High-Yielding Water-Soluble Asymmetric Cyanine Dyes for Labeling Applications

Article abstract of DOI:10.1021/acs.joc.0c01084

A simple and efficient microwave-assisted synthesis of asymmetric pentamethine cyanine dyes with various functional groups was developed, which allows high-yielding results. The synthesized dyes are modifiable and suitable for single-molecule imaging in biological and medical sciences by application of click chemistry or classic esterification and amidation.

Full text of DOI:10.1021/acs.joc.0c01084

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Article
High Yielding Water-Soluble Asymmetric
Cyanine Dyes for Labeling Applications
Natalia Wolf, Louise Kersting, Christoph Herok, Cornelius Mihm, and Juergen Seibel
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c01084 • Publication Date (Web): 20 Jul 2020
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The Journal of Organic Chemistry
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High Yielding Water-Soluble Asymmetric Cyanine Dyes for Label-
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Natalia Wolf, Louise Kersting, Christoph Herok, Cornelius Mihm, Juergen Seibel*.
Institute of Organic Chemistry, University of Wuerzburg, 97074 Wuerzburg, Germany.
Microwave irradiation, cyanine dyes, high yield, labeling applications, click chemistry.
ABSTRACT: A simple and efficient microwave-assisted synthesis of asymmetric pentamethine cyanine dyes with various functional
groups was developed that allows high yielding results. The synthesized dyes are modifiable and suitable for single molecule imaging
in biological and medicinal sciences by application of click-chemistry or classic esterification and amidation.
1. Introduction
Scheme 1. Proposed Mechanistic Pathway for the Synthesis
of Pentamethine Cyanine Dyes.
Water-soluble cyanine dyes are reliable fluorophores to illu-
minate processes in biological systems.1–18 In dependence of the
length of the polymethine chain, the fluorescence range extends
from the visible to the near IR region. This allows customized
applications in medicine and biological sciences.1–18 Further-
more, their ability to photo-switch makes them suitable for sto-
chastic super resolution microscopy methods like STORM and
dSTORM.19–21
The synthesis and purification of water-soluble cyanine dyes re-
mains challenging. To date, only a few example of asymmetric
pentamethine cyanine dyes with modifiable functional groups
have been reported.22–24 In this work, we present a microwave-
assisted, efficient and fast synthesis of water-soluble asymmet-
ric pentamethine cyanine dyes. Based on existing synthetic
routes, we focused on the optimization of the reaction condi-
tions, and hence the improvement of yields.
2. Results and Discussion
2.1. Choice of Solvent and Base
In order to optimize the synthesis, we suggested a formation
mechanism for pentamethine cyanine dyes. It follows a simple
imine-enamine pathway (Scheme 1) and requires a careful se-
lection of solvent and base. Initially, the enamine of the indo-
lium derivative attacks the imine of malonaldehyde dianilide.
The subsequent reaction of the resulting bisimine with a second
indolium derivative should involve a good leaving group. The
generation of a leaving group by acetylation of the imine results
in the irreversibility and thus the success of the reaction. In the
past, many different reaction conditions for the synthesis of wa-
ter-soluble cyanine dyes have been reported.21–33 Facing the
problem of solubility of the indolium salts, pyridine or alcohols
were commonly used as solvents. However, an excess of a nu-
cleophilic base promotes side reactions and decomposition of
the dye, which is in line with the low yields reported in the lit-
erature.25,26,31,33,35,38–41
We propose that a fast equilibrium between deprotonation and
protonation can accelerate the reaction. Those reaction condi-
tions are known from enzymatic reactions, i.e. serine proteases,
where the histidine acts as a proton donor and acceptor.42 Sev-
eral groups reported the use of acetic acid as solvent and potas-
sium acetate as base in combination with acetic anhydride,
where the yield depended on the buffer composition.30,36,43–45
2.2. Microwave Irradiation and Temperature
Due to the polarizability of each intermediate in the cyanine
formation, this reaction is particularly suitable for microwave
assistance.46–49 Polar molecules rotate with the electromagnetic
field and lose energy in collisions, leading to a mild and selec-
tive heating of the reaction medium. Independent of the combi-
nation of solvent and base in the synthesis of cyanine dyes, mi-
crowave heating optimizes results.24,50,51 In the last step of the
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cyanine formation, conventional conditions require reaction
N3
DMF
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52
46
79
69
94
96
91
2c
2d
2e
2f
times from 30 min up to three and a half hours.21–40 In compari-
son, exposing the reaction mixture to microwaves at 75 W and
110 °C significantly shortens the reaction time to 15 min (Table
2). It should be noted that cyanine dyes decompose at tempera-
tures above 120 °C, particularly in the presence of light.
1
2
3
4
5
6
7
8
CCH
CCH
NHTFA
NHTFA
SO3H
CN
DMF
DMF
butyronitrile
butyronitrile
neat
2g
2h
2i
2.3. Synthesis of Cyanine Dyes
neat
The conditions for the Fischer Indole synthesis of compound
1 were adapted from the conventional method,25 employing p-
hydrazinobenzenesulfonic acid hemihydrate and 3-methyl-2-
butanone as reactants in acetic acid. Using microwave irradia-
tion at 150 W and a temperature of 125 °C, the reaction was
accomplished within four hours and an overall yield of 52 %.
Hence, microwave irradiation reduced the reaction time and ad-
ditionally afforded the product with a higher degree of purity.
The most challenging part in the synthesis of cyanine dyes is
the formation of indolium salts through N-alkylation of indole-
nine 1. The examination of the 1H and 13C spectra in methanol-
d4 showed no exchange of protons with deuterium ions after
several hours. Furthermore, the 15N spectrum also indicates the
structure of an imine, as the detected signal has a shift of – 85.4
ppm and can be assigned to an imine according to increment
tables (compare supporting information). The reaction only
takes place at temperatures above 100 °C. However, tempera-
ture sensitive functional groups like alkynes and azides decom-
pose at high temperatures. We attained moderate yields for in-
dolium salts functionalized with non-polar chains when em-
ploying the respective bromides or iodides in DMF (Table 1,
compounds 2a  2e). The introduction of amines or carboxylic
acids requires a protection of these functional groups. The reac-
tion with TFA-protected 3-bromopropylamine and TFA-
protected 6-bromohexylamine in butyronitrile readily afforded
intermediates 2f and 2g. Compounds 2h – 2j were easily acces-
sible in excellent yields by reaction of 1 with neat 1,4-butane
sultone, 4-bromobutyronitrile or 6-bromohexanitrile, respec-
tively. The conversion of the nitrile function into a carboxylic
acid can be readily achieved by hydrolysis under acidic condi-
tions (AcOH/6 M HCl) at 125 W and 130 °C for 2 h without loss
of purity and yield.
9
CN
neat
2j
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The condensation of indolium salt 2h with malonaldehyde di-
anilide hydrochloride proceeded fast and with good yield for
both conventional heating and microwave irradiation and af-
forded hemicyanine 3 (Table 2). In contrast to conventional
heating, the symmetrical byproduct was not observed in the mi-
crowave-assisted method. Compound 3 precipitates upon addi-
tion of ethyl acetate and diethyl ether and can be used without
further purification.
We performed the synthesis of our asymmetric cyanine dyes
4a – 4k in a mixture of acetic acid, acetic anhydride and potas-
sium acetate (ratio 2:2:1) and under microwave irradiation
(75 W, 110 °C). Impressively, these conditions reduced the re-
action time to only 15 minutes. Following this protocol, we suc-
cessfully synthesized eleven water-soluble and asymmetric pen-
tamethine cyanine dyes with different chain lengths and func-
tional groups in good to excellent yields (Table 2).
Table 2. Synthesis of Asymmetric Cyanine Dyes under
Microwave-Assisted Conditions.
Table 1. General Synthesis of Indolium Salts.
Cyanine Dye
R
Time [min]
Yield [%]
N3
15
15
15
15
15
15
15
15
15
15
15
72
71
70
93
89
92
95
81
87
96
90
4a
4b
4c
4d
4e
4f
N3
N3
CCH
CCH
CN
CN
4g
4h
4i
CO2H
CO2H
NHTFA
NHTFA
Substance
R
Solvent
acetic acid
DMF
Yield [%]
4j
53
65
53
1
4k
N3
N3
2a
2b
DMF
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Dyes 4j and 4k were converted into the dyes 4l (n = 1,
on a Bruker Daltonics MicrOTOF Focus. For complex struc-
tures, the IUPAC name was abbreviated or replaced by trivial
names.
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R = NH2) and 4m (n = 4, R = NH2) by microwave-assisted hy-
drolysis. Acidic conditions are crucial for this deprotection
since nucleophilic bases lead to the decomposition of cyanine
dyes.
4.2. Synthesis of Side Chains
2.4. Derivatizations
General Procedure for Azido Alcohols (5). 3-Bromopro-
pan-1-ol, 6-bromohexan-1-ol or 9-bromononan-1-ol (1.0 eq)
were dissolved in H2O (5a) or a 1/1 mixture of H2O and MeCN
(5b, 5c) at a concentration of ~ 1 – 1.3 M. Sodium azide (2.0 eq)
was added and the mixture was stirred in an oil bath at 60 °C for
1 – 3 days. The mixture was extracted with Et2O, the combined
organic layers were dried over Na2SO4 and the solvent was re-
moved under reduced pressure. The product was purified by
column filtration (pentane/Et2O = 1:1).
Our water-soluble cyanine dyes carry a variety of chemically
modifiable functional groups. This renders them useful for cus-
tomized single molecule imaging in biological and medicinal
applications. The carboxylic acid-functionalized dyes 4h and 4i
are convertible into reactive NHS-esters and can be ligated to
proteins and antibodies. The amino-functionalized dyes 4l and
4m form stable amides and can be conjugated to NHS-esters of
proteins and antibodies as well. The azido and alkyne-function-
alized cyanine dyes 4a  4e are suitable for click-chemistry
based experiments.
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3-Azidopropan-1-ol (5a). Synthesized according to the
general procedure (276 mmol scale). Yield: 27.7 g (275 mmol,
99 %) of a colorless oil. 1H NMR (400 MHz, CDCl3)  3.68 (t,
3. Conclusions
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3J = 6.0 Hz, 2H, H-1), 3.40 (t, J = 6.6 Hz, 2H, H-3), 2.58 (s,
3
1H, OH), 1.7.8 (tt, J = 6.5 Hz, 2H, H-2) ppm. 13C{1H} NMR
We developed a simple and efficient microwave-assisted pro-
tocol that gives access to various water-soluble asymmetric pen-
tamethine cyanine dyes in high yields. In the light of previous
works, we systemically investigated the reaction process and
found that there is no need for inert conditions and long reaction
times. The right choice of leaving group, buffer conditions and
microwave irradiation is key to success. With our optimized re-
action conditions, we synthesized cyanine dyes with nitriles, al-
kynes, azides and amines as functional groups. These chemical
groups enable at least one further modification and generate a
library of water-soluble cyanine dyes that are suitable for cus-
tomized imaging applications.
(100 MHz, CDCl3)  59.7 (C-1), 48.4 (C-3), 31.4 (C-2) ppm.
15N NMR (60 MHz, CDCl3)  – 132.9 (N-5), – 170.0 (N-6),
– 310.3 (N-4) ppm. HRMS (ESI) m/z calcd for C3H7N3NaO
[MNa]: 124.0481; found: 124.0486 ( = 3.90 ppm).
6-Azidohexan-1-ol (5b). Synthesized according to the
general procedure (27.6 mmol scale). Yield: 3.67 g (25.6 mmol,
93 %) of a colorless oil. 1H NMR (400 MHz, CDCl3)  3.61 (t,
3
3J = 6.6 Hz, 2H, H-1), 3.25 (t, J = 6.9 Hz, 2H, H-6), 1.71 (s,
1H, OH), 1.61–1.54 (m, 4H, H-5, H-2), 1.40–1.36 (m, 4H, H-3,
H-4) ppm. 13C{1H} NMR (100 MHz, CDCl3)  62.7 (C-1), 51.4
(C-6), 32.6 (C-2), 28.9 (C-5), 26.6 (C-3), 25.4 (C-4) ppm. 15N
NMR (60 MHz, CDCl3)  – 132.6 (N-8), – 170.4 (N-9),
– 309.2 (N-7) ppm. HRMS (ESI) m/z calcd for C6H13N3NaO
[MNa]: 166.0951; found: 166.0952 ( = 0.71 ppm).
4. Experimental Section
4.1. General Information
9-Azidononan-1-ol (5c). Synthesized according to the
general procedure (112 mmol scale). Yield: 20.7 g (112 mmol,
All reagents were purchased from commercial sources and
used without further purification. Reactions were monitored by
TLC using pre-coated silica gel plates with a UV indicator
(UV254). The plates were either exposed to ultraviolet light
(254 nm) or stained with a solution of 3.7 mL para-anisaldehyde
in 135 mL EtOH, 5 mL conc. H2SO4, 1.5 mL glacial acetic acid.
Flash column chromatography was performed with silica gel
(particle size 0.032 – 0.062 mm). Reversed-phase column chro-
matography was carried out with RP18 silica gel (12 nm, S-15
µm). Microwave-assisted synthesis was performed in sealed
quartz vials with a µCHEMIST microPREP Microwave System
and a control unit. NMR spectra were recorded on a Bruker
Avance III HD 400 MHz spectrometer or a Bruker Avance III
HD 600 MHz spectrometer. Chemical shifts (δ) are reported in
parts per millions and coupling constants (J) are given in Hz.
The spectra were referenced to the residual solvent peak.
1
quantitative) of a colorless oil. H NMR (400 MHz, CDCl3)
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 3.64 (t, J = 6.6 Hz, 2H, H-1), 3.25 (t, 3J = 7.0 Hz, 2H, H-9),
1.63–1.53 (m, 4H, H-8, H-2), 1.36–1.31 (m, 10H, H-3, H-4, H-
5, H-6, H-7) ppm. 13C{1H} NMR (100 MHz, CDCl3)  63.2 (C-
1), 51.6 (C-9), 32.9 (C-2), 29.5 (1C, C-4/ C-6), 29.4 (1C, C-6/
C-4), 29.2 (C-5), 28.9 (C-8), 26.8 (C-7), 25.8 (C-3) ppm. 15N
NMR (60 MHz, CDCl3)  – 132.5 (N-11), – 170.6 (N-12),
– 309.0 (N-10) ppm. HRMS (ESI) m/z calcd for C9H19N3NaO
[MNa]: = 208.1420; found: 208.1422 ( = 0.66 ppm).
General Procedure for the Tosylation of Alcohols (6). In a
Schlenk flask under inert atmosphere, 1.0 eq of 3-azidopropan-
1-ol (5a), 6-azidohexan-1-ol (5b) or 9-azidononan-1-ol (5c)
were dissolved in anhydrous DCM (~ 1 – 2 M). Anhydrous
pyridine (1.2 eq) and p-toluenesulfonyl chloride (1.1 eq) were
added and the mixture was stirred at rt for 16 h. Hexane was
added and the mixture was filtered. The solvent was removed
under reduced pressure and the residue was dissolved in Et2O.
The organic phase was washed with 0.1 M aqueous CuSO4
solution and dried over Na2SO4. The solvent was removed under
reduced pressure and the crude product was purified by column
filtration (pentane/Et2O = 4:1).
1H:
δ (CDCl3) = 7.26 ppm,
d4) = 3.31 ppm],
13C{1H}:
[δ (AcOD-d4) = 2.04 ppm,
δ (MeCN-d3) = 1.94,
δ (MeOD-
[δ (AcOD-d4) = 20.0 ppm,
δ (D2O) = 4.79 ppm,
δ (MeCN-d3) = 118.26, δ (CDCl3) = 77.16 ppm, δ (MeOD-
d4) = 49.00 ppm]. 19F spectra were referenced to trifluoroacetic
1
acid. 15N spectra (obtained from the projection of the H-15N
HMBC spectra) were referenced to MeNO2. Structural assign-
ments were made with additional information from HMBC ex-
periments. HRMS spectra [electrospray ionization (ESI) and at-
mospheric pressure chemical ionization (APCI)] were recorded
3-Azidopropyl-p-toluenesulfonate (6a). Synthesized ac-
cording to the general procedure (158 mmol scale). Yield:
39.2 g (153 mmol, 97 %) of a colorless oil. 1H NMR (400 MHz,
CDCl3)  7.79–7.77 (m, 2H, H-2’), 7.37–7.34 (m, 2H, H-3’),
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4.09 (t, J = 5.5 Hz, 2H, H-1), 3.37 (t, J = 6.5 Hz, 2H, H-3),
2.44 (s, 3H, H-5’), 1.87 (tt, 3J = 6.0 Hz, 2H, H-2) ppm. 13C{1H}
NMR (100 MHz, CDCl3)  145.2 (C-1’), 132.8 (C-4’), 130.0
(2C, C-3’), 127.9 (2C, C-2’), 67.1 (C-1), 47.3 (C-3), 28.5 (C-2),
21.7 (C-5’) ppm. 15N NMR (60 MHz, CDCl3)  – 133.5 (N-5),
– 169.7 (N-6), – 311.5 (N-4) ppm. HRMS (ESI) m/z calcd for
C10H13N3NaO3S [MNa]: 278.0570; found: 278.0573
( = 1.0 ppm).
– 170.4 (N-9), – 309.2 (N-7) ppm. The spectroscopic data are in
agreement with those reported in the literature.53
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9-Azido-1-iodononane (7c). Synthesized according to the
general procedure (20.5 mmol scale). Yield: 5.57 g (18.9 mmol,
92 %) of a colorless oil. 1H NMR (400 MHz, CDCl3)  3.25 (t,
3
3J = 7.0 Hz, 2H, H-9), 3.18 (t, J = 7.0 Hz, 2H, H-1), 1.81 (tt,
3J = 7.2 Hz, 2H, H-2), 1.58 (tt, 3J = 7.1 Hz, 2H, H-8), 1.41–1.33
(m, 4H, H-3, H-7), 1.33–1.29 (m, 6H, H-4 – H-6) ppm. 13C{1H}
NMR (100 MHz, CDCl3)  51.6 (C-9), 33.6 (C-2), 30.5 (C-3),
29.3 (1C, C-4/ C-5/ C-6), 29.1 (1C, C-4/ C-5/ C-6), 28.9 (C-8),
28.5 (1C, C-4/ C-5/ C-6), 26.7 (C-7), 7.4 (C-1) ppm. 15N NMR
(60 MHz, CDCl3)  – 132.5 (N-11), – 170.6 (N-12), – 309.1 (N-
10) ppm. Elemental analysis calcd for C9H18IN3: C, 36.62; H,
6.15; N, 14.24. Found: C, 36.69; H, 6.24; N, 14.55.
6-Azidohexyl-p-toluenesulfonate (6b). Synthesized accord-
ing to the general procedure (75.1 mmol scale). Yield: 21.9 g
(73.6 mmol, 98 %) of a colorless oil. 1H NMR (600 MHz,
CDCl3)  7.76–7.74 (m, 2H, H-2’), 7.33–7.31 (m, 2H, H-3’),
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3
3
3.98 (t, J = 6.4 Hz, 2H, H-1), 3.19 (t, J = 6.9 Hz, 2H, H-6),
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2.41 (s, 3H, H-5’), 1.61 (tt, J = 6.9 Hz, 2H, H-2), 1.50 (tt,
3J = 7.2 Hz, 3J = 6.9 Hz, 2H, H-5), 1.32–1.27 (m, 4H, H-3, H-4)
ppm. 13C{1H} NMR (100 MHz, CDCl3)  144.8 (C-1’), 133.1
(C-4’), 129.9 (C-3’), 127.9 (C-2’), 70.4 (C-1), 51.2 (C-6), 28.7
6-Bromohexanamine Hydrobromide (8). Aqueous HBr so-
lution (19.5 mL, 256 mmol, 62 %wt, 33.4 g, 3.0 eq) was care-
fully added to 6-aminohexan-1-ol (10.0 g, 85 mmol, 1.0 eq) at
0 °C. The reaction mixture was stirred in an oil bath at 100 °C
for 24 h. As far as possible, the solvent was removed under re-
duced pressure. In order to eliminate remaining water, the resi-
due was dissolved in acetone and the solvent was removed by
rotary evaporation. The residue was washed with Et2O, dried
and used in the following step without further purification.
(C-2), 28.6 (C-5), 26.1 (C-4), 25.0 (C-3), 21.7 (C-5’) ppm. 15
N
NMR (60 MHz, CDCl3)  – 132.8 (N-8), – 170.6 (N-9), – 309.2
(N-7) ppm. HRMS (ESI) m/z calcd for C13H19N3NaO3S
[MNa]: 320.1039; found: 320.1039 ( = 0.10 ppm).
9-Azidononyl-p-toluenesulfonate (6c). Synthesized accord-
ing to the general procedure (111 mmol scale). Yield: 35.1 g
(103 mmol, 93 %) of a colorless oil. 1H NMR (400 MHz,
CDCl3)  7.77–7.75 (m, 2H, H-2’), 7.33–7.31 (m, 2H, H-3’),
4.00–3.97 (m, 2H, H-1), 3.22 (t, 3J = 6.8 Hz, 2H, H-9), 2.42 (s,
3H, H-5’), 1.61 (tt, 3J = 7.1 Hz, 2H, H-2), 1.55 (tt, 3J = 7.1 Hz,
2H, H-8), 1.30–1.28 (m, 4H, H-3, H-7), 1.25–1.22 (m, 6H, H-
4 – H-6) ppm. 13C{1H} NMR (100 MHz, CDCl3)  144.7 (C-
1’), 133.1 (C-4’), 129.9 (C-3’), 127.9 (C-2’), 70.7 (C-1), 51.4
(C-9), 29.2 (1C, C-4/ C-5/ C-6), 28.9 (1C, C-4/ C-5/ C-6), 28.8
(1C, C-4/ C-5/ C-6), 28.7 (2C, C-8, C-2), 26.6 (C-7), 25.2 (C-3),
21.6 (C-5’) ppm. 15N NMR (60 MHz, CDCl3)  – 132.7 (N-11),
– 170.9 (N-12), – 309.3 (N-10) ppm. HRMS (ESI) m/z calcd for
C16H25N3NaO3S [MNa]: 362.1509; found: 362.1504
( = 1.2 ppm).
1
Yield: 21.0 g (80.5 mmol, 94 %) of a colorless solid. H NMR
3
(400 MHz, CDCl3)  3.47 (t, J = 6.7 Hz, 2H, H-6), 2.94 (bt,
3
3J = 7.9 Hz, 2H, H-1), 1.88 (tt, J = 6.7 Hz, 2H, H-5), 1.68 (tt,
3J = 7.6 Hz, 2H, H-2), 1.56–1.48 (m, 2H, H-4), 1.47–1.39 (m,
2H, H-3) ppm. 13C{1H} NMR (100 MHz, CDCl3)  40.6 (C-1),
34.2 (C-6), 33.6 (C-5), 28.6 (C-2), 28.4 (C-4), 26.6 (C-3) ppm.
HRMS (ESI) m/z calcd for C6H15BrN [M+H]: 180.0382;
found: 180.0382 (∆ = 0.3 ppm).
N-(3-Bromopropyl)-2,2,2-trifluoroacetamide (9a). 3-Bro-
mopropylamine hydrobromide (50.0 g, 228 mmol, 1.0 eq) was
dissolved in anhydrous DCM (230 mL) and ethyl trifluoroace-
tate (30 mL, 35.7 g, 251 mmol, 1.1 eq) was added. Triethyla-
mine (34.8 mL, 25.4 g, 251 mmol, 1.1 eq) was added and the
reaction mixture was stirred at rt for 16 h. The solvent was re-
moved under reduced pressure and the residue was dissolved in
aqueous 1 M NaHCO3 solution (100 mL) and DCM (100 mL).
The aqueous phase was extracted with DCM (2 × 50 mL), the
combined organic layers were dried over Na2SO4 and the sol-
vent was removed under reduced pressure. The crude product
was purified by column chromatography (pentane → pen-
tane/Et2O = 1:1). Yield: 52.5 g (224 mmol, 98 %) of a colorless
solid. 1H NMR (400 MHz, CDCl3)  7.08 (s, 1H, NH), 3.52 (td,
General Procedure for Iodinated Azidoalkanes (7). 1.0 eq
of 3-azidopropyl-p-toluenesulfonate (6a), 6-azidohexyl-p-tol-
uenesulfonate (6b) or 9-azidononyl-p-toluenesulfonate (6c)
were dissolved in anhydrous acetone (~ 0.5 M). Sodium iodide
(1.5 eq) was added and the reaction mixture was stirred in an oil
bath at 60 °C for 16 h. Pentane was added and the mixture was
filtered. The solvent was removed under reduced pressure and
the crude product was purified by column filtration (pentane).
3-Azido-1-iodopropane (7a). Synthesized according to the
general procedure (153 mmol scale). Yield: 28.2 g (134 mmol,
87 %) of a colorless oil. 1H NMR (400 MHz, CDCl3)  3.42 (t,
3
3J = 6.5 Hz, 2H, H-1), 3.42 (t, J = 6.4 Hz, 2H, H-3), 2.15 (tt,
3J = 6.5 Hz, 2H, H-2) ppm. 13C{1H} NMR (100 MHz, CDCl3)
 157.8 (q, 2JC,F = 37.1 Hz, C-1’), 115.2 (q, 1JC,F = 287.5 Hz, C-
2’’), 38.7 (C-1), 31.3 (C-2), 30.1 (C-3) ppm. HRMS (ESI) m/z
calcd for C5H7BrF3NNaO [M+Na]: 255.9555; found:
255.9555 (∆ = 0.0 ppm).
3J = 6.4 Hz, 2H, H-3), 3.23 (t, J = 6.6 Hz, 2H, H-1), 2.02 (tt,
3
3J = 6.5 Hz, 2H, H-2) ppm. 13C{1H} NMR (100 MHz, CDCl3)
 = 51.5 (C-3), 32.4 (C-2), 2.5 (C-1) ppm. 15N NMR (60 MHz,
CDCl3)  = – 133.2 (N-5), – 169.6 (N-6), – 311.5 (N-4) ppm.
The spectroscopic data are in agreement with those reported in
the literature.52
N-(6-Bromohexyl)-2,2,2-trifluoroacetamide (9b). Tri-
fluoroacetic anhydride (5.86 mL, 8.85 g, 42.2 mmol, 1.1 eq)
was added to 6-bromohexanamine hydrobromide (8, 10.0 g,
38.3 mmol, 1.0 eq) at 0 °C. Triethylamine (5.84 mL, 4.26 g,
42.2 mmol, 1.1 eq) was added dropwise and the reaction mix-
ture was stirred for 2 h. Aqueous 1 M NaHCO3 solution (40 mL)
was added and the aqueous phase was extracted with DCM (2 ×
20 mL). The combined organic layers were dried over Na2SO4
and the solvent was removed under reduced pressure. The crude
product was purified by column chromatography (pentane →
6-Azido-1-iodohexane (7b). Synthesized according to the
general procedure (73.6 mmol scale). Yield: 15.5 g (61.4 mmol,
83 %) of a colorless oil. 1H NMR (400 MHz, CDCl3)  3.27 (t,
3J = 6.9 Hz, 2H, H-6), 3.19 (t, J = 7.0 Hz, 2H, H-1), 1.82 (tt,
3
3J = 7.0 Hz, 2H, H-2), 1.60 (tt, 3J = 7.0 Hz, 2H, H-5), 1.36–1.31
(m, 4H, H-3, H-4) ppm. 13C{1H} NMR (100 MHz, CDCl3)
 51.4 (C-6), 33.3 (C-2), 30.1 (C-3), 28.8 (C-5), 25.8 (C-4), 7.0
(C-1) ppm. 15N NMR (60 MHz, CDCl3)  – 132.6 (N-8),
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The Journal of Organic Chemistry
pentane/Et2O = 1:1). Yield: 8.60 g (31.1 mmol, 81 %) of a col-
orless oil. 1H NMR (400 MHz, CDCl3)  6.64 (s, 1H, NH), 3.39
°C (2h) or 150 W, 135 °C (2i, 2j). Then, EtOAc was added to
the reaction mixture and the precipitate was collected by filtra-
tion. The crude product was purified by reversed-phase column
1
2
3
4
5
6
7
8
3
3
(t, J = 6.7 Hz, 2H, H-6), 3.35 (td, J = 6.8 Hz, 2H, H-1), 1.84
(tt, 3J = 6.8 Hz, 2H, H-5), 1.59 (tt, 3J = 7.4 Hz, 2H, H-2), 1.48–
1.43 (m, 2H, H-4), 1.39–1.33 (m, 2H, H-3) ppm. 13C{1H} NMR
chromatography (RP18; H2O/TFA
H2O/MeCN/TFA = 50:50:0.1) and lyophilized.
1-(3-Azidopropyl)-2,3,3-trimethylindolium-5-sulfonate
=
100:0.1
→
2
(100 MHz, CDCl3)  = 157.4 (q, JC,F = 36.8 Hz, C-1’), 115.9
1
(2a). Synthesized according to the general procedure
(1.44 mmol scale). Yield: 302 mg (937 µmol, 65 %) of a beige
solid. 1H NMR (400 MHz, D2O)  8.09 (dd, 4J = 1.7 Hz,
5J = 0.4 Hz, 1H, H-4) 7.98 (dd, 3J = 8.5 Hz, 4J = 1.7 Hz, 1H, H-
6), 7.47 (dd, 3J = 8.5 Hz, 5J = 0.4 Hz, 1H, H-7), 4.59 (t,
3J = 7.3 Hz, 2H, H-1’), 3.55 (t, 3J = 6.1 Hz, 2H, H-3’), 2.23 (tt,
3J = 6.8 Hz, 2H, H-2’), 1.58 (s, 6H, H-3’’) ppm. 13C{1H} NMR
(100 MHz, D2O)  200.5 (C-2), 144.9 (C-5), 143.4 (C-7a),
143.1 (C-3a), 127.56 (C-6), 121.6 (C-4), 116.4 (C-7), 55.6 (C-
3), 48.8 (C-3’), 46.7 (C-1’), 26.8 (C-2’), 22.1 (C-3’’), 13.9
(quint, C-2’’; due to exchange with deuterium ions) ppm. 15N
NMR (60 MHz, AcOD-d4/D2O = 3:1)  – 134.6 (N-5’), – 167.8
(N-1), – 311.3 (N-4’) ppm. HRMS (ESI) m/z calcd for
C14H19N4O3S [MH]: 323.1172; found: 323.1169
(∆ = 1.0 ppm).
(q, JC,F = 287.6 Hz, C-2’), 39.9 (C-1), 33.7 (C-6), 32.5 (C-5),
28.8 (C-2), 27.7 (C-4), 25.9 (C-3) ppm. HRMS (ESI) m/z calcd
for C8H13BrF3NNaO [M+Na]: 298.0025; found: 298.0026
(∆ = 0.5 ppm).
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4.3. Synthesis of Indolium Salts
Potassium 2,3,3-Trimethylindolenine-5-sulfonate (1). In a
microwave vial, 4-hydrazinobenzenesulfonic acid (5.00 g,
26.6 mmol, 1.0 eq) and 3-methylbutan-2-one (8.48 mL, 6.87 g,
79.9 mmol, 3.0 eq) were dispersed in acetic acid (15 mL). The
reaction mixture was stirred in a microwave system at 125 °C
and at 150 W for 4 h. The mixture was cooled to 0 °C, the pre-
cipitate was collected by filtration and dried under vacuum. A
solution of KOH (3.17 g, 56.4 mmol, 1.0 eq) in 2-propanol
(100 mL) was added to a solution of the crude product (13.5 g,
56.4 mmol) in methanol (200 mL). The precipitate was ob-
tained by filtration and dried under removed pressure. Yield:
3.80 g (13.7 mmol, 52 %) of a yellow solid. 1H NMR
(400 MHz, MeOD-d4)  7.85 (dd, 4J = 1.7 Hz, 5J = 0.6 Hz, 1H,
1-(6-Azidohexyl)-2,3,3-trimethylindolium-5-sulfonate
(2b). Synthesized according to the general procedure
(1.44 mmol scale). Yield: 279 mg (766 µmol, 53 %) of a light
pink solid. 1H NMR (400 MHz, MeOD-d4)  8.05 (m, 1H, H-4),
7.90 (m, 2H, H-6, H-7), 4.55 (t, 3J = 7.9 Hz, 2H, H-1’), 3.31 (t,
3J = 6.7 Hz, 2H, H-6’; superimposed by the MeOD signal), 1.97
3
4
H-4), 7.82 (dd, J = 8.0 Hz, J = 1.7 Hz, 1H, H-6), 7.47 (dd,
3
(tt, J = 7.7 Hz, 2H, H-2’), 1.63 (m, 2H, H-5’), 1.61 (s, 6H, H-
3J = 8.0 Hz, J = 0.6 Hz, 1H, H-7), 2.33 (s, 3H, H-2’), 1.36 (s,
5
3’’), 1.58–1.49 (m, 4H, H-3’, H-4’) ppm. 13C{1H} NMR
(100 MHz, MeOD-d4)  199.6 (C-2), 148.5 (C-5), 143.42 (C-
3a), 143.38 (C-7a), 128.3 (C-6), 122.2 (C-4), 116.7 (C-7), 56.1
(C-3), 52.3 (C-6’), 49.5 (C-1’), 29.6 (C-5’), 28.6 (C-2’), 27.3
(C-4’), 27.2 (C-3’), 22.6 (C-3’’), 13.8 (sept, C-2’’; due to ex-
change with deuterium ions) ppm. 15N NMR (60 MHz, AcOD-
d4/D2O = 3:1)  – 132.7 (N-8’), – 165.5 (N-1), 171.9 (N-9’),
– 310.5 (N-7’) ppm. HRMS (ESI) m/z calcd for C17H25N4O3S
[MH]: 365.1642; found: 365.1637 (∆ = 1.4 ppm).
6H, H-3’) ppm. 13C{1H} NMR (100 MHz, MeOD-d4)  193.4
(C-2), 155.2 (C-7a), 146.9 (C-3a), 143.9 (C-5), 127.1 (C-6),
120.9 (C-4), 119.7 (C-7), 55.3 (C-3), 23.1 (C-3’), 15.4 (C-2’)
ppm. 15N NMR (40 MHz, MeOD-d4):  – 85.4 ppm.
HRMS (ESI) m/z calcd for C11H12NO3S– [M]–: 238.0543; found:
238.0542 (∆ = 0.5 ppm).
General Procedure for Indolium Derivatives (2).
Alkyne and Azide Chains: In a microwave vial, 1.0 eq of po-
tassium 2,3,3-trimethylindolenine-5-sulfonate (1) and 10 eq of
5-iodopentyne, 6-iodohexyne, 3-azido-1-iodopropane (7a), 6-
azido-1-iodohexane (7b) or 9-azido-1-iodononane (7c) were
dispersed in DMF (0.5 mL per 100 mg of indolenine 1). The re-
action mixture was stirred under microwave irradiation for 1.5 h
(75 W, 110 °C – 115 °C). EtOAc and Et2O were added and the
precipitate was collected by filtration. The crude product was
purified by reversed-phase column chromatography (RP18;
H2O/TFA = 100:0.1 → H2O/MeCN/TFA = 50:50:0.1) and ly-
ophilized.
Trifluoroacetamide Chains: In a microwave vial, 1.0 eq of po-
tassium 2,3,3-trimethylindolenine-5-sulfonate (1) and 10 eq of
N-(3-Bromopropyl)-2,2,2-trifluoracetamide (9a) or N-(6-bro-
mohexyl)-2,2,2-trifluoroacetamide (9b) were dispersed in bu-
tyronitrile (1 mL). The suspension was stirred under microwave
irradiation for 1.5 h (150 W, 135 °C). Then, EtOAc was added
and the precipitate was collected by filtration. The crude prod-
uct was purified by reversed-phase column chromatography
(RP18; H2O/TFA = 100:0.1 → H2O/MeCN/TFA = 50:50:0.1)
and lyophilized.
1-(9-Azidononyl)-2,3,3-trimethylindolium-5-sulfonate
(2c). Synthesized according to the general procedure (721 µmol
scale). Yield: 134 mg (400 µmol, 46 %) of a light pink solid. 1H
NMR (600 MHz, MeOD-d4)

8.05 (dd, 4J = 1.6 Hz,
5J = 0.4 Hz, 1H, H-4), 7.90 (dd, 3J = 8.4 Hz, 4J = 1.6 Hz, 1H, H-
6), 7.87 (dd, 3J = 8.4 Hz, 5J = 0.4 Hz, 1H, H-7), 4.53 (t,
3J = 7.9 Hz, 2H, H-1’), 3.28 (t, 3J = 6.8 Hz, 2H, H-9’), 1.95 (tt,
3J = 7.7 Hz, 2H, H-2’), 1.60–1.56 (m, 8H, H-3’’/ H-8’), 1.54–
1.49 (m, 2H, H-3’), 1.45–1.41 (m, 2H, H-7’), 1.40–1.36 (m, 6H,
H-4’, H-5’, H-6’) ppm. 13C{1H} NMR (100 MHz, MeOD-d4)
 199.4 (C-2), 148.5 (C-5), 143.41 (C-3a), 143.38 (C-7a), 128.3
(C-6), 122.2 (C-4), 116.6 (C-7), 56.1 (C-3), 52.4 (C-9’), 49.6
(C-1’), 30.3 (1C, C-5’/ C-6’), 30.1 (1C, C-6’/ C-5’), 30.8 (C-4’),
29.8 (C-8’), 28.8 (C-2’), 27.7 (C-7’), 27.6 (C-3’), 22.6 (C-3’’),
13.7 (sept, C-2’’; due to exchange with deuterium ions) ppm.
15N NMR (60 MHz, MeOD-d4)  – 132.6 (N-11’), – 165.3 (N-
1), 172.0 (N-12’), – 310.4 (N-10’) ppm. HRMS (ESI) m/z calcd
for C20H30N4NaO3S [MNa]: 429.1931; found: 429.1921
(∆ = 2.2 ppm).
Nitrile and Sulfonate Group Chains: Potassium 2,3,3-trime-
thylindolenine-5-sulfonate (1) was dispersed in neat 1,4-butane
sultone, 4-bromobutyronitrile or 6-bromohexanenitrile (2 mL
per 100 mg of 1). The suspension was placed in a microwave
system and stirred for 1.5 h. Microwave conditions: 125 W, 125
1-(Pentynyl)-2,3,3-trimethylindolium-5-sulfonate
(2d).
Synthesized according to the general procedure (721 µmol
1
scale). Yield: 115 mg (377 µmol, 52 %) of a beige solid. H
4
NMR (400 MHz, AcOD-d4/D2O = 3:1)  8.19 (d, J = 1.4 Hz,
1H, H-4), 8.09 (dd, 3J = 8.4 Hz, 4J = 1.4 Hz, 1H, H-6), 7.91 (d,
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3
3J = 8.4 Hz, 1H, H-7), 4.67 (bt, J = 7.5 Hz, 2H, H-1’), 2.47–
ppm. 19F NMR (377 MHz, AcOD-d4/D2O = 3:1)  – 76.38 (3F,
3
CF3-9’) ppm. HRMS (ESI) m/z calcd for C19H26F3N2O4S
[MH]: 435.1560; found: 435.1547 (∆ = 3.0 ppm).
1
2
3
4
5
6
7
8
2.42 (m, 3H, H-3’, H-5’), 2.22 (tt, J = 6.8 Hz, 2H, H-2’), 1.64
(s, 6H, H-3’’) ppm. 13C{1H} NMR (100 MHz, AcOD-
d4/D2O = 3:1)  200.1 (C-2), 146.4 (C-5), 143.1 (C-7a), 142.9
(C-3a), 128.2 (C-6), 122.0 (C-4), 116.2 (C-7), 83.1 (C-4’), 71.9
(C-5’), 55.8 (C-3), 48.1 (C-1’), 26.6 (C-2’), 22.3 (C-3’’), 15.9
(C-3’), 13.9 (quint, C-2’’; due to exchange with deuterium ions)
ppm. 15N NMR (60 MHz, AcOD-d4/D2O = 3:1)  – 167.1 ppm.
HRMS (ESI) m/z calcd for C16H20NO3S [MH]: 306.1158;
found: 306.1156 ( = 0.9 ppm).
1-(4-Sulfobutyl)-2,3,3-trimethylindolium-5-sulfonate (2h).
Synthesized according to the general procedure (3.61 mmol
scale). Yield: 1.27 g (3.38 mmol, 94 %) of a colorless solid. 1H
NMR (400 MHz, MeOD-d4)  8.12 (dd, 4J = 1.6 Hz,
5J = 0.5 Hz, 1H, H-4) 8.05 (dd, 3J = 8.4 Hz, 4J = 1.6 Hz, 1H, H-
6), 7.98 (dd, 3J = 8.4 Hz, 5J = 0.5 Hz, 1H, H-7), 4.57 (t,
3J = 7.9 Hz, 2H, H-1’), 2.90 (t, 3J = 7.2 Hz, 2H, H-4’), 2.14 (tt,
3J = 7.4 Hz, 2H, H-2’), 1.95 (tt, 3J = 7.4 Hz, 2H, H-3’), 1.63 (s,
6H, H-3’’) ppm. 13C{1H} NMR (100 MHz, MeOD-d4)  199.9
(C-2), 148.5 (C-5), 143.51 (C-3a), 143.49 (C-7a), 128.5 (C-6),
122.3 (C-4), 116.7 (C-7), 56.2 (C-3), 51.1 (C-4’), 49.2 (C-1’),
27.3 (C-2’), 23.2 (C-3’), 22.6 (C-3’’), 13.8 (quint, C-2’’; due to
exchange with deuterium ions) ppm. 15N NMR (60 MHz,
9
1-(Hexynyl)-2,3,3-trimethylindolium-5-sulfonate
Synthesized according to the general procedure (721 µmol
scale). Yield: 106 mg (332 µmol, 46 %) of a beige solid. H
(2e).
10
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
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53
54
55
56
57
58
59
60
1
4
NMR (400 MHz, AcOD-d4/D2O = 3:1)  8.17 (d, J = 1.5 Hz,
1H, H-4), 8.08 (dd, 3J = 8.5 Hz, 4J = 1.5 Hz, 1H, H-6), 7.91 (d,
3
3J = 8.5 Hz, 1H, H-7), 4.58 (bt, J = 7.8 Hz, 2H, H-1’), 2.33–
3
2.30 (m, 3H, H-4’, H-6’), 2.11 (tt, J = 7.8 Hz, 2H, H-2’), 1.71
AcOD-d4/D2O = 3:1)  – 166.2 ppm. HRMS (ESI) m/z calcd for
(tt, J = 7.2 Hz, 2H, H-3’), 1.64 (s, 6H, H-3’’) ppm. 13C{1H}
3

C15H20NO6S2
[MH]: 374.0738; found: 374.0748
NMR (100 MHz, AcOD-d4/D2O = 3:1)  199.6 (C-2), 146.2 (C-
5), 142.95 (C-3a), 142.94 (C-7a), 128.0 (C-6), 121.9 (C-4),
116.2 (C-7), 84.2 (C-5’), 70.7 (C-6’), 55.6 (C-3), 48.6 (C-1’),
26.9 (C-2’), 25.4 (C-3’), 22.2 (C-3’’), 17.8 (C-4’), 13.8 (quint,
C-2’’; due to exchange with deuterium ions) ppm. 15N NMR
(60 MHz, AcOD-d4/D2O = 3:1)  – 165.9 ppm. HRMS (ESI)
m/z calcd for C17H22NO3S [MH]: 320.1315; found: 320.1308
(∆ = 2.2 ppm).
1-(3-(2,2,2-Trifluoroacetamido)propyl)-2,3,3-trimethylin-
dolium-5-sulfonate (2f). Synthesized according to the general
procedure (1.44 mmol scale). Yield: 446 mg (1.14 mmol, 79 %)
of a colorless solid. H NMR (600 MHz, AcOD-d4/D2O = 3:1)
 8.18 (dd, 4J = 1.7 Hz, 5J = 0.4 Hz, 1H, H-4) 8.09 (dd,
3J = 8.5 Hz, 4J = 1.7 Hz, 1H, H-6), 7.90 (d, 3J = 8.4 Hz, 1H, H-
(∆ = 2.8 ppm).
1-(3-Cyanopropyl)-2,3,3-trimethylindolium-5-sulfonate
(2i). Synthesized according to the general procedure
(1.44 mmol scale). Yield: 423 mg (1.38 mmol, 96 %) of a col-
1
orless solid. H NMR (400 MHz, AcOD-d4/D2O = 3:1)  8.18
(d, 4J = 1.3 Hz, 1H, H-4) 8.07 (dd, 3J = 8.5 Hz, 4J = 1.3 Hz, 1H,
H-6), 7.92 (d, 3J = 8.5 Hz, 1H, H-7), 4.69 (t, 3J = 7.7 Hz, 2H, H-
1’), 2.81 (t, 3J = 7.0 Hz, 2H, H-3’), 2.41 (tt, 3J = 7.4 Hz, 2H, H-
2’), 1.65 (s, 6H, H-3’’) ppm. 13C{1H} NMR (100 MHz, AcOD-
d4/D2O = 3:1)  200.9 (C-2), 146.3 (C-5), 142.9 (2C, C-3a, C-
7a), 128.1 (C-6), 121.9 (C-4), 120.2 (C-4’), 116.1 (C-7), 55.9
(C-3), 47.7 (C-1’), 23.7 (C-2’), 22.3 (C-3’’), 14.8 (C-3’), 14.0
(quint, C-2’’; due to exchange with deuterium ions) ppm. 15N
NMR (60 MHz, AcOD-d4/D2O = 3:1)  – 138.2 (N-5’), – 168.7
(N-1) ppm. HRMS (ESI) m/z calcd for C15H19N2O3S [MH]:
307.1111; found: 307.1112 (∆ = 0.3 ppm).
1
3
3
7), 4.61 (t, J = 8.0 Hz, 2H, H-1’), 3.59 (t, J = 7.0 Hz, 2H, H-
3’), 2.31 (tt, J = 7.5 Hz, 2H, H-2’), 1.64 (s, 6H, H-3’’) ppm.
13C{1H} NMR (150 MHz, AcOD-d4/D2O = 3:1)  = 200.2 (C-
3
1-(5-Cyanopentyl)-2,3,3-trimethylindolium-5-sulfonate
(2j). Synthesized according to the general procedure
(1.44 mmol scale). Yield: 438 mg (1.31 mmol, 91 %) of a col-
2
2), 159.2 (q, JC,F = 37.6 Hz, C-5’), 146.2 (C-5), 142.9 (C-3a),
142.8 (C-7a), 128.1 (C-6), 121.9 (C-4), 116.5 (q,
1JC,F = 286.4 Hz, C-6’), 116.1 (C-7), 55.2 (C-3), 46.6 (C-1’),
37.4 (C-3’), 27.1 (C-2’), 22.2 (C-3’’), 13.8 (quint, C-2’’; due to
exchange with deuterium ions) ppm. 15N NMR (60 MHz,
AcOD-d4/D2O = 3:1)  – 167.7 (N-1), – 266.3 (N-4’) ppm.
19F NMR (565 MHz, AcOD-d4/D2O = 3:1)  – 76.17 (3F, CF3-
6’) ppm. HRMS (ESI) m/z calcd for C16H20F3N2O4S [MH]:
393.1090; found: 393.1080 (∆ = 2.6 ppm).
1-(6-(2,2,2-Trifluoroacetamido)hexyl)-2,3,3-trimethylin-
dolium-5-sulfonate (2g). Synthesized according to the general
procedure (1.08 mmol scale). Yield: 323 mg (743 µmol, 69 %)
of a light pink solid. 1H NMR (400 MHz, AcOD-d4/D2O = 3:1)
1
orless solid. H NMR (400 MHz, AcOD-d4/D2O = 3:1)  8.19
(d, 4J = 1.6 Hz, H-4) 8.10 (dd, 3J = 8.5 Hz, 4J = 1.6 Hz, 1H, H-
3
3
6), 7.90 (d, J = 8.5 Hz, 1H, H-7), 4.57 (t, J = 7.7 Hz, 2H, H-
1’), 2.51 (t, 3J = 6.9 Hz, 2H, H-5’), 2.08–2.02 (m, 2H, H-2’; su-
perimposed by the solvent signal), 1.79–1.71 (m, 2H, H-4’)
1.67–1.59 (m, 8H, H-3’’, H-3’), ppm. 13C{1H} NMR (100 MHz,
AcOD-d4/D2O = 3:1)  199.6 (C-2), 146.3 (C-5), 142.9 (2C, C-
7a, C-3a), 128.2 (C-6), 121.9 (C-4), 121.2 (C-6’), 116.3 (C-7),
55.7 (C-3), 48.8 (C-1’), 27.4 (C-2’), 25.9 (C-3’), 24.9 (C-4’),
22.3 (C-3’’), 16.8 (C-5’), 13.8 (quint, C-2’’; due to exchange
with deuterium ions) ppm. 15N NMR (60 MHz, AcOD-
d4/D2O = 3:1)  – 142.1 (N-5’), – 166.0 (N-1) ppm. HRMS
(ESI) m/z calcd for C17H23N2O3S [MH]: 335.1424; found:
335.1418 (∆ = 1.9 ppm).
4
3
4
 8.19 (d, J = 1.7 Hz, H-4) 7.90 (dd, J = 8.5 Hz, J = 1.7 Hz,
3
3
1H, H-6), 7.89 (d, J = 8.5 Hz, 1H, H-7), 4.54 (t, J = 7.3 Hz,
2H, H-1’), 3.33 (t, 3J = 7.1 Hz, 2H, H-6’), 1.99 (tt, 3J = 7.3 Hz,
2H, H-2’), 1.64 (s, 6H, H-3’’), 1.63–1.57 (m, 2H, H-5’), 1.55–
1.48 (m, 2H, H-3’), 1.46–1.38 (m, 2H, H-4’) ppm. 13C{1H}
NMR (100 MHz, AcOD-d4/D2O = 3:1)  199.4 (C-2), 158.8 (q,
2JC,F = 36.9 Hz, C-8’), 146.3 (C-5), 143.01 (C-7a), 142.99 (C-
3a), 128.2 (C-6), 122.0 (C-4), 116.7 (q, 1JC,F = 286.4 Hz, C-9’),
116.3 (C-7), 55.7 (C-3), 49.1 (C-1’), 40.1 (C-6’), 28.6 (C-5’),
28.0 (C-2’), 26.4 (2C, C-3’, C-4’), 22.3 (C-3’’), 13.8 (quint, C-
2’’; due to exchange with deuterium ions) ppm. 15N NMR
(60 MHz, AcOD-d4/D2O = 3:1)  – 165.6 (N-1), – 263.2 (N-7’)
1-(3-(Carboxypropyl)-2,3,3-trimethylindolium-5-sul-
fonate (2k). 1-(3-Cyanopropyl)-2,3,3-trimethylindolium-5-sul-
fonate (2i, 100 mg, 326 µmol, 1.0 eq) was dissolved in a mix-
ture of acetic acid (1 mL) and 6 M HCl (2 mL). The solution was
stirred under microwave irradiation for 2 h (125 W, 130 °C).
Then, the solvent was removed in vacuo and the residue was
dissolved in methanol. EtOAc and Et2O were added and the pre-
cipitate was collected by filtration. The product was dried under
vacuum and used in the following step without further purifica-
1
tion. Yield: 102 mg (313 µmol, 96 %) of a colorless solid. H
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4
NMR (400 MHz, AcOD-d4/D2O = 3:1)  8.21 (d, J = 1.7 Hz,
- and - protons with deuterium ions), 1H, H-), 8.31 (s (after
H-4) 8.13 (dd, 3J = 8.5 Hz, 4J = 1.7 Hz, 1H, H-6), 7.98 (d,
1
2
3
4
5
6
7
8
complete exchange of the - and - protons with deuterium
ions), 1H, H-), 7.91–7.87 (m, 4H, H-4, H-6), 7.43 (d,
3J = 8.9 Hz, 1H, H-7), 7.34 (d, 3J = 8.9 Hz, 1H, H-7), 6.73 (dd,
3J = 12.2 Hz, 1H, H-), 6.47 (d, 3J = 13.9 Hz, 1H, H-), 6.36 (d,
3J = 13.4 Hz, 1H, H-), 4.21–4.18 (m, 4H, H-1’, H-1’’), 3.52 (t,
3J = 6.1 Hz, 2H, H-3’’), 2.93 (t, 3J = 6.5 Hz, 2H, H-4’), 2.04 (tt,
3J = 6.7 Hz, 2H, H-2’’), 1.98–1.97 (m, 4H, H-2’, H-3’), 1.76 (s,
6H, H-3’’’), 1.75 (s, 6H, H-3’’’) ppm. 13C{1H} NMR
(100 MHz, MeOD-d4)  175.9 (C-2), 174.7 (C-2), 156.7 (C-),
155.9 (C-), 145.0 (C-7a), 144.8 (C-7a), 143.4 (C-5), 143.0 (C-
5), 142.8 (C-3a), 142.5 (C-3a), 128.1 (2C, C-6), 128.0 (C-),
121.3 (2C, C-4), 111.9 (C-7), 111.3 (C-7), 104.6 (2C, C-), 51.7
(C-4’), 50.7 (C-3), 50.4 (C-3), 49.7 (C-3’’), 45.1 (C-1’), 42.5
(C-1’’), 27.9 (C-3’’’), 27.8 (C-3’’’), 27.5 (C-2’’), 27.1 (C-2’),
3J = 8.5 Hz, 1H, H-7), 4.61 (t, J = 7.7 Hz, 2H, H-1’), 2.68 (t,
3
3J = 6.6 Hz, 2H, H-3’), 2.26 (tt, 3J = 7.3 Hz, 2H, H-2’), 1.65 (s,
6H, H-3’’), ppm. 13C{1H} NMR (100 MHz, AcOD-
d4/D2O = 3:1)  200.0 (C-2), 176.7 (C-4’), 146.4 (C-5), 143.2
(C-3a), 143.0 (C-7a), 128.3 (C-6), 122.1 (C-4), 116.3 (C-7), 55.8
(C-3), 48.3 (C-1’), 30.7 (C-3’), 23.1 (C-2’), 22.3 (C-3’’), 14.1
(quint, C-2’’; due to exchange with deuterium ions) ppm.
HRMS (ESI) m/z calcd for C15H20NO5S [MH]: 326.1057;
found: 326.1059 (∆ = 0.7 ppm).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
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56
57
58
59
60
1-(5-Carboxypentyl)-2,3,3-trimethylindolium-5-sulfonate
(2l). 1-(5-Cyanopentyl)-2,3,3-trimethylindolium-5-sulfonate
(2j, 200 mg, 598 µmol, 1.0 eq) was dissolved in a mixture of
acetic acid (1 mL) and 6 M HCl (2 mL). The solution was stirred
under microwave irradiation for 2 h (125 W, 130 °C). The sol-
vent was removed in vacuo, the residue was dissolved in H2O
(10 mL) and lyophilized. The product was used in the following
step without further purification. Yield: 209 mg (591 µmol,
99 %) of a colorless solid. 1H NMR (400 MHz, AcOD-
d4/D2O = 3:1)  8.16 (d, 4J = 1.7 Hz, H-4) 8.01 (dd, 3J = 8.5 Hz,

23.4 (C-3’) ppm. HRMS (ESI) m/z calcd for C32H39N5O9S3
[MH]: 732.1837; found: 732.1836 (∆ = 0.2 ppm).
Sulfo-(6-azidohexyl)pentamethine Cyanine (4b). Synthe-
sized according to the general procedure (573 µmol scale).
1
Yield: 316 mg (407 µmol, 71 %) of a blue solid. H NMR
3
4J = 1.7 Hz, 1H, H-6), 7.92 (d, J = 8.5 Hz, 1H, H-7), 4.56 (t,
3
(600 MHz, MeCN-d3/D2O = 3:1)  8.33 (d, J = 12.5 Hz, 2H,
4
4
3J = 7.7 Hz, 2H, H-1’), 2.39 (t, J = 7.3 Hz, 2H, H-5’), 2.03–
3
H-), 7.80 (d, J = 1.7 Hz, 1H, H-4), 7.79 (d, J = 1.7 Hz, 1H,
3
4
H-4), 7.75 (dd, J = 8.4 Hz, J = 1.7 Hz, 1H, H-6), 7.75 (dd,
1.97 (m, 2H, H-2’; superimposed by the solvent signal), 1.71–
1.66 (m, 2H, H-4’) 1.64 (s, 6H, H-3’’), 1.55–1.47 (m, 2H, H-3’)
3J = 8.4 Hz, 4J = 1.7 Hz, 1H, H-6), 7.31 (d, 3J = 8.4 Hz, 1H, H-
3
3
ppm. 13C{1H} NMR (100 MHz, AcOD-d4/D2O = 3:1)  199.4
(C-2), 178.2 (C-6’), 145.8 (C-5), 142.9 (2C, C-7a, C-3a), 127.8
(C-6), 121.6 (C-4), 116.3 (C-7), 55.5 (C-3), 48.8 (C-1’), 33.7
(C-5’), 27.6 (C-2’), 26.0 (C-3’), 24.3 (C-4’), 22.2 (C-3’’), 13.8
(quint, C-2’’; due to exchange with deuterium ions) ppm.
HRMS (ESI) m/z calcd for C17H24NO5S [MH]: 354.1370;
found: 354.1375 (∆ = 1.4 ppm).
7), 7.24 (d, J = 8.4 Hz, 1H, H-7), 6.58 (dd, J = 12.5 Hz, 1H,
H-), 6.26 (d, 3J = 13.5 Hz, 1H, H-), 6.24 (d, 3J = 13.5 Hz, 1H,
H-), 4.00–3.97 (m, 4H, H-1’, H-1’’), 3.24 (t, 3J = 6.1 Hz, 2H,
H-6’’), 2.81 (t, 3J = 7.2 Hz, 2H, H-4’), 1.82–1.80 (m, 4H, H-2’,
H-3’), 1.72 (tt, J = 7.3 Hz, 2H, H-2’’), 1.66 (s, 12H, H-3’’’),
1.51 (tt, J = 7.1 Hz, 2H, H-5’’), 1.36–1.35 (m, 4H, H-3’’, H-
3
3
4’’) ppm. 13C{1H} NMR (150 MHz, MeCN-d3/D2O = 3:1) 
174.9 (2C, C-2), 155.5 (C-), 155.4 (C-), 144.7 (C-7a), 144.7
(C-7a), 142.5 (C-3a), 142.4 (C-3a), 142.3 (C-5), 142.1 (C-5),
127.6 (C-6), 127.5 (C-6), 126.5 (C-), 120.8 (C-4), 120.7 (C-4),
111.9 (C-7), 111.7 (C-7), 104.5 (2C, C-), 51.9 (C-6’’), 51.4 (C-
4’), 50.2 (C-3), 50.1 (C-3), 44.9 (C-1’’), 44.8 (C-1’), 29.5 (C-
5’’), 27.7 (C-2’’), 27.6 (C-3’’’), 27.6 (C-3’’’), 26.9 (C-3’’), 26.7
(C-4’’), 26.6 (C-2’), 22.9 (C-3’) ppm. HRMS (ESI) m/z calcd
4.4. Synthesis of Pentamethine Cyanines
Sulfotetramethine Hemicyanine (3). In a microwave vial, 1-
(4-sulfobutyl)-2,3,3-trimethylindolium-5-sulfonate (2h, 1.09 g,
2.91 mmol, 1.0 eq) and malonaldehyde dianilide hydrochloride
(754 mg, 2.91 mmol, 1.0 eq) were dissolved in acetic anhydride
(5 mL) and acetic acid (5 mL). The reaction mixture was stirred
under microwave irradiation for 30 min (125 W, 135 °C). Then,
Et2O was added and the precipitate was collected by filtration.
In order to remove the by-product acetanilide, the crude product
was dissolved in methanol and triturated with EtOAc. Since
hemicyanine 3 is susceptible to hydrolysis and decomposes in
protic solvents, it was used in the following step without further
purification.
General Procedure for Pentamethine Cyanine Dyes (4). In
a microwave vial, indolium salt 2a – 2g or 2i – 2l (1.0 eq) and
sulfotetramethine hemicyanine (8, 1.0 eq) were dissolved in a
mixture of HOAc (1 mL) and Ac2O (1 mL). KOAc (500 mg)
was added and the suspension was stirred under microwave ir-
radiation for 15 min (75 W, 110 °C). The reaction mixture was
transferred to a 100 mL flask and EtOAc and Et2O were added.
The precipitate was collected by filtration. In order to remove
excess KOAc, the solid was dissolved in methanol and triturated
with EtOAc. The crude product was purified by reversed-phase
column chromatography (RP18; H2O/TFA = 100:0.1 →
H2O/MeCN/TFA = 50:50:0.1) and lyophilized.

for C35H44N5O9S3 [MH]: 774.2307; found: 774.2284
(∆ = 2.9 ppm).
Sulfo-(9-azidononyl)pentamethine Cyanine (4c). Synthe-
sized according to the general procedure (310 µmol scale).
1
Yield: 179 mg (219 µmol, 70 %) of a blue solid. H NMR
(600 MHz, MeOD-d4)  = 8.31 (d, 3J = 12.1 Hz, 2H, H-),
3
7.89–7.87 (m, 4H, H-4, H-6), 7.39 (d, J = 8.8 Hz, 1H, H-7),
7.33 (d, 3J = 8.2 Hz, 1H, H-7), 6.72 (dd, 3J = 12.5 Hz, 1H, H-),
3
3
6.42 (d, J = 13.7 Hz, 1H, H-), 6.35 (d, J = 13.6 Hz, 1H, H-
3
3
), 4.16 (t, J = 6.8 Hz, 2H, H-1’), 4.13 (t, J = 7.5 Hz, 2H, H-
1’’), 3.27 (t, 3J = 6.8 Hz, 2H, H-9’’), 2.92 (t, 3J = 7.0 Hz, 2H, H-
4’), 1.98–1.97 (m, 4H, H-2’, H-3’), 1.81 (tt, 3J = 7.5 Hz, 2H, H-
2’’), 1.76 (s, 6H, H-3’’’), 1.75 (s, 6H, H-3’’’), 1.56 (tt,
3J = 7.1 Hz, 2H, H-8’’),1.47–1.43 (m, 2H, H-3’’), 1.42–1.39 (m,
2H, H-4’’), 1.38–1.35 (m, 2H, H-7’’), 1.34–1.33 (m, 4H, H-5’’,
H-6’’) ppm. 13C{1H} NMR (150 MHz, MeOD-d4) 175.3 (C-
2), 175.2 (C-2), 156.3 (C-), 156.0 (C-), 145.0 (C-7a), 144.9
(C-7a), 143.2 (C-5), 143.1 (C-5), 142.6 (C-3a), 142.6 (C-3a),
128.1 (C-6), 128.0 (C-6), 127.9 (C-), 121.3 (C-4), 121.3 (C-4),
111.7 (C-7), 111.6 (C-7), 105.5 (C-), 105.3 (C-), 52.4 (C-
9’’), 51.6 (C-4’), 50.5 (2C, C-3), 45.1 (C-1’’), 45.0 (C-1’), 30.4
Sulfo-(3-azidopropyl)pentamethine Cyanine (4a). Synthe-
sized according to the general procedure (310 µmol scale).
Yield: 165 mg (225 µmol, 72 %) of a blue solid. H NMR
1
(400 MHz, MeOD-d4)  8.33 (s (after complete exchange of the
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(1C, C-5’’/ C-6’’), 30.2 (C-4’’), 30.0 (1C, C-5’’/ C-6’’), 29.8 142.3 (C-7a), 141.0 (C-3a), 140.6 (C-3a), 126.3 (2C, C-6), 126.2
1
2
3
4
5
6
7
8
(C-8’’), 28.4 (C-2’’), 27.8 (C-3’’’), 27.7 (C-7’’), 27.6 (C-3’’),
(C-), 120.4 (C-4’), 120.0 (2C, C-4), 110.9 (C-7), 109.9 (C-7),
104.6 (C-), 103.2 (C-), 50.7 (C-4’), 49.3 (C-3), 48.9 (C-3),
43.7 (C-1’), 42.5 (C-1’’), 27.3 (C-3’’’), 27.2 (C-3’’’), 26.0 (C-
2’), 23.0 (C-2’’), 22.1 (C-3’), 14.0 (C-3’’) ppm. HRMS (ESI)
27.1 (C-2’), 23.4 (C-3’) ppm. HRMS (ESI) m/z calcd for

C38H50N5O9S3
[MH]: 816.2776; found: 816.2787
(∆ = 1.4 ppm).

m/z calcd for C33H38N3O9S3 [MH]: 716.1776; found:
Sulfo-(pentynyl)pentamethine Cyanine (4d). Synthesized
according to the general procedure (164 µmol scale). Yield:
109 mg (152 µmol, 93 %) of a blue solid. 1H NMR (400 MHz,
MeOD-d4/D2O = 3:1)  8.36–8.27 (m, 2H, H-), 7.90–7.86 (m,
4H, H-4, H-6), 7.41 (d, 3J = 8.8 Hz, 1H, H-7), 7.36 (d,
3J = 8.2 Hz, 1H, H-7), 6.73 (dd, 3J = 12.4 Hz, 1H, H-), 6.47 (d,
3J = 13.7 Hz, 1H, H-), 6.42 (d, 3J = 13.6 Hz, 1H, H-), 4.23 (t,
3J = 7.3 Hz, 2H, H-1’’), 4.18 (t, 3J = 6.7 Hz, 2H, H-1’), 2.92 (t,
3J = 6.7 Hz, 2H, H-4’), 2.54 (t, 4J = 2.6 Hz, 1H, H-5’’), 2.37 (td,
716.1792 (∆ = 2.3 ppm).
Sulfo-(5-cyanopentyl)pentamethine Cyanine (4g). Synthe-
sized according to the general procedure (299 µmol scale).
1
Yield: 211 mg (283 µmol, 95 %) of a blue solid. H NMR
9
(600 MHz, MeOD-d4/D2O = 3:1) 8.32–8.29 (m, 2H, H-),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
3
7.89–7.87 (m, 4H, H-4, H-6), 7.40 (d, J = 8.9 Hz, 1H, H-7),
7.35 (d, 3J = 8.2 Hz, 1H, H-7), 6.75 (dd, 3J = 12.5 Hz, 1H, H-),
3
3
6.43 (d, J = 13.8 Hz, 1H, H-), 6.37 (d, J = 13.7 Hz, 1H, H-
), 4.18–4.14 (m, 4H, H-1’, H-1’’), 2.91 (t, 3J = 7.1 Hz, 2H, H-
4’), 2.48 (t, 3J = 7.1 Hz, 2H, H-5’’), 2.00–1.92 (m, 4H, H-2’, H-
3’), 1.86 (tt, 3J = 7.6 Hz, 2H, H-2’’), 1.75 (s, 12H, H-3’’’), 1.72
4
3
3J = 6.7 Hz, J = 2.6 Hz, 2H, H-3’’), 2.00 (tt, J = 7.6 Hz, 2H,
H-2’’), 1.98–1.97 (m, 4H, H-2’, H-3’), 1.76 (s, 12H, H-3’’’)
ppm. 13C{1H} NMR (100 MHz, MeOD-d4/D2O = 3:1)  175.8
(C-2), 174.8 (C-2), 156.6 (C-), 155.9 (C-), 145.0 (C-7a),
144.8 (C-7a), 143.4 (C-5), 143.0 (C-5), 142.8 (C-3a), 142.5 (C-
3a), 128.1 (2C, C-6), 128.0 (C-), 121.3 (2C, C-4), 111.9 (C-7),
111.3 (C-7), 105.9 (C-), 105.0 (C-), 83.8 (C-4’’), 71.5 (C-
5’’), 51.6 (C-4’), 50.7 (C-3), 50.4 (C-3), 45.1 (C-1’), 43.8 (C-
1’’), 27.9 (C-3’’’), 27.8 (C-3’’’), 27.2 (C-2’’), 27.1 (C-2’), 23.4
3
(tt, J = 7.8 Hz, 2H, H-4’’), 1.61–1.56 (m, 2H, H-3’’) ppm.
13C{1H} NMR (150 MHz, MeOD-d4/D2O = 3:1)  175.5 (C-2),
175.0 (C-2), 156.3 (C-), 156.0 (C-), 144.9 (C-7a), 144.8 (C-
7a), 143.2 (C-5), 143.0 (C-5), 142.7 (C-3a), 142.6 (C-3a), 128.1
(2C, C-6), 128.0 (C-), 121.3 (C-4), 121.2 (C-4), 121.0 (C-6’’),
111.8 (C-7), 111.6 (C-7), 105.6 (C-), 105.2 (C-), 51.7 (C-4’),
50.6 (C-3), 50.4 (C-3), 45.0 (C-1’), 44.8 (C-1’’), 27.9 (C-3’’’),
27.8 (C-3’’’), 27.6 (C-2’’), 27.1 (C-2’), 26.9 (C-3’’), 26.1 (C-
(C-3’), 16.5 (C-3’’) ppm. HRMS (ESI) m/z calcd for

C34H39N2O9S3
[MH]: 715.1823; found: 715.1803
(∆ = 2.8 ppm).
4’’), 23.4 (C-3’), 17.2 (C-5’’) ppm. HRMS (ESI) m/z calcd for

[MH]: 744.2089; found: 744.2066
Sulfo-(hexynyl)pentamethine Cyanine (4e). Synthesized
according to the general procedure (188 µmol scale). Yield:
122 mg (167 µmol, 89 %) of a blue solid. 1H NMR (400 MHz,
MeOD-d4/D2O = 3:1)  = 8.32–8.28 (m, 2H, H-), 7.90–7.87
C35H42N3O9S3
(∆ = 3.1 ppm).
Sulfo-(3-carboxypropyl)pentamethine Cyanine (4h). Syn-
thesized according to the general procedure (307 µmol scale).
3
1
(m, 4H, H-4, H-6), 7.40 (d, J = 8.8 Hz, 1H, H-7), 7.35 (d,
Yield: 184 mg (250 µmol, 81 %) of a blue solid. H NMR
3J = 8.2 Hz, 1H, H-7), 6.74 (dd, 3J = 12.5 Hz, 1H, H-), 6.44 (d,
(400 MHz, AcOD-d4/D2O = 3:1)  8.23–8.19 (m, 2H, H-),
3
3J = 13.7 Hz, 1H, H-), 6.37 (d, J = 13.6 Hz, 1H, H-), 4.17–
3
7.93–7.88 (m, 4H, H-4, H-6), 7.38 (d, J = 8.6 Hz, 1H, H-7),
4.14 (m, 4H, H-1’, H-1’’), 2.92 (t, 3J = 6.8 Hz, 2H, H-4’), 2.32
7.35 (d, 3J = 8.7 Hz, 1H, H-7), 6.71 (dd, 3J = 12.5 Hz, 1H, H-),
4
3
4
3
3
(t, J = 2.4 Hz, 1H, H-6’’), 2.30 (td, J = 6.8 Hz, J = 2.3 Hz,
2H, H-4’’), 1.98–1.89 (m, 6H, H-2’, H-3’, H-2’’), 1.76 (s, 12H,
6.39 (d, J = 13.0 Hz, 1H, H-), 6.30 (d, J = 13.0 Hz, 1H, H-
3
), 4.15 (m, 4H, H-1’, H-1’’), 3.02 (t, J = 7.0 Hz, 2H, H-4’),
H-3’’’), 1.67 (tt, J = 6.8 Hz, 2H, H-3’’), ppm. 13C{1H} NMR
3
2.58 (t, 3J = 6.8 Hz, 2H, H-3’’), 2.12 (tt, 3J = 6.9 Hz, 2H, H-2’’),
1.97–1.96 (m, 4H, H-2’, H-3’), 1.73 (s, 6H, H-3’’’), 1.72 (s, 6H,
H-3’’’) ppm. 13C{1H} NMR (100 MHz, AcOD-d4/D2O = 3:1) 
176.9 (C-4’’), 174.9 (C-2), 174.3 (C-2), 155.4 (C-), 155.0 (C-
), 144.6 (C-7a), 144.4 (C-7a), 142.2 (C-3a), 142.0 (C-3a),
140.7 (C-5), 140.5 (C-5), 127.5 (2C, C-6), 127.1 (C-), 120.6
(2C, C-4), 111.5 (C-7), 111.1 (C-7), 104.7 (C-), 104.3 (C-),
50.9 (C-4’), 49.8 (C-3), 49.6 (C-3), 44.4 (1C, C-1’/ C-1’’), 43.7
(1C, C-1’/ C-1’’), 30.8 (C-3’’), 27.4 (C-3’’’), 27.3 (C-3’’’), 26.2
(100 MHz, MeOD-d4/D2O = 3:1)  = 175.5 (C-2), 174.9 (C-2),
156.5 (C-), 156.0 (C-), 145.0 (C-7a), 144.8 (C-7a), 143.2 (C-
5), 143.1 (C-5), 142.7 (C-3a), 142.6 (C-3a), 128.1 (C-6), 128.0
(C-6), 127.9 (C-), 121.3 (C-4), 121.3 (C-4), 111.8 (C-7), 111.5
(C-7), 105.6 (C-), 105.2 (C-), 84.3 (C-5’’), 70.6 (C-6’’), 51.6
(C-4’), 50.6 (C-3), 50.5 (C-3), 45.0 (C-1’), 44.6 (C-1’’), 27.9
(C-3’’’), 27.8 (C-3’’’), 27.2 (C-2’’), 27.1 (C-2’), 26.6 (C-3’’),
23.4 (C-3’), 18.6 (C-4’’) ppm. HRMS (ESI) m/z calcd for

C35H41N2O9S3
[MH]: 729.1980; found: 729.2001
(C-2’), 22.3 (2C, C-3’, C-2’’) ppm. HRMS (ESI) m/z calcd for
(∆ = 2.9 ppm).

C33H39N2O11S3
(∆ = 4.2 ppm).
[MH]: 735.1721; found: 735.1691
Sulfo-(3-cyanopropyl)pentamethine Cyanine (4f). Synthe-
sized according to the general procedure (78.3 µmol scale).
Sulfo-(5-carboxypentyl)pentamethine Cyanine (4i). Syn-
1
Yield: 52.0 mg (72.4 µmol, 92 %) of a blue solid. H NMR
thesized according to the general procedure (567 µmol scale).
3
1
(400 MHz, MeOD-d4/D2O = 3:1)  8.35 (d, J = 12.4 Hz, 1H,
Yield: 376 mg (492 µmol, 87 %) of a blue solid. H NMR
3
H-), 8.30 (d, J = 12.7 Hz, 1H, H-), 7.91–7.86 (m, 4H, H-4,
(400 MHz, AcOD-d4/D2O = 3:1)  8.22–8.20 (m, 2H, H-),
3
3
3
H-6), 7.45 (d, J = 8.9 Hz, 1H, H-7), 7.34 (d, J = 8.4 Hz, 1H,
7.92–7.89 (m, 4H, H-4, H-6), 7.36 (d, J = 8.3 Hz, 1H, H-7),
3
3
7.32 (d, 3J = 8.4 Hz, 1H, H-7), 6.71 (dd, 3J = 12.3 Hz, 1H, H-),
H-7), 6.74 (dd, J = 12.4 Hz, 1H, H-), 6.50 (d, J = 13.8 Hz,
1H, H-), 6.35 (d, 3J = 13.4 Hz, 1H, H-), 4.21 (t, 3J = 7.1 Hz,
4H, H-1’, H-1’’), 2.93 (t, 3J = 6.9 Hz, 2H, H-4’), 2.66 (t,
3J = 7.1 Hz, 2H, H-3’’), 2.15 (tt, 3J = 7.1 Hz, 2H, H-2’’), 1.98–
1.97 (m, 4H, H-2’, H-3’), 1.76 (s, 12H, H-3’’’) ppm. 13C{1H}
NMR (100 MHz, DMSO-d6)  174.0 (C-2), 172.6 (C-2), 155.1
(C-), 154.2 (C-), 144.9 (C-5), 144.4 (C-5), 142.5 (C-7a),
3
3
6.36 (d, J = 13.5 Hz, 1H, H-), 6.31 (d, J = 13.4 Hz, 1H, H-
), 4.14–4.12 (m, 4H, H-1’, H-1’’), 3.02 (t, 3J = 6.9 Hz, 2H, H-
3
4’), 2.37 (t, J = 7.3 Hz, 2H, H-5’), 1.96 (m, 4H, H-2’, H-3’),
3
1.85 (tt, J = 7.5 Hz, 2H, H-2’’), 1.73 (s, 6H, H-3’’’), 1.72 (s,
6H, H-3’’’), 1.68 (tt, 3J = 7.6 Hz, 2H, H-4’’), 1.51–1.43 (m, 2H,
H-3’’) ppm. 13C{1H} NMR (100 MHz, AcOD-d4/D2O = 3:1) 
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The crude product was purified by reversed-phase column chro-
178.4 (C-6’’), 174.6 (2C, C-2), 155.2 (2C, C-), 144.6 (C-7a),
1
2
3
4
5
6
7
8
matography (RP18; H2O/TFA = 100:0.1 → H2O/MeCN/TFA =
144.5 (C-7a), 142.1 (2C, C-3a), 140.6 (2C, C-5), 127.5 (2C, C-
6), 126.9 (C-), 120.6 (2C, C-4), 111.3 (2C, C-7), 104.4 (2C, C-
), 50.9 (C-4’), 49.7 (2C, C-3), 44.4 (1C, C-1’/ C-1’’), 44.3 (1C,
C-1’/ C-1’’), 33.9 (C-5’’), 27.3 (2C, C-3’’’), 27.2 (C-2’’), 26.3
(C-2’), 26.2 (C-3’’), 24.7 (C-4’’), 22.3 (C-3’) ppm. HRMS (ESI)
50:50:0.1) and lyophilized. Yield: 75.8 mg (107 µmol, 86 %) of
1
a blue solid. H NMR (600 MHz, D2O/ MeOD-d4 = 4:0.1) 
8.06–7.99 (m, 2H, H-), 7.84 (d, 4J = 1.4 Hz, 1H, H-4), 7.82 (d,
3
4J = 1.4 Hz, 1H, H-4), 7.77 (d, J = 8.3 Hz, 2H, H-6), 7.34 (d,
3J = 8.3 Hz, 1H, H-7), 7.24 (d, 3J = 8.5 Hz, 1H, H-7), 6.57 (dd,
3J = 12.5 Hz, 1H, H-), 6.33 (d, 3J = 13.9 Hz, 1H, H-), 6.16 (d,
3J = 13.6 Hz, 1H, H-), 4.12–4.08 (m, 4H, H-1’, H-1’’), 3.12 (t,
3J = 7.9 Hz, 2H, H-3’’), 2.97 (t, 3J = 7.4 Hz, 2H, H-4’), 2.14 (tt,
3J = 7.7 Hz, 2H, H-2’’), 1.94 (tt, 3J = 7.4 Hz, 2H, H-2’), 1.86 (tt,
3J = 7.3 Hz, 2H, H-3’), 1.63 (s, 6H, H-3’’’), 1.61 (s, 6H, H-3’’’)
ppm. 13C{1H} NMR (150 MHz, D2O/ MeOD-d4 = 4:0.1) 
176.2 (C-2), 173.7 (C-2), 156.3 (C-), 155.0 (C-), 145.0 (C-
7a), 144.8 (C-7a), 143.0 (C-3a), 142.4 (C-3a), 140.7 (C-5),
139.8 (C-5), 127.5 (2C, C-6), 127.2 (C-), 120.8 (C-4), 120.7
(C-4), 112.3 (C-7), 111.1 (C-7), 105.9 (C-), 103.9 (C-), 51.3
(C-4’), 50.4 (C-3), 49.7 (C-3), 44.9 (C-1’), 41.8 (C-1’’), 37.8
(C-3’’), 27.9 (C-3’’’), 27.6 (C-3’’’), 26.6 (C-2’), 25.6 (C-2’’),
m/z calcd for C35H43N2O11S3 [MH]: 763.2034; found:

763.2023 (∆ = 1.5 ppm). The spectroscopic data are in agree-
ment with those reported in the literature.26
Sulfo-(3-trifluoroacetamido)propyl)pentamethine Cya-
nine (4j). Synthesized according to the general procedure
(255 µmol scale). Yield: 197 mg (245 µmol, 96 %) of a blue
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
solid. H NMR (600 MHz, MeOD-d4/D2O = 3:1)  8.33 (d,
3J = 12.5 Hz, 1H, H-), 8.30 (d, J = 12.6 Hz, 1H, H-), 7.91–
3
7.86 (m, 4H, H-4, H-6), 7.43 (d, 3J = 8.9 Hz, 1H, H-7), 7.29 (d,
3J = 8.3 Hz, 1H, H-7), 6.72 (dd, 3J = 12.4 Hz, 1H, H-), 6.46 (d,
3J = 13.9 Hz, 1H, H-), 6.31 (d, 3J = 13.6 Hz, 1H, H-), 4.19 (t,
3J = 7.1 Hz, 2H, H-1’), 4.15 (t, 3J = 7.5 Hz, 2H, H-1’’), 3.44 (t,
3J = 7.2 Hz, 2H, H-3’’), 2.91 (t, 3J = 7.0 Hz, 2H, H-4’), 2.07 (tt,
3J = 7.4 Hz, 2H, H-2’’), 2.00–1.95 (m, 4H, H-2’, H-3’), 1.76 (s,
6H, H-3’’’), 1.75 (s, 6H, H-3’’’) ppm. 13C{1H} NMR
(150 MHz, MeOD-d4/D2O = 3:1)  176.2 (C-2), 174.4 (C-2),
159.2 (C-4’’), 156.9 (C-), 155.9 (C-), 144.9 (C-7a), 144.7 (C-
7a), 143.5 (C-5), 142.9 (C-5), 142.8 (C-3a), 142.4 (C-3a), 128.1
(2C, C-6), 128.0 (C-), 121.4 (C-4), 121.3 (C-4), 117.5 (C-5’’),
112.0 (C-7), 111.0 (C-7), 106.0 (C-), 104.7 (C-), 51.6 (C-4’),
50.8 (C-3), 40.3 (C-3), 45.2 (C-1’), 42.6 (C-1’’), 38.2 (C-3’’),
27.9 (C-3’’’), 27.8 (C-3’’’), 27.5 (C-2’’), 27.1 (C-2’), 23.4 (C-
3’) ppm. 19F NMR (376 MHz, MeOD-d4/ D2O = 3:1, double sig-
nal set)  76.49, 76.44 ppm. HRMS (ESI) m/z calcd for

22.6 (C-3’) ppm. HRMS (ESI) m/z calcd for C32H40N3O9S3
[MH]: 706.1932; found: 706.1939 (∆ = 0.9 ppm).
Sulfo-(6-aminohexyl)pentamethine Cyanine (4m). In a mi-
crowave vial, sulfo-(6-(trifluoroacetamido)hexyl)pentamethine
cyanine (4k, 100 mg, 118 µmol, 1.0 eq) was dissolved in 6 M
HCl (1 mL) and EtOH (2 mL). The solution was stirred under
microwave irradiation for 30 min (150 W, 110 °C). EtOAc and
Et2O were added and the precipitate was collected by filtration.
The crude product was purified by reversed-phase column chro-
matography (RP18; H2O/TFA = 100:0.1 → H2O/MeCN/TFA =
50:50:0.1) and lyophilized. Yield: 82.3 mg (110 µmol, 93 %) of
a blue solid. 1H NMR (400 MHz, MeOD-d4/D2O = 3:1)  8.23–
8.21 (m, 2H, H-), 7.88–7.85 (m, 4H, H-4, H-6), 7.38 (d,
3J = 8.6 Hz, 1H, H-7), 7.33 (d, 3J = 8.4 Hz, 1H, H-7), 6.74 (dd,
3J = 12.5 Hz, 1H, H-), 6.41 (d, 3J = 13.7 Hz, 1H, H-), 6.36 (d,
3J = 13.6 Hz, 1H, H-), 4.15 (t, 3J = 6.9 Hz, 2H, H-1’), 4.13 (t,
3J = 7.1 Hz, 2H, H-1’’), 2.94 (t, 3J = 7.8 Hz, 2H, H-6’’), 2.93 (t,
3J = 7.2 Hz, 2H, H-4’), 1.98–1.93 (m, 4H, H-2’, H-3’), 1.83 (tt,
3J = 7.5 Hz, 2H, H-2’’), 1.73 (s, 12H, H-3’’’), 1.66 (tt,
3J = 7.5 Hz, 2H, H-5’’), 1.47–1.45 (m, 4H, H-3’’, H-4’’) ppm.
13C{1H} NMR (100 MHz, MeOD-d4/D2O = 3:1)  175.3 (C-2),
175.0 (C-2), 155.9 (C-), 155.7 (C-), 145.0 (C-7a), 144.8 (C-
7a), 142.7 (C-3a), 142.6 (C-3a), 142.3 (C-5), 142.2 (C-5), 128.0
(C-6), 127.9 (C-6), 127.8 (C-), 121.2 (C-4), 121.1 (C-4), 111.8
(C-7), 111.7 (C-7), 105.3 (C-), 105.1 (C-), 51.6 (C-4’), 50.3
(C-3), 50.3 (C-3), 44.8 (2C, C-1’, C-1’’), 40.5 (C-6’’), 28.1 (C-
5’’), 28.0 (C-2’’), 27.9 (2C, C-3’’’), 27.0 (1C, C-3’’/ C-4’’),
26.9 (C-2’), 26.9 (1C, C-3’’/ C-4’’), 23.2 (C-3’) ppm. HRMS
2
C34H38F3N3O10S3 [MH]2: 400.58412; found: 400.58434
(∆ = 0.55 ppm).
Sulfo(-6-(trifluoroacetamido)hexyl)pentamethine
Cya-
nine (4k). Synthesized according to the general procedure
(230 µmol scale). Yield: 175 mg (207 µmol, 90 %) of a blue
solid. 1H NMR (400 MHz, MeOD-d4/D2O = 3:1)  8.30 (d,
3J = 12.4 Hz, 2H, H-), 7.91–7.87 (m, 4H, H-4, H-6), 7.40 (d,
3J = 9.2 Hz, 1H, H-7), 7.35 (d, 3J = 8.4 Hz, 1H, H-7), 6.73 (dd,
3J = 12.4 Hz, 1H, H-), 6.42 (d, 3J = 13.7 Hz, 1H, H-), 6.35 (d,
3J = 13.7 Hz, 1H, H-), 4.17–4.11 (m, 4H, H-1’, H-1’’), 3.28 (t,
3J = 7.1 Hz, 2H, H-6’’), 2.93 (t, J = 7.1 Hz, 2H, H-4’), 1.98–
3
1.96 (m, 4H, H-2’, H-3’), 1.80 (tt, 3J = 7.6 Hz, 2H, H-2’’), 1.75
(s, 12H, H-3’’’), 1.60 (tt, 3J = 7.2 Hz, 2H, H-5’’), 1.53–1.47 (m,
2H, H-3’’), 1.45–1.38 (m, 2H, H-4’’) ppm. 13C{1H} NMR
(100 MHz, MeOD-d4/D2O = 3:1)  175.2 (C-2), 175.2 (C-2),
158.9 (C-7’’), 156.3 (C-), 156.1 (C-), 145.0 (C-7a), 144.9 (C-
7a), 143.1 (2C, C-5), 142.7 (C-3a), 142.6 (C-3a), 128.1 (2C, C-
6), 128.0 (C-), 121.3 (2C, C-4), 117.6 (C-8’’), 111.7 (C-7),
111.6 (C-7), 105.3 (2C, C-), 51.7 (C-4’), 50.5 (C-3), 50.5 (C-
3), 45.0 (C-1’’), 44.9 (C-1’), 40.5 (C-6’’), 29.7 (C-5’’), 28.3 (C-
2’’), 27.8 (2C, C-3’’’), 27.4 (C-4’’), 27.3 (C-3’’), 27.0 (C-2’),
23.4 (C-3’) ppm. 19F NMR (376 MHz, MeOD-d4/ D2O = 3:1) 

(ESI) m/z calcd for C35H46N3O9S3 [MH]: 748.2402; found:
748.2378 (∆ = 1.9 ppm).
ASSOCIATED CONTENT
Supporting Information
2
77.24 ppm. HRMS (ESI) m/z calcdd for C37H44F3N3O10S3
The Supporting Information is available free of charge on the
ACS Publications website, including NMR and ESI spectra.
(pdf)
[MH]2: 421.60759; found: 421.60822 (∆ = 1.48 ppm).
Sulfo-(3-aminopropyl)pentamethine Cyanine (4l). In a mi-
crowave vial, sulfo-(3-(trifluroacetamido)propyl)pentamethine
cyanine (4j, 100 mg, 124 µmol, 1.0 eq) was dissolved in 6 M
HCl (1 mL) and EtOH (2 mL). The solution was stirred under
microwave irradiation for 30 min (150 W, 110 °C). EtOAc and
Et2O were added and the precipitate was collected by filtration.
AUTHOR INFORMATION
Corresponding Author
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Page 10 of 11
and Its Application for Bioimaging. Chem. Commun. 2010, 46
(47), 8953–8955.
Luo, S.; Zhang, E.; Su, Y.; Cheng, T.; Shi, C. A Review of NIR
Dyes in Cancer Targeting and Imaging. Biomaterials 2011, 32
(29), 7127–7138.
*Juergen Seibel – Institute of Organic Chemistry, University of
Wuerzburg, 97074 Wuerzburg, Germany; Email:
seibel@chemie.uni-wuerzburg.de
1
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(13)
(14)
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Guo, Z.; Park, S.; Yoon, J.; Shin, I. Recent Progress in the
Development of Near-Infrared Fluorescent Probes for
Bioimaging Applications. Chem. Soc. Rev. 2014, 43 (1), 16–29.
Yin, J.; Kwon, Y.; Kim, D.; Lee, D.; Kim, G.; Hu, Y.; Ryu, J.-H.;
Yoon, J. Preparation of a Cyanine-Based Fluorescent Probe for
Highly Selective Detection of Glutathione and Its Use in Living
Cells and Tissues of Mice. Nat. Protoc. 2015, 10 (11), 1742–
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Sun, W.; Guo, S.; Hu, C.; Fan, J.; Peng, X. Recent Development
of Chemosensors Based on Cyanine Platforms. Chem. Rev. 2016,
116 (14), 7768–7817.
Leisle, L.; Chadda, R.; Lueck, J. D.; Infield, D. T.; Galpin, J. D.;
Krishnamani, V.; Robertson, J. L.; Ahern, C. A. Cellular
Encoding of Cy Dyes for Single-Molecule Imaging. Elife 2016,
5, e19088.
Liu, Y.; Zhou, J.; Wang, L.; Hu, X.; Liu, X.; Liu, M.; Cao, Z.;
Shangguan, D.; Tan, W. A Cyanine Dye to Probe Mitophagy:
Simultaneous Detection of Mitochondria and Autolysosomes in
Live Cells. J. Am. Chem. Soc. 2016, 138 (38), 12368–12374.
Oshikawa, Y.; Furuta, K.; Tanaka, S.; Ojida, A. Cell Surface-
Anchored Fluorescent Probe Capable of Real-Time Imaging of
Single Mast Cell Degranulation Based on Histamine-Induced
Coordination Displacement. Anal. Chem. 2016, 88 (3), 1526–
1529.
Natalia Wolf – Institute of Organic Chemistry, University of
Wuerzburg, 97074 Wuerzburg, Germany
Louise Kersting – Institute of Organic Chemistry, University
of Wuerzburg, 97074 Wuerzburg, Germany
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Christoph Herok – Institute of Organic Chemistry, University
of Wuerzburg, 97074 Wuerzburg, Germany
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Cornelius Mihm – Institute of Organic Chemistry, University
of Wuerzburg, 97074 Wuerzburg, Germany
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script.
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ACKNOWLEDGMENT
This work was supported by the German Research Foundation
(DFG, TRR 166 ReceptorLight), and SE1410/6-1, SE1410/6-2.
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