Approach to a Substituted Heptamethine Cyanine Chain by the Ring Opening of Zincke Salts

Article abstract of DOI:10.1021/jacs.9b02537

Cyanine dyes play an indispensable and central role in modern fluorescence-based biological techniques. Despite their importance and widespread use, the current synthesis methods of heptamethine chain modification are restricted to coupling reactions and nucleophilic substitution at the meso position in the chain. Herein, we report the direct transformation of Zincke salts to cyanine dyes under mild conditions, accompanied by the incorporation of a substituted pyridine residue into the heptamethine scaffold. This work represents the first general approach that allows the introduction of diverse substituents and different substitution patterns at the C3′-C5′ positions of the chain. High yields, functional tolerance, versatility toward the condensation partners, and scalability make this method a powerful tool for accessing a new generation of cyanine derivatives.

Full text of DOI:10.1021/jacs.9b02537

Article
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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Approach to a Substituted Heptamethine Cyanine Chain by the Ring
Opening of Zincke Salts
†
,†
̌
̌
́
́
Lenka Stackova, Peter Stacko,* and Petr Klan*
Department of Chemistry and RECETOX, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic
S
* Supporting Information
ABSTRACT: Cyanine dyes play an indispensable and central
role in modern fluorescence-based biological techniques.
Despite their importance and widespread use, the current
synthesis methods of heptamethine chain modification are
restricted to coupling reactions and nucleophilic substitution
at the meso position in the chain. Herein, we report the direct
transformation of Zincke salts to cyanine dyes under mild
conditions, accompanied by the incorporation of a substituted
pyridine residue into the heptamethine scaffold. This work
represents the first general approach that allows the introduction of diverse substituents and different substitution patterns at
the C3′−C5′ positions of the chain. High yields, functional tolerance, versatility toward the condensation partners, and
scalability make this method a powerful tool for accessing a new generation of cyanine derivatives.
INTRODUCTION
■
Since their first synthesis in 1856, cyanine dyes have become
invaluable fluorophores in contemporary chemistry and
biology.1 The defining feature of the cyanines is an odd-
numbered polyene linking two nitrogen-containing hetero-
cycles, related to both their intriguing photophysical properties
and unique chemical reactivities.2 Within this family, heptame-
thine cyanines dyes (Cy7, Figure 1a), containing seven carbon
atoms in the linker, are particularly valued for their absorption
and emission maxima located in the center of the near-infrared
(NIR) window (∼800 nm).3 In biology, the presence of
endogenous chromophores, such as hemoglobin and water, and
optical scattering limit the depth of light penetration into tissues.
Light absorption in the phototherapeutic window (600−1000
nm), where these effects are minimized, thus ensures a broad
scope of biological applications.
The expanding medical significance of Cy7 dyes is
demonstrated by FDA-approved indocyanine green (ICG)
with hundreds of clinical trials emerging even after 50 years of its
clinical use (Figure 1b).4 Another promising derivative, IR800-
CW, has been explored for a number of fluorescence-guided
surgery applications (Figure 1b).5−7 Furthermore, cyanines
Figure 1. (a) Heptamethine cyanine dyes (Cy7). The numbering of the
heptamethine chain is depicted in red. (b) Indocyanine green. (c) IR-
800CW.
have been used in a number of other applications, including pH
sensing,8,9 analyte-responsive sensing,2,10 glutathione11 or
reactive oxygen species12−14 visualization, single-molecule
The introduction of electron-withdrawing nitrile,24 amide,25 or
triazole26 moieties in the chain meso position improves the dye
photostability as a result of decreased reactivity toward singlet
oxygen. The installation of the 2,2,6,6-tetramethylpiperidinyl-
oxyl (TEMPO)-bearing substituent at the C4′ position resulted
fluorescence and super-resolution imaging,15−17 photodynamic
therapy,18,19 NIR photouncaging,20,21 and others.22 These
applications take advantage not only of their excellent
photophysical properties but also the distinct chemical reactivity
of the cyanine polyene itself.23
The nature of heptamethine chain substituents has a profound
influence on both the photophysical and photochemical
properties of the Cy7 dyes as well as their chemical stability.
Received: March 7, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/jacs.9b02537
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Article
a
Scheme 1. Chain Modification, Schiff Base Preparation, and Our Current Strategy
a
(a) Conventional approach to the heptamethine chain modification involving palladium-catalyzed coupling reactions (left) and SRN1 reactions
with N, O, or S nucleophiles (right). (b) Custom Schiff base preparation via cyclohexene formylation. (c) Our current strategy involving the ring
opening of pyridinium (Zincke) salts and subsequent incorporation of the original substituted pyridine residue (red) into the heptamethine chain.
a
Scheme 2. Typical Zincke Reaction (Route A) and Utilization of Its Intermediates in the Synthesis of Cy7 Dyes (Route B)
a
The original substituted pyridinium residue (red) is transferred and incorporated into heptamethine scaffold 5.
in an ∼33-fold increase in the quantum yield of singlet oxygen
formation compared to that of ICG sensitization.27 Bioconju-
gation of cyanines commonly achieved via the attachment of
C4′-O-aryl or C4′-S-alkyl linkers often suffers from poor
chemical stability of the conjugate toward biologically relevant
nucleophiles, such as glutathione or cellular proteins.28−30 One
way to circumvent this issue is to employ C4′-O-alkyl-
substituted cyanines which have so far been accessible only by
Smiles rearrangement of C4′-N-methylethanolamine precur-
sors.31 In addition, an elegant design of the C4′-dialkylamine
linker bearing a carbamate functionality allowed us to harness
the undesired photooxidiation in a NIR photouncaging
process.20,21
Despite the widespread utilization of cyanines and the evident
importance of polyene substitution, the strategies used to
modify the heptamethine chain remain surprisingly under-
developed and limited. A majority of the current methods start
from the C4′-chloro-substituted cyanine and rely on an electron-
transfer-mediated SRN1 reaction with N, O, or S nucleo-
philes13,25,32−38 or palladium-catalyzed Suzuki39 or Sonoga-
shira40 coupling reactions (Scheme 1a). The other option
involves the preparation of a custom Schiff base intermediate
from a substituted cyclohexene in the Vilsmeier−Haack reaction
(Scheme 1b). However, this procedure gives moderate yields
only for methyl- and phenyl-substituted cyclohexenes presum-
ably because of harsh conditions employed in the synthesis.41,42
Besides a cyclopentyl or cyclohexyl ring embedded within the
heptamethine scaffold, no modifications at the C3′ and C5′
positions have been reported to date. Therefore, essentially only
the C4′-position modifications are available for modification to
date, imposing fundamental restrictions on the design of new
Cy7 fluorophores. Herein, we report the first general synthesis
method to access Cy7 dyes possessing diverse substituents and
substitution patterns at the C3′−C5′ positions of a heptame-
thine chain under mild conditions (Scheme 1c).
B
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Article
confirmed in a reaction of 1a and aniline using HPLC and UV−
vis spectroscopy by comparison with an authentic sample of 3
(Figures S154 and S155). Conversely, the use of a strong
electron-withdrawing (EWG) 4-cyano substituent led to only a
marginal increase in the yield compared to that of an unanalyzed
reaction (34%, entry 5). On the contrary, weakly electron
accepting 4- or 3-bromo substituents dramatically improved the
yield to 72 and 73%, respectively (entries 4 and 7). Decreasing
the aniline amount to 1 equiv had no negative effect on the yield,
whereas its further decrease to substoichiometric amounts or
reducing the number of equivalents of sodium acetate led to
lower yields of 5a (entries 6−10). N-Phenylpyridinium was
observed as a major side product upon the temperature increase
as a result of intramolecular Schiff base cyclization.43
The dependence of cyanine formation yields on the nature of
aniline substituents implies a delicate interplay of electronic
effects. An aniline reagent is required to be electron-rich enough
to facilitate pyridinium ring opening and, at the same time, be
sufficiently electron-poor to activate the resulting imine for a
facile condensation with the heterocycle. Thus, while 4-
methoxyaniline expedites a rapid cleavage of the pyridinium
ring, corresponding electron-rich imine 3 reacts too slowly in the
subsequent condensation (and vice versa for 4-cyanoaniline).
Established empirically, 3- and 4-bromoaniline facilitated both
steps with a desirable efficiency and thus the highest yields.
Following the optimization, we sought to investigate the
scope of this method and its tolerance toward various
substituents and substitution patterns. A variety of substituted
pyridinium salts 1a−t were examined in the reaction with 4a,
and the results are summarized in Table 2. In most cases, the
pure products were isolated directly by filtration of the
precipitate from the reaction mixture. The products were, in
some cases, contaminated with 2,4-dinitroaniline, which was
removed by rapid column chromatography.
RESULTS AND DISCUSSION
■
Typically, the Zincke reaction is used to convert pyridines to N-
pyridinium salts by the reaction of N-2,4-dinitrophenyl-
pyridinium (Zincke salt) with primary amines at elevated
temperatures (Scheme 2, route A).43 We envisioned that, under
specific conditions, intermediate 2 produced by the ring opening
of pyridinium salt 1, as suggested in the synthesis of
benzothiazole Cy7 derivatives with an unsubstituted heptame-
thine chain,44 could instead be intercepted by another
equivalent of aniline as a nucleophile, and resulting intermediate
3 would undergo a condensation with quaternized heterocycle 4
to give the final cyanine 5 in a one-pot tandem fashion (Scheme
2, route B). In such a manner, the substituents on the pyridine
residue would be transferred and incorporated into the cyanine
as a part of the heptamethine scaffold. We also hypothesized that
the ring opening of very electron-deficient pyridinium salts
could also be performed directly with 4 without the need for any
auxiliary nucleophile.
We synthesized a number of pyridinium salts 1a−v on a
multigram scale by the reaction of 2,4-dinitrophenyl (DNP)
tosylate or triflate with substituted pyridines at an elevated
temperature (Supporting Information). The salts were isolated
in high purity simply by filtration and proved to be benchtop-
stable for months. Subjecting 1a to a reaction with heterocycle
4a in the presence of sodium acetate (6 equiv) and in the
absence of aniline at room temperature overnight provided
corresponding cyanine 5a in 24% yield (Table 1, entry 1). The
Table 1. Optimization of the Preparation of Cyanine 5a from
a
Zincke Salt 1a
The method is versatile, and it tolerates both electron-
withdrawing and electron-donating groups on the pyridinium
ring. The pyridinium salts with EWGs reacted faster and usually
with higher yields (vide infra) than those with EDGs. A
decreased reactivity of pyridines with mediocre EDGs (1b, 1m)
under the optimized conditions was compensated for by a higher
loading of 4-bromoaniline (3 equiv). On the other hand, 1n did
not undergo the ring-opening reaction under these conditions,
presumably because of a strong resonance electron-donating
effect of the methoxy substituent. We reasoned that employing a
less polar solvent, such as ethanol or pyridine, would lead to less
favorable solvation of the pyridinium salt, thus promoting the
pyridinium ring opening. Indeed, when the reaction was carried
out in pyridine, cyanine 5n was successfully isolated in 40%
yield. On the contrary, strong EWGs (1d, 1g, and 1k) did not
actually demand the presence of aniline as an auxiliary
nucleophile, and the pyridinium ring opening could be achieved
efficiently in its absence. Our observations are consistent with
the mechanism of the Zincke reaction and the subsequent
condensation, both of which involve a nucleophilic attack and
therefore require a sufficiently electron-deficient substrate
(Scheme 2).
b
entry
R-PhNH2
aniline
AcONa
yield
1
2
3
4
5
6
7
8
9
10
6 equiv
6 equiv
6 equiv
6 equiv
6 equiv
6 equiv
6 equiv
4 equiv
6 equiv
3 equiv
24%
47%
<2%
73%
34%
72%
73%
60%
35%
22%
4-H
3 equiv
3 equiv
3 equiv
3 equiv
1.2 equiv
1.2 equiv
1.2 equiv
0.2 equiv
0.2 equiv
4-MeO
4-Br
4-CN
4-Br
3-Br
3-Br
4-Br
4-Br
a
Reaction conditions: 1a (0.5 mmol), 4a(1.5 mmol), aniline, and
sodium acetate in methanol (5 mL) were stirred at room temperature
b
for 16 h. Isolated yields are given.
addition of excess aniline (3 equiv) to facilitate ring opening of
the pyridinium salt improved the yield to 47% (entry 2).
Encouraged by this finding, we examined the electronic effects of
the aniline substituents on the reaction yield. The use of a strong
electron-donating (EDG) 4-methoxy substituent led to rapid
pyridinium ring opening as evidenced by the development of a
deep-red coloration of the reaction mixture (λmax = 485 nm),45
whereas the corresponding cyanine was produced only in trace
amounts (entry 3). The presence of intermediate 3 was further
Both C3′-alkyl- and C3′-aryl-substituted pyridinium salts
gave cyanines 5b and 5c in very good yields. Substrates bearing
halogens also efficiently afforded products 5d−5f in yields of
49−90%, increasing in the order of increasing halogen
electronegativity. Various functional derivatives of carboxylic
acids, such as esters (5g and 5h), carboxylic acid (5i), amide
(5j), and nitrile (5k), as well as an acyl derivative (5l) were well
C
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a b c d e
, , , ,
Table 2. Substrate Scope of the Cyanine Synthesis from Zincke Salts
a
Reaction conditions: Zincke salts 1a−t (0.35 mmol), 4a (1.05 mmol), 4-bromoaniline (0.42 mmol), and sodium acetate (2.1 mmol) in methanol
b
(4 mL) were treated at 20 °C for 16 h. Isolated yields are given. Counteranions are omitted for clarity. 1.05 mmol (3 equiv) of 4-bromoaniline was
used. Reaction time was 6 h. Performed without 4-bromoaniline. Pyridine was used as a solvent.
c
d
e
tolerated (40−86%) and provided a family of cyanines
inaccessible by any of the currently known methods. The
hydroxymethyl- and methoxy-substituted pyridiniums could
also be converted to the corresponding cyanines (5m and 5n),
albeit with lower yields, thanks to their decreased reactivity (vide
supra). Accounting for frequent utilization of cyanine dyes as
D
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a b
,
Table 3. Synthesis of Cy7 Analogues with Different Heterocyclic Ends
a
Reaction conditions: Zincke salt 1a, 1c, 1e−f, or 1i (0.35 mmol), 4b−f (1.05 mmol), 4-bromoaniline (0.42 mmol), and sodium acetate (2.1
b
mmol) in methanol (4 mL) were treated at rt for 16 h. Isolated yields are given. 1.05 mmol (3 equiv) of 4-bromoaniline and a catalytic amount of
Et3N instead of sodium acetate were used in pyridine as a solvent.
bioconjugated fluorescent tags and probes, we decided to test
the tolerance of the protocol toward the presence of azide and
terminal alkynyl substituents. Neither of those two substituents
appended to the pyridinium via an amidic linker interfered with
the reaction, and desired cyanines 5o and 5p were formed in 61
and 63% yields, respectively. The reactivity of 5o and 5p in a
click reaction with phenylacetylene and benzyl azide employing
a Cu/TBTA/ascorbate catalytic system46 was investigated. The
cyanines were fully converted to corresponding click products
13 and 14, respectively, as confirmed by 1H NMR and HRMS
spectroscopy (Figures S156−S158).
Subsequently, we evaluated the reactivity of disubstituted
pyridinium salts. All 3,4-disubstituted pyridiniums were trans-
formed to cyanines 5q−5s in 47−65% yields. In contrast, the
reactivity of 3,5-disubstituted pyridiniums was substituent-
dependent. 3-Bromo-5-methyl-substituted pyridinium was
converted to 5t in 54% yield, whereas neither 3,5-dimethylpyr-
idinium (1u) nor 5-methylpyridinium-3-carboxylate (1v)
reacted under these conditions. It is unlikely that such a lack
of reactivity can be attributed solely to electronic effects because
the substituents individually did not prevent the transformation.
Instead, we assume that one of the pyridine ortho positions is
required to be sterically accessible for a nucleophilic attack of the
aniline (Scheme 2). Indeed, the A values in this series decrease in
the order of COOCH3 > CH3 > Br (1.7, 1.27, and 0.38 kcal
mol−1, respectively).47,48
the reaction was assessed by performing the preparation of 5g on
a multigram scale. Cyanine 5g was successfully prepared in a 2.2
g amount in a single batch without any detrimental effects on the
purity or yield of the product. In fact, this large-scale reaction
was accomplished with a higher yield (92%) than that obtained
on a smaller scale (75%, Table 2).
Furthermore, we probed the generality of the reaction toward
various heterocycles (Table 3). Using N-methyl-2-methylqui-
nolinium 4b as the condensation partner resulted in the
formation of cyanine 10 in 47% yield, thus offering easy access to
an underexplored class of quinolinium-based Cy7 dyes. In the
case of benzoxazolium-based heterocycles, the formation of the
corresponding cyanine was initially observed; however, the
product decomposed in the course of the reaction. On the other
hand, cyanines 8 and 11 were formed from a benzothiazolium-
based heterocycle in yields comparable to those of their
indolium-derived analogues (5a and 5e). Employing indolium-
containing N-propyl sulfonate, which is routinely used to
improve the solubility of cyanines in aqueous media, provided
cyanine 7 in 45% yield, comparable to that of 5i. Heterocycles
with an additional benzene ring fused in different positions of 4a
were also very well tolerated and afforded corresponding
cyanines 9 and 12 in 87 and 47% yields, respectively.
The photophysical properties, absorption (λmax(abs)) and
emission (λmax(em)) maxima, and molar absorption coefficients
(εmax) were determined in methanol as a solvent for all of the
synthesized derivatives (Table 4). The absorption spectra
exhibit one major absorption band with maxima in the range
of 707−815 nm, relatively small Stokes shifts, and large molar
absorption coefficients, typical for Cy7 dyes (Figures S4 and S5).
EWGs in the C4′ position (5g, λmax(abs) = 778 nm; 5k,
λmax(abs) = 805 nm; and 5l, λmax(abs) = 771 nm) are responsible
for a bathochromic shift of the maxima compared to that of the
unsubstituted derivative 5a (λmax(abs) = 740 nm). This is in
The accommodation of various sensitive functional groups,
including ester, amide, nitrile, azide, alkyne, and unprotected
hydroxyl and carboxylic moieties at different positions of the
Cy7 chain, highlights the mildness and synthetic utility of this
protocol. In addition, a number of the described cyanines can be
employed in further reactions including coupling chemistry,39,40
“click” chemistry,49 and Staudinger ligation.50 To further
demonstrate the applicability of this method, the scalability of
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Table 4. Photophysical Properties of the Prepared Cyanine
Dyes in Methanol
CONCLUSIONS
■
The utilization of the Zincke salts ring opening in the cyanine
synthesis reported herein represents the first general method for
the derivatization of the heptamethine chain. Using this
approach, the substituted pyridine backbone is transferred and
incorporated into a cyanine dye as a part of the heptamethine
chain. A wide variety of substituents can thus be installed at
different positions of the chain, and different heterocycles can be
employed as condensation partners under mild conditions and
in high yields and purities. This method constitutes a very
powerful addition to the limited arsenal of tools available for the
synthesis of substituted cyanines and promises the emergence of
more complex cyanine-based molecules tailored for specific
applications.
a
cyanine
λmax(abs)/nm
λmax(em)/nm
ϵmax
5a
5b
5c
5d
5e
5f
5g
5h
5i
740
736
738
761
736
733
778
707
748
735
805
771
728
753
735
735
770
799
815
735
754
758
782
815
749
769
760
765
788
762
237 000
273 000
152 000
209 000
205 000
233 000
150 000
139 000
240 000
205 000
215 000
137 000
190 000
180 000
224 000
269 000
202 000
288 000
148 000
269 000
172 000
212 000
183 000
204 000
161 000
141 000
760
b
750
779
763
833
804
753
782
764
766
798
828
847
766
784
786
814
848
774
5j
5k
5l
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.9b02537.
5m
5n
5o
5p
5q
5r
5s
5t
7
8
9
10
11
12
■
S
Materials and methods; synthesis; and NMR, absorption,
and emission spectra (PDF)
c
AUTHOR INFORMATION
■
Corresponding Authors
*stacko.peter@gmail.com
*klan@sci.muni.cz
ORCID
c
792
823
́
Petr Klan: 0000-0001-6287-2742
a
b
c
ϵmax/mol−1 dm3 cm−1. Not determined. PBS (10 mM, pH 7.4, I =
Author Contributions
100 mM).
†These authors contributed equally.
Notes
accordance with previously reported 4′-bromo and 4′-iodo-
substituted Cy7 dyes, which also possess absorption maxima
(λmax(abs) = 770 nm) bathochromically shifted by 30 nm
compared to that of 5a.51 On the other hand, the presence of the
electron-donating methoxy group on C4′ exhibits a hypsochro-
mic shift of the maximum to 693 nm.51 A more electron-
donating substituent in this position, such as the alkylamino
moiety, has been shown to exhibit an even larger hypsochromic
shift of the maximum to ∼626−670 nm, respectively.25,37
Acylation of the 4-amino (EDG) derivative to its acetamido
(EWG) analogue results in a significant reverse bathochromic
shift from ∼650 to ∼800 nm.25
The effects of the substituents at the C3′ position are much
more subtle and rather unambiguous (Table 4). The presence of
an EWG can induce both hypsochromic (5h, λmax(abs) = 707
nm) and bathochromic shifts (5d, λmax(abs) = 761 nm).
Interestingly, the identical group exhibits either a bathochromic
or a hypsochromic effect on the absorption maximum in
different positions on the heptamethine chain. For instance,
derivatives 5g and 5h bearing an ester functionality at the C4′
and C3′ positions showed absorption maxima at 778 and 708
nm, respectively. In general, EWGs in the C4′ position and
EDGs in the C3′ position result in a bathochromic shift of the
absorption maxima, whereas substitution in the C4′ position
exhibits a much stronger influence on the photophysical
properties. The Stokes shifts of ∼25−35 nm seem to be
unaffected by the nature or position of the substituents. A
thorough investigation to understand the substituent effects on
the photophysical properties of the cyanine dyes is currently
being carried out in our laboratory.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This project has received funding from the European Union’s
Horizon 2020 Research and Innovation Program under Marie
Skłodowska-Curie, and it is cofinanced by the South Moravian
Region under grant agreement no. 665860 (SoMoPro III
̌
Program - Project VLAMBA, Nr. 6SA17811, P.S.). This
publication reflects only the authors’ views, and the EU is not
liable for any use that may be made of the information contained
herein. Support for this work was provided by the Czech Science
Foundation (GA18-12477S, P.K.) and RECETOX Research
Infrastructure (LM2015051 and CZ.02.1.01/0.0/0.0/16_013/
0001761). We acknowledge Lukas Maier (Masaryk University)
for assistance with NMR analysis and Miroslava Bittova
(Masaryk University) for HRMS analysis
́
̌
́
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