Synthesis and optical properties of near-infrared meso-phenyl-substituted symmetric heptamethine cyanine dyes

Article abstract of DOI:10.3390/molecules23020226

Heptamethine cyanine dyes are a class of near infrared fluorescence (NIRF) probes of great interest in bioanalytical and imaging applications due to their modifiability, allowing them to be tailored for particular applications. Generally, modifications at the meso-position of these dyes are achieved through Suzuki-Miyaura C-C coupling and SRN1 nucleophilic substitution of the chlorine atom at the meso-position of the dye. Herein, a series of 15 meso phenyl-substituted heptamethine cyanines was synthesized utilizing a modified dianil linker. Their optical properties, including molar absorptivity, fluorescence, Stokes shift, and quantum yield were measured. The HSA binding affinities of two representative compounds were measured and compared to that of a series of trimethine cyanines previously synthesized by our lab. The results indicate that the binding of these compounds to HSA is not only dependent on hydrophobicity, but may also be dependent on steric interferences in the binding site and structural dynamics of the NIRF compounds.

Full text of DOI:10.3390/molecules23020226

molecules
Article
Synthesis and Optical Properties of Near-Infrared
meso-Phenyl-Substituted Symmetric Heptamethine
Cyanine Dyes
Andrew Levitz 1,† , Fahad Marmarchi 1,† and Maged Henary 1,2,
ID
*
1
Department of Chemistry, Georgia State University, 50 Decatur St., Atlanta, GA 30303, USA;
alevitz1@student.gsu.edu (A.L.); fjrjes1@student.gsu.edu (F.M.)
Center for Diagnostics and Therapeutics, Georgia State University, Petit Science Center,
2
1
00 Piedmont Ave SE, Atlanta, GA 30303, USA
*
†
Correspondence: mhenary1@gsu.edu; Tel.: +1-404-413-5566; Fax: +1-404-413-5505
These authors contributed equally to this work.
Received: 27 November 2017; Accepted: 20 January 2018; Published: 24 January 2018
Abstract: Heptamethine cyanine dyes are a class of near infrared fluorescence (NIRF) probes of
great interest in bioanalytical and imaging applications due to their modifiability, allowing them
to be tailored for particular applications. Generally, modifications at the meso-position of these
dyes are achieved through Suzuki-Miyaura C-C coupling and SRN1 nucleophilic substitution of
the chlorine atom at the meso-position of the dye. Herein, a series of 15 meso phenyl-substituted
heptamethine cyanines was synthesized utilizing a modified dianil linker. Their optical properties,
including molar absorptivity, fluorescence, Stokes shift, and quantum yield were measured. The HSA
binding affinities of two representative compounds were measured and compared to that of a series
of trimethine cyanines previously synthesized by our lab. The results indicate that the binding of
these compounds to HSA is not only dependent on hydrophobicity, but may also be dependent on
steric interferences in the binding site and structural dynamics of the NIRF compounds.
Keywords: heptamethine cyanine dyes; absorbance; fluorescence; NIRF; physiochemical properties;
HSA binding
1
. Introduction
Heptamethine cyanine dyes are a class of near infared fluorescence (NIRF) probes that have shown
great potential in numerous applications due to their versatility, low toxicity, narrow absorption band,
and high extinction coefficients [ ]. These dyes are comprised of two terminal nitrogen-containing
1–6
heterocycles linked together by a conjugated polymethine chain. The heterocycles act as both electron
donors and acceptors creating an electron deficient system throughout the molecule, allowing for long
wavelength absorption [
cartilage, bone, endocrine gland, biomolecular labeling and much more all serving as contrast agents
to aid in surgical application [ 11 17]. Modifications to the cyanine dye scaffold can alter optical
7–10]. Heptamethine cyanines have been used in medical imaging targeting of:
5
, –
properties, solubility, and allow for specific tailoring of dyes for their desired application.
A main contributing factor in the successful application of heptamethine cyanine dyes is their
modifiability. Most commonly cyanines are modified by the use of different heterocycles and with
different substituents on the nitrogen atom of the heterocycle. One point of modification that has not
been thoroughly investigated is the central (meso) carbon of the methine bridge. Many derivatives
described in literature have been made by replacing the chlorine atom at this position via a SRN
mechanism [12 14]. The most common method of carbon-coupling at the meso-position thus far
has been done by first synthesizing a heptamethine cyanine dye containing a chlorine atom at the
1
–
Molecules 2018, 23, 226; doi:10.3390/molecules23020226
www.mdpi.com/journal/molecules

Molecules 2018, 23, 226
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meso-position followed by Suzuki-Miyaura coupling [18
–20]. While this method is successful in
synthesizing many scaffolds, it requires tedious purification and the use of an expensive palladium
catalyst [12 14]. Although the phenyl-substituted dianil linker has been previously synthesized, it has
–
not been thoroughly investigated for its effects on the optical properties on the NIRF dye [21,22].
Many of these cyanine dyes are administered via IV injection in which the dyes are transported
to their target through the bloodstream [23]. Human serum albumin (HSA) is the most abundant
protein in human blood plasma, serving an important role of transporting substances throughout
the body [23
compounds [23
delivery [15]. HSA contains four binding pockets and does not require biomolecular ligand specificity,
increasing its versatility and usefulness in medical research [23 26]. It is well described in literature
–
25]. It is synthesized in the liver and has great binding capacity for hydrophobic
,26]. HSA has been a widely studied protein because of its importance in drug
–
that HSA binds to hydrophobic entities. A unique attribute of HSA is that it forms reversible covalent
bonds with the binding agent, this allows for stable complex formation; however, since the bonds are
reversible also allows for localization and deposit [27].
Our lab has previously designed and synthesized a series of trimethine dyes and studied
their hydrophobicity and its effect on their interactions with HSA [28]. In this paper, a series of
heptamethine cyanines with varying degrees of hydrophobicity containing a meso-phenyl substituent
were synthesized through the use of a phenyl-substituted dianil linker. This method not only allows for
a more facile synthesis, but a wider array of dyes can be made and can serve for various applications.
The effect of the phenyl ring on the dyes hydrophobicity, optical properties, and binding to HSA was
studied and compared to the results from our previous study [28].
2
. Results and Discussion
2
.1. Synthesis
As shown in Scheme 1, the synthesis began with a Fischer indole cyclization by refluxing
4-substituted phenylhydrazines 1 overnight with 3-methyl-2-butanone in glacial acetic acid.
After cooling to room temperature, the reaction mixture was neutralized and the substituted indoles
were extracted with dichloromethane to give brown oils. The oils were dissolved in acetonitrile and
refluxed overnight with various alkyl halides to yield quaternary ammonium salts . In parallel
to salt formation, a phenyl-substituted dianil compound was synthesized through a Vilsmeier
Haack formylation with 1-phenylcyclohexene ( ). The ends of the dianil linker were capped with
aniline for stability to yield dianil compound 21 22]. Various quaternary ammonium salts and
dianil compound are then condensed in a 2:1 ratio in acetic anhydride to yield the final phenyl
2
3
4
5
[
,
3
5
substituted heptamethine cyanines
with methanol.
6. Pure compounds were obtained in good yield by simply washing
Scheme 1. Synthetic routes of heptamethine dyes containing a phenyl ring at the meso-position.

Molecules 2018, 23, 226
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The synthetic route described in Scheme 1 provided a new carbon-carbon linked substituent
position at the meso center adding to the versatility of heptamethine cyanine. The phenyl group was
added before the dye was made. This allowed for an efficient method of preparation of the dye, which
required no catalysts or complex purification methods and allowed for a wider array hydrophobic
compounds to be made.
Once the dyes have been successfully synthesized, the optical properties were measured, and
representative dyes 6a and 6k were studied for their binding affinity to HSA. The optical properties
were compared to commercially available heptamethine dyes Cy-7 and IR-780 due to the similar
absorbance and emission wavelengths. The binding studies of compounds 6a and 6k were compared
to MHI-06, a dye previously reported as a strong HSA binding agent [28]. Figure 1 shows the three
dye structures of the standards used in our study.
Figure 1. The structures of the three NIR standards used for the study.
2
.2. Optical Properties
As described in Scheme 1, fifteen final NIRF contrast agents were synthesized using the dianil
linker to yield symmetrical heptamethine cyanines 6a
sets of five. Dyes 6a all contain a methyl substituent off the nitrogen of the heterocycle with varying
substitutions at the six position of the heterocyclic ring.
Dyes 6f and 6k contain ethyl and butyl N-alkyl substituents, respectively. In comparison to
–o. The compounds are broken down to three
–
e
–
j
–o
Cy-7, the optical properties of the new compounds were found to be superior (Table 1). The addition
of the cyclohexene ring provided rigidity to the compounds, increasing the molar absorptivity and
−
1
−1
quantum yield by 60,000 M
·
cm and 5%, respectively, for 6a
[
29]. To determine the effects the
30]. Although the
phenyl ring had on the optical properties, the dyes were compared to IR-780
[
molar absorptivity of the studied dyes were within the same range as the commercially available dye,
the quantum yield was dramatically increased with the introduction of the electron rich phenyl ring,
observing a 23%–47% increase in quantum yield. The chlorine atom at the meso-position of dye IR-780
promotes intersystem crossing due to the heavy atom effect, and allows for the molecule to relax in
non-radiative means and decreases the fluorescence [31].
Table 1. Spectral Characteristics of dyes Cy-7, IR-780 and 6a–o. All optical properties of the dyes were
measured in ethanol.
Dye
λmax (nm)
λemission (nm)
Stokes Shift (nm)
ε (L·mol−1·cm−1
)
Φ (%)
Molecular Brightness (M−1·cm−1
)
Cy-7
IR-780
753
779
759
765
767
782
798
760
769
770
786
797
763
772
773
789
800
775
799
774
780
783
802
810
781
785
786
804
810
780
787
788
805
812
22
20
15
15
16
20
12
21
16
16
18
13
17
15
15
16
12
200,000
274,000
265,700
261,000
275,600
249,500
255,400
263,900
286,600
282,900
143,500
231,800
198,500
123,400
239,200
249,900
226,600
28
8.0
31
34
35
10
16
39
38
42
12
17
45
47
48
11
17
56,000
20,800
82,000
88,700
96,500
25,000
40,900
102,300
109,300
119,200
17,200
39,400
89,300
58,000
113,600
27,100
38,500
6a
6
6
b
c
6
6
d
e
6f
6
g
6h
6
i
6j
6
6
k
l
6
6
m
n
6o

Molecules 2018, 23, 226
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Increasing the length of N-alkyl substituents from a methyl to ethyl did not result in any significant
changes in optical properties, but an increase in size to the butyl group generally lowered the molar
absorptivity. Dyes containing hydrogen 6a, 6f, 6k, and halogens 6b, 6c, 6g, 6h, 6l, 6m at the 6
position of the heterocycle displayed absorption λmax values of 759–773 nm with redshifts from the
hydrogen to the halogens from 6 nm to 10 nm which has previously been described [32]. Absorption
spectra of representative compound 6m was shown in Figure 2. All 15 compounds have Stokes
shifts ranging from 12 to 21 with the benz[e]indolenine containing compounds 6e
shortest Stokes shifts. All three methoxy-substituted compounds 6d 6i 6n had redshifted absorption
λmax values from 16 nm to 26 nm and lower quantum yields than the other compounds while
dyes 6e 6j 6o were redshifted 37–39 nm around 800 nm due to the increased conjugation of the
, 6j, 6o having the
,
,
,
,
benz[e]indolenine heterocycle.
1
0
0
0
0
.8
.6
.4
.2
0
600
650
700
750
800
850
900
Wavelengh (nm)
1
µM
1.5µM
2µM
2.5µM
3µM
4µM
Figure 2. Absorption spectra of dye 6m in ethanol.
The emission data of all the dyes followed the same trends, whereby compounds containing
the hydrogen 6a 6f 6k, and halogen 6b 6c 6g 6h 6l 6m substituents had the highest quantum
yields, and the longer N-alkyl chains increased the quantum yield by 4%–8% from methyl to ethyl and
%–7% from ethyl to butyl. Emission spectra of representative compound 6m was shown in Figure 3.
There was no significant difference in quantum yield between the hydrogen and halogens within the
same set. Compounds 6e 6j 6o containing the benz[e]indolenine heterocycle and 6d 6i 6n containing
,
,
,
,
,
,
,
5
,
,
,
,
the methoxy substituent displayed lower quantum yields at 16%–17% and 10%–12%, respectively,
which is consistent with previous reports [12].
50
40
30
20
10
0
765
785
805
825
845
865
885
Wavelengh (nm)
0
.1 μM
0.15 μM
0.2 μM
0.25 μM
0.3 μM
0.4 μM
Figure 3. Emission spectra of dye 6m in ethanol with excitation wavelength of 750 nm.

Molecules 2018, 23, 226
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Although molar absorptivity and quantum yield are important properties of fluorophores,
in regards to application, the molecular brightness gives a more useful indication of the
dye utility. Molecular brightness takes into account both molar absorptivity and quantum
yield [33,34]. Dyes that have high quantum yield, but do not absorb light efficiently (low molar
absorptivity) are still not emitting as many photons and are less useful for fluorescent applications.
N-alkyl substituents from a methyl to ethyl increased the molecular brightness by approximately
−
1
−1
for the hydrogen-6a
20,000 M
·
cm
, 6f, 6k, and halogen-6b, 6c, 6g, 6h, 6l, 6m substituted
compounds while the butyl compounds showed lower molecular brightness. Due to the low
quantum yield of the benz[e]indolenine heterocycle 6e
,
6j
,
6o and the methoxy 6d
,
6i
,
6n substituted
compounds, the two sets showed the lowest molecular brightness of 38,000–40,000 M−1·cm and
−1
−
1
−1
1
7,000–20,000 M ·cm , respectively.
2
.3. Physiochemical Properties
In our previous study of trimethine cyanine dyes it was shown that MHI-06 (Figure 1) bound
6
−1
HSA with an affinity of 1.0
×
10 M
[28]. In that study of the binding affinity of trimethine cyanines,
a trend was observed correlating hydrophobicity to the binding affinity. As the dyes became more
hydrophobic greater binding affinity was observed. However, the correlation did not hold when the
large N-phenylpropyl side chain was introduced in the trimethine series [28]. It was hypothesized that
the binding affinity decreased due to the increased size of the N-phenylpropyl side chain hindering
the dye from entering the HSA binding pocket. The heptamethine cyanines 6a–o synthesized in this
work were tailored to be hydrophobic in order to observe if binding increases due to increase of
hydrophobicity or decreases due to the size and steric hindrance.
The physicochemical properties were calculated using ChemAxon for the 15 heptamethine dyes
synthesized and compared to our pervious reported compound MHI-06 (Table 2) [28]. Physicochemical
trends were observed in each series with the same heterocyclic substituents as well as with the same
N-alkyl side chain. Compounds 6d
due to its ability to form hydrogen bonds. Slightly higher values were observed for compounds
with the hydrogen-substitued 6a 6f 6k. A 1.2 and 1.5 increase of logD was observed from the
hydrogen to the chloro-and bromo-substituted compounds, respectively. As expected, the series with
the benz[e]indolenine heterocycle 6e 6j 6o had the highest logD values due to the addition of another
phenyl ring. All heptamethine cyanines 6a had significantly higher logD values compared to that of
, 6i, 6n with the methoxy substituent showed the lowest logD,
,
,
,
,
–
o
MHI-06 due to the presence of the phenyl substituent at the meso-position and the increased size of the
hydrocarbon skeleton. The dipole moments decreased as the length of the alkyl chain increased from
methyl to butyl, with methoxy-substituted dyes having significantly higher values. In comparison to
MHI-06 most of the compounds, especially that of the methyl series 6a–e, had a higher dipole moment,
but for 6k which had fairly similar results to MHI-06 at 2.85 and 2.48, respectively. The number of
rotatable bonds increased by 2 from methyl to ethyl and by 4 from ethyl to butyl. Only the methoxy
heterocyclic substitution affected the number of rotatable bonds with an additional rotatable bond for
each methoxy in compounds 6d
exception of those containing the methoxy substituent had a total polar surface area (TPSA) of 6.25,
hydrogen bond donors, and 1 hydrogen bond acceptor (ChemAxon). Dyes 6d 6i 6n containing the
, 6i, 6n. MHI-06 had eight rotatable bonds. All of the dyes with the
0
,
,
methoxy substituent had higher TPSA at 24.71 and three hydrogen bond acceptors. The presence of the
polar oxygen increases the TPSA, and increases the number of hydrogen bond that can form by two.
In summary, the newly synthesized heptamethine dyes 6a–o were significantly more hydrophobic
then MHI-06, but had a much larger volume. Although hydrophobicity plays a key role in binding
to HSA, reversibly the size of the compound could inhibit the ability of its binding to HSA pocket.
Compounds 6a and 6k were tested for their ability to bind to HSA, to further determine if other
factors than hydrophobicity, play an important role in HSA binding allowing a better understanding of
binding nature of the larger heptamethine cyanines to HSA. The HSA binding spectra for 6a is shown
below (Figure 4) and binding data of 6k is shown in the supplemental information (6k HSA binding).

Molecules 2018, 23, 226
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Table 2. Physicochemical properties (in silico) of dyes MHI-06 and 6a–o calculated using ChemAxon.
The data calculated (at pH 7.4) include: logD, polarizability, dipole moment (debye), number of
3
3
rotatable bonds, volume (Å ), molecular surface area (Å ), and molar mass (g/mol).
Dye
logD
Polarizability Dipole Moment
Rot. Bonds
Volume
Molec. Surface Area
Molar Mass
MHI-06
4.97
6.07
7.28
7.61
5.75
8.05
6.75
7.99
8.32
6.47
8.76
8.72
9.92
10.25
8.40
10.69
59.45
65.57
69.15
70.67
70.51
80.82
69.26
72.84
74.35
74.2
84.51
76.65
80.23
81.71
81.59
91.9
2.48
8.35
12.3
13.02
27.66
11.87
3.2
4.81
4.97
25.29
4.59
2.85
4.63
4.96
26.66
3.96
8
4
4
4
6
4
6
6
6
441.44
519.85
547.45
556.15
569.71
604.27
552.14
579.48
588.47
603.83
638.63
620.01
647.91
656.64
672.63
708.29
693.216
814.001
845.982
854.732
907.778
936.392
874.685
906.491
915.629
969.908
999.272
996.744
1029.597
1038.143
1094.66
1124.8
584.54
652.66
721.55
810.46
712.72
752.78
680.72
749.6
838.51
740.77
7803.84
736.81
805.71
894.62
796.88
836.95
6a
6
6
b
c
6
6
d
e
6f
6
g
6h
i
8
6
6
6
k
l
10
10
10
12
10
6
6
m
n
6o
300
250
200
150
100
50
0
750
770
790
810
830
850
870
890
Wavelengh (nm)
0
μM
0.025 μM
0.05 μM
0.075 μM
0.1 μM
0.2 μM
Figure 4. The emission specra of 6a (0.2 µM) binding with various concentration of HSA, in PBS buffer
at excitation wavelength of 740 nm.
2
.4. HSA Binding
The formation of a dye/substrate conjugate was studied with HSA in phosphate buffered saline
PBS), pH 7.4. Previous research by Kim et al. suggested that cyanine dyes bind HSA in with a 1:1
stoichiometry which was confirmed by our and other research groups using trimethine cyanine
dyes [ 28 35 37]. The binding interactions were studied by measuring the changes in emission
intensities at a fixed concentration of dye with varying micromolar concentrations of HSA and using
(
3
, , –
−
1
−1
a double reciprocal plot of [HSA] vs.
∆F , where
∆F is the change in emission intensity of the
Dye/HSA conjugate, that should give a linear relationship. The binding affinity is then calculated by
dividing the intercept by the slope of the line. Our lab has previously shown that both the N-alkyl
substituents and the heterocyclic ring of the cyanines have profound effects on overall conjugation
with the biomolecule [20]. It has also previously been shown that cyanine dyes aggregate in polar
solvents such as PBS buffer due to strong intermolecular van der Waals interactions between the
heterocycles that cause the dyes to form H-aggregates [38]. Generally, organic solvents are used to
disrupt this aggregate formation, but organic solvents cannot be used in the presence of HSA due to
their ability to denature the biomolecule. Conjugate formation with HSA disrupts the aggregation

Molecules 2018, 23, 226
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increasing monomer formation and thereby increasing fluorescence emission of the dyes. It was
1
−1
determined that heptamethine dye 6a binds HSA with an affinity of 8
×
10 M . This is 5 orders
of magnitude lower than the previously tested trimethine cyanines, MHI-06, which bound on the
6
−1
[28]. This further confirms our previous hypothesis that the binding affinity
order of 1
×
10 M
of the dyes is not only hydrophobicity dependent, but dependent on steric interferences in the HSA
binding site. It also confirms that the delocalized cationic nature of the dyes may has no electrostatic
interference with the binding cavity as this heptamethine cyanine displays increased delocalization
over the previously tested trimethine cyanines. Albumin is known to bind a variety of compounds
including fatty acids, nucleic acids, and oligoproteins therefore this information on the steric specificity
of these binding sites is of potential interest when developing methods to study them [28].
3
. Experimental Section
3
.1. General Information
All chemicals and solvents were of American Chemical Society grade or HPLC purity and were
used as received. HPLC grade ethanol were purchased from Sigma-Aldrich (St. Louis, MO, USA).
All other chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA) or Acros Organics
(
Pittsburgh, PA, USA). The reactions were followed using silica gel 60 F254 thin layer chromatography
1
13
plates (Merck EMD Millipore, Darmstadt, Germany). The H NMR and C NMR spectra were
obtained using high quality Kontes NMR tubes (Kimble Chase, Vineland, NJ, USA) rated to 500 MHz
1
and were recorded on an Avance spectrometer (Bruker, Billerica, MA; 400 MHz for H and 100 MHz for
1
3
C) in DMSO-d , acetone-d CD Cl-d . High-resolution accurate mass spectra (HRMS) were obtained
6
6
3
3
at the Georgia State University Mass Spectrometry Facility using a Q-TOF micro (ESI-Q-TOF) mass
spectrometer (Waters, Milford, MA, USA). All compounds tested were >95% pure.
A solution of POCl (11 mL, 117.66 mmol) in dichloromethane (10 mL) was added dropwise to
3
◦
a solution of DMF (13 mL, 167.89 mmol) in dichloromethane (13 mL) at 0 C for 30 min under inert
conditions. Then 1-phenylcyclohexene (4, 5.5 mL, 32.81 mmol) was dissolved in dry dichloromethane
(
5 mL) and added dropwise to the solution which was then refluxed for 3 h. The solution was allowed
to cool to room temperature and then poured over 500 mL of ice/water. Aniline (9 mL, 98.57 mmol) in
ethanol (9 mL) was added to cap the ends. The crude solid was collected and washed with diethyl ether
and hexanes. Resulting in the dianil linker
In parallel, substituted hydrazines
5 as a pure compound and used without further purification.
1
(4.0 g, 22.25 mmol) were reacted with 3-methylbutanone
◦
(3 mL, 28.04 mmol) in acetic acid and heated to a 100 C for 24 h. The solution was then neutralized
using sodium bicarbonate and extracted using dichloromethane; affording substituted indolenine
heterocycles
2
which was dried under reduced pressure. The heterocycles
2
were then reacted with
were precipitated
◦
an alkyl halide in acetonitrile at 100 C for 12–18 h. The quaternary ammonium salts
with diethyl ether, and collected.
3
The various salts
3
(2 molar eq), the dianil linker
5
(1 molar eq), and sodium acetate (2 molar
◦
eq) were dissolved in acetic anhydride and heated to 60 C for 2–3 h. The crude product was then
precipitated with diethyl ether, collected, and washed with methanol to yield heptamethine dyes
pure sample.
6 as
3
.2. Characterization
0
1
,3,3-Trimethyl-2-((E)-2-((E)-6-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1 -biphenyl]
◦
1
-2-yl)vinyl)-3H-indol-1-ium iodide, 6a: Yield 77%; m.p. > 260 C; H NMR (CDCl )
δ
1.11 (s, 12H),
.95 (m, 2H), 2.68 (t, J = 6.0 Hz, 4H), 3.58 (s, 6H), 6.17 (d, J = 14.0 Hz, 2H), 7.16 (m, 4H), 7.25 (d, J = 8.4 Hz,
H), 7.34 (m, 4H), 7.46 (d, J = 7.6 Hz, 2H), 7.62 (m, 3H). 13C NMR (CDCl3)
21.3, 24.5, 27.3, 31.5, 48.5,
3
1
2
δ
1
00.7, 111.3, 122.7, 124.9, 128.5, 128.8, 129.0, 129.6, 131.0, 139.1, 140.9, 143.3, 147.3, 161.5, 172.1. HRMS
+
(ESI) m/z: calcd. for C H N 525.3264, obsd 525.3241.
38
41
2

Molecules 2018, 23, 226
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0
-Chloro-2-((E)-2-((E)-6-(2-((E)-5-chloro-1,3,3-trimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1 -biphenyl]
5
◦
1
-
2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide, 6b: Yield 78%; m.p. > 260 C; H NMR (CDCl )
δ
1.17
3
(
2
s, 12H), 2.07 (m, 2H), 2.74 (t, J = 6.4 Hz, 4H), 3.65 (s, 6H), 6.12 (d, J = 14.0 Hz, 2H), 7.05 (d, J = 8.4 Hz,
13
H), 7.17 (m, 6H), 7.30 (t, J = 8.4 Hz, 2H), 7.56 (m, 3H); C NMR (CDCl3)
δ
21.1, 25.0, 27.5, 32.5, 48.5,
1
00.8, 111.3, 122.5, 128.2, 128.5, 128.7, 129.4, 130.2, 133.0, 138.7, 141.5, 142.1, 148.2, 163.0, 171.4. HRMS
+
(ESI) m/z: calcd. for C H Cl N 593.2485, obsd 593.2475.
38
39
2
2
0
-Bromo-2-((E)-2-((E)-6-(2-((E)-5-bromo-1,3,3-trimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1 -biphenyl]
5
◦
1
-
2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide, 6c: Yield 75%; m.p. > 260 C; H NMR (CDCl )
δ
1.12
3
(
2
s, 12H), 2.08 (m, 2H), 2.76 (t, J = 6.4 Hz, 4H), 3.66 (s, 6H), 6.15 (d, J = 14.0 Hz, 2H), 6.99 (d, J = 8.4 Hz,
H), 7.15 (d, J = 14.0 Hz, 2H), 7.21 (dd, J = 7.6 Hz, 2H), 7.46 (dd, J = 8.4 Hz, 2H), 7.56 (m, 3H). 13C NMR
(
1
CDCl3)
δ
21.0, 25.0, 27.5, 32.5, 48.4, 100.9, 111.7, 117.7, 125.3, 128.2, 128.5, 129.4, 131.5, 133.2, 138.7,
+
42.0, 142.4, 148.1, 162.9, 171.2. HRMS (ESI) m/z: calcd. for C H Br N 681.1475, obsd 681.1475.
38
39
2
2
5
-Methoxy-2-((E)-2-((E)-6-(2-((E)-5-methoxy-1,3,3-trimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-
0
◦
1
[
1,1 -biphenyl]-2-yl)vinyl)-1,3,3-trimethyl-3H-indol-1-ium iodide, 6d: Yield 73%; m.p. 238–240 C; H NMR
CDCl3) 1.17 (s, 12H), 2.06 (m, 2H), 2.70 (t, J = 6.4 Hz, 4H), 3.61 (s, 6H), 3.82 (s, 6H), 6.03 (d, J = 14.0 Hz,
H), 6.76 (s, 2H), 6.86 (d, J = 8.8 Hz, 2H), 7.04 (d, J = 8.4 Hz, 2H), 7.10 (d, J = 14.0 Hz, 2H), 7.21
21.2, 24.9, 27.5, 32.2, 48.5, 55.9, 99.8, 109.1, 110.9,
(
δ
2
(
d, J = 6.8 Hz, 2H), 7.55 (m, 3H); 13C NMR (CDCl3)
δ
1
12.8, 128.0, 128.4, 129.5, 131.4, 136.5, 139.1, 142.2, 146.8, 157.8, 161.3, 171.0. HRMS (ESI) m/z: calcd. for
+
C H N O 585.3476, obsd 585.3469.
40
75
2
2
1
5
,1,3-Trimethyl-2-((E)-2-((E)-6-((E)-2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)-3,4,
0
◦
1
,6-tetrahydro-[1,1 -biphenyl]-2-yl)vinyl)-1H-benzo[e]indol-3-ium iodide, 6e: Yield 63%; m.p. > 260 C; H
1.50 (s, 12H), 2.11 (m, 2H), 2.79 (t, J = 6.4 Hz, 4H), 3.77 (s, 6H), 6.16 (d, J = 14.0 Hz, 2H),
.30 (m, 2H), 7.44 (m, 4H), 7.55 (t, J = 7.6 Hz, 2H), 7.66 (m, 3H), 7.92 (m, 6H); 13C NMR (CDCl3)
21.2,
NMR (CDCl3)
7
2
δ
δ
5.0, 27.1, 32.5, 50.2, 99.9, 110.5, 121.9, 124.7, 127.5, 128.0, 128.3, 128.6, 129.5, 130.1, 130.5, 131.7, 132.2,
+
1
32.9, 139.0, 140.2, 147.1, 162.0, 173.3. HRMS (ESI) m/z: calcd. for C H N 625.3577, obsd 625.3570.
46
45
2
0
-Ethyl-2-((E)-2-((E)-6-(2-((E)-1-ethyl-3,3-dimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1 -biphenyl]
1
-
◦
1
2-yl)vinyl)-3,3-dimethyl-3H-indol-1-ium iodide, 6f; Yield: 70%; m.p. > 260 C; H NMR (acetone-d )
δ
6
1
7
2
.11 (s, 12H), 1.24 (t, J = 7.2 Hz, 6H), 1.96 (m, 2H), 2.97 (t, 4H), 4.14 ( m, 4H) 6.20 (d, J = 14 Hz, 2H),
.18 (m, 4H), 7.35 (m, 4H), 7.47 (d, J = 7.6 Hz, 2H), 7.61 (m, 3H); 13C NMR (acetone-d6)
δ
12.5, 21.3,
4.6, 27.4, 39.8, 40.0, 40.2, 48.7, 100.2, 111.2, 123.0, 125.0, 128.6, 129.0, 129.1, 129.6, 131.1, 141.1, 142.1,
+
1
47.7, 171.2. HRMS (ESI) m/z: calcd. for C H N 553.3577, obsd 553.1566.
40
45
2
5
-Chloro-2-((E)-2-((E)-6-(2-((E)-5-chloro-1-ethyl-3,3-dimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-
0
◦
1
[
1,1 -biphenyl]-2-yl)vinyl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium iodide, 6g; Yield 64%; m.p. > 260 C; H
NMR (CDCl3) 1.16 (s, 12H), 1.38 (t, J = 7.2 Hz, 6H), 2.07 (t, J = 5.6, 2H), 2.73 (t, J = 6.0 Hz, 4H),
.142 (m, 4H), 6.10 (d, J = 14.0 Hz, 2H), 7.07 (d, J = 8.4 Hz, 2H), 7.28 (m, 8H) 7.59 (m, 3H); 13C NMR
δ
4
(
1
CDCl3)
δ
12.2, 21.1, 25.0, 27.5, 40.7, 48.6, 100.1, 111.4, 122.6, 128.3, 128.7, 128.8, 129.4, 130.3, 132.6, 138.7,
+
40.5, 142.4, 148.3, 163.1, 170.5. HRMS (ESI) m/z: calcd. for C H Cl N 621.2798, obsd 621.2788.
40
43
2
2
5
-Bromo-2-((E)-2-((E)-6-(2-((E)-5-chloro-1-ethyl-3,3-dimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-
0
◦
1
[1,1 -biphenyl]-2-yl)vinyl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium iodide, 6h; Yield 71%; m.p. > 260 C; H
NMR (CDCl3) 1.18 (s, 12H), 1.41 (t, J = 7.2 Hz, 6H), 2.10 (t, J = 6.4 Hz, 2H), 2.75 (t, 5.6 Hz, 4H),
δ
1
3
4
2
1
.16 (m, 4H), 6.13 (d, J = 14.4 Hz, 2H), 7.22 (m, 10H), 7.59 (m, 3H); C NMR (CDCl3) δ 12.2, 21.1, 25.1,
7.5, 40.1, 48.6, 100.3, 111.7, 117.7, 125.5, 128.3, 128.6, 129.4, 131.6, 133.0, 138.8, 141.0, 142.7, 148.2, 162.9,
70.3. HRMS (ESI) m/z: calcd. for C H Br N 709.1788, obsd 709.1780.
+
40
43
2
2

Molecules 2018, 23, 226
9 of 12
1
[
-Ethyl-2-((E)-2-((E)-6-(2-((E)-1-ethyl-5-methoxy-3,3-dimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-
0 ◦
1,1 -biphenyl]-2-yl)vinyl)-5-methoxy-3,3-dimethyl-3H-indol-1-iumiodide, 6i; Yield 61%; m.p. > 260 C;
1
H NMR (acetone-d6)
δ
1.22 (s; 12H), 1.34 (t, J = 7.2 Hz, 6H), 2.02 (m, 2H), 2.73 (t, J = 6.0 Hz, 4H),
.84 (s, 6H), 4.21 (m, 4H), 6.23 (d, J = 14 Hz, 2H), 6.23 (d, J = 2.8 Hz, 2H) 6.94 (d, J = 2.4 Hz 2H)
.22 (s, 2H) 7.28 (m, 6H) 7.66 (m, 3H); 13C NMR (acetone-d6)
11.6, 21.3 24.5, 26.8, 39.0, 48.8, 55.4, 99.1,
3
7
δ
1
09.0, 111.2, 113.4, 128.0, 128.7, 129.5, 130.3, 135.5, 139.4, 142.8, 145.0, 158.1, 161.0, 170.6. HRMS (ESI)
+
m/z: calcd. for C H N O 613.3789, obsd 613.3777.
42
49
2
2
3
-Ethyl-2-((E)-2-((E)-6-((E)-2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)-3,4,5,6
0
-tetrahydro-[1,1 -biphenyl]-2-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium iodide, 6j
;
Yield 65%;
◦
1
m.p. > 260 C; H NMR (CDCl )
2
δ 1.46 (t, J = 6.8 Hz, 6H), 1.51 (s, 12H), 2.13 (t, J = 5.6 Hz, 2H),
3
13
.79 (t, J = 6.0 Hz, 4H), 4.28 (m, 4H), 6.17 (d, J = 14.0 Hz, 2H), 7.45 (m, 13H), 7.94 (m, 6H); C NMR
(
1
CDCl3)
δ
99.3, 110.4, 122.0, 124.8, 127.6, 128.1, 128.3, 128.7, 129.5, 130.1, 130.7, 131.7, 132.0, 133.3, 139.1,
+
39.3, 147.3, 162.0, 172.4. HRMS (ESI) m/z: calcd. for C H Cl N 593.2485, obsd 593.2475.
48
49
2
2
0
,3,3-Trimethyl-2-((E)-2-((E)-6-(2-((E)-1,3,3-trimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1 -biphenyl]
1
-
◦
2-yl)vinyl)-3H-indol-1-ium iodide, 6k; yield 68%; m.p. > 260 C; H-NMR (CDCl ) δ 1.04 (t, J = 7.2 Hz,
3
1
6H), 1.23 (s, 12H), 1.49 (m, 4H), 1.80 (m, 4H), 2.11 (t, J = 6.0 Hz, 2H), 2.745 (s, 4H), 4.04 (t, J = 7.2 Hz,
4
H), 6.10 (d, J = 14 Hz, 2H), 7.07 (d, J = 11.6 Hz, 2H), 7.22 (m, 6H), 7.33 (t, J = 7.2 Hz, 2H), 7.61 (m, 3H);
13
C NMR (CDCl ) 14.0, 20.5, 21.3, 25.0, 27.7, 29.4, 110.4, 122.1, 124.8, 128.2, 128.6, 129.6, 140.8, 142.3.
3
+
HRMS (ESI) m/z: calcd. for C H N 609.4203, obsd 609.4198.
44
53
2
1
-Butyl-2-((E)-2-((E)-6-(2-((E)-1-butyl-5-chloro-3,3-dimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetra-hydro-
0
1
[
(
2
1,1 -biphenyl]-2-yl)vinyl)-5-chloro-3,3-dimethyl-3H-indol-1-ium iodide, 6l: Yield 67%; m.p. > 260; H NMR
CDCl3) 1.03 (t, J = 7.2 Hz, 6H), 1.191 (s, 12H), 1.47 (m, 4H), 1.78 (t, J = 6.8 Hz, 4H), 2.12 (s, 2H),
.75 (s, 4H), 4.02 (s, 4H), 6.09 (d, J = 12.4 Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H), 7.28 (m, 8H), 7.86 (m, 3H); 13
14.0, 20.4, 21.3, 25.0, 27.7, 29.3, 44.7, 48.6, 100.4, 111.3, 122.7, 128.3, 128.6, 129.6, 130.2,
δ
C
NMR (CDCl3)
δ
+
1
38.7, 141.0, 142.4, 148.5, 171.0. HRMS (ESI) m/z: calcd. for C H Cl N 677.3424, obsd 677.3421.
44 51 2 2
0
-Butyl-2-((E)-2-((E)-6-(2-((E)-1-butyl-3,3-dimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro-[1,1 -biphenyl]
1
-
◦
1
2-yl)vinyl)-3,3-dimethyl-3H-indol-1-ium iodide, 6m: Yield 65%; m.p. > 260 C; H NMR (CDCl ),
δ
3
1
.04 (t, J = 7.2 Hz, 6H), 1.20 (s, 12H), 1.51 (m, 4H), 1.81 (m, 4H), 2.13 (t, J = 5.2 Hz, 2H), 2.75 (s, 4H),
4
.04 (t, J =7.2 Hz, 4H), 6.10 (d, J = 14.0 Hz, 2H), 7.07 (d, J = 8 Hz, 2H), 7.20 (m, 8H), 7.61 (m, 3H);
13
C NMR (CDCl3)
δ
14.0, 20.5, 21.3, 25.0, 27.7, 29.4, 44.4, 48.6, 100.0, 110.3, 122.1, 124.7, 128.2, 128.6,
+
129.6, 132.1, 138.8, 140.8, 142.3, 148.4, 162.9, 171.4. HRMS (ESI) m/z: calcd. for C H Br N 765.2414,
44
51
2
2
obsd 765.2422.
1
-Butyl-2-((E)-2-((E)-6-(2-((E)-1-butyl-5-methoxy-3,3-dimethylindolin-2-ylidene)ethylidene)-3,4,5,6-tetrahydro
0
◦
-
[1,1 -biphenyl]-2-yl)vinyl)-5-methoxy-3,3-dimethyl-3H-indol-1-ium iodide, 6n: Yield 66%; m.p. > 260 C;
H NMR (CDCl3)
1
δ
1.01 (t, J = 6.8 Hz, 6H), 1.17 (s, 12H), 1.46 (m, 4H), 1.80 (m, 4H), 2.07 (s, 2H),
.68 (s, 4H), 3.82 (s, 6H), 4.03 (t, J = 7.2 Hz, 4H), 6.00 (d, J = 14.0 Hz, 2H) 6.78 (s, 2H), 6.88 (d, J = 8.4 Hz,
H), 7.03 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 14.0 Hz, 2H), 7.21 (d, J = 6.8 Hz, 2H), 7.57 (m, 3H); 13C NMR
2
2
(
1
CDCl3)
δ
13.9, 20.4, 21.3, 24.8, 27.7, 29.4, 44.6, 48.7, 56.0, 99.4, 109.1, 111.1, 113.1, 128.1, 128.6, 129.4,
+
30.9, 135.9, 139.0, 142.4, 146.9, 157.9, 161.4, 170.6. HRMS (ESI) m/z: calcd. for C H N O 669.4415,
46
57
2
2
obsd 669.4404.
3
6
-Butyl-2-((E)-2-((E)-6-((E)-2-(3-butyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)-3,4,5,
0
-tetrahydro-[1,1 -biphenyl]-2-yl)vinyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium iodide, 6o
:
Yield 70%;
1.03 (t, J = 7.2 Hz, 6H), 1.52 (m, 16H), 1.86 (m, 4H), 2.16 (t, J = 6.0 Hz,
H), 2.79 (s, 4H), 4.17 (s, 4H), 6.15 (d, J = 14.4 Hz, 2H), 7.34 (m, 6H), 7.45 (d, J = 7.6 Hz, 2H), 7.53 (m,
H), 7.68 (t, J = 2.8 Hz, 3H), 7.93 (m, 6H); 13C NMR (CDCl3)
14.0, 20.4, 21.4, 25.0, 27.2, 29.7, 44.6, 50.4,
◦
1
m.p. >260 C; H NMR (CDCl )
2
2
δ
3
δ

Molecules 2018, 23, 226
10 of 12
9
9.6, 110.6, 122.0, 124.8, 127.6, 128.1, 128.4, 128.7, 129.6, 130.1, 130.6, 131.7, 133.2, 139.0, 139.7, 147.3,
+
1
72.8. HRMS (ESI) m/z: calcd. for C H N 709.4516, obsd 709.4540.
52 57 2
3
.3. Stock Solutions
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.
3
.4. Method of Determining Molar Absorptivity and Fluorescence Quantum Yield
Stock solutions were used to prepare six dilutions of dyes in ethanol and the standard 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
4
00 nm to 900 nm. The emission spectrum of each sample was measured in triplicate with a 750 nm
excitation wavelength.
For molar absorptivity, the absorbance at the wavelength of maximum absorbance (λmax) was
determined and the absorbance of each sample at λmax was plotted as a function of dye concentration.
The linear regression equation was computed using Microsoft Excel.
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 (φ , 13.2% [29]) as per Equation (1):
S
2
2
φ = φ * Grad /Grad ∗ η /η
(1)
D
S
D
S
S
D
3
.5. HSA Binding Study
−
5
−5
A stock solution of 6a (4
×
10 M) and HSA (4
×
10 M; Sigma Aldrich, St. Louis, MO, USA)
M) were made by
L dye solution with PBS buffer solution with and without HSA to a total volume of 4000
in a fluorescence cuvette to make working solutions of 2 M dye. Fluorescence spectra were measured
in duplicate with excitation at 740 nm and slit widths of 5 nm for both excitation and emission.
were prepared in PBS buffer. Fluorescence titration with HSA concentrations (0–2
mixing 35
µ
µ
µL
µ
4
. Conclusions
A series of 15 phenyl-substituted heptamethine cyanines was synthesized in good yields and
1
13
characterized by H and C NMR. Their optical properties including molar absorptivity, fluorescence,
Stokes shift, and quantum yield were measured. The optical properties followed similar trends
to previously published cyanine dye MHI-06. The binding affinity of one of these heptamethine
cyanine dyes to HSA was measured to be 5 orders of magnitude lower than our previous synthesized
trimethine cyanines further confirming the hypothesis that the binding affinity of the dyes is not
only hydrophobicity dependent, but dependent on steric interferences in the binding site [28].
Because albumin is known to bind a variety of compounds, this information on the steric specificity of
these binding sites is of potential interest when developing methods to study them.
Supplementary Materials: Supplementary materials are available online.
Acknowledgments: M.H., A.L. and F.M. would like to thank the Department of Chemistry at Georgia State
University for their support and the funds provided for the Ph.D. program for A.L. and Master’s program for
F.M. M.H., A.L. and F.M. would like to thank Ariana Laskey for her contribution in the synthesis of some the
compounds. M.H. wishes to thank the NIH/NIBI R01EB022230, the Brains and Behavior Seed Grant, the Atlanta
Clinical & Translational Science Institute for the Healthcare Innovation Program Grant, and the Georgia Research
Alliance for the Ventures Phase 1 Grant.

Molecules 2018, 23, 226
11 of 12
Author Contributions: M.H. designed the research and all authors wrote the paper. F.M. and A.L. performed
experiments. All authors discussed the results and commented on the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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