved fluorescence quantum yields when compared with ICG and IR-820.

Full text of DOI:10.1080/17415993.2013.778258

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Promising near-infrared non-targeted
probes: benzothiazole heptamethine
cyanine dyes
Okoh Adeyi Okoha, Roger H. Bisbyb, Clare L. Lawrencec, Carole E.
Rolphc & Robert B. Smitha
a Centre for Material Sciences, University of Central Lancashire,
Preston PR1 2HE, UK
b School of Environment and Life Sciences, University of Salford,
Salford M5 4WT, UK
c School of Pharmacy and Biomedical Sciences, University of
Central Lancashire, Preston PR1 2HE, UK
Published online: 10 Apr 2013.
To cite this article: Okoh Adeyi Okoh, Roger H. Bisby, Clare L. Lawrence, Carole E. Rolph & Robert
B. Smith (2014) Promising near-infrared non-targeted probes: benzothiazole heptamethine cyanine
dyes, Journal of Sulfur Chemistry, 35:1, 42-56, DOI: 10.1080/17415993.2013.778258
To link to this article: http://dx.doi.org/10.1080/17415993.2013.778258
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Journal of Sulfur Chemistry, 2014
Vol. 35, No. 1, 42–56, http://dx.doi.org/10.1080/17415993.2013.778258
Promising near-infrared non-targeted probes: benzothiazole
heptamethine cyanine dyes
OkohAdeyi Okoh,a Roger H. Bisby,b Clare L. Lawrence,c Carole E. Rolphc and Robert B. Smitha*
aCentre for Material Sciences, University of Central Lancashire, Preston PR1 2HE, UK; bSchool of Environ-
ment and Life Sciences, University of Salford, Salford M5 4WT, UK; cSchool of Pharmacy and Biomedical
Sciences, University of Central Lancashire, Preston PR1 2HE, UK
(Received 31 December 2012; final version received 17 February 2013)
A series of benzothiazole heptamethine cyanine dyes have been synthesized and their photophysical prop-
erties evaluated in relation to their structural features. These have been compared against two classical
probes of this type: Indocyanine Green (IGC) and New Indocyanine Green (IR-820). Growth inhibitory
studies were also performed using a eukaryotic, unicellular organism, fission yeast Schizosaccharomyces
pombe. Herein we highlight some potentially interesting candidates with improved fluorescence quantum
yields when compared with ICG and IR-820.
S
Cl
S
N+
R X–
N
R
S
S
N+
X–
N
R
R
Keywords: benzothiazole; cyanine dyes; fluorescence; growth inhibition; near infrared
1. Introduction
The detection, imaging and quantification of biomolecules using fluorescent probes is an area
of increasing interest and importance (1–4). With dyes that fluoresce in the visible region, auto-
fluorescence of a sample matrix may interfere (5) The use of near-infrared (NIR) probes (700–
1000 nm) has generated a vast array of interest, as these can be efficiently used to visualize and
investigate in-vivo molecular targets as most tissues generate little NIR fluorescence (6). Other
useful characteristics of probes operating in NIR region are that they often have low phototoxicity,
*Corresponding author. Email: rbsmith@uclan.ac.uk
© 2013 Taylor & Francis

Journal of Sulfur Chemistry 43
advantageous for cell and tissue imaging (7) and deeper light penetration in tissues is achieved in
the NIR.
Two classical NIR probes are Indocyanine Green (IGC) and New Indocyanine Green (IR-820).
ICG is commercially available and approved by the United States Food and Drug Administration
for use in clinical applications, in particular, evaluating blood flow (8–11) and clearance (11–13).
It also has the potential as a tool for fluorescence-guided management and treatment of cancer
(14, 15). One of the main drawbacks of ICG is its low fluorescence quantum yield (16–18). IR-
820 is structurally related to ICG except with the incorporation of a rigid cyclohexane moiety
providing improved in vitro and in vivo stability. However, it is documented that IR-820 has a
notably lower quantum yield than ICG (19).
The literature reports that benzothiazole heptamethine cyanine dyes featuring two benzothia-
zole rings linked by a polymethine chain possess the greatest potential for use in visualization
of cellular changes occurring in vivo due to their excellent biocompatibility (20). Patonay and
Antoine (21) have previously reported that heptamethine cyanine dyes bearing sulfur atoms within
their heterocyclic ring system bind preferentially to human serum albumin binding sites and fur-
thermore, the sulfur atom on the heterocyclic ring system could enhance the specificity of the
dye molecule. Waggoner and coworkers (22, 23) have also reported that the presence of sulfonate
groups on the aromatic rings of the dyes helps to increase aqueous solubility and thereby reduces
aggregation.
Motivated by this, we report the synthesis and photophysical properties of a series of struc-
turally related non-targeting NIR benzothiazole heptamethine cyanine dyes. These contain either
a chain that is partially rigidified by the inclusion of a central cyclic ring (compounds 3a–g) or the
simple heptamethine conjugated chain (compounds 5a–g). Our intention is to compare the pho-
tophysical (absorption and fluorescence spectra, fluorescence quantum yields and Stokes shifts)
and biological properties of the dyes bearing a sulfur moiety (3 and 5a–g) against those of the
Figure 1. The structures of IR-820, ICG, OA1 and OA2.

44 O.A. Okoh et al.
standard fluorophores ICG and IR-820. As shown in Figure 1, the established ICG (linear chain)
and IR-820 (rigidified chain) dyes have terminal dimethyl-indolyl groups with an additional fused
benzene ring. Two further analogs of IR-820 and ICG (OA1 and OA2, respectively) have thus
been synthesized, which lack the additional fused ring (also shown in Figure 1). This allows the
direct evaluation of the effects of replacement of the dimethyl-indolyl group by the benzothiazole
ring on spectroscopic and biological properties.
The impact of this research is timely, highlighting the development of cost-effective molecu-
lar probes with enhanced photophysical and toxicity characteristics when compared against the
current clinical standard ICG and IR820. The synthesis of the linear heptamethine dyes (5a–g)
is in itself significant. This in situ cascade reaction is a modification of the Zincke reaction and
provides an elegant approach to the linear dyes which can be tailored to develop more structurally
sophisticated cyanine dyes.
2. Results and discussion
2.1. Synthesis
Synthesis of both the linear and rigid cyanine dyes (3a–g and 5a–g) was straightforward and
required no harsh or unusual synthetic methodology. The starting benzothiazolium salts were
preparedbythealkylationofbenzothiazoleineithertolueneoracetonitrileusingthecorresponding
alkyl halides or sulfones to afford the benzothiazolium salts (1a–g) (Scheme 1). The iminium
salt (2) was prepared by Vilsmeier–Haack formylation of cyclohexanone (26) (Scheme 1). The
rigid cyanine dyes (3a–g) and linear cyanine dyes (5a–g) were prepared using an aldol-like
condensation of the benzothiazolium salts and the iminium salt, in the presence of sodium acetate
as the base. The reaction mixture was heated under reflux in ethanol overnight (24) to produce the
crude dyes (3a–g) (Scheme 2). The linear cyanine dyes (5a–g) were produced using a standard
method to produce the 5-anilino-N-phenyl-2,4-pentadienylideniminium chloride (25) followed
by the in situ addition of 1a–g in the presence of sodium acetate as the base (Scheme 3). The
reaction mixture was stirred in ethanol overnight to produce the crude dyes (5a–g) (26). All the
crude dyes were purified by column chromatography using silica gel to obtain the pure compounds
(3a–g and 5a–g). It should be noted that the N-alkylations for dyes 3a–g are the same as 5a–g;
these groups are shown in Scheme 1 under R.
Scheme 1. The synthetic route to the alkylated salts (1 a–g) and the Vilsmeier–Haack iminium
salt (2).

Journal of Sulfur Chemistry 45
Scheme 2. The synthetic route to the rigid benzothiazole heptamethine cyanine dyes (3a–g).
a = Me
b = Et
c = n-Pr
d = n-Bu
e = CH2(CH2)2SO3
N+
S
–
S
(1 a-g)
–
Aniline
EtOH
Cl
NO2
+
+
NaOAc
EtOH
N
N
H
N
N
H
–
–
X
Cl
–
R
R
f = CH2(CH2)3SO3
g = Bn
Intermediate
5 (a-g)
NO2
4
Scheme 3. The synthetic route to the rigid benzothiazole heptamethine cyanine dyes (5a–g).
2.2. Photophysical properties
The photophysical properties of the benzothiazole heptamethine cyanine dyes used in this study
are summarized in Tables 1 and 2. Stock solutions of dyes were prepared in methanol. The
absorption and fluorescence spectra of each of the dyes were measured sequentially to reduce
photobleaching and solubility issues. The fluorescence quantum yields of the dyes were calculated
using the relative method, i.e. integrated fluorescence peak area versus fraction of light absorbed
at the excitation wavelength were plotted for both the standards and cyanine dyes (27). It is well
noted that rigidification of the polymethine chain is an established approach for improving the
chemical and photostability of NIR dyes (28).
Table 1. Photophysical and growth inhibition data for rigid benzothiazole heptamethine cyanine dyes (3a–g), IR-820
and OA1 in MeOH. Quantum yields 10%, λmax 1 nm.
Compounds
R
Fluorescence studies
Absorption Fluorescence Fluorescence
Growth inhibition studies
Stokes shift Minimum growth
Code
λmax (nm) emission (nm) quantum yield (ꢀ)
(nm)
inhibition (μM) Log P
3a
3b
3c
3d
3e
3f
Me
Et
n-Pr
796
796
797
796
798
800
804
820
782
812
812
814
813
815
817
820
840
802
0.087
0.087
0.091
0.093
0.066
0.082
0.093
0.032
0.085
16
16
17
17
17
17
16
20
20
0.66a
0.63a
3.923
4.675
5.68
0.60a
n-Bu
0.05a
6.799
CH2(CH2)2SO−3
CH2 (CH2)3SO−3
Bn
18.43b
17.71b
1.01b
11.78b
17.2b
−2.424
−1.883
9.176
1.441
−0.877
3g
IR-820 Commercial dye
OA1
CH2(CH2)3SO−3
Notes: aNo visible yeast growth observed at this concentration of dye.
bHighest possible concentration tested, visible yeast growth observed.

46 O.A. Okoh et al.
Table 2. Photophysical and growth inhibition data for linear benzothiazole heptamethine cyanine dyes (5a–g)
ICG and OA2 in MeOH. Quantum yields 10%, λmax 1 nm.
Compounds
R
Fluorescence studies
Growth inhibition studies
Stokes shift Minimum growth
Absorption Fluorescence
Fluorescence
Code
λmax (nm) λmax (nm) quantum yield (ꢀ)
(nm)
inhibition (μM) Log P
5a
5b
5c
5d
5e
5f
Me
Et
n-Pr
756
759
761
761
762
762
765
785
747
783
785
786
787
789
789
791
814
775
0.165
0.161
0.176
0.181
0.146
0.166
0.229
0.072
0.13
27
26
25
26
27
27
26
29
28
1.21b
0.57a
2.805
3.557
4.562
0.54a
n-Bu
0.06a
5.681
CH2(CH2)2SO−3
CH2(CH2)3SO−3
Bn
16.58b
15.85b
4.62b
−1.733
−1.192
5.776
1.591
−0.728
5g
ICG Commercial dye
12.92b
15.36b
OA2 CH(CH2)3SO−3
Notes: aNo visible yeast growth observed at this concentration of dye.
bHighest possible concentration tested, visible yeast growth observed.
2.2.1. Absorption and fluorescence spectra
This study focused on the structural diversity of the NIR dyes and the results obtained from
this experiment show that the rigid benzothiazole heptamethine cyanine dyes (3a–g) exhibited
absorption spectra maxima at longer wavelengths than the linear dyes (5a–g), with all absorbing
in the NIR region between 747 and 820 nm. Tables 1 and 2 demonstrate that rigidification shifts
the absorption and fluorescence maxima approximately 30–40 nm to the red when comparing 3a
(796 and 812 nm, respectively) with 5a (756 and 783 nm).
This increase is not unusual and can also be seen when comparing ICG (785 and 814 nm)
with IR-820 (820 and 840 nm). It is also interesting to note that the replacement of a 3,3-
dimethylindolenine ring with a benzathiazole also shifts the absorption and fluorescence maxima
deeper into the red as shown by comparing OA1 (782 and 802 nm) with 3f (800 and 817 nm) and
OA2 (747 and 775 nm) with 5f (762 and 789 nm). Indeed, this comparison is seen throughout
both series in Tables 1 and 2.
The similarities in the absorbance wavelength of the dyes could be attributed to the fact that
the N-donor and N-acceptor substituting groups are too far away from the chromophore and as
such have very little influence on the spectroscopic properties of the dyes.
2.2.2. Fluorescent quantum yields
An interesting observation is that the rigid benzothiazole heptamethine cyanine dyes (3a–g)
demonstrated an approximate three-fold increase in quantum yield when compared with IR-
820 but showed comparable quantum yields when compared with OA1. The linear benzothiazole
heptamethine dyes (5a–g) show a two-fold increase in quantum yield when compared with ICG
as well as an approximate 1.5-fold increase when compared with OA2. We also note that rigidi-
fication of the polymethine chain reduces the quantum yields for fluorescence by approximately
50%, as shown by comparing 5a (ꢀ = 0.165) with 3a (ꢀ = 0.087). This reduction in quantum
yield is also seen when comparing ICG (ꢀ = 0.072) with IR-820 (ꢀ = 0.032) and when com-
paring OA1 (ꢀ = 0.085) with OA2 (ꢀ = 0.13). The lower quantum yield in methanol for IR820

Journal of Sulfur Chemistry 47
relative to that for ICG is also consistent with the shorter fluorescence lifetime of 250 ps for IR820
compared with 510 ps for ICG (29). It is interesting to note that the introduction of a N-benzyl
group to the non-restricted linear polymethine chain increases the quantum yields for fluorescence
by comparing 5g (ꢀ = 0.229) with 3g (ꢀ = 0.093).
Due to these interesting quantum yields, the benzothiazole heptamethine cyanine dyes (3a–g
and 5a–g) show potential for clinical applications when compared with the clinical standard ICG.
2.2.3. Stokes shifts
We note the Stokes shifts for the rigid dyes 3a–g show comparable shifts (16–20 nm) when
compared against IR-820 and OA1. This is also the case for the unrestricted dyes 5a–g (25–29 nm)
upon comparison with ICG and OA2.
2.3. Growth inhibitory screening
Apart from the photophysical characteristics of the dyes, the solubility and growth inhibitory
properties are important characteristics which determine the practical use of these fluorophores
(3a–g and 5a–g) in living cells. The effects of the newly synthesized dyes on cell growth were
compared with ICG, IR-820, OA1 and OA2. Dimethyl sulfoxide (DMSO), ICG, IR820, OA1 and
OA2 were used as controls throughout the screening process.
Comparative growth inhibitory studies were performed using Schizosaccharomyces pombe, an
eukaryotic microorganism previously used as an important and valuable screening tool for the
identification of toxicity (30, 31). This is important should these dyes show potential for clinical
applications. Using S. pombe as a model for mammalian cells is advantageous as many cellular
processes are highly conserved between the two (30, 31). Cell permeability and growth inhibition
can be tested simultaneously and S. pombe strains are low in cost, easy to prepare and the results
obtained are highly reproducible (30).
The growth inhibitory screening procedure involved the inoculation of S. pombe wild-type cells
(NJ2 h− ura4-D18 leu1-32 ade6-M210 his7-366) (32) into yeast extract broth (YE), as previously
reported (33). The culture was then incubated overnight at 30◦C with shaking at 200 rpm. Stock
solutions of the dyes were prepared in 20% DMSO in YE. DMSO and YE were used as controls
for the experiment. 3 × 104 yeast cells were transferred into the wells of a 96-well plate. A 1:2
serial dilution of the dyes was then performed. The well plates were visually inspected for growth
after 24 h of incubation at 30◦C. Growth was indicated by full or partial appearance of yeast on
the bottom of the wells. The minimum growth inhibitory concentration of the dyes was estimated
to be the concentration of the compound in the well before yeast growth was first seen. The
experiment was repeated three times to ensure reproducibility of the results.
For a number of dyes, no growth was observed at the lowest concentration tested. These are
included in Tables 1 and 2, where the lowest concentration tested is indicated. In addition, a
number of dyes had limited solubility; the values within the tables indicate the highest possible
concentration tested. Wells containing 10% DMSO and only YE showed 100% yeast growth.
The results in Tables 1 and 2 show that most of the dyes which exhibited no growth inhibitory
effects were the dyes bearing the sulfonic acid group (3e–f and 5e–f) on their N-donor and
N-acceptor systems. Most of the dyes that exhibited growth inhibition were the dyes bearing
the alkyl or benzyl group at their N-donor and N-acceptor system (3a–d, f and 5a–d, f). We
therefore suggest that the growth inhibitory effect of these dyes could be attributed to the nature
of substituent on the N-donor and N-acceptor system and using a virtual method, the log P
(the base 10 logarithm was used throughout) values were obtained (log P values were obtained
from http://www.molinspiration.com) and these are shown in Tables 1 and 2. The presence of the

48 O.A. Okoh et al.
sulfonic acid group on the dyes tends to increase their solubility and reduce their growth inhibitory
characteristics, possibly through suppressing membrane permeability and cellular uptake. The
dyes bearing the sulfonic groups are structurally very similar to ICG, IR820, OA1 and OA2.
The presence of the alkyl group tends to reduce the solubility and increase the growth inhibitory
characteristic of the dyes due to their lipophilic nature. Our findings also indicated that the linear
benzothiazole heptamethine cyanine dyes (5a–g) were more soluble and more bio-assimilable
than the rigid benzothiazole heptamethine cyanine dyes (3a–g).
3. Conclusions
In summary, a series of benzothiazole heptamethine cyanine dyes have been synthesized and their
photophysical properties have been evaluated by placing emphasis on their structural diversity
and nature of the substituent on the N-donor and N-acceptor system. It is clear to see that both
series of benzothiazole dyes (3a–g and 5a–g) show notably improved fluorescence quantum yield
in comparison to ICG, IR820, OA1 and OA2. It is also clear to see that each of the dyes show
increased bathochromic shift when compared against OA1 and OA2. However, the fused benzyl
rings on ICG and IR-820 lead to an increased bathochromic shift by approximately 20 nm into red.
The growth inhibitory characteristics of the dyes were also affected by the nature of substituent
on the N-donor and N-acceptor system. It is noted that compounds 3e–f and 5e–f showed low
growth inhibitory effects and are comparable to ICG, IR820, OA1 and OA2. To conclude, the
benzothiazole heptamethine cyanine dyes reported herein show huge potential as new fluorescent
probes.
4. Experimental
4.1. General information
1H and 13C NMR spectra were measured on either a Bruker DPX 250 MHz, 300 MHz or a
Bruker Avance 400 MHz spectrometer with tetramethylsilane (TMS) as an internal standard for
1H NMR and deuteriochloroform (CDCl3, 77.23 ppm) and deuteriodimethylsulfoxide (d6-DMSO,
39.52 ppm) for 13C NMR unless otherwise stated. All chemical shifts are quoted in δ (ppm) and
coupling constants in Hertz (Hz) using the high-frequency positive convention. The abbreviations
used for the multiplicity of the NMR signals are: s = singlet, d = doublet, t = triplet, q = quartet,
quin = quintet, sex = sextet, m = multiplet, dd = doublet of doublet, td = triplet of doublets,
dm = doublet of multiplets, brs = broad singlet, etc. Low- and high-resolution mass spectra were
obtained using electrospray ionization mass spectrometry on a hybrid linear ion trap-Fourier
transform mass spectrometer. The ultraviolet absorptions were recorded on a WPA Lightwave II
UV/Visible spectrophotometer. IR spectra were recorded on a Specac ATR with a He/Ne-633 nm
laser. Thin layer chromatography was carried out on Machery-Nagel polygram Sil/G/UV254 pre-
coated plates. Melting point (Mp) analysis was carried out in capillary tubes using the Griffin
Mp apparatus. All chemicals, solvents and silica gel were obtained from Sigma Aldrich and used
without further purification.
4.2. Photophysical information
Fluorescence quantum yields (ꢀ) were measured using a fluorimeter based on an Innovative Pho-
tonic Solutions 785 nm diode laser, operating with a power output of 8 mW, an Andor Shamrock

Journal of Sulfur Chemistry 49
SR-303i spectrograph and anAndor iDus CCD detector (model DU420A-BR-DD). Fluorescence
was detected at right angles without any filters. The spectral response of the system was cor-
rected following the method outlined by Kosch et al. (34). Absorbance values (A) of solutions at
785 nm were measured using a Perkin Elmer Lambda 25 spectrophotometer. Corrected fluores-
cence spectra for a series of solutions of increasing concentration were rescaled in energy and
the integrated intensity was plotted versus 1–10−A, with A (785 nm) ≤0.1. The quantum yields
were obtained from the relative slopes of such plots compared with that from solutions of ICG
in DMSO, for which a quantum yield of ꢀ = 0.13 has been reported (35). For determinations in
different solvents, the refractive index (n2) correction was applied.
4.3. Synthesis
Below is the general synthetic procedure for salts 1a–g. Full spectroscopic data are given for all
compounds.
4.3.1. 2-Methyl-3-(methyl)-benzothiazolium iodide (1a)
To a solution of 2-methylbenzothiazole (0.94 g, 6.30 mmol) in toluene (50.0 ml) was added
iodomethane (10.5 ml, 168 mmol) and with constant stirring, the solution was refluxed for 18 h.
Upon cooling, the precipitate produced was filtered under pressure, washed with n-hexane and
1
dried in vacuo to yield 1a (1.57 g, 87%) as a white solid. H NMR (d6-DMSO, 250 MHz) δ
8.52 (d, J = 8.0 Hz, 1H, Ar-H), 8.35 (d, J = 9.0 Hz, 1H, Ar-H), 7.95 (t, J = 7.0 Hz, 1H, Ar-H),
7.81 (t, J = 9.0 Hz, 1H, Ar-H), 4.23 (s, 3H, N-CH3), 3.11 (s, 3H, C-CH3). 13C NMR (d6-DMSO,
62.8 MHz) δ 179.2, 141.2, 129.8, 129.2, 123.4, 118.5, 83.2, 19.1. IR (ATR) 2965 (CH), 1548
(C N), 1441, 1336, 1210, 1131, 1011, 764, 717, 579 cm−1. MS (ESI) m/z: 164 [M+]. Mp
=
227–231◦C, Literature Mp 225-226 ◦C (36).
4.3.2. 2-Methyl-3-(ethyl)-benzothiazolium iodide (1b)
Yield: 1b (0.78 g, 41%) as a white solid. 1H NMR (d6-DMSO, 250 MHz) δ 8.49 (d, J = 7.0 Hz,
1H, Ar-H), 8.40 (d, J = 7.0 Hz, 1H, Ar-H), 7.83 (t, J = 8.0 Hz, 1H, Ar-H), 7.79 (t, J = 7.0 Hz,
1H, Ar-H), 4.75 (q, J = 7.0 Hz, 2H, N-CH2), 3.19 (s, 3H, N-C-CH3), 1.45 (t, J = 9.0 Hz, 3H,
C-CH3). 13C NMR (d6-DMSO, 62.8 MHz) δ 176.8, 140.4, 129.1, 128.1, 124.7, 116.6, 44.7, 16.7,
m−1
=
13.2. IR (ATR) 2912 (CH), 1613 (C N), 1513, 1445, 1329, 1270, 1203, 1100, 777, 713, 670 c
.
MS (ESI) m/z: 178 [M+]. Mp 196–200◦C, Literature Mp 198-199◦C (37).
4.3.3. 2-Methyl-3-(propyl)-benzothiazolium iodide (1c)
Yield: 1c (1.62 g, 81%) as a white solid. 1H NMR (d6-DMSO, 400 MHz) δ 8.51 (d, J =
6.0 Hz, 1H, Ar-H), 8.42 (d, J = 6.0 Hz, 1H, Ar-H), 7.93 (t, J = 9.0 Hz, 1H, Ar-H), 7.84 (t,
J = 6.0 Hz, 1H, Ar-H), 4.74 (t, J = 9.0 Hz, 2H, N-CH2-CH2), 3.25 (s, 3H, N-C-CH3), 1.93
(q, J = 6.0 Hz, 2H, CH2-CH2), 1.05 (t, J = 6.0 Hz, 3H, CH2-CH3). IR (ATR) 2919 (CH), 1607
(C N), 1518, 1449 cm−1. MS (ESI) m/z: 192.21 [M+]. Mp 199–203◦C.
=
4.3.4. 2-Methyl-3-(butyl)-benzothiazolium iodide (1d)
Yield: 1d (3.65 g, 33%) as a cream solid. 1H NMR (d6-DMSO, 300 MHz) δ 8.48 (d, J = 6.0 Hz,
1H, Ar-H), 8.37 (d, J = 6.0 Hz, 1H, Ar-H), 7.92 (t, J = 6.0 Hz, 1H, Ar-H), 7.83 (t, J = 6.0 Hz,

50 O.A. Okoh et al.
1H, Ar-H), 4.74 (t, J = 6.0 Hz, 2H, N-CH2-CH2), 3.23 (s, 3H, N-C-CH3), 1.86–1.71 (m, 2H,
N-CH2-CH2-CH2), 1.49-1.31 (m, 2H, N-CH2-CH2-CH2), 0.96 (t, J = 9.0 Hz, 3H, CH2-CH3).
13C NMR (75.4 MHz, CDCl3): δ 177.5, 141.3, 129.5, 128.5, 125.1, 117.3, 49.5, 30.2, 19.7, 17.4,
14.0. IR (ATR) 2951(CH), 1577 (C N), 1439, 1378, 1280, 948, 764 cm−1. MS (ESI) m/z: 206.09
=
[M+]. Mp 189–193◦C, Literature Mp 186–187◦C (38).
4.3.5. 2-Methyl-3-(4-sulfopropyl)-benzothiazolium (1e)
1
Yield: 1e (15.3 g, 91%) as a white solid. H NMR (d6-DMSO, 250 MHz) δ 8.23–7.80 (m, 2H,
Ar-H), 7.28 (t, J = 7.0 Hz, 1H, Ar-H), 7.67 (t, J = 7.0 Hz, 1H, Ar-H), 4.72 (t, J = 7.0 Hz, 2H,
N-CH2), 3.08 (s, 3H, N-C-CH3), 3.02 (t, J = 7.0 Hz, 2H, SO3-CH2), 2.28–2.20 (m, 2H, CH2-
CH2-CH2). IR (ATR) 2917 (CH), 1511 (C N), 1413, 1353, 1263, 1032, 756, 663 cm−1. MS
=
(ESI) m/z: 272.20 [M+]. Mp 258–262◦C, Literature Mp 276–278◦C (39).
4.3.6. 2-Methyl-3-(4-sulfobutyl)-benzothiazolium (1f)
1
Yield: 1f (9.29 g, 52%) as a white solid. H NMR (D2O, 400 MHz) δ 8.06 (d, J = 8.0 Hz, 1H,
Ar-H), 8.02 (d, J = 8.0 Hz, 1H, Ar-H), 7.76 (t, J = 6.0 Hz, 1H, Ar-H), 7.66 (t, J = 7.0 Hz, 1H,
Ar-H), 4.65 (t, J = 8.0 Hz, 2H, N-CH2), 3.06 (s, 3H, N-C-CH3), 2.87 (t, J = 7.0 Hz, 2H, SO3-
CH2), 2.01–1.96 (m, 2H, N-CH2-CH2-CH2), 1.83–1.78 (m, 2H, CH2-CH2-CH2-SO3). 13C NMR
(D2O, 62.8 MHz) δ 175.9, 140.9, 129.6, 128.9, 128.4, 123.6, 116.4, 50.0, 48.9, 26.3, 21.4, 16.2.
IR (ATR) 2947 (CH), 1523 (C N), 1443, 1343, 1283, 1175, 1032, 796, 777, 714, 683 cm−1. MS
=
(ESI) m/z: 286 [M+]. Mp 258–262◦C, Literature Mp 294◦C (40).
4.3.7. 2-Methyl-3-(2-benzyl)-benzothiazolium bromide (1g)
Yield: 1g (0.13 g, 7%) as a pink solid. 1H NMR (d6-DMSO, 250 MHz) δ 8.47 (d, J = 7.0 Hz, 1H,
Ar-H), 8.12 (d, J = 7.0 Hz, 1H, Ar-H), 7.45-7.40 (m, 2H, Ar-H), 7.32–7.11 (m, 5H, Ar-H), 6.12
(s, 2H, CH2-Ph), 3.21 (s, 3H, C-CH3). 13C NMR (d6-DMSO, 62.8 MHz) δ ppm 186.4, 178.4,
140.9, 132.7, 129.5, 129.2, 128.1, 126.9, 124.8, 117.0, 51.8, 17.2. IR (ATR) 2938 (CH), 1578
(C N), 1455, 1339, 1021, 808, 748, 577 cm−1. MS (ESI) m/z: 249 [M+]. Mp 94–98◦C, Literature
=
Mp 93◦C (41).
4.3.8. N-[5-Anilino-3-chloro-2,4-(propane-1,3-diyl)-2,4-pentadiene]-anilinium chloride (2)
At 0◦C, phosphorus oxychloride (11.0 ml, 120 mmol) was added dropwise from a pressure-
equalizing funnel to anhydrous DMF (13.0 ml, 170 mmol) with stirring. After 30 min, cyclo-
hexanone (5.50 ml, 53.0 mmol) was added and the mixture was heated under reflux for 1 h. The
reaction was cooled to 20◦C and with stirring, a mixture of aniline/EtOH [1:1 (v/v), 180 ml] was
added dropwise to the reaction mixture. The reaction was continued for 30 min at 20◦C with vig-
orous stirring, and then the deep purple mixture was poured into H2O/HCl [10:1 (v/v), 110 ml].
Crystals were allowed to form for 2 h in an ice bath. The reaction was filtered, washed with cold
H2O and Et2O, and then dried in vacuo to yield 2 (14.8 g, 97%) as a dark purple solid. 1H NMR
(d6-DMSO, 250 MHz) δ 11.2 (s, 2H, NH), 8.55 (d, J = 14.0 Hz, 2H, CHalkene), 7.68 (d, J = 5.0 Hz
4H, Ar-H), 7.44 (t, J = 7.0 Hz 4H, Ar-H), 3.03 (t, J = 6.0 Hz, 4H, CH2-CH2), 2.05–1.98 (m, 2H,
CH2-CH2). IR (ATR) 1609 (C N), 1562, 1457, 1267, 1179, 752, 683, 565 cm−1. MS (ESI) m/z:
=
323.22 [M+]. Mp 226–231◦C.
Below is the general synthetic procedure for rigid cyanine dyes 3a–g and OA1. Full
spectroscopic data are given for all compounds along with chromatography details where required.

Journal of Sulfur Chemistry 51
4.3.9. 2-[(E)-2-[(3E)-2-Chloro-3-{2-[(2Z)-3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]
ethylidene}cyclohex-1-en-1-yl]ethenyl]-3-methyl-1,3-benzothiazol-3-ium iodide (3a)
To a solution of 1a (0.29 g, 1.0 mmol) in EtOH (20.0 ml) was added anhydrous sodium acetate
(0.08 g, 1.00 mmol) and 2 (0.18 g, 0.50 mmol). With constant stirring, the reaction was heated
under reflux for 3.5 h. Upon cooling, green crystals precipitated from the reaction mixture. These
were washed with n-hexane and dried in vacuo to yield the cyanine dye 3a (0.30 g, 51%) as green
crystals. 1H NMR (d6-DMSO, 400 MHz) δ 7.95 (d, J = 7.0 Hz, 2H, Ar-H), 7.70 (d, J = 13.0 Hz,
2H, CHalkene), 7.70 (d, J = 8.0 Hz, 2H, Ar-H), 7.52–7.48 (m, 2H, Ar-H), 7.34 (t, J = 8.0 Hz,
2H, Ar-H), 6.47 (d, J = 13.0 Hz, 2H, CHalkene), 3.85 (s, 6H, N-CH3), 2.66 (t, J = 6.0 Hz, 4H,
=
CH2-CH2), 1.08–1.03 (m, 2H, CH2-CH2-CH2). IR (ATR) 2936 (CH), 1652 (C N), 1506, 1423,
1384, 905, 849, 563 cm−1. MS (ESI) m/z: 463 [M+]. UVabs λmax = 796 nm. Mp 255–260◦C.
4.3.10. 2-[(E)-2-[(3E)-2-Chloro-3-{2-[(2Z)-3-ethyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]
ethylidene}cyclohex-1-en-1-yl]ethenyl]-3-ethyl-1,3-benzothiazol-3-ium iodide (3b)
1
Yield: cyanine dye 3b (0.37 g, 60%) as a green solid. H NMR (d6-DMSO, 400 MHz) δ 8.01
(d, J = 14.0 Hz, 2H, CHalkene), 7.66 (d, J = 7.0 Hz, 2H, Ar-H), 7.46–7.38 (m, 4H, Ar-H), 7.30–
7.25 (m, 2H, Ar-H), 6.35 (d, J = 14.0 Hz, 2H, CHalkene), 4.28 (t, J = 7.0 Hz, 4H, N-CH2-CH2),
2.63–2.59 (m, 4H, CH2-CH2), 2.51–2.48 (m, 2H, CH2-CH2-CH2), 1.51 (t, J = 7.0 Hz, 6H, CH3-
=
CH2). IR (ATR) 2957 (CH), 1552 (C N), 1509, 1478, 1397, 1282, 1249, 1208, 1034, 913, 828,
742, 579 cm−1. MS (ESI) m/z: 491 [M+]. UVabs λmax = 796 nm. Mp 246–250◦C, Literature Mp
248–249◦C (39).
4.3.11. 2-[(E)-2-[(3E)-2-Chloro-3-{2-[(2Z)-3-propyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]
ethylidene}cyclohex-1-en-1-yl]ethenyl]-3-propyl-1,3-benzothiazol-3-ium iodide (3c)
Purified by flash chromatography (eluent, chloroform:methanol = 8:2). Yielded the cyanine dye
1
3c (0.81 g, 31%) as a green solid. H NMR (d6-DMSO, 400 MHz) δ 7.82 (d, J = 8.0 Hz, 2H,
Ar-H), 7.63 (d, J = 13.0 Hz, 4H, CHalkene), 7.39 (t, J = 7.0 Hz, 2H, Ar-H), 7.20 (t, J = 7.0 Hz,
2H, Ar-H), 6.38 (d, J = 13.0 Hz, 2H, CHalkene), 4.25 (t, J = 6.0 Hz, 4H, N-CH2-CH2), 2.51–2.47
(m, 4H, CH2-CH2), 1.63–1.57 (m, 6H, CH2), 0.85 (t, J = 7.0 Hz, 6H, CH2-CH3). IR (ATR) 2950
(CH), 1528 (C N), 1496, 1387, 1274, 1216, 1055, 983, 844, 740 cm−1. MS (ESI) m/z: 519.15
=
[M+]. UVabs λmax = 797 nm. Mp 245–249◦C.
4.3.12. 3-Butyl-2-[(E)-2-[(3E)-3-{2-[(2Z)-3-butyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]
ethylidene}-2-chlorocyclohex-1-en-1-yl]ethenyl]-1,3-benzothiazol-3-ium iodide (3d)
Purified by flash chromatography (eluent, chloroform:methanol = 8:2). Yielded the cyanine dye
3d (0.38 g, 19%) as a green solid. 1H NMR (d6-DMSO, 400 MHz) δ 7.99 (d, J = 6.0 Hz, 2H, Ar-
H), 7.83 (d, J = 15.0 Hz, 2H, CHalkene), 7.77 (d, J = 6.0 Hz, 2H, Ar-H), 7.59 (t, J = 6.0 Hz, 2H,
Ar-H), 7.43 (t, J = 9.0 Hz, 2H, Ar-H), 6.52 (d, J = 12.0 Hz, 2H, CHalkene), 4.47 (t, J = 6.0 Hz,
4H, N-CH2), 2.67–2.60 (m, 4H, CH2-CH2), 1.84-1.80 (m, 2H, CH2-CH2), 1.74 (t, J = 9.0 Hz, 4H,
CH2-CH2), 1.44 (q, J = 6.0 Hz, 4H, CH2-CH3), 0.95 (t, J = 7.0 Hz, 6H, CH3-CH2). IR (ATR)
2937 (CH), 1578 (C N), 1499, 1451, 1390, 1323, 1213, 1273, 1213, 1051, 902, 739 cm−1. MS
=
(ESI) m/z: 547.18 [M+]. UVabs λmax = 796 nm. Mp 212–216◦C.

52 O.A. Okoh et al.
4.3.13. 2-[(E)-2-[(3E)-2-Chloro-3-{2-[(2Z)-3-(3-sulfonatopropyl)-2,3-dihydro-1,3-
benzothiazol-2-ylidene]ethylidene}cyclohex-1-en-1-yl]ethenyl]-3-(3-sulfonatopropyl)-
1,3-benzothiazol-3-ium (3e)
Purified by flash chromatography (eluent, dichloromethane:methanol = 9 : 1). Yielded the cya-
nine dye 3e (0.10 g, 9%) as a green solid. 1H NMR (d6-DMSO, 400 MHz) δ 7.88 (d, J = 13.0 Hz,
2H, CHalkene), 7.69–7.60 (m, 4H, Ar-H), 7.43 (t, J = 7.0 Hz, 2H, Ar-H), 7.25 (t, J = 7.0 Hz, 2H,
Ar-H), 6.56 (d, J = 13.0 Hz, 2H, CHalkene), 4.51 (t, J = 7.0 Hz, 4H, N-CH2-CH2), 2.55–2.45 (m,
8H, SO3-CH2), 1.97 (t, J = 6.0 Hz, 4H, CH2-CH2), 1.76-1.70 (m, 2H, CH2-CH2). IR (ATR)
3050 (CH), 1527 (C N), 1499, 1454, 1324, 1208, 983 cm−1. MS (ESI) m/z: 679.07 [M+]. UVabs
=
λmax = 775 nm. Mp 240–244◦C.
4.3.14. 2-[(E)-2-[(3E)-2-Chloro-3-{2-[(2Z)-3-(4-sulfonatobutyl)-2,3-dihydro-1,3-
benzothiazol-2-ylidene]ethylidene}cyclohex-1-en-1-yl]ethenyl]-3-(4-sulfonatobutyl)-
1,3-benzothiazol-3-ium (3f)
Purified by flash chromatography (eluent, chloroform:methanol = 9:1). Yielded the cyanine dye
1
3f (0.30 g, 49%) as a green solid. H NMR (d6-DMSO, 400 MHz) δ 7.98 (d, J = 13.0 Hz, 2H,
CHalkene), 7.83–7.68 (m, 4H, Ar-H), 7.58–7.56 (m, 2H, Ar-H), 7.54–7.49 (m, 2H, Ar-H), 6.57 (d,
J = 13.0 Hz, 2H, CHalkene), 4.46 (t, J = 7.0 Hz, 4H, N-CH2-CH2), 3.34 (s, 1H, CH2), 2.70–2.65
(m, 4H, CH2-CH2), 2.09 (s, 1H, CH2), 1.76–1.50 (m, 12H, SO3-CH2). IR (ATR) 3393 (CH), 1651
(C N), 1499, 1392, 1324, 1276, 1217, 1127, 1011, 852, 796, 743, 564 cm−1. MS (ESI) m/z: 707
=
[M+]. UVabs λmax = 800 nm. Mp 247–251◦C.
4.3.15. 3-Benzyl-2-[(E)-2-[(3E)-3-{2-[(2Z)-3-benzyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]
ethylidene}-2-chlorocyclohex-1-en-1-yl]ethenyl]-1,3-benzothiazol-3-ium bromide (3g)
Purified by flash chromatography (eluent, chloroform:methanol = 9:1). Yielded the cyanine dye
1
3g (1.50 g, 27%) as a green solid. H NMR (d6-DMSO, 400 MHz) δ 7.94 (d, J = 7.0 Hz, 2H,
Ar-H), 7.74 (d, J = 13.0 Hz, 2H, CHalkene), 7.62 (d, J = 8.0 Hz, 2H, Ar-H), 7.44 (t, J = 7.0 Hz,
2H, Ar-H), 7.33 (d, J = 6.0 Hz, 4H, Ar-H), 7.27–7.13 (m, 8H, Ar-H), 6.52 (d, J = 13.0 Hz, 2H,
CHalkene), 5.70 (s, 4H, N-H-Ph), 3.12 (s, 2H, CH2), 2.57 (s, 2H, CH2), 1.67 (s, 2H, CH2). IR
(ATR) 1543 (C N), 1506, 1363, 1225, 1099, 896, 784, 668, 597 cm−1. MS (ESI) m/z: 615.06
=
[M+]. HRMS (FAB): Calculated for C38H32ClN2S2 [M+] 615.1689, found 615.1682. UVabs
λmax = 804 nm. Mp 254–258◦C.
4.3.16. 2-[(E)-2-[(3E)-2-Chloro-3-{2-[(2E)-3,3-dimethyl-1-(4-sulfonatobutyl)-2,3-dihydro-
1H-indol-2-ylidene]ethylidene}cyclohex-1-en-1-yl]ethenyl]-3,3-dimethyl-1-(4-
sulfonatobutyl)-3H-indol-1-ium (OA1)
Purified by flash chromatography (eluent, chloroform:methanol = 7:3). Yielded the cyanine dye
OA1 (1.01 g, 46%) as a shiny purple solid. 1H NMR (d6-DMSO, 400 MHz) δ 8.21 (d, J = 14.0 Hz,
2H, CHalkene), 7.67 (d, J = 7.0 Hz, 2H,Ar-H), 7.39–7.31 (m, 4H,Ar-H), 7.12–7.06 (m, 2H,Ar-H),
6.31 (d, J = 14.0 Hz, 2H, CHalkene), 4.89 (t, J = 5.0 Hz, 4H, N-CH2-CH2), 3.01–2.96 (m, 4H,
CH2-CH2), 2.68–2.60 (m, 4H, CH2-CH2), 1.70–1.61 (m, 8H, CH2-CH2-CH2-SO−3 ), 1.50 (s, 12H,
=
C-CH3), 1.30–1.09 (m, 2H, CH2-CH2). IR (ATR) 2921 (CH), 1541 (C N), 1240, 1004, 889, 707,
565 cm−1. MS (ESI) m/z: 727 [M+]. UVabs λmax = 770 nm. Mp 253–257◦C.

Journal of Sulfur Chemistry 53
4.3.17. N-(2,4-Dinitrophenyl) pyridinium chloride (4)
Pydrine (3.00 ml, 37.9 mmol) was dissolved in acetone (17.0 ml) and stirred, while 2,4-
dinitrochlorobenzene (7.67 g, 37.9 mmol) was added to the reaction mixture. The mixture was
heated under reflux overnight and cooled to room temperature. The precipitate produced was fil-
tered under suction and washed with n-hexane to obtain N-(2,4-dinitrophenyl) pyridinium chloride
(4, 8.10 g, 97%) as a pale yellow powder. 1H NMR (d6-DMSO, 250 MHz) δ 9.43 (d, J = 6.0 Hz,
2H, Ar-H), 9.21 (s, 1H, Ar-H), 8.95 (t, J = 7.0 Hz, 2H, Ar-H), 8.50-8.41 (m, 3H, Ar-H). 13C NMR
(d6-DMSO, 68.2 MHz) δ 149.1, 148.9, 146.1, 143.1, 138.8, 132.0, 130.3, 128.1, 121.4. IR (ATR)
2971 (CH), 1540 (C N), 1474, 1341, 1266, 1154, 943, 855, 739 cm−1. MS (ESI) m/z: 186.24
=
[M+]. Mp 197–201◦C.
Below is the general synthetic procedure for linear cyanine dyes 5a–g. Full spectroscopic data
are given for all compounds along with chromatography details where required.
4.3.18. 3-Methyl-2-[(1E,3E,5E)-7-[(2Z)-3-methyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]
hepta-1,3,5-trien-1-yl]-1,3-benzothiazol-3-ium iodide (5a)
To a solution of N-(2,4-dinitrophenyl) pyridinium chloride (0.22 g, 0.80 mmol) in EtOH (10.0 ml)
wasadded 5a (0.58 g, 2.00 mmol)andaniline(0.19 g, 2.00 mmol).Thereactionmixturewasstirred
for 5 min, and then followed by the addition of sodium acetate (0.41 g, 5 mmol). The reaction was
stirred at room temperature overnight and 20.0 ml n-hexane added. The precipitate produced was
filtered under suction and washed with n-hexane. The crude product was purified by silica gel
column chromatography (eluent solvent, chloroform:methanol = 9:1) to obtain cyanine dye 5a
(0.32 g, 78%) as a green solid. 1H NMR (d6-DMSO, 400 MHz) δ 7.95 (d, J = 7.0 Hz, 2H, Ar-H),
7.67 (d, J = 8.0 Hz, 2H, Ar-H), 7.53 (t, J = 7.0 Hz, 2H, Ar-H), 7.43-7.35 (m, 5H, Ar-H), 6.54 (d,
J = 13.0 Hz, 2H, CHalkene), 6.48 (t, J = 12.0 Hz, 2H, CHalkene), 3.77 (s, 6H, N-CH3). IR (ATR)
1543 (C N), 1518, 1356, 1232, 1081, 887, 764, 615 cm−1. MS (ESI) m/z: 389.07 [M+]. UVabs
=
λmax = 756 nm. Mp 210–214◦C.
4.3.19. 3-Ethyl-2-[(1E,3E,5E)-7-[(2Z)-3-ethyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]hepta-
1,3,5-trien-1-yl]-1,3-benzothiazol-3-ium iodide (5b)
Purified by flash chromatography (eluent, chloroform:methanol = 9:1). Yielded the cyanine dye
1
5b (0.20 g, 46%) as a green solid. H NMR (d6-DMSO, 400 MHz) δ 7.95 (d, J = 7.0 Hz, 2H,
Ar-H), 7.68 (d, J = 6.0 Hz, 2H, Ar-H), 7.54 (t, J = 6.0 Hz, 2H, Ar-H), 7.43–7.32 (m, 5H, Ar-H),
6.58 (d, J = 13.0 Hz, 2H, CHalkene), 6.45 (t, J = 13.0 Hz, 2H, CHalkene), 4.36 (q, J = 6.0 Hz, 4H,
=
N-CH2-CH3), 1.30 (t, J = 7.0 Hz, 6H, CH3-CH2). IR (ATR) 2971 (CH), 1579 (C N), 1507, 1396,
1059, 983, 830, 738 cm−1. MS (ESI) m/z: 417.07 [M+]. UVabs λmax = 759 nm. Mp 216–221◦C.
4.3.20. 3-Propyl-2-[(1E,3E,5E)-7-[(2Z)-3-propyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]
hepta-1,3,5-trien-1-yl]-1,3-benzothiazol-3-ium iodide (5c)
Purified by flash chromatography (eluent, chloroform:methanol = 9:1). Yielded the cyanine dye
5c (0.30 g, 66%) as a shiny green solid. 1H NMR (d6-DMSO, 400 MHz) δ 7.95 (d, J = 7.0 Hz,
2H, Ar-H), 7.71 (d, J = 8.0 Hz, 2H, Ar-H), 7.53 (d, J = 7.0 Hz, 2H, Ar-H), 7.44 (d, J = 13.0 Hz,
2H, CHalkene), 7.37 (d, J = 8.0 Hz, 2H, Ar-H), 6.60 (d, J = 12.0 Hz, 2H, CHalkene), 6.40 (t, J =
13.0 Hz, 2H, CHalkene), 4.27 (t, J = 7.0 Hz, 4H, N-CH2-CH2), 1.75 (q, J = 7.0 Hz, 4H, CH2-
=
CH3), 0.97 (t, J = 7.0 Hz, 6H, CH2-CH3). IR (ATR) 2960 (CH), 1579 (C N), 1500, 1393, 1324,

54 O.A. Okoh et al.
970, 805, 743 cm−1. MS (ESI) m/z: 445.12 [M+]. HRMS (FAB): Calculated for C27H29N2S2I
[M+] 445.1766, found 445.1758. UVabs λmax = 761 nm. Mp 193–197◦C.
4.3.21. 3-Butyl-2-[(1E,3E,5E)-7-[(2Z)-3-butyl-2,3-dihydro-1,3-benzothiazol-2-
ylidene]hepta-1,3,5-trien-1-yl]-1,3-benzothiazol-3-ium iodide (5d)
Purified by flash chromatography (eluent, chloroform:methanol = 9:1). Yielded the cyanine dye
1
5d (0.21 g, 44%) as a shiny purple green solid. H NMR (d6-DMSO, 300 MHz) δ 7.98 (d, J =
9.0 Hz, 2H, Ar-H), 7.71 (d, J = 9.0 Hz, 2H, Ar-H), 7.57 (t, J = 12.0 Hz, 2H, CHalkene), 7.47–7.35
(m, 5H, Ar-H), 6.60 (d, J = 12.0 Hz, 2H, CHalkene), 6.49 (d, J = 12.0 Hz, 2H, CHalkene), 4.33 (t,
J = 6.0 Hz, 4H, N-CH2-CH2), 1.71 (quin, J = 6.0 Hz, 4H, CH2-CH2-CH2), 1.47–1.37 (m, 4H,
CH2-CH2-CH3), 0.95 (t, J = 6.0 Hz, 6H, CH2-CH3). 13C NMR (d6-DMSO, 75.4 MHz) δ ppm
196.4, 170.8, 141.9, 128.5, 125.7, 125.3, 123.4, 113.7, 79.6, 60.2, 49.0, 29.9, 21.2, 19.8, 14.5,
14.1. IR (ATR) 2951 (CH), 1501 (C N), 1451, 1391, 1320, 1051, 918, 821, 737 cm−1. MS (ESI)
=
m/z: 473.14 [M+]. UVabs λmax = 761 nm. Mp 176–180◦C.
4.3.22. 3-(3-Sulfonatopropyl)-2-[(1E,3E,5E)-7-[(2Z)-3-(3-sulfonatopropyl)-2,3-dihydro-1,3-
benzothiazol-2-ylidene]hepta-1,3,5-trien-1-yl]-1,3-benzothiazol-3-ium (5e)
Purified by flash chromatography (eluent, chloroform:methanol = 7:3). Yielded the cyanine dye
1
5e (0.22 g, 47%) as a green solid. H NMR (d6-DMSO, 400 MHz) δ 7.95 (d, J = 7.0 Hz, 2H,
Ar-H), 7.79 (d, J = 8.0 Hz, 2H, Ar-H), 7.55 (t, J = 8.0 Hz, 2H, Ar-H), 7.44–7.34 (m, 5H, Ar-H),
6.69 (d, J = 13.0 Hz, 2H, CHalkene), 6.44 (t, J = 12.0 Hz, 2H, CHalkene), 4.48 (t, J = 8.0 Hz, 4H,
N-CH2-CH2), 2.58 (t, J = 7.0 Hz, 4H, SO3-CH2-CH2), 2.03 (quin, J = 8.0 Hz, 4H, CH2-CH2-
CH2). IR (ATR) 3398, 2361 (CH), 1581 (C N), 1504, 1405, 1329, 1091, 993, 749 cm−1. MS
=
(ESI) m/z: 603.07 [M+]. HRMS(FAB): Calculated for C27H27N2O6S4 [M+] 603.0757, found
603.0742. UVabs λmax = 762 nm. Mp 225–229◦C.
4.3.23. 3-(4-Sulfonatobutyl)-2-[(1E,3E,5E)-7-[(2Z)-3-(4-sulfonatobutyl)-2,3-dihydro-1,3-
benzothiazol-2-ylidene]hepta-1,3,5-trien-1-yl]-1,3-benzothiazol-3-ium (5f)
Purified by flash chromatography (eluent, chloroform:methanol = 7:3). Yielded the cyanine dye
1
5f (0.23 g, 43%) as a dark green solid. H NMR (d6-DMSO, 400 MHz) δ 7.94 (d, J = 8.0 Hz,
2H, Ar-H), 7.78 (d, J = 8.0 Hz, 2H, Ar-H), 7.53 (t, J = 8.0 Hz, 2H, Ar-H), 7.43-7.33 (m, 5H,
Ar-H), 6.68 (d, J = 13.0 Hz, 2H, CHalkene), 6.42 (d, J = 13.0 Hz, 2H, CHalkene), 4.06–3.87 (m,
4H, N-CH2-CH2), 2.56 (t, J = 8.0 Hz 4H, SO3-CH2-CH2), 2.41–2.06 (m, 4H, CH2-CH2), 1.91–
=
1.65 (m, 4H, CH2-CH2). IR (ATR) 3405, 2361 (CH), 1683 (C N), 1505, 1400, 1328, 1074, 978,
742 cm−1. MS (ESI) m/z: 631.84 [M+]. UVabs λmax = 762 nm. Mp 228–232◦C.
4.3.24. 3-Benzyl-2-[(1E,3E,5E)-7-[(2Z)-3-benzyl-2,3-dihydro-1,3-benzothiazol-2-ylidene]
hepta-1,3,5-trien-1-yl]-1,3-benzothiazol-3-ium bromide (5g)
Purified by flash chromatography (eluent, chloroform:methanol = 8:2). Yielded the cyanine dye
5g (0.300 g, 60%) as a green solid. 1H NMR (d6-DMSO, 400 MHz) δ 8.02 (d, J = 7.0 Hz, 2H,
Ar-H), 7.65 (d, J = 8.0 Hz, 2H, Ar-H), 7.52–7.36 (m, 11H, Ar-H), 7.33 (t, J = 8.0 Hz, 2H, Ar-H),
7.26 (d, J = 7.0 Hz, 4H, Ar-H), 6.64 (d, J = 13.0 Hz, 2H, CHalkene), 6.31 (t, J = 12.0 Hz, 2H,
=
CHalkene), 5.64 (s, 4H, Ph-CH2). IR (ATR) 2979 (CH), 1582 (C N), 1504, 1459, 1328, 1114,
994, 814, 732 cm−1. MS (ESI) m/z: 541.03 [M+]. HRMS (FAB): Calculated for C35H29N2S2.Br
[M+] 541.1766, found 541.1756. UVabs λmax = 765 nm. Mp 209–213◦C.

Journal of Sulfur Chemistry 55
4.3.25. 2-[(1E,3E,5E)-7-[(2E)-3,3-Dimethyl-1-(4-sulfonatobutyl)-2,3-dihydro-1H-indol-2-
ylidene]hepta-1,3,5-trien-1-yl]-3,3-dimethyl-1-(4-sulfonatobutyl)-3H-indol-1-ium
(OA2)
Purified by flash chromatography (eluent, chloroform:methanol = 7:3). Yielded the cyanine dye
OA2 (0.380 g, 73%) as a green solid. 1H NMR (d6-DMSO, 400 MHz) δ 7.90 (t, J = 13.0 Hz, 2H,
CHalkene), 7.74 (s, 1H, Ar-H), 7.57 (d, J = 7.0 z, 2H, Ar-H), 7.41–7.35 (m, 4H, Ar-H), 7.23 (t,
J = 8.0 Hz, 2H, Ar-H), 6.58 (t, J = 12.0 Hz, 2H, CHalkene), 6.44 (d, J = 14.0 Hz, 2H, CHalkene),
4.06 (t, J = 7.0 Hz, 4H, N-CH2-CH2), 2.47 (t, J = 7.0 Hz, 4H, SO3-CH2-CH2), 1.74–1.68 (m,
=
8H, CH2-CH2), 1.62 (s, 12H, C-CH3). IR (ATR) 2924 (CH), 1597 (C N), 1509, 1317, 1158,
966, 741 cm−1. MS (ESI) m/z: 653.29 [M+]. Mp 221–225◦C.
Acknowledgements
We are pleased to acknowledge the financial support from the Centre for Material Sciences, School of Forensic and
Investigative Sciences, University of Central Lancashire. This paper is written in dedication of the late Mrs Esther Otuh.
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