A microwave-assisted and environmentally benign approach to the synthesis of near-infrared fluorescent pentamethine cyanine dyes

Article abstract of DOI:10.1016/j.dyepig.2014.07.035

A time-efficient and eco-conscious microwave methodology was developed and applied to synthesize a systematic library of pentamethine cyanine dyes and their corresponding precursors. The synthesis outlined herein drastically reduced the reaction pathway for pentamethine carbocyanine dye syntheses from days to min, as well as producing increased yields (89-98%) to the conventional heating method (18-64%). Twelve examples of pentamethine cyanine dyes were synthesized by means of microwave-assisted organic synthesis which provided excellent yield in expedited reaction time and were obtained using facile isolation methods. Furthermore, three cyanines were prepared with a novel methylene dioxy heterocyclic structure which imparted an approximately 40 nm bathochromic shift compared to unsubstituted counterparts; these results were shown to be in agreement with DFT calculations and HOMO-LUMO energy differences.

Full text of DOI:10.1016/j.dyepig.2014.07.035

Dyes and Pigments 113 (2015) 27e37
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
A microwave-assisted and environmentally benign approach to the
synthesis of near-infrared fluorescent pentamethine cyanine dyes
,
b
,
, b, c, *
Eric A. Owens a c, Nicholas Bruschi a, Joseph G. Tawney a, Maged Henary a
a Department of Chemistry, Georgia State University, 100 Piedmont Road SE, Atlanta, GA 30303, USA
b The Center for Diagnostics and Therapeutics, Georgia State University, 100 Piedmont Road SE, Atlanta, GA 30303, USA
c The Center for Biotechnology and Drug Design, Georgia State University, 100 Piedmont Road SE, Atlanta, GA 30303, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A time-efficient and eco-conscious microwave methodology was developed and applied to synthesize a
systematic library of pentamethine cyanine dyes and their corresponding precursors. The synthesis
outlined herein drastically reduced the reaction pathway for pentamethine carbocyanine dye syntheses
from days to min, as well as producing increased yields (89e98%) to the conventional heating method
(18e64%). Twelve examples of pentamethine cyanine dyes were synthesized by means of microwave-
assisted organic synthesis which provided excellent yield in expedited reaction time and were ob-
tained using facile isolation methods. Furthermore, three cyanines were prepared with a novel methy-
lene dioxy heterocyclic structure which imparted an approximately 40 nm bathochromic shift compared
to unsubstituted counterparts; these results were shown to be in agreement with DFT calculations and
HOMO-LUMO energy differences.
Received 20 May 2014
Received in revised form
23 June 2014
Accepted 28 July 2014
Available online 7 August 2014
Keywords:
Pentamethine cyanine dye
Microwave assisted organic synthesis
(MAOS)
© 2014 Elsevier Ltd. All rights reserved.
Aqueous reaction methodology
Dioxolane
Fluorescence
Density functional theory
1. Introduction
Near-infrared (NIR, 640e900 nm) fluorescent chromophores
have garnered considerable research interest for biomolecular la-
Mother Nature provides our atmosphere to breathe, food
required to eat and beautiful scenery to enjoy. Protecting and
nurturing our environment should be a task shared by all in the
research community. There are many methods for helping to pre-
serve our precious natural resources which include the following:
(1) using green solvents, (2) reducing heating times to conserve
energy, (3) increasing atom efficiency by using higher yielding re-
action conditions and (4) eliminating wasteful purification steps by
using optimized methods that reduce side-products. These princi-
ples have been applied to the synthesis of many classes of com-
pounds that are interesting to a broad population of the scientific
beling because of their unique red-shifted optical properties [1e-
4]. The majority of fluorescent sensors have shown to emit light
in the visible region (400e600 nm), which forces unwanted
competition with background noise that arises from inherent bio-
molecular auto-fluorescence. This competition disrupts the mean-
ingful signal and can lead to extreme difficulty in signal delineation
often with undesirable results [5]. To avoid the problems associated
with visible-light-emitting fluorophores, molecules which absorb
and emit light in the near-infrared region have been of significant
interest to the scientific community. [1e3,5e7]
Specifically among the NIR emitting dyes, immense interest has
been placed in the particular class of chromophores known as
cyanine dyes, and they have shown extensive applications in cancer
imaging, nucleic acid detection, biomolecular labeling, photo-
graphic processes, information storage and dye lasers [7e10].
Possessing relatively high molar absorption coefficients and a broad
range of tunable fluorescence wavelengths (600e900 nm) cyanine
dyes have been synthesized to emit light in the NIR range while
maintaining biological efficacy [4,5,8e11]. Chiefly among these
fluorophores, pentamethine cyanine dyes have shown significant
promise for image guided surgery using NIR light [1]. Specifically,
we reported several pentamethine cyanine dyes that have been
community. Correspondingly, we have chosen to develop
a
completely benign synthetic route for the synthesis of near-
infrared fluorescent compounds that have shown excellent prom-
ise in biological imaging, solar-cell technology, as chemodosimeters
in sensing biologically relevant species and for non-covalent la-
beling of biomolecules.
* Corresponding author. Department of Chemistry, Georgia State University, 100
Piedmont Road SE, Atlanta, GA 30303, USA. Tel.: þ1 404 413 5566 (office).
E-mail address: mhenary1@gsu.edu (M. Henary).
http://dx.doi.org/10.1016/j.dyepig.2014.07.035
0143-7208/© 2014 Elsevier Ltd. All rights reserved.

28
E.A. Owens et al. / Dyes and Pigments 113 (2015) 27e37
shown to specifically locate various tissues of clinical importance
during image-guided surgery [1]. Expanding upon the synthesis of
pentamethine cyanine dyes as biomolecular imaging agents has
been a focus of many bioorganic research labs worldwide [12,13].
The design and synthesis of various substituted pentamethine
cyanines has been achieved in a combinatorial manner by changing
the N-alkyl substituent or functionalizing different positions of the
heterocyclic backbone [2,4]. It has been observed that minor
structural alterations elicits a drastic biological response which
makes synthesis of highly varied chemical structures very impor-
tant [1]. This provides a significant rationale for developing a fast,
facile and effective method for generating a library of pentamethine
cyanines with high purity levels.
matrix [9]. The pentamethine class of cyanine dyes has not been
optimized using MAOS. Utilizing a green approach, an entire syn-
thetic pathway has been designed to prepare pentamethine cya-
nines with diverse chemical structures. Specifically, in the final
synthetic step, using the CEM Discover LabMate several different
substituted pentamethine cyanine dyes have been synthesized
within 20 min in analytical purity without column purification. In
comparison to the conventional heating method, the syntheses
described herein provide drastically decreased reaction times and
comparable or increased yields (89e98% in the final step, 80e91%
overall).
2. Results and discussion
Synthesis of various pentamethine cyanine dyes with slightly
altered connectivity has been shown to take hours to days by the
conventional oil bath heating method with lengthy, difficult col-
umn chromatography that generates environmentally polluting
solvent waste [2,6,14]. Cyanine dyes are sensitive to the acidic na-
ture of silica gel, photodegrade during purification steps and cleave
in the basic solutions required for their synthesis (Fig. 1); therefore,
expediting the reaction time and increasing purity levels is of
highly important. Recently, the implementations of microwave
chemistry has helped speed reactions and decrease the environ-
mentally harmful waste common in organic labs; it has received
interest throughout the scientific community and it is very desir-
able to conduct synthetic protocol under microwave irradiation.
In order to implement this technology in cyanine synthesis, we
have harnessed the ability of our reaction mixture to absorb mi-
crowave energy. Electromagnetic irradiation of molecules results
in rapid, volumetric heating caused by the dielectric effect which
yields the final compounds without the need for column purifi-
cation that may jeopardize the chemical integrity of the com-
pounds from photodegradation or decomposition on the silica
2.1. Eco-friendly synthesis of pentamethine cyanine fluorophores
The common synthetic method for the preparation of pen-
tamethine cyanines begins with the formation of the terminal
heterocyclic moieties. The four heterocycles used to afford the
compounds were synthesized according to Scheme 1 and Equation
(2). We optimized each step for the synthesis beginning with
heterocyclic formation and begins when substituted phenyl-
hydrazines are refluxed in glacial acetic acid with 3-methyl-2-
butanone to afford 1 and 2. This reaction affords the desired
indolenine compounds in good yield, but this method requires
extended periods of reaction time exceeding 24 h and excess acetic
acid as solvent. Our green microwave method employs water as an
environmentally conscious solvent and a catalytic (0.1 mol eq.)
amount of sulfuric acid to achieve quantitative conversion in
10 min. The second synthetic step is the quaternization of the
indolenine nitrogen atom. A classical method for this synthesis is
mixing the two reagents in acetonitrile and refluxing until the
reaction goes to completion. The volatile nature of short chain
Fig. 1. The reaction intermediates and postulated degradation products that form during either the classical heating method of synthesis or purification of cyanine dyes that lead to
difficulty in obtaining analytically pure compounds for biological testing.

E.A. Owens et al. / Dyes and Pigments 113 (2015) 27e37
29
Scheme 1. The new, green microwave assisted method for preparation of alkylated salts 3e11 based off of the indolenine heterocyclic structure.
alkyl halides limits the reaction scope and many molar equivalents
should be used to completely react with the heterocyclic starting
materials. This leads to low atom efficiency and the use of an un-
necessarily high quantity of alkylating agent. Our optimized
alkylation is effective at a 1.2 molar equivalency of alkyl iodide
(Me, iodomethane, Et, iodoethane, Pr, iodopropane) without the
use of harmful solvents at a drastically reduced reaction time. After
20 min, the corresponding quaternary salts precipitate in >98%
yield as a fine power and can be utilized in the next reaction
without purification procedures.
Achieving new heterocyclic building blocks for the synthesis of
cyanine dyes has been limited by the number of commercially avail-
able hydrazine hydrochlorides. The synthesis of hydrazines with a
Equation 2. The preparation of the fused dioxolane heterocycle 13.3
number of interesting substitutions has remained scarce throughout
€
the literature; however, utilizing
a
modified BischlereMohlau
In order to develop a green-method for the formation of this
dioxolane, we began by examining the mechanism of this reaction.
From a mechanistic point-of-view, this reaction is very interesting
(Scheme 2). In basic conditions, the anilino-amine is highly
nucleophilic substituting the tertiary bromine alpha to the
carbonyl of 12 through an S1N pathway. Monoalkylation of the
arylamine is successful due to high steric hindrance from the
dimethyl groups. Treatment of this intermediate with para-tolue-
nesulfonic acid in the presence of excess catalytic aniline yields the
dehydrated arylimine intermediate that undergoes loss of aniline
through electrophilic aromatic substitution and re-aromatization
yields the heterocyclic precursor 13. We postulated that this may
work satisfactorily using eco-friendly conditions through micro-
wave irradiation and successfully replaced the organic solvents
with the ionic liquid [BMIM][PF6] and we maintained the use of
potassium carbonate as the base. The first part of the reaction was
completed using microwave irradiation (150 W) at 150 ꢀC followed
by treatment with para-toluene sulphonic acid as the catalytic
activation of the carbonyl and cyclization of the ring. The alkylation
of 13 was performed using the methodology previously employed
in Scheme 1 and the salts 14e16 were obtained in moderate yield
with classical reflux methods and excellent yield using MAOS
(Equation (1)).
approach, we can achieve the synthesis of an acidic methylene proton
with the quaternized nitrogen through a facile microwave-assisted
approach (accessing the scope of this reaction using additional het-
erocycles are explored in another paper, Owens et al. manuscript in
preparation). Our model compound was the dioxolane structure.
The initial precursor to the dioxolane heterocyclic unit, 3-
bromo-3-methylbutan-2-one (shown in Equation (1)), was classi-
cally prepared beginning with bromination of 3-methyl-2-
butanone under acidic conditions at 0 ꢀC. The high volatility and
dangerous nature of working with molecular bromine requires the
use of an alternate and environmentally friendly route. Applying
the the bromine “on water” method reported by the Iskra lab [15],
we achieved selective and high yielding bromination of 3-methy-2-
butanone to afford 12.
2.2. The final synthetic StepdOptimization
In the final synthetic step, shown in Scheme 3, two equivalents
of individual monocationic salt were allowed to react with malo-
naldehyde bis(phenylimine)monohydrochloride in the presence of
sodium acetate and acetic anhydride open vessel classical oil-
heating causes a wide variety of decomposition and incomplete
reaction products and results in a substantial net loss of product,
yielding between 18% and 64%. In order to optimize the
Equation 1. The preparation the brominated ketone 12.2
The cyclic product 13 is formed through treatment of the
commercially available 3,4-(methylenedioxy)aniline with inter-
mediate 12 in DMF with potassium carbonate followed by addition
of para-toluenesulfonic acid in toluene at 125 ꢀC (Equation (2)).

30
E.A. Owens et al. / Dyes and Pigments 113 (2015) 27e37
€
Scheme 2. The proposed mechanism based off of an adaptation of the BischlereMohlau method for indole formation.
microwave-assisted reaction temperature, we first anticipated that
the optimum reaction temperatures would be most heavily
dependent on the length of the N-indolenyl substituent. We per-
formed the synthesis of a representative fluorophore with various
alkyl lengths on the indolenyl nitrogen to obtain the correct
temperature to employ for all of the pentamethine cyanines
Equation (3).
As the alkyl length increases, the insulation properties of the
compound also increase. In order to determine the optimal tem-
perature, the systematic set of dyes 17e19 were tested at a range of
temperatures. The isolated yield for each of the reactions are shown
as a function of temperature in Fig. 2 and shows a correlation as the
alkyl group becomes longer, the most desirable reaction tempera-
ture also increases.
The medium length N-substituted ethyl dye 18 was chosen as a
model for discussing optimal reaction temperatures. As shown in
Table 1, multiple trials were conducted varying temperature by
10 ꢀC increments starting with 90 ꢀC while alternating reaction
time for 10 and 20 min. Conducting the reaction at temperatures
between 50 ꢀC and 90 ꢀC showed comparatively low yields. The
optimum temperature was observed as a maximum and heating
above this temperature resulted in undesirable byproducts and
decomposition of the product, which decreased yield shown in
Fig. 2 this method was applied to the synthesis of compounds 17
and 19. Methyl substituted dye, 17 was shown to reach a maximum
yield at 80 ꢀC, ethyl 18 at 130 ꢀC and propyl 19 at 140 ꢀC. Continuing
Equation 3. The preparation of the alkylated fused dioxolane heterocycles 14e16.4
Scheme 3. The microwave assisted preparation of a systematic set of pentamethine cyanines with variance in heterocyclic and N-alkyl groups.

E.A. Owens et al. / Dyes and Pigments 113 (2015) 27e37
31
2.3. Microwave assisted organic synthesis application to a
systematic library of pentamethine cyanines
After optimizing the reaction conditions for each of the three
sets of pentamethine cyanines (Me, Et and Pr), we subjected the
other structures with heterocyclic variation to the same reaction
conditions and isolated the compounds using precipitation and
facile recrystallization techniques. Notably, the reaction time,
shown in Table 2, is drastically reduced in some cases more than 13-
fold. Additionally, the purified yield increased, especially for com-
pound 23 which showed an increase from 32% to 98%; corre-
spondingly, compound 28, which was difficult to purify and obtain
an 18% overall yield using classical methods, precipitated as a
nearly analytically pure solid after initial workup in 89% yield. The
conventional synthetic method affords numerous byproducts
which are not efficiently removed from the desired compound;
however, the microwave methods developed herein have avoided
the undesirable side reactions which have led to much reduced
solvent waste with substantially less time and hazardous solvent
waste spent on purification.
Fig. 2. Isolated yield (%) versus reaction temperature (ꢀC) versus the three pentam-
ethine carbocyanine dyes with varied lengths of the N-alkyl groups 17 (methyl, blue),
18 (ethyl, red), and 19 (propyl, green). Reactions were held at 20 min for the isolated
yields appearing in this table. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
the reaction time past 20 mine25 min and 30 min also resulted in
reduced percentage isolated yield.
2.4. Heterocyclic variation alters the optical properties of the
We suspected that the increasing N-alkyl chain length was
corresponded to a decrease in overall dipole moment of the
corresponding indolinium salt, causing a reduced microwave
energy absorption which required an increase in thermal energy
to attain a comparable reaction yield [9]. We became intrigued
with this observation and performed DFT calculations at the BLYP
6-31G* level beginning with the conformer of the lowest energy.
The electrostatic maps qualitatively show less polarization and
the decrease in dipole moment corresponds directly to the
decrease in microwave energy absorbed by the compound. In
order for the molecule to react in the microwave, a higher
amount of energy is required. The salt containing a methyl group
has a dipole moment of 3.39 debye which is more than double
the dipole moment of the salt with a propyl substituent which
was calculated to be only 1.69 debye (Fig. 3). This is in agreement
with the thermal energy required to form the final product with
methyl and propyl; the methyl salt required only 80 ꢀC to form
the final compound in optimized yield whereas the propyl
compound was optimized at 140 ꢀC.
cyanine fluorophores
The effect of changing the N-alkyl chain length of cyanine dyes
has been shown by our laboratory to minimally alter optical
properties [2]; therefore, we will not concentrate on re-evaluating
this phenomenon. However, while keeping the alkyl groups con-
stant, differences in absorbance, emission, molar absorptivity
(extinction coefficient) and relative quantum yield were observed
for dyes containing heterocyclic variations, including chlorine,
bromine and methylene dioxy groups (Table 3). The effect of
chlorine 20 and bromine 23 was almost negligible in methanol
along with except for an increased Stoke's Shift from 22 nm to 32/
34 nm (Table 3). The addition of the methylene dioxy group 26 was
shown to red shift the dye 35 nm (Fig. 4), where the Stokes' Shift
was observed to increase to 43 nm. Extinction coefficients for all
dyes were shown to be favorably large, (85,100 to
222,800 Mꢁ1 cmꢁ1) while quantum yields ranged from 30.5 to 9.5%
(Table 3). The quantum yield of the dioxy compound 26 is lower due
to the possibility for non-radiative energy decay associated with
the non-aromatic substituents on the heterocyclic ring.
In order to confirm the observed red-shift of the methylene-
dioxy substituted compounds, and knowing the N-alkyl substitu-
tion does not significantly alter the optical properties of the chro-
mophore, we performed density functional theory calculations on
the synthesized compounds 17 and 26 to look at the HOMO and
LUMO levels to determine energetic differences between the
compounds. The calculations were performed at the BLYP 6-31G*
level starting from the most energetically stable state. The DFT
calculations reveal a larger energy gap for compound 17 (Egap
(eV) ¼ 1.41 eV) predicting a blue-shifted absorption spectrum
compared to the small energy barrier for compound 26 (Egap
(eV) ¼ 1.11 eV) which displays a 41 nm bathochromic shift. These
results corroborate the observed bathochromic shift of compound
26 and we can use similar computational methods for predicting a
red or blue shift before synthesizing additional compounds (Fig. 5).
Table 1
Optimization data for pentamethine carbocyanine dye 18 with an ethyl group.
Trial
Temp (ꢀC)
Reaction time (min)
Yield (%)
1
2
3
4
5
6
7
8
50
50
90
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
25
30
25
31
32
36
38
38
40
47
73
81
89
95
76
72
70
68
35
25
91
84
90
100
100
110
110
120
120
130
130
140
140
150
150
170
170
130
130
9
10
11
12
13
14
15
16
17
18
19
20
3. Conclusions
The complete synthetic route for various substituted pentam-
ethine carbocyanine dyes was completed using microwave-based
green eco-conscious methods including the preparation of a novel
methylene dioxy heterocyclic structure. Optimal reaction condi-
tions were found at a constant reaction time of 20 min for all dyes

32
E.A. Owens et al. / Dyes and Pigments 113 (2015) 27e37
Fig. 3. DFT calculation results showing a decrease in the dipole moment as alkyl length increases.
Table 2
resulting in the red-shifted optical profile. As NIR fluorophores
continue to garner increasing interest in the scientific community,
the optimized synthetic protocol will help to increase efficiency
while simultaneously reducing the environmental impact.
Microwave synthesis versus conventional synthesis.
Dye
structure
M.W.
time
(min)
Conventional
time (min)
Microwave
yielda
Conventional
yielda
17
18
19
20
20
20
20
20
20
20
20
20
20
20
20
20
120
180
220
190
180
250
240
270
340
480
480
480
98%
95%
96%
95%
96%
94%
98%
98%
96%
90%
92%
89%
64%
62%
61%
36%
58%
64%
32%
60%
55%
27%
21%
18%
4. Experimental
4.1. Synthesis
21
22b
23
The chemical reagents used in the synthesis of these compounds
were obtained from Acros Organics (Geel, Belgium), Alfa Aesar and
Matrix Scientific. Microwave irradiation was completed using a
Discover LabMate apparatus interfaced to a PC using the provided
Synergy software to monitor reaction temperature, pressure,
wattage and simultaneous cooling. Scale-up reactions of indolenine
salts were performed using the open-vessel accessories and the
24
25b
26b
27b
28b
a
The percentage value refers to the purified yield.
These compounds are novel.
b
with increasing alkyl lengths resulting in increased optimal reaction
temperatures. The benefits associated with our methodology
include decreased reaction time with significantly improved yield
and outstanding purity (based on 1H NMR spectra) without the need
for lengthy purification methods. Additionally, the red-shifted
compounds dioxymethylene compounds were shown, through
DFT calculations, to have small HOMO-LUMO energy differences
Table 3
The optical properties of propyl substituted dyes 17, 20, 23 and 26 with respect to
different substituents on the heterocyclic backbone. The data presented was ob-
tained in methanol from 1 mM stock solutions made in DMSO.
Dye
labs (nm)
lem(nm)
ε(Mꢁ1 cmꢁ1
)
F (%)
17
20
23
26
644
640
640
679
666
674
672
722
212, 500
222, 800
210, 100
85, 100
30.5
29.4
16.9
9.5
Fig. 4. Near infrared (NIR) absorbance of fluorohores in methanol at
concentration.
a 2.0 mM

E.A. Owens et al. / Dyes and Pigments 113 (2015) 27e37
33
Fig. 5. In silico DFT calculations depicting the HOMO and LUMO for each compound 17 and 26 with corresponding energy levels (eV) shown below the structures.
same protocol as detailed in the experimental with similar yield. All
classical method reactions were kept under a positive pressure of
nitrogen during the entirety of the reaction. Microwave-assisted
reaction vessels were purged with nitrogen before the reaction
was initially subjected to irradiation. The reactions were followed
using silica gel 60 F254 thin layer chromatography plates (Merck
EMD Millipore, Darmstadt, Germany) with 5% methanol in DCM as
the mobile phase. Open column chromatography was utilized for
the purification of the final compounds needing additional purifi-
cation using 60e200 u, 60 A classic column silica gel (Dynamic
Adsorbents, Norcross, GA). The 1H NMR and 13C NMR spectra were
obtained using high quality Kontes NMR tubes (Kimble Chase,
Vineland, NJ) rated to 500 MHz and were recorded on a 400 MHz
organic layer was extracted, dried over magnesium sulfate and
concentrated to afford the final compounds in 76% and 81% yield,
respectively.
4.3. Microwave synthesis of indolenine compounds 1 and 2
Heterocyclic phenylhydrazine hydrochloride (1 g) was added
to a 10-mL microwave vessel equipped with a micro magnetic
stir bar along with 3-methyl-2-butanone (1.2 mol eq.). Deionized
water (3 mL) and sulfuric acid (0.1 mol eq.) was added to the
reaction mixture. The reaction vessel was securely capped and
then subjected to microwave irradiation for 10 min at 100 ꢀC
allowing for the complete transformation of the starting mate-
rial. The water was decanted off and a saturated solution of so-
dium bicarbonate (3 mL) was added. The flask was sonicated for
10 min to quench the reactive acid. The liquid was poured off and
the resulting oil was dissolved in minimum dichloromethane
dried over sodium sulfate for 30 min, gravity filtered and
concentrated to yield the corresponding products in quantitative
yield.
Bruker Avance (at either 400 MHz or 100 MHz for 1H NMR and 13
C
NMR, respectively) spectrometer using DMSO-d6 or CDCl3 con-
taining tetramethylsilane (TMS) as an internal calibration standard.
High-resolution accurate mass spectra (HRMS) were obtained
either at the Georgia State University Mass Spectrometry Facility
using a Waters Q-TOF micro (ESI-Q-TOF) mass spectrometer or
utilizing a Waters Micromass LCT TOF ES þ Premier Mass Spec-
trometer. Liquid chromatography utilized a Waters 2487 single
wavelength absorption detector with wavelengths set between 640
and 700 nm depending on the dye's photophysical properties. The
4.4. Classical synthesis of indolenine salts 3e11
column used in LC was
a Waters Delta-Pak 5 mm 100 Å
Individual indolenine derivative 1e3 was added to a 50 mL
round bottom flask with a magnetic stir bar along with appropriate
alkyl iodide (3 mol eq) and acetonitrile (10 mL). The low boiling
point of the alkyl iodide limits the reaction temperature; also,
some of the alkylating agent will evaporate during the reactions,
higher equivalents are needed in the classical methods. The reac-
tion mixtures were heated to reflux for 48e72 h. After TLC indi-
cated a consumption of the starting materials, the reaction was
allowed to cool and the mixture was concentrated under reduced
pressure. Diethyl ether (25 mL) was added to the flask resulting in
an oily residue. Acetone (5 mL) was added to the residue and the
reaction mixture was sonicated for 10 min resulting in a free
flowing solid which was filtered, washed with diethyl ether and
hexanes and dried under vacuum. The compounds were obtained
in 58e79% yield.
3.9 ꢂ 150 mm reversed phase C18 column. Evaporative light scat-
tering detection analyzes trace impurities that cannot be observed
by alternate methods; a SEDEX 75 ELSD (Olivet, France) was uti-
lized in tandem with liquid chromatography to confirm purity.
2,3,3-trimethylindolenine was commercially obtained and used
in the alkylation reaction without purification.
4.2. Classical synthesis of indolenine compounds 1 and 2
Substituted phenylhydrazine hydrochloride heterocyclic
starting materials (1 g) were added to an oven dried and nitrogen
flushed round bottom flask. Acetic acid (15 mL) was added to the
flask and the reaction mixture was heated using an oil bath to
reflux. Ketone, 3-methyl-2-butanone (1.5 mol eq.), was added to
the reaction mixture. The mixture was allowed to heat for
12e48 h until TLC indicated a consumption of starting materials.
After the reaction was completed, the acetic acid and excess ke-
tone was removed under high vacuum rotary evaporation at
elevated temperature. The resulting reddish oil was dissolved in
dichloromethane and a saturated aqueous solution of sodium
bicarbonate was added carefully to the organic layer. The organic
layer was washed with sodium bicarbonate (3 ꢂ 10 mL), the
4.5. Microwave synthesis of indolenine salts 3e11
Individual indolenine derivative, 2,3,3-trimethlyindolenine,1, or
2 was added to a 10-mL microwave vessel equipped with a micro
magnetic stir bar along with appropriate alkyl iodide (1.2 mol eq.).
The reaction vessel was securely capped and then subjected to
microwave irradiation for 20 min allowing for the complete

34
E.A. Owens et al. / Dyes and Pigments 113 (2015) 27e37
transformation of the heterocyclic starting material to the corre-
sponding salt. The resulting residue was filtered, dried overnight on
vacuum and the corresponding salts were obtained in 98% þ yield
in excellent purity as determined by NMR (1H and 13C)
spectroscopy.
After concentration under reduced pressure, the resulting oil was
vacuum distilled (52 ꢀC, ~25 mmHg) to afford the final compound
in 91% yield.
4.8. Classical cyclization
Salts 3, 4, 6, 7, 9 and 10 are previously published compounds and
the data agrees with the literature values. [16]
4.8.1. 6,7,7-trimethyl-7H-[1,3]dioxolo[4,5-f]indole (13)
A solution of DMF, (8 mL), aniline (1.56 g, 11.38 mmol), and
potassium carbonate (1.05 g, 6.8 mmol) was brought to 45 ꢀC in a
two-neck round bottom flask with small magnetic stir-bar. 3-
Bromo-3-methylbutan-2-one (12, 1.86 g, 1 mL) was added to the
solution overnight with a syringe pump (Kd Scientific, Model 100).
After addition was completed, solution was stirred for 24 h at 45 ꢀC
and monitored by TLC. After TLC showed that the starting material
was consumed, dimethylformamide was evaporated in vacuo, and
concentrate was extracted with toluene (5 ꢂ 20 mL) and was
washed with deionized water. Toluene was concentrated to an
amount of 10e20 mL, and p-methyltoluenesulfonic acid (0.110 g,
0.1 mol eq.) was added to solution. Solution was allowed to reflux
for 24 h, until TLC showed an absence of starting material. Toluene
was evaporated in vacuo, and dichloromethane (20 mL) was added
to the reaction mixture. Solution was washed with a saturated
solution of sodium carbonate (5 ꢂ 50 mL) until the organic layer
was a red/brown color. DCM was removed under reduced pressure
and the resulting red/brown oil was recovered and the crude 13
(obtained in 57% yield) was used without purification.
4.5.1. 2,3,3-Trimethyl-1-propyl-3H-indol-1-ium iodide (5)
1H NMR (400 MHz, DMSO-d6)
d
: 1.00 (t, J ¼ 7.20 Hz, 3H), 1.56
(s, 6H) 1.80e1.98 (m, 2H), 2.88 (s, 3H), 4.47 (t, J ¼ 7.45 Hz, 2H)
7.63 (d, J ¼ 5.05, J ¼ 3.28 Hz, 2H), 7.82e7.92 (m, 1H), 7.96e8.07
(m, 1H), 13C NMR (100 MHz, DMSO-d6)
d: 10.76, 14.22, 20.78,
22.06, 48.83, 54.14, 115.53, 123.51, 128.90, 129.37, 141.06, 141.82,
196.54.
4.5.2. 5-Chloro-2,3,3-trimethyl-1-propyl-3H-indol-1-ium iodide
(8)
1H NMR (400 MHz, CDCl3)
d
: 1.05 (t, J ¼ 8.00 Hz, 3H) 1.65 (s, 6H)
1.93e2.10 (m, 2H) 3.09 (s, 3H) 4.62 (t, J ¼ 8.0 Hz, 2H) 7.48e7.60 (m,
2H) 7.79 (d, J ¼ 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6)
d: 11.22,
14.77, 21.22, 22.35, 49.52, 54.92, 118.01, 123.29, 127.41, 132.32,
140.94, 144.64, 197.61.
4.5.3. 5-Bromo-2,3,3-trimethyl-1-propyl-3H-indol-1-ium iodide
(11)
1H NMR (400 MHz, DMSO-d6)
d
: 0.99 (t, J ¼ 7.20 Hz, 3H) 1.57 (s,
6H) 1.86 (d, J ¼ 7.33 Hz, 2H) 2.81e2.91 (m, 3H) 4.37e4.55 (m, 2H)
7.86 (s, 1H) 8.00 (d, J ¼ 8.59 Hz, 1H) 8.21 (s, 1H), 13C NMR (100 MHz,
4.9. Microwave assisted cyclization
DMSO-d6) d: 11.24, 14.86, 21.24, 22.17, 22.36, 49.56, 54.92, 118.03,
123.29, 127.41, 132.32, 140.94, 144.64, 182.07, 188.62, 197.60.
4.9.1. 6,7,7-trimethyl-7H-[1,3]dioxolo[4,5-f]indole (13)
A
solution of [BMIM][PF6] (4 mL), potassium carbonate
4.6. Classical bromination
(1.2 mol eq.) and aniline (1.2 mol eq.) was added to a microwave
vessel (10 mL). 3-Bromo-3-methylbutan-2-one (1.0 mol eq.) was
added to the microwave vessel. The contents were subjected to
microwave irradiation at 150 W, 150 ꢀC for 20 min. Approximately
90% of the reactive aniline (100% of the butanone was consumed)
was allowed to go to completion leaving an additional amount to
participate in the imine formation seen in the mechanism. Para-
toluene sulphonic acid was added to the vessel (0.2 mol eq.) and
the reaction was heated to 100 ꢀC for 20 min. The brown solution
was dissolved in DCM (10 mL) and washed with saturated solution
of sodium bicarbonate (3 ꢂ 15 mL). The organic layer was dried over
magnesium sulfate and was evaporated under reduced pressure to
afford the final heterocyclic dioxolane. The compound was ob-
tained in 92% yield.
3-bromo-3-methyl-2-butanone (12): A mixture of 3-methyl-
butan-2-one (25.00 mL, 233.65 mmol) and acetic acid (38.00 mL)
was added to a three-neck oven-dried round bottom flask and
maintained at a temperature of 5 ꢀC. Next, both molecular bromine
(1.2 mL, 23.4 mmol) and acetic acid (5.0 mL) was combined in an
addition funnel and added dropwise to the round bottom flask.
When all of the bromine was added the reaction mixture was
allowed to stir overnight at room temperature. To the resulting
mixture, water (10 mL) was added, and then transferred to a sep-
aratory funnel where another 10 mL of water was added and the
organic layer was extracted with diethyl ether (3 ꢂ 15 mL). The
compound was then washed with cold, saturated sodium bicar-
bonate (4 ꢂ 10 mL), dried over anhydrous sodium sulfate, and the
reaction mixture was concentrated in vacuo to leave yellow oil. The
crude product was further purified via vacuum distillation (52 ꢀC,
~25 mmHg) to yield a colorless oil, 3.14 g (81%). 1H NMR (400 MHz,
4.10. Classical alkylation
The classical alkylation of 6,7,7-trimethyl-7H-[1,3]dioxolo[4,5-f]
indole was achieved using identical procedures found above and
the respective yields are found below.
CDCl3) d: 1.824 (s, 6H), 2.401 (s, 3H).
4.7. Eco-friendly bromination
4.7.1. 3-bromo-3-methyl-2-butanone (12)
4.11. Microwave assisted alkylation
3-methylbutan-2-one (20.1 mmol) was added to water (10 mL) a
round bottom flask at room temperature. The round bottom flask
was enveloped in aluminum foil to prevent light-induced in-
teractions. A 48% aqueous solution of HBr (30.2 mmol) was added
to the stirring mixture. After 10 min of stirring at room tempera-
ture, a solution of H2O2 (30% in water, 40.2 mmol) was added to the
reaction mixture. The clear solution was allowed to stir for 18 h.
After full consumption of the starting materials, the reaction was
quenched by adding excess saturated sodium bicarbonate solution
and the final brominated compound was extracted in diethyl ether.
Dioxyindolenine was added to a 10-mL microwave vessel
equipped with a micro magnetic stir bar along with appropriate
alkyl iodide (1.2 mol eq.). The reaction vessel was securely capped
and then subjected to microwave irradiation for 20 min allowing
for the complete transformation of the heterocyclic starting ma-
terial to the corresponding salt. The resulting residue was filtered,
dried overnight on vacuum and the corresponding salts were ob-
tained in 98% yield in excellent purity as determined by NMR (1H
and 13C) spectroscopy.

E.A. Owens et al. / Dyes and Pigments 113 (2015) 27e37
35
4.11.1. 5,6,7,7-Tetramethyl-7H-[1,3]dioxolo[4,5-f]indol-5-ium iodide
(14)
Classical Yield 60%, Microwave Yield 95%. 1H NMR (400 MHz,
DMSO-d6) : 1.48 (s, 6H) 2.70 (br. s., 3H) 3.90 (s, 3H) 6.19 (s, 2H) 7.48
(s, 1H) 7.64 (s, 1H), 13C NMR (100 MHz, DMSO-d6)
: 15.43, 21.48,
4.13.1. 1,3,3-Trimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2-
ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium iodide (17)
1H NMR (400 MHz, DMSO-d6)
d 1.71 (s, 12H), 3.63 (s, 6H), 6.28
d
(d, J ¼ 16.0 Hz, 2H), 6.65 (t, J ¼ 12.0 Hz, 1H), 7.24 (t, J ¼ 8.0 Hz, 2H),
7.29 (d, J ¼ 8.0 Hz, 2H), 7.39 (t, J ¼ 8.0 Hz, 2H), 7.48 (d, J ¼ 8.0 Hz,
2H), 8.25 (t, J ¼ 12.0 Hz, 2H).
d
23.05, 53.61, 55.34, 101.44, 103.34, 125.76, 128.64, 129.34, 139.90,
145.50, 146.91, 147.91, 187.11.
4.13.2. 1-Ethyl-3,3-trimethyl-2-((1E,3E,5E)-5-(1,3,3-
trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium
iodide (18)
4.11.2. 5-Ethyl-6,7,7-trimethyl-7H-[1,3]dioxolo[4,5-f]indol-5-ium
(15)
1H NMR (400 MHz, DMSO-d6)
d
1.27 (t, J ¼ 4.0, 6H), 1.68 (s, 12H),
Classical Yield 53%, Microwave Yield 94%. 1H NMR (400 MHz,
4.15 (d, J ¼ 8.0, 4H), 6.57 (t, J ¼ 12.0, 1H), 7.26 (t, J ¼ 4.0, 2H), 7.39 (s,
DMSO-d6)
d
: 1.48 (s, 6H) 2.70 (br. s., 3H) 3.90 (s, 3H) 6.19 (s, 2H) 7.48
4H), 7.63 (d, J ¼ 8.0, 2H), 8.34 (t, J ¼ 12.0, 2H).
(s, 1H) 7.64 (s, 1H), 13C NMR (100 MHz, DMSO-d6)
d: 13.28, 14.33,
22.37, 43.71, 54.33, 97.77, 103.21, 104.52, 134.88, 137.31, 148.54,
149.29, 194.54.
4.13.3. 1-Propyl-3,3-dimethyl-2-((1E,3E,5E)-5-(1-propyl,3,3-
dimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium
iodide (19)
4.11.3. 6,7,7-Trimethyl-5-propyl-7H-[1,3]dioxolo[4,5-f]indol-5-ium
(16)
1H NMR (400 MHz, DMSO-d6)
d
0.95 (t, J ¼ 4.0 Hz, 6H), 1.69 (s,
12H), 4.08 (m, J ¼ 4.0 Hz, 4H), 6.35 (d, J ¼ 16.0 Hz, 2H), 6.60 (t,
J ¼ 12.0 Hz, 1H), 7.25 (d, J ¼ 8.0 Hz, 2H), 7.42 (s, 4H), 7.64 (d,
J ¼ 8.0 Hz, 2H), 8.34 (t, J ¼ 12.0 Hz, 2H). HR-MS calculated for
[C31H39N2]þ 439.3108 found 439.3093.
Classical Yield 45%, Microwave Yield 92%. 1H NMR (400 MHz,
DMSO-d6)
d
: 0.85e1.06 (m, 3H) 2.79 (s, 6H) 4.38 (br. s., 1H) 6.20 (s,
4H) 7.51 (s, 1H) 7.75 (s, 1H), 13C NMR (100 MHz, DMSO-d6)
d: 11.14,
14.50, 21.35, 22.55, 49.39, 54.36, 97.65, 103.23, 104.44, 135.27,
137.24, 148.55, 149.34, 195.03.
4.13.4. 2-((1E,3Z,5E)-3-Chloro-5-(1,3,3-trimethylindolin-2-ylidene)
penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium iodide (20)
1H NMR (400 MHz, DMSO-d6)
d 1.69, (s, 12H), 3.58 (s, 6H), 6.29
4.12. General classical synthesis for NIR dyes 17e25 and 26e28
(d, J ¼ 12.0 Hz, 2H), 6.55 (t, J ¼ 12.0 Hz, 1H), 7.39 (d, J ¼ 8.0 Hz, 2H),
7.46 (d, J ¼ 8.0 Hz, 2H), 7.81 (s, 2H), 8.33 (t, J ¼ 12.0 Hz, 2H).
A mixture of indolium salt (40 mg, 0.154 mmol), bis-iminium
salt (20 mg, 0.077 mmol), NaOAc (17 mg, 0.23 mmol) and acetic
anhydride (1 mL) was added to an oven dried round bottom flask
with stirring bar. The reaction mixture was heated using a standard
oil bath for a particular reaction time (refer to Table 1). After
starting materials were consumed, the reaction was allowed to cool
to room temperature. Diethyl ether was added to the round bottom
flask resulting in an oily metallic blue residue. The diethyl ether was
decanted and the oil was dissolved in a minimal amount of meth-
anol followed by the addition of diethyl ether (50 mL) resulting in
the formation of light blue crystals, which were filtered. The crys-
tals were dissolved in dichloromethane leaving unreacted sodium
acetate on the funnel. The dichloromethane was removed in vacuo.
Many compounds needed additional purification methods. Silica-
gel column chromatography eluting with 2e5% methanol in
dichloromethane afforded the various pentamethine cyanines in
their respective yield.
4.13.5. 2-((1E,3Z,5E)-3-Chloro-5-(1-ethyl-3,3-dimethylindolin-2-
ylidene)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium
iodide (21)
1H NMR (400 MHz, DMSO-d6)
d 1.25 (s, 6H), 1.69 (s, 12H), 4.13 (s,
4H), 6.33 (d, J ¼ 12.0 Hz, 2H), 6.58 (t, J ¼ 12.0 Hz, 1H), 7.46 (m,
J ¼ 12.0 Hz, 2H), 7.89 (s, 2H), 8.35 (t, J ¼ 12.0 Hz, 2H).
4.13.6. 2-((1E,3Z,5E)-3-Chloro-5-(1-propyl-3,3-dimethylindolin-2-
ylidene)penta-1,3-dien-1-yl)-1-propyl-3,3-dimethyl-3H-indol-1-
ium iodide (22)
1H NMR (400 MHz, DMSO-d6)
d
0.94 (t, J ¼ 8.0 Hz, 6H), 1.70 (s,
16H), 4.07 (t, J ¼ 8.0 Hz, 4H), 6.35 (d, J ¼ 12.0 Hz, 2H), 6.65 (t,
J ¼ 12.0 Hz,1H), 7.45 (s, 4H), 7.83 (s, 2H), 8.35 (t, J ¼ 12.0 Hz, 2H). 13
C
NMR (100 MHz, DMSO-d6) d: 0.57, 11.39, 20.80, 27.43, 45.33, 49.57,
104.08, 113.08, 123.50, 128.75, 129.48, 141.53, 143.65, 154.93, 173.20.
HR-MS calculated for [C31H37N2Cl2]þ 507.2328 found 507.2315.
4.13. General microwave assisted synthesis for NIR dyes
4.13.7. 2-((1E,3Z,5E)-3-Bromo-5-(1,3,3-trimethylindolin-2-ylidene)
penta-1,3-dien-1-yl)-1,3,3-trimethyl-3H-indol-1-ium iodide (23)
A mixture of indolium salt (40 mg, 0.154 mmol), bis-iminium
salt (20 mg, 0.077 mmol), NaOAc (17 mg, 0.23 mmol) and acetic
anhydride (1 mL) was placed in sealed microwave vessel with
stirring bar. Sealed vessel was placed in single-mode microwave
(CEM Discover) on standard power setting, at indicated tempera-
ture, see Table 1 for 20 min resulting in increased pressure
(40e100 psi). Reaction mixture was diluted with diethyl ether
(10e20 mL) and filtered in vacuo. Solid was washed twice with
diethyl ether (5 mL). A clean filter flask was attached to the funnel
and the resulting solid was dissolved with dichloromethane
(10e15 mL) leaving unreacted sodium acetate crystals on the filter
funnel. The filtrate was transferred to a clean round bottom flask
and dichloromethane was removed with a rotary evaporator.
Metallic blue/green crystals were formed after solvent removal
resulting in the yield of individual compounds reported in Table 2.
Compounds 17e21, 23 and 24 have been previously reported by
conventional method [16].
1H NMR (400 MHz, DMSO-d6)
d 1.91 (s, 12H), 3.82 (s, 6H), 6.36
(d, J ¼ 8.0 Hz, 2H), 7.16 (d, J ¼ 8.0 Hz, 2H, 7.26e7.31 (m, 4H),
7.39e7.44 (m, 4H), 8.93 (d, J ¼ 12.0 Hz, 2H).
4.13.8. 2-((1E,3Z,5E)-3-Bromo-5-(1-ethyl-3,3-dimethylindolin-2-
ylidene)penta-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indol-1-ium
iodide (24)
1H NMR (400 MHz, DMSO-d6)
d
1.25 (m, J ¼ 4.0 Hz, 6H), 1.69 (s,
12H), 4.12 (m, J ¼ 8.0 Hz, 4H), 6.30 (d, J ¼ 16.0 Hz, 2H), 6.58 (t,
J ¼ 8.0 Hz,1H), 7.37 (t, J ¼ 8.0 Hz, 2H), 7.59 (d, J ¼ 8.0 Hz, 2H), 7.94 (s,
2H), 8.35 (d, J ¼ 12.0 Hz, 2H).
4.13.9. 2-((1E,3Z,5E)-3-Bromo-5-(1-propyl-3,3-dimethylindolin-2-
ylidene)penta-1,3-dien-1-yl)-1-propyl-3,3-dimethyl-3H-indol-1-
ium iodide (25)
1H NMR (400 MHz, DMSO-d6)
J ¼ 12.0 Hz, 16H), 4.06 (t, J ¼ 4.0 Hz, 4H), 6.35 (d, J ¼ 12.0 Hz, 2H),
d
0.94 (t, J ¼ 4.0 Hz, 6H), 1.70 (m,

36
E.A. Owens et al. / Dyes and Pigments 113 (2015) 27e37
6.65 (t, J ¼ 12.0 Hz, 1H), 7.40 (d, J ¼ 8.0 Hz, 2H), 7.59 (d, J ¼ 8.0 Hz,
2H), 7.95 (s, 2H), 8.35, (t, J ¼ 12.0 Hz, 2H). 13C NMR (100 MHz,
Eppendorf). The stock solutions were vortexed for 30 s, and sub-
sequently sonicated for 20 min to ensure complete dissolution.
DMSO-d6) d: 11.39, 20.79, 27.43, 45.29, 49.56, 104.09, 113.53, 117.49,
126.26, 126.60, 131.59, 141.95, 143.97, 154.97, 173.06. HR-MS calcu-
4.17. Method of determining molar absorptivity
lated for [C31H37N2Br2]þ 595.1318 found 595.1311.
Stock solutions were used to prepare six to seven samples in
methanol with various concentrations such that absorbance values
remained less than 1. Samples were prepared in 5.00 mL of DMSO
having used 5e50 uL, 10e100 uL, and 100e1000 uL Micropipettes,
(Eppendorf). Samples were prepared in disposable glass test tubes,
(10 mL, Fischer Scientific) and vortexed for 20 s to ensure dissolu-
tion. Appropriate measurements were chosen to ensure the absor-
bance values were less than 1.0 but greater than 0.1. Five
measurements were taken and the recorded absorbance values
versus concentration were plotted and the linear regression was
determined to obtain the molar absorptivity according to Beer's law.
4.13.10. 5,7,7-Trimethyl-6-((1E,3E,5Z)-5-(5,7,7-trimethyl-5H-[1,3]
dioxolo[4,5-f]indol-6(7H)-ylidene)penta-1,3-dien-1-yl)-7H-[1,3]
dioxolo[4,5-f]indol-5-ium iodide (26)
1H NMR (400 MHz, DMSO-d6)
d 1.63 (s,12H), 3.54 (s, 6H), 6.06 (s,
4H), 6.18 (d, J ¼ 16.0 Hz, 2H), 6.46 (t, J ¼ 16.0 Hz, 1H), 7.13 (s, 2H),
7.28 (s, 2H), 8.16 (t, J ¼ 15 Hz, 2H). HR-MS calculated for
[C29H31O4N2]þ 471.2278 found 471.2268.
4.13.11. 5-Ethyl-7,7-dimethyl-6-((1E,3E,5Z)-5-(5-ethyl-7,7-
dimethyl-5H-[1,3]dioxolo[4,5-f]indol-6(7H)-ylidene)penta-1,3-
dien-1-yl)-7H-[1,3]dioxolo[4,5-f]indol-5-ium iodide (27)
1H NMR (400 MHz, DMSO-d6)
d
1.24 (t, J ¼ 4.0 Hz, 6H), 1.63 (s,
4.18. Method of determining quantum yield
12H), 4.08 (m, J ¼ 4.0 Hz, 4H), 6.07 (s, 4H), 6.22 (d, J ¼ 16.0 Hz, 2H),
6.48 (t, J ¼ 12.0 Hz, 1H), 7.18 (s, 2H), 7.32 (s, 2H), 8.20 (t, J ¼ 12.0 Hz,
2H). 13C NMR (100 MHz, DMSO-d6)
d 12.68, 27.55, 49.34, 94.24,
Rhodamine 800, (99.9% Fluorescence grade, Sigma Aldrich) was
used as a standard within the given range of absorbance maxima,
(648e682 nm). Samples of the dyes and their respective standards
were prepared from stock solutions such that their absorbance
maxima were less than 0.1. The absorbance and fluorescence
spectra of each sample were obtained concurrently to minimize
experimental error from photobleaching. Each dye was diluted
accordingly, to ensure fluorescence was below 1000 units.
102.17, 102.95, 104.30, 135.15, 136.19, 145.50, 148.05, 152.99, 172.22.
HR-MS calculated for [C31H35O4N2]þ 499.2591 found 499.2577.
4.13.12. 5-Propyl-7,7-dimethyl-6-((1E,3E,5Z)-5-(5-propyl-7,7-
dimethyl-5H-[1,3]dioxolo[4,5-f]indol-6(7H)-ylidene)penta-1,3-
dien-1-yl)-7H-[1,3]dioxolo[4,5-f]indol-5-ium iodide (28)
1H NMR (400 MHz, DMSO-d6)
d
0.93 (t, J ¼ 8.0 Hz, 6H), 1.63 (m,
16H), 4.01 (s, 4H), 6.07 (s, 4H), 6.24 (d, J ¼ 16.0 Hz, 2H), 6.50 (t,
J ¼ 12.0 Hz, 1H), 7.21 (s, 2H), 7.32 (s, 2H), 8.20 (t, J ¼ 16.0 Hz, 2H). 13
C
Acknowledgments
NMR (100 MHz, DMSO-d6)
d: 11.35, 20.89, 27.69, 49.06, 49.32,
94.49, 102.19, 103.42, 104.20, 135.01, 136.77, 145.50, 148.04, 152.81,
172.78. HR-MS calculated for [C33H39O4N2]þ 527.2904 found
527.2883.
M.H. would like to thank the Brains and Behavior seed grant
from the Neuroscience Institute at Georgia State University. EAO
was supported through a fellowship with the Center for Diagnostics
and Therapeutics and JGT received his undergraduate fellowship
through the Molecular Basis of Disease program at GSU.
4.14. Computational experimental
Spartan Computational Modeling Software was utilized to draw
the chemical structures and calculations were performed using
Density Functional Theory at the B3LYP level. Chemical structures
were minimized to the conformer of the lowest energy and calcu-
lations were performed using appropriately applied charges in a
vacuum.
References
[1] Ashitate Y, Kim SH, Tanaka E, Henary M, Choi HS, Frangioni JV, et al. Two-
wavelength near-infrared fluorescence for the quantitation of drug anti-
platelet effects in large animal model systems. J Vasc Surg 2012;56:171e80.
[2] Chapman G, Henary M, Patonay G. The effect of varying short-chain alkyl
substitution on the molar absorptivity and quantum yield of cyanine dyes.
Anal Chem Insights 2011;6:29e36.
[3] Mehranpour AM, Hashemnia S, Maghamifar R. Synthesis and characterization
4.15. Analytical instrumentation
of new g-substituted pentamethine cyanine dyes. Synth Commun 2010;40:
3594e602.
Absorbance spectra were measured using a Varian Cary 50
UVeVisible Spectrophotometer interfaced to a PC, with a spectral
bandwidth of 2 nm. Fluorescence spectra for the pentamethine
cyanine dyes were obtained using a Shimadzu RF-1501 Spectro-
fluorophotometer (Shimadzu Scientific Instruments, Colombia,
MD) interfaced to a PC, with spectral bandwidths for both excita-
tion and emission set to 10 nm and the sensitivity set to “high”.
Disposable absorbance and fluorescence cuvettes were used with a
pathlength of 1.00 cm. All calculations were performed on Micro-
soft Excel 2010, (Microsoft Corporation, Redmond, WA).
[4] Patonay G, Salon J, Sowell J, Strekowski L. Noncovalent labeling of bio-
molecules with red and near infrared dyes. Molecules 2004;9:40e9.
[5] Luo S, Zhang E, Su Y, Cheng T, Shi C. A review of NIR dyes in cancer targeting
and imaging. Biomaterials 2011;32:7127e38.
[6] Nanjunda R, Owens EA, Mickelson L, Alyabyev S, Kilpatrick N, Wang S, et al.
Halogenated pentamethine cyanine dyes exhibiting high fidelity for G-quad-
ruplex DNA. Bioorg Med L Chem 2012;20:7002e11.
[7] Oushiki D, Kojima H, Terai T, Arita M, Hanaoka K, Urano Y, et al. Development
and application of a near-infrared fluorescence probe for oxidative stress
based on differential reactivity of linked cyanine dyes.
J Am Chem Soc
2010;132:2795e801.
[8] Mujumdar RB, Ernst LA, Mujumdar SR, Lewis CJ, Waggoner AS. Cyanine dye
labeling reagents: sulfoindocyanine succinimidyl esters. Bioconjugate Chem
1993;4:105e11.
[9] Levitus M, Ranjit S. Cyanine dyes in biophysical research: the photophysics of
polymethine fluorescent dyes in biomolecular environments. Q Rev BIOPHYS
2011;44:123e51.
4.16. Stock solutions
[10] Escobedo JO, Rusin O, Lim S, Strongin RM. NIR dyes for bioimaging applica-
tions. Curr Opin Chem Biol 2010;14:64e70.
[11] Shao F, Yuan H, Josephson L, Weissleder R, Hilderbrand SA. Facile synthesis of
monofunctional pentamethine carbocyanine fluorophores. Dyes Pigments
2011;90:119e22.
Stock solutions of the dyes in 1 mM concentration were pre-
pared by weighing the dye on a 5-digit analytical balance directly
into a brown glass vial and adding DMSO, (98.5% Spectroscopy
grade, Sigma Aldrich) with an auto pipette (100e1000 uL,

E.A. Owens et al. / Dyes and Pigments 113 (2015) 27e37
37
[12] Yang X, Shi C, Tong R, Qian W, Zhau HE, Wang R, et al. Near IR
heptamethine cyanine dyeemediated Cancer imaging. Clin Cancer Res
2010;16:2833e44.
[14] Samanta A, Vendrell M, Das R, Chang Y-T. Development of photostable near-
infrared cyanine dyes. Chem Commun 2010;46:7406e8.
ꢀ
[15] Podgorsek A, Stavber S, Zupan M, Iskra J. Bromination of ketones with
[13] Ebert B, Riefke B, Sukowski U, Licha K. Cyanine dyes as contrast agents for
near-infrared imaging in vivo: acute tolerance, pharmacokinetics, and fluo-
rescence imaging. J Biomed Opt 2011;16. 066003-1-9.
H2O2eHBr “on water”. Green Chem 2007;9:1212e8.
[16] Zhifei D, Bixian P. Synthesis and characterization of indodicarbocyanines.
Dyes Pigments 1998;36:169e75.