Introduction of various substitutions to the methine bridge of heptamethine cyanine dyes: Via substituted dianil linkers

Article abstract of DOI:10.1039/c8pp00218e

The unique optical properties of cyanine dyes have prompted their use in numerous applications. Heptamethine cyanines are commonly modified on the methine bridge after synthesis of a meso-chlorine containing cyanine. Herein, a series of heptamethine cyanines containing modified methine bridges were synthesized using substituted dianil linkers. Their optical properties including, molar absorptivity, fluorescence, and quantum yield were measured as well as their hydrophobic effects in polar buffer solution. It was shown that dyes containing cyclopentene in the methine bridge or a phenyl ring in the meso position display increased molar absorptivity while the increased flexibility of the dye containing a cycloheptene in the methine bridge prevented fluorescence.

Full text of DOI:10.1039/c8pp00218e

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Introduction of various substitutions to the
methine bridge of heptamethine cyanine dyes Via
substituted dianil linkers†
Cite this: DOI: 10.1039/c8pp00218e
Andrew Levitz,
Fahad Marmarchi and Maged Henary
*
The unique optical properties of cyanine dyes have prompted their use in numerous applications.
Heptamethine cyanines are commonly modified on the methine bridge after synthesis of a meso-chlorine
containing cyanine. Herein, a series of heptamethine cyanines containing modified methine bridges were
synthesized using substituted dianil linkers. Their optical properties including, molar absorptivity, fluor-
escence, and quantum yield were measured as well as their hydrophobic effects in polar buffer solution.
It was shown that dyes containing cyclopentene in the methine bridge or a phenyl ring in the meso posi-
tion display increased molar absorptivity while the increased flexibility of the dye containing a cyclohep-
tene in the methine bridge prevented fluorescence.
Received 29th May 2018,
Accepted 10th September 2018
DOI: 10.1039/c8pp00218e
rsc.li/pps
literature dealing with modification at the 5 position of this
cyclohexene ring. This position is not conjugated into the dye
Introduction
Over the last decade, research interest in cyanine dyes has scaffold and therefore allows for modification of the dye struc-
heightened due to their use in a wide range of applications ture with little effect on the dyes optical profile. Nucleophilic
spanning analytical, biological and biomedical research fields.1 substitution of the chlorine atom at the meso-position of the
This broad class of dyes are distinguished from other dyes in methine bridge has allowed for conjugation to targeting
that they possess two nitrogen containing heterocycles con- ligands or biomolecules3,4 through an SRN1 reaction, and most
nected by a conjugated methine bridge.2–4 The heterocycles act recently, carbon–carbon coupling at the meso-position has been
as both electron donors and acceptors creating an electron explored utilizing an adapted Suzuki–Miyaura method,1,16,17 but
deficient system throughout the molecule that gives cyanines a this method uses an expensive palladium catalyst, has generally
wide range of absorption and fluorescence from the visible only been applied to water soluble cyanine dyes, and requires
to infrared regions. They are characterized as having narrow difficult purification of the product and unreacted chloro
absorption bands and high extinction coefficients.5 These version by column chromatography.
unique properties along with the excellent safety profile of indo-
Herein, the synthesis of a series of substituted heptamethine
cyanine green (ICG) in humans6 and the ease of which cyanines cyanines has been described using substituted dianil linkers.
can be modified have allowed the dyes to be used in numerous Substitutions include methyl, phenyl, chloro, and bromo
applications.7,8 Specifically, near-infrared (NIR) absorbing and groups at the meso-position as well as phenyl and t-butyl groups
fluorescing cyanine dyes have gained attention in biomedical at the 5-position of the commonly used cyclohexene in hepta-
imaging9–12 for the low background signal in this spectral methine cyanines. Cyclopentene and cycloheptene have also
region and in dye-sensitized solar cells (DSSCs) to allow for use been explored and how these methine bridge substitutions
of the red/near-IR part of the spectrum of sunlight.13,14
affect the dyes optical properties is studied. The use of these
Heptamethine cyanine dyes have most commonly been substituted dianil linkers as an alternative to C–C coupling can
modified with substituents on the heterocycles. Cyclohexene make for a more facile condensation to the final cyanine dyes.
rings are often introduced into a methine bridge to increase
rigidity and make the dyes more stable,15 but there is a lack of
Results and discussion
Department of Chemistry, Center for Diagnostics and Therapeutics,
Georgia State University, Atlanta, GA 30303, USA. E-mail: mhenary1@gsu.edu;
Fax: +1 404-413-5505; Tel: +1 404-413-5566
Synthesis
As described in Scheme 1, a series of substituted hepta-
methine cyanines has been synthesized by a condensation
reaction between two quaternary ammonium salts and a dianil
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8pp00218e
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Scheme 1 Synthesis of meso-substituted heptamethine cyanines.
linker. The key step in the synthesis is the formation of the more stable and less reactive dianil linker. The increased rigid-
dianil linkers which can individually be used to form large ity causes the absorption maximum to red shift to 803 nm and
libraries of cyanine compounds with substitutions on the could also cause the dye to slightly twist out of cyanine’s
methine chain. These dianil linkers are formed by reaction of general planar structure. Correspondingly, the addition of
a substituted cycloketone 1 or 1-substituted cycloalkene 2 with these cyclic rings generally involves the addition of a chlorine
a Vilsmeier–Haack reagent, generated from phosphorous oxy- substitutent at the meso-position, but it has been reported that
chloride and N,N-dimethylformamide, which gives a dialde- alkyl or aryl substituents such as the methyl or phenyl substi-
hyde that is later capped with aniline for stability to give dianil tutions described herein further increase photostability.21
linkers 3. These dianil linkers 3 are then condensed with qua-
Suzuki–Miyaura coupling has become a useful tool for con-
ternary ammonium salts in acetic anhydride in the presence of jugating the dyes to targeting ligands or biomolecules using
sodium acetate. The dyes are then purified either by washing water soluble cyanines.1,16 While there has been success
with methanol, dissolution in a minimal amount of methanol synthesizing these dyes through carbon–carbon coupling with
followed by precipitation in ether or column chromatography a meso-chlorine atom, a meso-bromine atom should give better
(1–5% methanol/DCM).
success. These reactions generally have lower yields due to the
Using this classical method to synthesize heptamethine need to separate the unreacted meso-halogenated dye from the
cyanines, many of these dyes were able to be purified solely by C–C coupled dye. Typically compounds 4g and 4h would be
washing with methanol and were produced in good yields. synthesized by first preparing 4a and then reacting it with
Cyanines have been widely tested for biomedical imaging methyl- or phenyl-boronic acid with a palladium catalyst,1,22
applications. Our lab has shown that small modifications in but by using this classical method with a substituted dianil
the structure of these compounds can cause them to have linker the Suzuki–Miyaura coupling step can be avoided. In
specific targeting.9,11,18,19 While modifications to the hetero- addition to removing a step and the need for an expensive
cycles and N substituents have been widely explored, changes catalyst, this method allows for application to both water
to the methine bridge have not. The combination of these soluble and insoluble compounds. Modifying the dianil
changes substantially increases the number of structures that linkers and synthesizing these compounds in a larger scale
can be made, further enhancing the modifiability of cyanine allows for the facile synthesis of a large number of substituted
dyes for their desired application.
cyanines by one step.
Heptamethine cyanines undergo rapid photooxidation in
Chloro-substituted cyanines are weakly or non-reactive in
solution.20 Ring systems have been introduced to hepta- comparison to iodo- or bromo-derivatives. Suzuki coupling
methine cyanine dyes for added stability,21 but only cyclohex- often requires strong base which is not suitable for use with
ene rings have been explored in detail. In this work cyclopen- sensitive heptamethine cyanines because they undergo hydro-
tene and cylcloheptene have also been introduced as shown in lysis or decomposition in strong base. The addition of a
Scheme 1. Column chromatography was needed to purify com- second heteroatom in the quaternary ammonium heterocycle,
pound 4b and it was synthesized in the lowest yield at 53%. such as benzothiazolium, further activates the C-2 position by
The cyclopentene ring is more planar potentially creating a inductive effect making it even more vulnerable to nucleophi-
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lic addition. Suzuki coupling has been shown to fail with these log D above 6 the ability of the phenyl ring to rotate into and out
compounds.1 This approach provides a convenient way to of plane with the rest of the dye can act to prevent stacking while
prepare a wide array of functionally different heptamethine the t-butyl in compound 4e is locked in a single conformation.
cyanines by reacting a variety of Vilsmeier reagents such as 3 The lack of aggregation observed in the rest of these compounds
with different quaternary ammonium salts and produces increases their attractiveness as biological probes.
various dyes in good yields.
Optical properties
Hydrophobicity
Because it is important for structural changes to retain the
Cyanine dyes are known to aggregate, but it was previously favorable optical properties of cyanine dyes, the absorbance
reported that the t-butyl group in compound 4e could be used and fluorescence of all compounds in Scheme
1 were
to prevent aggregation and make solar cells more efficient.13 measured. The absorption shifts in ethanol caused by the
Cyanines with heterocycles containing dimethylindolenine different substitutions from the dianil linkers can be seen in
have a more pronounced H-band that tends to become ampli- Fig. 1. The λmax varies from 759 nm to 805 nm in the dyes con-
fied when the dyes aggregate.23 Generally this aggregation, taining a meso-phenyl ring and a cyclopentene in the methine
caused by polar solvents such as water or PBS buffer, can be chain, respectively. The λmax of heptamethine cyanines with an
prevented using small amounts of organic solvents (i.e. 2% open chain or with the most common meso-chlorine substi-
DMSO or ethanol) although more hydrophobic dyes have tution is generally about 780 nm. Absorption shifts in cyanines
required as much as 30% DMSO to completely break up the are generally seen when different heterocycles are used that
aggregation.24 Hydrophobicity studies of the synthesized dyes have more interaction with the conjugated system.7 When the
shown in Scheme 1, were carried out in ethanol/PBS buffer methine chain substitutions are changed, the most notable
mixtures. As the polarity of the solvent decreased with increas- absorption shifts come from the alternate conjugation
ing ethanol concentration from 1% to 35%, a general amplifi- pathway in the meso-phenyl compound 4h and the strain
cation of the monomeric band in the absorption spectra was caused by the cyclopentene ring on the methine chain in com-
observed (Fig. 4). In dyes that are aggregating as the polarity pound 4b.
decreases there is also an increase in the ratio of monomeric
Optical properties of the dyes are shown in Table 1. The
band to the aggregate (H-band) until all aggregation is broken molar absorptivity of these compounds ranges from
up and the ratio becomes steady. The increased absorbance is 78 000–256 000. Interestingly, the two compounds 4b and 4h
related to the higher molar absorptivity of these compounds in that were furthest from the usual 780 nm absorbance
organic solvents compared to more polar ones and aggregation maximum measured in heptamethine cyanines also showed
is only observed if the ratio of the 2 peaks differs as the solvent the highest molar absorptivities, over 250 000. The meso-
polarity changes. As explained by our group previously, phenyl group in 4h and the strain put on the methine chain by
this aggregation is predominantly due to a plane-to-plane the cyclopentene in 4b can prevent cis–trans isomerization
arrangement.25
increasing the molar absorptivity.
Changes in the hydrophobic nature of the dyes can make
Subsequently, the lowest molar absorptivities were observed
them less biocompatible regarding uptake and in vivo transpor- in the compounds with the cycloheptene 4c and the t-butyl 4e.
tation. Of these newly synthesized heptamethine cyanines only The cycloheptene can have the opposite effect of the cyclopen-
compound 4e displayed aggregation beyond 2% ethanol as tene allowing for more rotation at the C2–C3 bond (Fig. 2) while
shown in Fig. 4. As shown in Table 1 this compound has a calcu- the t-butyl can cause increased aggregation in polar solvents.
lated log D value above 6. While compounds 4d and 4h also have Stokes shifts ranged from 12–20 nm, but the most interesting
Fig. 1 Absorption spectra of 4 μM heptamethine cyanines in ethanol (Right side is magnified).
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Table 1 Summary of optical properties of dyes 4a–h in ethanol and calculated log D values
Absorbance
λmax (nm)
Fluorescence
λmax (nm)
Molar absorptivity
Stokes
shift (nm)
Fluorescence
quantum yield (%)
Dye
(M−1 cm−1
)
log D
4a
4b
4c
4d
4e
4f
780
803
781
777
776
779
768
759
798
230 500
256 762
102 014
171 369
78 827
170 611
147 331
264 657
18
12
—
16
19
18
20
15
0.21
4.79
4.34
5.23
6.21
6.11
4.96
4.80
6.07
815
0.14
a
a
793
795
797
788
774
0.24
0.22
0.18
0.08
0.31
4g
4h
a Not fluorescent.
lead to rigidity enhancing the stability of the molecule in the
excited state, ultimately increasing the quantum yield. A trend
is observed with decreasing quantum yield as the rigidity of
the cyclic system increases with the addition of carbons. The
cycloheptene containing dye shows no fluorescence.
Fig. 2 Numbering of methine bridge carbons.
Conclusion
find from fluorescence studies was that compound 4c, contain-
ing a cycloheptene in the methine chain, was not fluorescent
(Fig. 3). The increased flexibility due to the seventh carbon in
the cyloheptene ring must allow for the excited energy to be
lost through motion in the ring rather than fluorescence.
Quantum yields of the synthesized dyes range from 8% to
31%. The dyes with halogens at the meso-position show
similar quantum yields ranging from 18% in the bromine con-
taining dye 4f to 21–24% in the 3 dyes with chlorine in the
meso-position 4a,d,e. The t-butyl and phenyl substitutions at
the 5 position of the cyclohexene have little effect on quantum
yield as they are not conjugated into the system. The dye with
a meso-methyl group 4g had the lowest quantum yield at 8%
due to the its ability to relax through rotation of the methyl
position while the dye with the meso-phenyl 4h had the
highest quantum yield at 31%. The long wavelengths exhibited
by cyanine dyes are caused by the delocalization of electrons
throughout the molecule. Cyclic structures within the dyes
Several heptamethine cyanine derivatives were synthesized
from modified dianil linkers. The simple general procedure is
advantageous for carbon–carbon coupling over previous
Suzuki–Miyaura methods in that a large number of structurally
diverse fluorophores can be synthesized after a single step
without the use of palladium catalysts. This one-pot method
can also eliminate the cumbersome steps of protecting/depro-
tecting amino or hydroxy groups usually applied to reactions
that required conjugation to meso-chlorine substituted dyes. In
addition, the procedure is applicable to hydrophobic hetero-
cyclic salts well as water soluble ones. It was shown that dyes
containing cyclopentene in the methine bridge or a meso-
phenyl display increased molar absorptivity. The dye contain-
ing a cycloheptene in the methine bridge did not fluoresce.
The compound containing a t-butyl showed lower molar
absorptivity and large amounts of aggregation in PBS buffer.
These structural substitutions can be useful in the many appli-
cations of cyanines.
Fig. 3 Fluorescence spectra of 0.4 μM heptamethine cyanines in ethanol (Left side: dyes were excited at 760 nm. Right side: dye 4h was excited at
750 nm due to blueshifted fluorescence).
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Fig. 4 Absorption spectra of 4a, 4c, 4d, and 4e as a function of solvent hydrophobicity (% v/v ethanol to PBS buffer) at constant dye concentration
of 4 μM.
ively, while stirring still at 0 °C. After the solution becomes
yellow, the slurry is heated at 100 °C for 2 h. The reaction
mixture is then cooled to room temperature and aniline in
Experimental
General information
All chemicals and solvents were of American Chemical Society ethanol is added slowly in an ice bath. The reaction mixture
grade or HPLC purity and were used as received. All chemicals is then poured into ice and concentrated HCl is added to give
were purchased from Fisher Scientific (Pittsburgh, PA, USA), a burgundy precipitate. The powder is then filtered and
Sigma-Aldrich (Saint Louis, MO) and Acros Organics. Melting washed with ether and hexanes and used without further
points (mp, open Pyrex capillary) were measured on a Thomas purification.
Hoover apparatus and are uncorrected. 1H and 13C NMR
General procedure for synthesis of heptamethine dyes 4a–h.
spectra were recorded on BrukerAvance (400 MHz) spectro- The quaternary ammonium salt (200 mg, 2 mol eq.) was
meter. Absorption spectra were recorded on a Varian Cary 50 stirred in acetic anhydride (3 mL) followed by addition of
UV-Visible Spectrophotometer (Santa Clara, CA) in ethanol sodium acetate (2 mol eq.) and dianil linker 3 (1 mol eq.). The
using VWR disposable two-sided polystyrene cuvettes path reaction was heated at 80 °C for 2 h. The reactions were moni-
length 1 cm. Fluorescence emission analyses were performed tored closely using regular phase thin layer chromatography
using a Shimadzu RF-5301PC Spectrofluorophotometer (Kyoto, with a mobile phase of DCM/MeOH (99 : 1) as well as UV-Vis-
Japan) in ethanol using Sigma-Aldrich disposable polystyrene NIR spectrophotometer with methanol as the solvent to visual-
fluorimeter cuvettes of pathlength 1 cm. Excitation was ize the absorption band at ∼780 nm. Upon completion of the
achieved at 760 nm (750 nm for 4h) with slit widths of 5 mm. reaction the mixtures were allowed to cool to room tempera-
Microsoft Excel 2010 was used for all calculations.
ture before the dye was precipitated in diethyl ether (50 mL).
The pure products were obtained after washing with methanol,
dissolving the dyes in acetonitrile (1 mL) and precipitating
Synthesis
General procedure for synthesis of dianil linkers 3a–h. with ether (50 mL), or column chromatography with eluent
Under nitrogen atmosphere, DMF was added dropwise to from 1–5% methanol/DCM.
phosphorous oxychloride keeping the reaction temperature at
2-((E)-2-((E)-2-Chloro-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)
or below 5 °C. Cycloketones or 1-substitutedcyclohexenes in ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-
dry dichloromethane were added to this solution, respect- 1-ium iodide, 4a: was synthesized as previously described.26
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1H NMR (400 MHz, DMSO-d6) δ 1.67 (s, 12 H), 1.85 (m, 2 H), 3 H), 2.62 (t, J = 5.6 Hz, 4 H), 3.72 (s, 6 H), 6.16 (d, J = 13.6 Hz,
2.72 (t, J = 6.0 Hz, 4 H), 3.68 (s, 6 H), 6.30 (t, J = 14.4 Hz, 2 2 H), 7.16 (d, J = 8.0 Hz, 2 H), 7.23 (d, J = 7.6 Hz, 2 H), 7.38 (m,
H), 7.28 (m, 2 H), 7.44 (m, 4 H), 7.62 (d, J = 7.2 Hz, 2 H), 8.25 4 H), 8.05 (d, J = 13.2 Hz, 2 H). 13C NMR (100 MHz, CDCl3)
(d, J = 14.4 Hz, 2 H).
δ ppm 15.1, 20.9, 25.7, 28.3, 32.2, 100.6, 110.3, 122.1, 124.8,
2-((E)-2-((E)-2-Chloro-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene) 128.7, 132.1, 140.5, 142.4, 156.0, 171.5. HRMS m/z: calc. for
ethylidene)cyclopent-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1- C33H39N2+ 463.3108, obsd 463.3091.
ium iodide, 4b. Yield 53%; mp >260 °C; 1H NMR (400 MHz,
1,3,3-Trimethyl-2-((E)-2-((E)-6-(2-((E)-1,3,3-trimethylindolin-2-
CDCl3) δ 1.73 (s, 12 H), 3.10 (m, 4 H), 3.77 (s, 6 H), 6.13 (d, J = ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1′-biphenyl]-2-yl)vinyl)-3H-
14.0 Hz, 2 H), 7.16 (d, J = 8.0 Hz, 2 H), 7.24 (t, J = 8.0 Hz, 2 H), indol-1-ium iodide, 4h. Yield 77%; mp >260 °C; 1H NMR
7.40 (m, 4 H), 7.83 (d, J = 14.0 Hz, 2 H); 13C NMR (100 MHz, (400 MHz, CDCl3) δ 1.11 (s, 12 H), 1.95 (m, 2 H), 2.68 (t, J =
CDCl3) δ 27.1, 28.0, 32.6, 49.0, 102.9, 110.6, 122.0, 125.1, 128.8, 6.0 Hz, 4 H), 3.58 (s, 6 H), 6.17 (d, J = 14.0 Hz, 4 H), 7.16 (m,
136.8, 138.3, 140.9 142.9, 151.8, 171.4. HRMS m/z: calc for 4 H), 7.25 (d, J = 8.0 Hz, 2 H), 7.34 (m, 4 H), 7.46 (d, J = 8.0 Hz,
C31H34N2Cl+ 469.2405, obsd 469.2387.
2 H), 7.62 (m, 3 H). 13C NMR (100 MHz, CDCl3) δ 21.3, 24.5,
2-((E)-2-((E)-2-Chloro-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene) 27.3, 31.5, 48.5, 100.7, 111.3, 122.7, 124.9, 128.5, 128.8, 129.0,
ethylidene)cyclohept-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1- 129.6, 131.0, 139.1, 140.9, 143.3, 147.3, 161.5, 172.1. HRMS
ium iodide, 4c. Yield 79%; mp 139–141 °C; 1H NMR (400 MHz, m/z: calc. for C38H41N2+ 525.3264, obsd 525.3241.
CDCl3) δ 1.74 (s, 12 H), 1.91 (m, 4 H), 2.83 (m, 4 H), 3.79 (s, 6
Stock solutions
H), 6.31 (d, J = 14.4 Hz, 2 H), 7.19 (d, J = 7.2 Hz, 2 H), 7.26 (t,
J = 7.2 Hz, 2 H), 7.38 (d, J = 7.2 Hz, 2 H), 7.44 (t, J = 7.2 Hz, 2
H), 8.37 (d, J = 14.4 Hz, 2 H); 13C NMR (100 MHz, CDCl3)
δ 23.9, 26.9, 28.0, 32.8, 49.2, 101.4, 110.7, 122.0, 125.2, 128.8,
131.2, 142.8, 147.2, 154.7, 173.1. HRMS m/z: calc for
C33H38N2Cl+ 497.2718, obsd 497.2698.
Stock solutions of the dyes and standard were prepared by
weighing the solid on a 5-digit analytical balance in an amber
vial and adding solvent via a class A volumetric pipette to a
final concentration of 1.0 mM. The vials were vortexed for 20 s
and then sonicated for 15 min to ensure complete dissolution.
The stock solutions were stored in a dark freezer at 4 °C when
not in use. Working solutions were prepared just prior to use
by dilution of the stock to final concentrations.
2-((E)-2-((E)-4-Chloro-5-(2-((E)-1,3,3-trimethylindolin-2-ylidene)
ethylidene)-1,2,5,6-tetrahydro-[1,1′-biphenyl]-3-yl)vinyl)-1,3,3-tri-
methyl-3H-indol-1-ium iodide, 4d. Yield 72%; mp 171–173 °C;
1H NMR (400 MHz, CDCl3) δ 1.68 (s, 12 H), 2.70 (m, 2 H), 3.04
(m, 1 H), 3.15 (m, 2 H), 3.65 (s, 6 H), 6.32 (d, J = 14.0 Hz, 2 H),
7.29 (m, 3 H), 7.41 (m, 6 H), 7.48 (d, J = 7.2 Hz, 2 H), 7.63 (d,
J = 7.6 Hz, 2 H), 8.31 (d, J = 14.0 Hz, 2 H). 13C NMR (100 MHz,
CDCl3) δ 27.8, 32.0, 33.6, 38.7, 49.3, 102.4, 111.9, 122.8, 125.6,
126.3, 127.1, 127.9, 129.0, 141.5, 143.3, 145.4, 147.7, 173.3;
HRMS m/z: calc. for C38H40N2Cl+ 559.2875, obsd 559.2855.
2-((E)-2-((E)-5-(tert-Butyl)-2-chloro-3-(2-((E)-1,3,3-trimethylindolin-
2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-
indol-1-ium iodide, 4e. Yield 76%, mp 171–173; 1H NMR
(400 MHz, DMSO-d6) δ 1.07 (s, 9 H), 1.54 (m, 2 H), 1.67 (s,
12 H), 2.18 (m, 2 H), 2.97 (m, 2 H), 3.71 (s, 6 H), 6.32 (d, J =
13.6 Hz, 2 H), 7.29 (m, 2 H), 7.44 (m, 4 H), 7.62 (d, J = 7.2 Hz, 2
H), 8.25 (d, J = 13.6 Hz, 2 H). 13C NMR (100 MHz, DMSO-d6)
δ 27.7, 32.1, 32.7, 49.3, 102.0, 111.8, 122.8, 125.6, 126.8, 129.0,
141.4, 143.2, 173.1. HRMS m/z: calc. for C38H40N2Cl+ 539.3188,
obsd 539.3187.
Method of determining molar absorptivity and fluorescence
quantum yield
Stock solutions were used to prepare six dilutions of dyes in
ethanol with concentrations ranging from 1 μM to 4 μM using
a class A volumetric pipette in order to maintain absorption
between 0.1 and 1.0. The dye solutions were diluted ten-fold
for fluorescence in order to minimize inner filter effect. The
absorbance spectra of each sample was measured in triplicate
from 550–900 nm. The emission spectrum of each sample was
measured in triplicate with a 760 nm (750 nm for 4h) exci-
tation wavelength.
For molar absorptivity, the absorbance at the wavelength of
maximum absorbance (λmax) was determined and the absor-
bance of each sample at λmax was plotted as a function of dye
concentration. The linear regression equation was computed
using Microsoft Excel.
2-((E)-2-((E)-2-Bromo-3-(2-((E)-1,3,3-trimethylindolin-2-ylidene)
ethylidene)cyclohex-1-en-1-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-
ium iodide, 4f. Yield 74%; mp >260 °C; 1H NMR (400 MHz,
DMSO-d6) δ 1.69 (s, 12 H), 1.85 (m, 2 H), 3.91 (m, 4 H), 3.69 (s,
6 H), 6.23 (d, J = 14.4 Hz, 2 H), 7.29 (m, 2 H), 7.44 (m, 4 H),
7.63 (d, J = 7.2 Hz, 2 H), 8.26 (d, J = 14.0, 2 H). 13C NMR
(100 MHz, DMSO-d6) δ 27.5, 31.9, 49.3, 102.6, 111.9, 122.8,
125.6, 128.4, 129.0, 141.4, 143.3, 144.9, 146.7, 173.1. HRMS
m/z: calc for C32H36N2Br+ 527.2056, obsd 527.2040.
The fluorescence quantum yields were determined relative
to the indocyanine green standard utilizing the gradient from
the plot of integrated fluorescence intensity vs. absorbance
(Grad) and the published quantum yield of the standard
(φS, 13.2%27) as per eqn (1).
2
φD ¼ φS ꢀ GradD=GradS ꢀ ηS2=ηD
:
ð1Þ
Hydrophobicity studies
1,3,3-Trimethyl-2-((E)-2-((E)-2-methyl-3-(2-((E)-1,3,3-trimethyl-
indolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1- Hydrophobic characteristics of the dyes were assessed by
ium iodide, 4g. Yield 71%; mp 217–219 °C; 1H NMR (400 MHz, acquiring absorbance spectra of the dyes in varying ratios of
CDCl3) δ ppm 1.73 (s, 12 H), 1.92 (t, J = 5.6 Hz, 2 H), 2.43 (s, ethanol-buffer mixture (in the range of 1–35% ethanol) using
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PBS buffer pH 7.4. The concentration of ethanol was slowly
increased until there were no apparent changes in the ratio
between the monomeric peak and H-band.
imaging/radiography system, Arthritis Rheum., 2010, 62,
2322–2327.
7 E. Soriano, C. Holder, A. Levitz and M. Henary, Benz[c,d]
indolium-containing Monomethine Cyanine Dyes: Synthesis
and Photophysical Properties, Molecules, 2015, 21, E23.
8 C. Gibas, N. Kretschy and M. M. Somoza, Comparison of
the Sequence-Dependent Fluorescence of the Cyanine Dyes
Cy3, Cy5, DyLight DY547 and DyLight DY647 on Single-
Stranded DNA, PLoS One, 2014, 9, e85605.
Log D calculations
Log D values were predicted using MarvinSketch 5.9
(ChemAxon, Budapest, Hungary). Values were derived from cal-
culations by Viswanadhan,28 Klopman29 and the PHYSPROP
database at pH 7.4 and the three were averaged.
9 H. Hyun, H. Wada, K. Bao, J. Gravier, Y. Yadav, M. Laramie,
M. Henary, J. V. Frangioni and H. S. Choi,
Phosphonated Near-Infrared Fluorophores for Biomedical
Imaging of Bone, Angew. Chem., Int. Ed., 2014, 53, 10668–
10672.
Conflicts of interest
There are no conflicts to declare.
10 H. Wada, H. Hyun, C. Vargas, J. Gravier, G. Park, S. Gioux,
J. V. Frangioni, M. Henary and H. S. Choi, Pancreas-
Targeted NIR Fluorophores for Dual-Channel Image-
Guided Abdominal Surgery, Theranostics, 2015, 5, 1–11.
11 H. Hyun, M. H. Park, E. A. Owens, H. Wada, M. Henary,
H. J. M. Handgraaf, A. L. Vahrmeijer, J. V. Frangioni and
H. S. Choi, Structure-inherent targeting of near-infrared
fluorophores for parathyroid and thyroid gland imaging,
Nat. Med., 2015, 21, 104–U109.
12 C. N. Njiojob, E. A. Owens, L. Narayana, H. Hyun,
H. S. Choi and M. Henary, Tailored near-infrared contrast
agents for image guided surgery, J. Med. Chem., 2015, 58,
2845–2854.
13 A. Otsuka, K. Funabiki, N. Sugiyama, H. Mase, T. Yoshida,
H. Minoura and M. Matsui, Design and Synthesis of Near-
infrared-active Heptamethine–Cyanine Dyes to Suppress
Aggregation in a Dye-sensitized Porous Zinc Oxide Solar
Cell, Chem. Lett., 2008, 37, 176–177.
14 P. K. D. Duleepa Pitigala, M. M. Henary, E. A. Owens,
A. G. UnilPerera and K. Tennakone, Excitonic photovoltaic
effect in a cyanine dye molecular assembly electronically
coupled to n- and p-type semiconductors, J. Photochem.
Photobiol., A, 2016, 325, 39–44.
Acknowledgements
The authors would like to thank the Department of Chemistry
at Georgia State University for the support of the Ph.D. dis-
sertation of AL and the MSc. thesis of FM. This study was
supported by grants to MH from the Georgia State University
Brains and Behavior Seed Grant, the Atlanta Clinical and
Translational Science Institute Healthcare Innovation Seed
Grant, and the Georgia Research Alliance Ventures Phase 1
Grant. MH would like thank the U.S. Department of Health
and Human Services, National Institutes of Health National
Institute of Biomedical Imaging and Bioengineering (Grant
Number: RO1EB022230).
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