Ultrabright and Serum-Stable Squaraine Dyes

Article abstract of DOI:10.1021/acs.jmedchem.0c00617

Highly stable symmetric and asymmetric squaraine fluorophores have been synthesized featuring an internal salt bridge between a quaternary ammonium cation and the central oxycyclobutenolate ring of the chromophore. Some of our newly synthesized symmetric and asymmetric compounds display increased molar absorptivity, quantum yield in serum, and thermal/photochemical stability over previously reported squaraine-based dyes. Consequently, both classes show great promise in resurfacing the normal environment-labile squaraine dyes as novel imaging agents and scaffolds for fluorescence sensing. Furthermore, incorporating a covalent attachment point away from the conjugated system allows for biological tagging applications without disturbing the optimum optical characteristics of the newly designed fluorophore.

Full text of DOI:10.1021/acs.jmedchem.0c00617

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Article
Ultra-Bright and Serum-Stable Squaraine Dyes
Yogesh Yadav, Eric A Owens, Shinsuke Nomura, Takeshi Fukuda,
Baek, Yoonji, Satoshi Kashiwagi, Hak Soo Choi, and Maged Henary
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00617 • Publication Date (Web): 04 Aug 2020
Downloaded from pubs.acs.org on August 4, 2020
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Ultra-Bright and Serum-Stable Squaraine Dyes
Yogesh Yadav1#, Eric Owens,1,2# Shinsuke Nomura,3,4 Takeshi Fukuda,3 Yoonji Baek,3
Satoshi Kashiwagi,3 Hak Soo Choi,3,* Maged Henary1,2*
1Department of Chemistry, Georgia State University, 2Center for Diagnostics and Therapeutics,
145 Piedmont Avenue S.E., Atlanta, GA 30303, United States of America
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3Gordon Center for Medical Imaging, Department of Radiology, Massachusetts General Hospital
and Harvard Medical School, Boston, MA 02114, United States of America
4Department of Surgery, National Defense Medical College, Tokorozawa, Saitama 359-8513,
Japan
#Equal contribution
*Corresponding Authors: mhenary1@gsu.edu; Tel: +1 404-413-5566 (MH)
hchoi12@mgh.harvard.edu; Tel: +1 617-726-5784 (HSC)
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Abstract
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Highly stable symmetric and asymmetric squaraine fluorophores have been synthesized featuring
an internal salt bridge between a quaternary ammonium cation and the central oxycyclobutenolate
ring of the chromophore. Some of our newly synthesized symmetric and asymmetric compounds
display increased molar absorptivity, quantum yield in serum, and thermal/photochemical stability
over previously reported squaraine based dyes. Consequently, both classes show great promise in
resurfacing the normally environment-labile squaraine dyes as novel imaging agents and scaffolds
for fluorescence sensing. Furthermore, incorporating a covalent attachment point away from the
conjugated system allows for biological tagging applications without disturbing the optimum
optical characteristics of the newly designed fluorophore.
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Keywords: Fluorescence, squaraine, salt bridge, stability, quantum yield, density functional
theory, extinction coefficient, optical brightness.
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Introduction
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Biological sensors and imaging agents that absorb and fluoresce within the near-infrared (NIR)
region of the electromagnetic spectrum continue to garner sustained research interest due to their
inherent optical clarity for in vivo and in vitro use. The NIR region features a reduction in light
scattering, low light absorption by biomolecules and the removal of fluorescence signal
competition arising from biological sources. Designing a NIR-absorbing and fluorescing
compound must satisfy an array of diverse and crucial characteristics, including, (1) the compound
must display absorbance and fluorescence above 630 nm which is red-shifted enough to be easily
filtered from lower wavelength biomolecular autofluorescence, (2) a sufficient extinction
coefficient and quantum yield in serum/water to be used in living/aqueous systems, (3) sustained
water solubility to avoid micro-crystallization and reduction in optical performance, and (4) high
photo- and chemical stability to retain absorbance and fluorescence abilities in harsh conditions
such as covalently labeling a biomolecule with a NIR-dye (Figure 1 - top). Many fluorophore
subclasses, including fluorescein,1,2 BODIPY,3-5 and aminocoumarin6,7 based fluorophores (Figure
1 - bottom) satisfy some of these characteristics, and they have been explored with great interest.
One particularly interesting class of NIR fluorescent compounds centers on an
oxocyclobutenolate ring and are called squaraine dyes.8-10 These compounds are characterized by
extremely high fluorescence quantum yield (20-30%) which is highly desirable for increasing
meaningful signal; however, since their initial discovery, they have been reported by various
researchers to be unstable due to their highly chemically and photolytically labile
oxocyclobutenolate ring which quickly diminishes their widespread utility.11 The delocalized
cation between the terminal heterocyclic rings helps to resolve this but adds only minor stability
to the overall structure; various other strategies such as dye polymerization,12 rotaxane-protection
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of the squaraine-core,13 polymer-incorporation,14 or dextran enveloping15 have been employed to
harness the characteristically remarkable quantum yield of these compounds. All of these methods
have been shown to impart a subtle increase on the stability by protecting the electrophilic core of
the chromophore, but these low-yielding (10%) and tedious synthesis and purification processes
have not offered a substantial breakthrough in increasing squaraine dye stability or overall utility.
In addition, these fluorophores cannot be utilized for many specific labeling techniques that require
a relatively small molecular size — such as imaging in vivo or in biomolecular labeling which
renders squaraine compounds useless in these increasingly demanding applications.16-18 Many
efforts have been exhausted in the attempt to increase the stability of squaraine fluorophores and
herein, we report a facile scaffold for producing the most stable squaraine compounds synthesized
with unparalleled brightness in serum as determined by their measured extinction coefficients (ɛ),
quantum yield (Φ) and optical brightness (ɛ x Φ). Using our generic fluorophore platform, we are
able to harness the optical prowess of squaraine-based NIR fluorophores without sacrificing
stability.
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Figure 1. (top) General overview of covalently labeling a biomolecule with a NIR-dye (bottom)
commonly used fluorophores with benefits, in green, and negatives, in red, associated with their
in vivo use.
Results and Discussion
We initiated this finding by synthesizing water-soluble squaraine fluorophores (Scheme 1)
as potential probes for image guided surgery. To begin, we utilized Fischer indole synthesis, which
proceeds well in acetic acid, to install various halogens on the ring for influencing physicochemical
descriptors for in vivo studies. Alkylating the heterocyclic nitrogen with a propyl based quaternary
ammonium side chain is followed by the corresponding dicationic salt which is allowed to react
with squaric acid under dean-stark conditions. Having the final fluorophores in hand we initially
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confirmed that we successfully installed improved water solubility groups and we are investigating
the optical profile and the ability of our squaraine dyes to survive the intravenous injection.
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Scheme 1. Synthesis of squaraine dyes 9-12 containing quaternary ammonium propyl bromide
O
R
R
HCl
R
BrTMAB
NH2
ACN
reflux
72h
N
N
H
AcOH
reflux
N
Br-
N
5:
R = H
Br-
1:
R = H
Br-
N
6
: R = F
2
: R = F
BrTMAB
Br
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8:
R = Cl
R = Br
3:
4:
R = Cl
R = Br
R
O
O
N
Br-
O
O
N
HO
OH
quinoline
n-BuOH/benzene
dean-stark
N
Br-
N
reflux, 15h
9:
R = H
10
11:
12:
: R = F
R = Cl
R = Br
R
Similarly, to the above synthesis, we prepared additional analogs containing only a single
quaternary ammonium arm, shown in Scheme 2. The synthetic route begins with methylation and
subjecting the precursor 13 to a reaction with diisopropyl squarate. Careful reaction monitoring
and using triethyl amine afford the single heterocyclic adduct, which is then reacted with an
ethanolic sodium hydroxide solution to afford the intermediate 15 for reaction with a second
heterocyclic equivalent (5-8) under identical reaction conditions, to result in the final asymmetric
dyes 16-19 in high yields.
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Scheme 2. Final asymmetric compound 16-19 preparation.
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O
OH
O
O
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O
I-Me
ACN
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N
N
1. EtOH, NEt3
2. EtOH, NaOH
N
I
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N
O
N
I
5-8
Salts
quinoline
N
O
n-BuOH/benzene
dean-stark
16:
17
18:
19:
R = H
: R = F
R = Cl
R = Br
reflux, 12h
R
After the synthesis of the asymmetric compounds 16-19 successfully, we began to realize
that we could, in fact, use these new fluorophores for in vivo experiments. Biological experiments
often require a reactive handle for covalent attachment of a fluorophore, and at this point, our
compounds lacked a reactive functionality; therefore, using the chemistry optimized in Scheme 2,
we prepared the derivative 22 (Scheme 3) bearing the reactive carboxylic acid functionality for
further covalent modification. Having confirmed that we could synthesize a biologically relevant
squaraine dye, we fully examined the optical profile in various solvents (ethanol,
dimethylsulfoxide, phosphate buffered saline and fetal bovine serum) to confirm the near-infrared
properties and ensure our scaffold still featured the positive properties of squaraine dyes.
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Scheme 3. Final asymmetric compound 22 preparation.
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OH
O
O
O
O
O
O
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N
N
Br
9
1. EtOH, NEt3
2. EtOH, NaOH
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HOOC
HOOC
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N
O
5
N
Salt
Br-
quinoline
N
n-BuOH/benzene
dean-stark
O
reflux, 15h
22
COOH
Importantly for future in vivo experimentation/applications, all dyes dissolve immediately
in aqueous systems without the need for a co-solvent and show near-infrared absorption and
fluorescence characteristics with common Stokes Shift values (8-15 nm). The sharpness of the
absorption band in serum is highly remarkable as other fluorophores display an aggregation which
reduces their fluorescence quantum yield and, correspondingly, their in vivo applicability.19, 20 We
found that our fluorophores display extinction coefficient values in serum to be greater than
200,000 M-1cm-1 (Table 1S) probably due to the rigidity associated with the electrostatic
interaction between the ammonium cation and the partially negative central oxygen atoms (Figure
3). Common intramolecular salt bridge distances are less than 4 Å as ionic interactions are no
longer effective at longer distances. Observing the energy-minimized structures and measuring
the point-to-point distances of compounds 10 and 17, we can qualitatively see this interaction
occurring. DFT calculations suggest that the quaternary ammonium arms fold back onto the
central core of the electrophilic chain to within 4 Å. In Figure 3, we show these intramolecular
distances and the electrostatic maps associated with both the symmetric and asymmetric
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fluorophores.
All distances for the compounds’ quaternary ammonium nitrogen and
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corresponding central oxygen are less than 3.7 Å with representative compounds 10 and 17 shown
as examples. This adds rigidity and stability by locking the conformational state of the squaraine
fluorophore which minimizes the relaxation pathways that compete with fluorescence. We suspect
that this rigidization of the conjugated system of the compounds owes to the increase in quantum
yield as some of the measured values approach unity (compound 16  in FBS = 45%). We noted,
however, that bromine incorporation reduced the overall quantum yield, especially in serum. This
could be due to a heavy atom stabilization of the triplet state or increased hydrophobicity and
diminishment of fluorescence due to micro-aggregation.
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Finally, the paramount issue associated with squaraine dyes is their inherent instability. It
was reported by Johnson et al.21 that squaraine-rotaxane compound was well-demonstrated as
stable NIR probe compared to the commercial and widely used Cy5. To determine the stability of
our squaraine fluorophores bearing an internal salt bridge, each compound was incubated in FBS
o
at 37 C and irradiated using a high energy light for 72 h. The optical half-life recognized by
absorbance reaching half of the original value for each of the compounds 9 and 16 was not reached
after 72 h; however, the industry standard, Cy521 (which is a more stringent test as it is inherently
more stable than previously known squaraine dyes) was depleted of >75% of the absorption
measurements after the same timeframe. We noted an increase in stability of the symmetric version
over the asymmetric compound, however, the difference was not substantial as even the
asymmetric compound is more stable than Cy5.
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Figure 2. (left) The absorption and fluorescence spectra for compounds 9-12, 16-19 and 22 in FBS
supplemented with HEPES buffer at pH = 7.4 and warmed to physiological temperature (37 oC).
(right) The absorption-based stability in light and under heat (37 oC) in FBS buffer for compounds
9, and 16 and industry standard Cy5 as representative examples.
Figure 3. (left) DFT calculations (Wavefunction, Spartan, ‘10, B3LYP 6-311++G**) confirmed
the presence of a salt-bridge between the quaternary ammonium cation and the delocalized
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negative charge across the central oxygens in both the symmetric and asymmetric compounds
(right) schematic cartoon showing the locked nature of the double bonds which is thought to
contribute to improved absorptivity, quantum yield and stability.
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Next, we examined the effect of surface charges on the biodistribution and clearance of
squaraines in vivo. To this end, we injected 25 nmol of the individual compounds 9, 16 or 22 into
25 g CD-1 mice and monitored their behaviors using the intraoperative FLARE imaging system
up to 4 hours (Figure 4). Two positive (+2) charged compound 9 (logD = -5.15, pH 7.4) was
cleared through both renal and hepatobiliary routes with an overall distribution in most tissues and
organs due to the strong positive charges (Figure 4A).22, 23 Interestingly, +1 charged compound 16
with increased hydrophobicity (logD = -1.46, pH 7.4) and +1/-1 charged compound 22 (logD =
0.12, pH 7.4) showed a major uptake in the liver but minimum to no signals in the kidneys and
bladder, indicating predominant excretion through the hepatobiliary route (Figure 4B).
Histologically, kidney tissue showed NIR fluorescence signal for compound 9 in the cortex but
not in the medulla after injection of a squaraine dye (Figure 4C), indicating little renal clearance.
Consistently, liver tissue showed diffuse signal in hepatocytes, indicating the uptake and clearance
of squaraine dyes through the hepatobiliary route (Figure 4C). Compounds 16 and 22 show
decreased uptake in the chest wall and head/neck area, which suggests that we could reproducibly
control the biodistribution and clearance of squaraines in vivo by introducing different surface
charges to the backbone molecule as appropriate.
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Figure 4. Biodistribution and clearance of compounds 9, 16 and 22. 25 nmol of each fluorophore
was injected intravenously into 25 g CD-1 mice 4 h prior to imaging and resection. (A)
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Intraoperative NIR fluorescence imaging of mice and resected organs 4 h post-injection. Scale bars
= 5 mm (B) Quantitative analysis of signal-to-background ratio (SBR) for major organs against
muscle at 4 h post-injection. (C) Histological evaluation of biodistribution of compound 9 in major
organs. Shown are H&E and NIR fluorescence images of the resected kidney and liver 4 h post-
intravenous injection. Abbreviations used are: Bl, bladder; BD, bile duct; Du, duodenum; Ga,
gallbladder (arrowhead); He, heart; In, intestine; Ki, kidneys; Li, liver; Lu, lungs; Mu, muscle; Pa,
pancreas; Sp, spleen. Scale bars = 200 µm.
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Conclusions
To conclude, we have synthesized and evaluated the ability of an intramolecular salt bridge to
protect the central oxocyclobutenolate ring of squaraine fluorophores and increase the molar
absorptivity and quantum yield in serum owing to increased rigidity associated with the locked
structure. The asymmetric scaffold was modified to include a hexanoic acid moiety optimized for
biological labeling experiments which require high extinction coefficients and quantum yield in
the NIR. The biodistribution and clearance of squaraines in vivo were achieved by introducing
different surface charges to the backbone of the compounds. These findings offer a facile solution
to squaraine stability while finally increasing their in vivo appeal.
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Experimental Section
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All starting reagents were obtained commercially from Sigma Aldrich or Matrix Scientific and
were used without purification. Nuclear magnetic resonance experiments were performed on a
Bruker Avance 400 MHz spectrometer and processed using Topspin 3.1. Chemical shift values are
reported versus tetramethylsilane as an internal standard purchased in the commercially obtained
deuterated solvents (Cambridge Isotope Laboratory). The reactions were followed using silica gel
60 F254 thin layer chromatography plates (Merck EMD Millipore, Darmstadt, Germany). Open
column chromatography was utilized for the purification of all final compounds using 60-200u,
60A, classic column silica gel (Dynamic Adsorbents, Norcross, GA). UV-Vis/NIR absorption
spectra were recorded on a Varian Cary 50 spectrophotometer. 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 + Premier Mass Spectrometer. Liquid chromatography utilized a Waters 2487 single
wavelength absorption detector with wavelengths set between 640 and 700 nm depending on the
particular photophysical properties. The column used in LC was a Waters Delta-Pak 5 µM 100 A
3.9 x 150 mm reversed phase C18 column. Evaporative light scattering detection analyzes trace
impurities that cannot be observed by alternate methods; a SEDEX 75 ELSD was utilized in
tandem with liquid chromatography to confirm purity (> 95% as determined by LC-ELSD-MS).
The precursors and the final compounds were purified using column chromatography and the
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purity of all synthesized compounds was confirmed with HNMR, 13CNMR and ESI-HRMS.
These analytical methods used to determine the purity of compounds to be ≥ 95%.
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H
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Br
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Br
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2,3,3-Trimethyl-1-(3-(trimethylammonio)propyl)-3 H-indol-1-ium bromide (5): (2.52 g,
61%); mp 197-199 °C. 1H NMR (400 MHz, DMSO-d6): δ 1.58 (s, 6H), 2.41 (m, 2H), 2.99 (s, 3H),
3.17 (s, 9H), 3.98 (t, J = 7.6 Hz, 2H), 4.57 (t, J = 7.6 Hz, 2H), 7.45 (m, 2H), 7.86 (d, J = 5.2 Hz,
13
1H), 8.19 (d, J = 5.2 Hz, 1H). C NMR (100 MHz, DMSO-d6): δ 21.3, 22.0, 25.4, 44.6, 52.5,
61.9, 64.9, 115.6, 123.5, 129.0, 129.4, 140.9, 141.8, 197.8. HRMS (ESI) calculated for [C17H28N2]+
m/z 260.2174, found m/z 260.2177.
F
N
Br
N
Br
6
5-Fluoro-2,3,3-trimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (6):
Yield 90%; mp 237-240 °C; 1H NMR (400 MHz, D2O): δ 1.56 (s, 6H), 2.47 (t, J = 8.8 Hz, 2H),
3.17 (s, 9H), 3.65 (tt, J = 8.4 Hz, J = 8.4 Hz, 2H), 4.53 (t, J = 8.0 Hz, 2H), 7.34 (t, J = 9.2 Hz, 1H),
7.82 (dd, J = 8.8 Hz, J = 4.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6): δ 12.2, 20.0, 20.7, 43.3,
52.1, 53.2, 61.5, 115.9, 123.3, 128.8, 134.3, 138.2, 142.3, 197.9. HRMS (ESI) calculated for
[C17H27FN2]+ m/z 278.2194, found m/z 278.2182.
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Cl
7
8
9
N
10
11
12
13
14
15
16
17
18
19
20
21
22
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25
26
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60
Br
N
Br
7
5-Chloro-2,3,3-trimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (7):
Yield 82%; mp 215–217 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.65 (s, 6H), 2.56 (t, J = 8.0 Hz,
2H), 3.25 (s, 9H), 3.73 (tt, J = 8.0 Hz, J = 8.0 Hz, 2H), 4.64 (t, J = 8.0 Hz, 2H), 4.80 (s, 3H), 7.69
(dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 7.84–7.92 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ 12.2,
20.0, 20.7, 43.3, 52.1, 53.2, 61.5, 115.9, 123.3, 128.8, 134.3, 138.2, 142.3, 197.9. HRMS (ESI)
calculated for [C17H27ClN2]2+ (m/z) 294.1956, found m/z 294.1910
Br
N
Br
N
Br
8
5-Bromo-2,3,3-trimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium bromide (8):
Yield 81%; mp 225–227 °C; 1H NMR (400 MHz, DMSO-d6): δ 1.57 (s, 6H), 2.27 (s, 9H), 3.02 (s,
3H), 3.83 (t, J = 8.0 Hz, 2H), 4.57 (t, J = 8.0 Hz, 2H), 7.61 (m, 2H), 7.88 (t, J = 5.6 Hz, 1H). 13C
NMR (100 MHz, DMSO-d6): δ 15.2, 22.0, 25.5, 30.6, 52.3, 54.3, 63.9, 115.6, 123.5, 128.8, 129.3,
140.9, 141.7, 197.7. HRMS (ESI) calculated for [C17H27BrN2]2+ (m/z) 338.1320, found 338.1305
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General procedure for the synthesis of symmetric squaric acid fluorophores: A mixture of the
corresponding indolenine salt (1.0 mol. eq.) and squaric acid (0.5 mol. eq.) and quinoline (5.0 mol.
eq.) was refluxed in a mixture of n-butanol and benzene (40 mL, 1:1) with azeotropic distillation
using dean stark apparatus for 15 h. The reaction mixture was monitored by Vis/NIR
spectrophotometry to observe the completion of the reaction and by measuring the new wavelength
(~ 640 nm in ethanol) of the formed NIR dyes. The solvent was removed under reduced pressure.
The residue was stirred in ethyl acetate (100 mL). The precipitated product was filtered, washed
with ethyl acetate, acetone and dried under vacuum. The crude material was dissolved in methanol
(10 mL), and poured into ethyl acetate (100 mL). The precipitated product was filtered and dried
under vacuum. This precipitation procedure was repeated once more and the obtained pure product
was dried under vacuum.
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H
N
Br-
O
N
N
Br-
O
N
H
9
Mono((Z)-4-((3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-2-yl) methylene)-2-
((E)-(3,3-dimethyl-1-(3-(trimethylammonio)propyl)indolin-2-ylidene) methyl)-3-oxocyclobut-1-
enolate) tribromide (9) Yield 70%. mp >260oC 1H NMR (400 MHz, DMSO) δ 1.71 (s, 6H, CH3),
2.15 (s, 2H, CH2), 3.12 (s, 9H, CH3), 3.57-3.61 (s, 2H, CH2), 4.16 (s, 2H, CH2), 5.86 (s, 1H, CH),
7.20 (t, J = 7.2 Hz, 1H, ArH), 7.38 (t, J = 8.0 Hz, 1H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.55
(d, J = 7.2 Hz, 1H, ArH); 13C NMR (100 MHz, DMSO) δ 21.0, 27.1, 49.3, 53.0, 63.2, 86.8, 110.8,
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122.7, 124.3, 128.4, 141.8, 142.4, 169.6, 180.4, 181.0. HRMS (m/z) for C38H52N4O22+ calculated
5
6
298.3046, found 298.3036.
7
8
F
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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42
43
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45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
Br-
O
N
N
Br-
O
N
F
10
Mono((Z)-4-((5-fluoro-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-2-
yl)methylene)-2-((E)-(5-fluoro-3,3-dimethyl-1-(3-(trimethylammonio)propyl)indolin-2-
ylidene)methyl)-3-oxocyclobut-1-enolate) tribromide (10) Yield 64%; mp >260oC 1H NMR (400
MHz, DMSO) δ 1.71 (s, 12H, CH3), 2.13 (s, 4H, CH2), 3.10 (s, 18H, CH3), 3.55 (s, 4H, CH2), 4.14
(s, 4H, CH2), 5.83 (s, 2H, CH), 7.21-7.25 (s, 2H, Ar-H), 7.46 (brs, 2H, Ar-H), 7.53-7.54 (m, 2H,
ArH); 13C NMR (100 MHz, DMSO) δ 20.7, 26.5, 40.8, 49.5, 53.3, 63.3, 86.4, 110.4, 110.6, 111.7,
111.8, 115.0, 115.2, 138.1, 143.8, 143.9, 159.0, 161.4, 170.9, 175.8, 182.7. HRMS (m/z) for
C38H50F2N4O22+ calculated 316.2046, found 316.2065.
Cl
N
Br-
O
N
N
Br-
O
N
Cl
11
Mono((Z)-4-((5-chloro-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-2-
yl)methylene)-2-((E)-(5-chloro-3,3-dimethyl-1-(3-(trimethylammonio)propyl)indolin-2-
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9
ylidene)methyl)-3-oxocyclobut-1-enolate) tribromide (11) Yield 75%; mp >260oC 1H NMR (400
MHz, DMSO) δ 1.71 (s, 12H, CH3), 2.13 (s, 4H, CH2), 3.10 (s, 18H, CH3), 3.54 (s, 4H, CH2), 4.15
(s, 4H, CH2), 5.86 (s, 2H, CH), 7.43-7.49 (s, 4H, Ar-H), 7.70 (s, 2H, Ar-H); 13C NMR (100 MHz,
DMSO) δ 21.0, 26.9, 49.0, 49.5, 53.1, 63.3, 87.3, 112.2, 123.1, 128.3, 128.8, 141.4, 143.9, 169.6,
10
11
12
13
14
15
16
17
18
19
20
21
22
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24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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43
44
45
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
2+
180.9, 181.2. HRMS (m/z) for C38H50Cl2N4O2 , calculated 332.1634, found 316.1629.
Br
N
Br-
O
N
N
Br-
O
N
Br
12
Mono((Z)-4-((5-bromo-3,3-dimethyl-1-(3-(trimethylammonio)propyl)-3H-indol-1-ium-2-yl)
methylene)-2-((E)-(5-bromo-3,3-dimethyl-1-(3-(trimethylammonio)propyl)indolin-2-ylidene)
methyl)-3-oxocyclobut-1-enolate) tribromide (12) Yield 62%; mp >260oC H NMR (400 MHz,
1
DMSO) δ 1.70 (s, 12H, CH3), 2.12 (s, 4H, CH2), 3.09 (s, 18H, CH3), 3.54 (s, 4H, CH2), 4.14 (s,
13
4H, CH2), 5.85 (s, 2H, CH), 7.43-7.55 (s, 4H, Ar-H), 7.80 (s, 2H, Ar-H); C NMR (100 MHz,
DMSO) δ 20.9, 26.9, 49.5, 53.0, 63.1, 87.3, 112.8, 116.5, 125.9, 131.2, 141.8, 144.2, 159.4, 162.3,
2+
169.3, 180.9. HRMS (m/z) for C38H50Br2N4O2 , calculated 377.2035, found 378.2039.
OH
O
OH
O
O
N
O
N
HOOC
15
21
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Synthesis
of
(Z)-2-hydroxy-3-oxo-4-((1,3,3-trimethyl-3H-indol-1-ium-2-yl)methylene)
5
6
7
8
cyclobut-1-enolate iodide (15) and (Z)-4-((1-(5-carboxypentyl)-3,3-dimethyl-3H-indol-1-ium-2-
yl)methylene)-2-hydroxy-3-oxocyclobut-1-en-1-olate (21). Individual salt (4.0 mol), 3,4-
diisopropoxycyclobutane-1,2-dione (4.0 mol) and triethylamine (1.0 mL) was heated in EtOH for
3 hr. After the reaction was completed, the reaction was cooled and the solvent was removed under
reduced pressure. The crude product was purified by silica gel column chromatography
(CH2Cl2/MeOH). Compound (1.0 mol) was dissolved in ethanol (10 mL) and NaOH (40%, 0.5
mL) was added. The mixture was heated at 70oC for 30 min. After the reaction was completed, the
mixture was cooled and 2N HCl (5 mL) was added. The product was purified by silica gel column
chromatography (MeOH/DCM 1:9). These intermediates were used in the next steps without
characterization.
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General procedure for the synthesis of asymmetric squaric acid fluorophores: A mixture of
the corresponding indolenine salt 5-8 (1.0 mol. eq.) and mono squaric acid modified compound 15
or the corresponding indolenine salt 5 (1.0 mol. eq.), compound 21 (1.0 mol. eq.) and quinoline
(5.0 mol. eq.) was refluxed in a mixture of n-butanol and benzene (40 mL, 1:1) with azeotropic
distillation using dean stark apparatus for 12 h. The reaction mixture was monitored by Vis/NIR
spectrophotometry to observe the completion of the reaction and by measuring the new wavelength
(~ 630 nm in ethanol) of the formed NIR dyes. The solvent was removed under reduced pressure.
The residue was stirred in ethyl acetate (100 mL). The precipitated product was filtered, washed
with ethyl acetate, acetone and dried under vacuum. The crude material was dissolved in methanol
(10 mL), and poured into ethyl acetate (100 mL). The precipitated product was filtered and dried
under vacuum. This precipitation procedure was repeated once more and the obtained pure product
was dried under vacuum.
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N
O
N
I
N
O
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15
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19
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21
22
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H
16
Mono((Z)-2-((E)-(3,3-dimethyl-1-(3-(trimethylammonio)propyl)indolin-2-ylidene) methyl)-3-oxo-
4-((1,3,3-trimethyl-3H-indol-1-ium-2-yl)methylene)cyclobut-1-enolate mono bromide mono
iodide (16) Yield 37%; mp >198-200oC 1H NMR (400 MHz, MeOD) δ 1.76-1.77 (m, 12H, CH3),
2.34 (brs, 2H, CH2), 3.22 (s, 9H, CH3), 3.63-3.67 (m, 5H, CH3&CH2), 4.23 (brs, 2H, CH2), 5.89
(s, 1H, CH), 5.99 (s, 1H, CH), 7.21-7.26 (m, 2H, Ar-H), 7.32-7.43 (m, 4H, Ar-H), 7.46-7.50 (m,
13
2H, Ar-H), C NMR (100 MHz, MeOD) δ 20.6, 25.6, 26.1, 30.3, 40.0, 49.0, 49.4, 52.5, 52.8,
62.3, 63.3, 109.8, 110.3, 121.9, 122.0, 123.9, 124.6, 128.0, 129.2, 130.0, 135.2, 141.3, 141.8, 142.6,
+
144.6, 147.5, 182.7. HRMS (m/z) for C33H40N3O2 , calculated 510.8955, found 510.8995.
N
O
N
I
N
O
F
17
Mono((Z)-2-((E)-(5-fluoro-3,3-dimethyl-1-(3-(trimethylammonio)propyl)indolin-2-
ylidene)methyl)-3-oxo-4-((1,3,3-trimethyl-3H-indol-1-ium-2-yl)methylene)cyclobut-1-enolate)
1
mono bromide monoiodide (17) Yield 43%; H NMR (400 MHz, MeOD) δ 1.76 (s, 12H, CH3),
2.32 (s, 2H, CH2), 3.22 (s, 9H, CH3), 3.62-3.70 (m, 5H, CH3 & CH2), 4.20 (s, 2H, CH2), 5.85 (s,
1H, CH), 5.99 (s, 1H, CH), 7.13 (t, J = 8.0 Hz, 1H, ArH), 7.25-7.29 (m, 2H, Ar-H), 7.32-7.36 (m,
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9
2H, Ar-H), 7.40-7.43 (m, 1H, Ar-H), 7.50 (t, J = 7.6 Hz, 1H, ArH); 13C NMR (100 MHz, MeOD)
δ 20.5, 25.5, 26.1, 30.3, 40.1, 52.5, 63.5, 85.3, 86.2, 109.8, 110.1, 110.2, 110.3, 114.0, 114.3, 121.8,
124.7, 127.9, 138.2, 141.9, 142.7, 169.0, 173.4, 173.9, 177.8, 183.0. HRMS (m/z) for
+
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60
C33H39FN3O2 , calculated 528.3021, found 528.2086.
N
O
N
I
N
O
Cl
18
Mono((Z)-2-((E)-(5-chloro-3,3-dimethyl-1-(3-(trimethylammonio)propyl)indolin-2-
ylidene)methyl)-3-oxo-4-((1,3,3-trimethyl-3H-indol-1-ium-2-yl)methylene)cyclobut-1-enolate)
1
mono bromide monoiodide (18) Yield 31%; H NMR (400 MHz, MeOD) δ 1.75 (s, 12H, CH3),
2.30-2.31 (m, 2H, CH2), 3.23 (s, 9H, CH3), 3.63-3.65 (m, 2H, CH2), 3.71 (s, 3H, CH3), 4.17-4.21
(m, 2H, CH2), 5.84 (s, 1H, CH), 6.01 (s, 1H, CH), 7.26-7.36 (m, 4H, ArH), 7.40-7.42 (m, 1H,
Ar-H), 7.44-7.46 (m, 1H, Ar-H), 7.49-7.51 (m, 1H, Ar-H); 13C NMR (100 MHz, MeOD) δ 20.4,
25.5, 26.2, 30.4, 40.0, 49.7, 52.5, 63.5, 85.5, 86.6, 110.5, 121.9, 122.5, 124.9, 127.8, 128.0, 128.8,
+
141.0, 142.0, 142.6, 143.1, 168.0, 173.9, 183.0. HRMS (m/z) for C33H39ClN3O2 , calculated
543.1150, found 543.1108.
N
O
N
I
N
O
Br
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Mono((Z)-2-((E)-(5-bromo-3,3-dimethyl-1-(3-(trimethylammonio)propyl)indolin-2-
5
6
7
8
ylidene)methyl)-3-oxo-4-((1,3,3-trimethyl-3H-indol-1-ium-2-yl)methylene)cyclobut-1-enolate)
mono bromide monoiodide (19) Yield 36%; mp 204-206 oC 1H NMR (400 MHz, MeOD) δ 1.75-
1.76 (m, 12H, CH3), 2.31 (s, 2H, CH2), 3.23 (s, 9H, CH3), 3.71-3.72 (m, 5H, CH3 & CH2), 4.17 (s,
2H, CH2), 5.84 (s, 1H, CH), 6.01 (s, 1H, CH), 7.28-7.32 (m, 2H, Ar-H), 7.35-7.37 (m, 1H, Ar-H),
7.40-7.43 (m, 1H, Ar-H), 7.49-7.51 (m, 2H, Ar-H), 7.58-7.61 (m, 1H, Ar-H); 13C NMR (100 MHz,
MeOD) δ 20.4, 25.5, 26.1, 30.4, 39.9, 49.6, 52.5, 63.3, 85.5, 86.6, 110.6, 111.1, 116.0, 121.9,
124.9, 125.4, 128.0, 130.8, 141.4, 142.0, 142.5, 173.6, 178.2, 182.9. HRMS (m/z) for
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+
C33H39BrN3O2 , calculated 588.6220, found 588.6233
N
O
N
Br-
N
O
22
COOH
(Z)-2-((E)-(1-(5-carboxypentyl)-3,3-dimethylindolin-2-ylidene)methyl)-3-oxo-4-((1,3,3-trimethyl-
1
3H-indol-1-ium-2-yl)methylene)cyclobut-1-enolate, iodide (22) Yield 43%; mp 166-168oC H
NMR (400 MHz, CDCl3) δ 1.70-1.91 (m, 18H, CH3&CH2), 2.45 (s, 2H, CH2), 3.58 (s, 3H, CH3),
4.04 (s, 2H, CH2), 5.89 (s, 1H, CH), 6.08 (s, 1H, CH), 6.97-7.02 (m, 2H, Ar-H), 7.16-7.17 (m, 2H,
Ar-H), 7.28-7.38 (m, 4H, Ar-H); 13C NMR (100 MHz, CDCl3) δ 23.8, 25.7, 26.8, 26.9, 34.3, 43.6,
49.1, 49.2, 86.5, 87.1, 109.3, 109.5, 122.1, 122.2, 123.8, 123.9, 127.8, 142.1, 142.2, 142.9, 170.7,
170.9, 176.7, 177.6, 183.4. HRMS (m/z) for C38H48N3O4, calculated 610.3675, found 610.3635
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Optical property analyses. All optical measurements were performed in various solvents,
including ethanol (EtOH), dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS, pH 7.4)
and at 37 oC in 100% fetal bovine serum (FBS) buffered with 50 mM HEPES, pH 7.4. Absorbance
and fluorescence emission spectra of the series of NIR fluorophores were measured using Varian
Cary 50 absorbance spectrophotometer (190–1100 nm) and Shimadzu RF-5301PC
spectrofluorometer (350–1000 nm). For fluorescence quantum yield (QY) measurements,
rhodamine 800 in absolute ethanol (QY = 28%) was used as a calibration standard, under
conditions of matched absorbance at 620 nm.
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60
Stability studies. The stability experiments were performed to determine the photo bleaching and
heat decomposition of the fluorophores in serum. We determined this by measuring the
absorbance at the wavelength of maximum absorption over 72 h time period. The light condition
involved a cuvette containing individual contrast agent (0.01 mm in FBS) was affixed 250 mm
away from a 15W F15T8 broad spectrum bulb being irradiated using a portable lamp at room
temperature. Similarly, the heat was based measurements were conducted in sealed centrifuge
tubes at physiological temperatures (37 oC) for the indicated time periods and measurements were
taken intermittently.
Molecular modeling analyses. In silico calculations of chemical structures were performed using
the geometric conformation of the lowest energy and distances were calculated Spartan DFT
calculations at the B3LYP level.
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Animal models and intraoperative imaging. Animals were housed in an AAALAC-certified
facility and all procedures were performed according to the approved institutional protocol by
Massachusetts General Hospital (protocol #2016N000136). CD-1 mice (Charles river weight, 19–
21 g; age, 6 weeks; male) were purchased from Charles River Laboratories (Wilmington, MA) and
were given free access to food and water. Mice were fed with using chlorophyll-free mouse chow
(VWR international) 5 days prior to imaging study to minimize autofluorescence. To investigate
the biodistribution of each squaraine fluorophore, mice were imaged using the FLARE imaging
system.19,20 The objective lens was positioned at 25 cm from the operative field. NIR fluorescence
and bright-field images were simultaneously obtained using a 660 nm excitation laser at a power
of 2 mW/cm2 through 710 ± 25 nm emission filter and white light (400-650 nm; 40,000 lux). To
accurately confirm the biodistribution of these fluorophores, camera exposure time was adjusted
appropriately. Intraoperative fluorescent imaging was performed under isoflurane anesthesia. Four
hours prior to imaging, 25 nmol (250 µM, 100 µL) of each squaraine fluorophore was systemically
injected via retro-orbital sinus under isoflurane anesthesia. Upon imaging, the limbs were taped,
and the skin from the pubic bone to the neck was incised in the middle. Then, the salivary gland
was exposed. After taking images of the salivary gland, it was split between the left lobe and the
right lobe, and the muscle in front of the trachea was incised to expose the thyroid gland for
fluorescent images. Animals were sacrificed after time-course NIR fluorescence imaging for ex
vivo biodistribution study. The abdominal wall was incised, and then the chest wall and diaphragm
were incised to thoracotomy. The excretion route of the fluorophore was identified from the
abdominal image. After in vivo imaging, the major organs including heart, lung, liver, pancreas,
spleen, kidney, duodenum, intestine, and muscle were excised. NIR signals from individual
resected tissues were imaged using the FLARE imaging system.
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Quantitative analysis of NIR signals. The fluorescence intensity of a region of interest (ROI)
over each organ/muscle was quantified using ImageJ version 1.52p. The signal-to-background
ratio (SBR) was calculated as SBR = target signal/muscle signal.
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NIR fluorescence microscopy. Major organs were harvested 4 h post-intravenous injection of
squaraine dyes, trimmed and embedded in Tissue-Tek OCT compound (Fisher Scientific). Frozen
sections of 10 µm thickness were cut with a cryostat until the region of interest was reached (Leica,
Germany). After fluorescence images were acquired on a fluorescence microscope, the sections
were stained with hematoxylin and eosin (H&E). Fluorescence and bright-field imaging was
conducted using a Nikon TE2000 microscope (Nikon, Melville, NY) equipped with a custom filter
set for NIR imaging (Chroma Technology, Brattleboro, VT). Images were acquired on an Orca-
AG CCD camera (Hamamatsu, Bridgewater, NJ) with a custom filter set (Chroma, Brattleboro,
VT) composed of a 650 ± 22 nm excitation filter, a 675 nm dichroic mirror, and a 710 ± 25 nm
emission filters (Chroma Technology).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge
1H NMR and 13C NMR, mass spectroscopy analyses and optical properties of the final squaraine
dyes 9-12 and 16-19 are attached.
The Molecular-formula strings of the reported compounds are included (CSV)
Notes
The authors declare no competing financial interest
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Acknowledgements
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M.H. and E.A.O would like to thank the Center for Diagnostics and Therapeutics at Georgia State
University for their support and additionally the dissertation fellowship provided to E.A.O. M.H.
wishes to thank the Brains and Behavior Seed Grant, the Health Innovation Platform Grant, and
the Georgia Research Alliance Venture Grant Phase 1 for their support. MH thanks Mr. Emmanuel
Buabeng for his help on the optical properties. This study was supported by the US NIH grant
NIBIB #R01-EB022230.
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ABBREVIATIONS USED
N/A
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