Near-Infrared Illumination of Native Tissues for Image-Guided Surgery

Article abstract of DOI:10.1021/acs.jmedchem.6b00038

Our initial efforts to prepare tissue-specific near-infrared (NIR) fluorescent compounds generated successful correlation between physicochemical properties and global uptake in major organs after systemic circulation and biodistribution. Herein, we focus on the effects on biodistribution based on modulating electronic influencing moieties from donating to withdrawing moieties at both the heterocyclic site and through meso-substitution of pentamethine cyanine fluorophores. These selected modifications harnessed innate biodistribution pathways through the structure-inherent targeting, resulting in effective imaging of the adrenal glands, pituitary gland, lymph nodes, pancreas, and thyroid and salivary glands. These native-tissue contrast agents will arm surgeons with a powerful and versatile arsenal for intraoperative NIR imaging in real time.

Full text of DOI:10.1021/acs.jmedchem.6b00038

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Article
Near-Infrared Illumination of Native Tissues for Image-Guided Surgery
Eric A. Owens, Hoon Hyun, Tyler L. Dost, Jeong Heon Lee, GwangLi
Park, Dang Huan Pham, Min Ho Park., Hak Soo Choi, and Maged Henary
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00038 • Publication Date (Web): 21 Apr 2016
Downloaded from http://pubs.acs.org on May 6, 2016
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NearꢀInfrared Illumination of Native Tissues for ImageꢀGuided Surgery
Eric A. Owens1,#, Hoon Hyun2,3,#, Tyler L. Dost1,#, Jeong Heon Lee2, GwangLi Park2, Dang Huan Pham3,
Min Ho Park4, Hak Soo Choi2,*, Maged Henary1,*
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1Center for Diagnostics and Therapeutics, Center for Biotechnology and Drug Design, Department of
Chemistry, Georgia State University, Petit Science Center, 100 Piedmont Ave SE, Atlanta, GA 30303
2Division of Hematology/Oncology, Department of Medicine, Beth Israel Deaconess Medical Center
and Harvard Medical School, Boston, MA 02215
3Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju 501ꢀ746,
South Korea
4Department of Surgery, Chonnam National University Medical School, Gwangju 501ꢀ746, South Korea
#Authors contributed equally to this work
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Abstract: Our initial efforts to prepare tissueꢀspecific nearꢀinfrared (NIR) fluorescent compounds
generated successful correlation between physicochemical properties and global uptake in major organs
after systemic circulation and biodistribution. Herein, we focus on the effects on biodistribution based
on modulating electronic influencing moieties from donating to withdrawing moieties at both the
heterocyclic site and through mesoꢀsubstitution of pentamethine cyanine fluorophores. These selected
modifications harnessed innate biodistribution pathways through the structureꢀinherent targeting
resulting in effective imaging of the adrenal glands, pituitary gland, lymph nodes, pancreas, thyroid and
salivary glands. These nativeꢀtissue contrast agents will arm surgeons with a powerful and versatile
arsenal for intraoperative NIR imaging in real time.
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Keywords: Imageꢀguided surgery, pentamethine cyanine, nearꢀinfrared fluorescence, electron
donating/withdrawing, endocrine/exocrine.
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Introduction
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During endocrine surgeries and intricate surgical resections, surgeons mostly rely on their naked
eye and experience during oftenꢀlengthy procedures to avoid sensitive glands and tissues.1ꢀ3 Neglecting
to carefully navigate the surgical field can lead to poor patient prognosis even morbidity.4ꢀ11 Endocrine
and exocrine tissues regulate the body’s hormone levels and any perturbation, especially transection, can
jeopardize the patient’s ability to recover after surgery and maintain proper hormone levels.12ꢀ15 The
important task of avoiding sensitive tissues is often difficult as these small and imperative glands are
often obscured by blood and surrounding tissues. Surgeons require an imaging modality that can allow
them to visualize these tissues during cancer resection surgeries to improve the surgical success rate,
lower overall fatalities and advance patient prognoses.
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Nearꢀinfrared (NIR) fluorescence guided resection of cancerous tissues has demonstrated
significant promise with increasing advancements being reported over the recent years.15ꢀ27
Unfortunately, research efforts have been overwhelmingly concentrated on developing cancerꢀspecific
NIRꢀfluorophores with optimal optical, physicochemical and targeting properties. This only satisfies
oneꢀhalf of the main objective: the ability to simultaneously image the surgical field with both a diseaseꢀ
targeted fluorophore and a nativeꢀtissue targeted fluorophore of distinct wavelengths that can both be
simultaneously and separately detected for direct dualꢀtarget imaging in real time.
Significant advancements have been made to develop NIRꢀfluorescent compounds that target
sensitive endogenous tissue and help surgeons avoid these hormonal glands in real time without
changing the overall look of the surgical field. The class of NIRꢀfluorescent cyanine dyes has shown
excellent promise in the area of NIRꢀfluorescence image guided surgery for in vivo tumor targeting.
Cyanine chromophores are broadly defined by having two nitrogenꢀcontaining heterocycles that are
connected through an electron deficient polymethine bridge with a delocalized monocation conjugated
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between both nitrogen atoms. The wavelengths associated with these compounds are heavily dependent
on the length of the polymethineꢀbridge and the terminal heterocyclic moieties. Polymethine cyanines
are chemically stable NIRꢀfluorescent compounds, and we have investigated the pentamethine class of
compounds previously16,20 and herein for laying the groundwork for endogenous tissue targeting. The
heptamethine cyanine class (~800 nm fluorescence) has been more extensively explored for disease
targeting through the development of zwitterionic decorated heptamethine cyanines linked with a
propinoic acid and conjugated with a small cyclic peptide (cRGDꢀZW800ꢀ1).10,21,22 The longer NIR
wavelengths (i.e. 800 nm) are more easily detected against the background and usually reserved to
detect occult cancerous metastasis due to the minimal autofluorescence. We plan to further exploit the
700 nm channel (pentamethine cyanines) for native tissue imaging for dual channel intraoperative
imaging.
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Toward achieving this goal, we have continued to harness the structureꢀinherent tissue affinity to
determine the innate biodistribution and targeting by modulating overall molecular characteristics such
as hydrophobicity (determined through LogD calculations), total polar surface area, molecular
size/weight and the electronic contributions (electron donating/withdrawing characteristics).15,24,26 The
compounds synthesized and analyzed herein vary by their heterocyclic moieties (electron donating or
withdrawing) and various halogenation at the central carbon atom of the polymethine chain which alters
the net dipole, volume, polarization and even the overall shape of the compounds. We expect these
selected modifications to offer biological perturbation toward understanding the molecular
characteristics for targeting specific tissues. Correlating these molecular descriptors with in vivo
biodistribution in expected to result in a clinically significant paradigm in the quest of translating NIRꢀ
targeted contrast agents into the clinic, and every analyzed compound offers another evidentiary piece to
the puzzle.
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Results and Discussion
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Synthesis of Pentamethine Fluorophores. Since it has been previously reported that halogenated
hydrocarbons localize in higher concentration within endocrine and exocrine glands compared to their
nonꢀhalogenated counterparts,16 we decided to probe the effect of halogenation on pentamethine
cyanines for endocrine and exocrine tissue targeting. For a direct comparison, we synthesized electronꢀ
donating counterparts that could offer insight into how potential electronic factors present in the
compounds may influence biodistribution. We selected methoxy and methyleneꢀdioxy heterocyclic
groups for a direct comparison to the halogenated counterparts and fully expected perturbation in the
biological distribution profile, but we could not predict the results of these modifications. Understanding
the effects that altering physicochemical properties imparts on the distribution adds important
fundamental knowledge about future targetingꢀability for this and many other classes of imaging agents.
We have developed several optimized synthetic routes for maximizing the structural diversity in
the pentamethine cyanine pharmacophore. The compounds with neutral effects and electron with
drawing groups are synthesized starting with the corresponding phenyl hydrazine salts being refluxed in
the presence of 3ꢀmethylꢀ2ꢀbutanone in acidic conditions. This reaction furnishes the indolenines 2ꢀ5 in
excellent yield. We then alkylated the obtained oil using iodomethane in acetonitrile, which yields the
cationic salt 6ꢀ10 bearing an acidic methylene proton. We also separately react aniline with a warm
ethanolic solution of either mucochloric or mucobromic acid to obtain the pentamethine precursor 12 or
13 with 11 being commercially obtained. These two compounds are individually combined in the
presence of acetic anhydride and either triethylamine (fluorineꢀcontaining compounds) or sodium
acetate and are heated to afford the brilliant blue pentamethine fluorophores.
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The electron donatingꢀcontaining methoxyꢀsubstituted compounds are prepared through an
identical synthetic route; however, the final step requires a longer reaction time at a slightly elevated
temperature, due to the methoxy group electron donating effects which increases the pKa of the
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methylene proton resulting in reduced reactivity of the methylene carbon. We
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complementary set of compounds bearing heterocyclic donating groups features the methylene dioxy
ring shown in compounds 36ꢀ38. These compounds are prepared using an alternate synthetic strategy
beginning with the acidic bromination of 3ꢀmethylꢀ2ꢀbutanone followed by SN1 replacement of the
tertiary bromine by the methylene dioxyaniline compound. Upon heating in the presence of acid
catalyst, paraꢀtoluenesulphonic acid, the compound forms the cyclic methylene dioxy 2,3,3ꢀ
trimethylindolenine ring through a largely unexplored BischlerꢀMöhlau method that can be alkylated
using aforementioned conditions for the formation of compound 35. The final methylene dioxy
compounds 36ꢀ38 were synthesized using similar chemistry as the methoxy group.
Optical and Physicochemical Properties. The optical properties, shown in Table 1, were performed in
several solvents to determine the in vivo success for the fluorophores. We can see that the compounds
with hydrogen, chlorine, bromide and methoxy groups (14ꢀ25, 31ꢀ33) exhibit sharp and NIR absorbance
bands with very high molar absorptivity all being greater than 100,000 Mꢀ1cmꢀ1which suggests that these
fluorophores will satisfactorily absorb NIR wavelengths in vivo and will be compatible with the
intraoperative imaging system. As expected, the methylenedioxy compounds exhibited slightly lower
molar absorptivity due to a broadening of the absorption profile which results from a more widely
dispersed energy subꢀlevels. Similarly, the trifluoromethyl substituted compounds are very poor at
absorbing light at their wavelength of maximum absorbance as indicated by their low molar absorptivity
values in Table 1. We attribute this to the high electron withdrawing characteristics of the trifluromethyl
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groups which lowers the overall probability of electron movement across the methineꢀbridge connecting
the azaꢀheterocycles. The in vivo success of these compounds also depends of the quantum yield, and
more importantly the molecular brightness (determined as the product of extinction coefficient and
quantum yield) of the compounds in serum; therefore, we examined the quantum yield of these
fluorophores in fetal bovine serum (FBS). Compounds 14ꢀ28, 31ꢀ33 exhibit high quantum yield values
in serum which is very appealing. All of the synthesized compounds have sufficiently high molecular
brightness values in serum with many compounds exhibiting molecular brightness values of >20,000.
We observed an overall trend for the majority of compounds that as the size of the central halogen
increases, the molecular brightness decreases. When analyzing the minimized threeꢀdimensional
structures, we see that the mesoꢀhalogenation perturbs the transꢀpseudoꢀalkene angles and the elongated
structure of the fluorophore which actually shortens the overall length and perturbs the conjugated
bridge by requiring the compound to bend to accommodate this central substitution. This is an
interesting finding since upon first glance, it seems that adding larger groups to the middle of the
compound would result in an elongation of the compound simply because it would force the neighboring
hydrogen atoms apart. Another potential explanation for the decrease in molecular brightness is the
heavy atom effect that can effectively stabilize the triplet state allowing for a higher probability for
competing decay processes of the excited state (i.e. phosphorescence or energy transfer) compared to the
nonꢀhalogenated fluorophores .
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Photostability studies were performed on a select number of contrast agents to observe their
performance when continuously irradiated and can be seen in Figure 1. The selected compounds
featuring a hydrogen atom at the meso position of the polymethine bridge showed little decomposition
after being exposed to light for 48 hours, however, when chlorine or bromine replace the hydrogen,
there is noticeable degradation of up to 35% of the original absorbance. Only a minimal 4% absorbance
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was lost for each of the first compounds in each set; furthermore, this data helps conclude that these
compounds would be applicable for longꢀduration surgical resections with the ability to absorb and
fluoresce NIRꢀlight for high contrast imaging with a halfꢀlife of >48h for the entire set studied. The
studies were compared to the same concentration of dyes kept in dark containers, but the same distance
from the lamp to determine if thermal degradation contributed as well. We found an absence in
measurable degradation for the samples kept in the dark which leads us to conclude that heat from the
lamp imparts a negligible effect and the absorbance decrease comes from irradiation.
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StructureꢀInherent Targeting Ability of NIR Fluorophores. As native tissue targeting remains
predominantly understudied, we focused our attention to the salivary, thyroid, pituitary, adrenal glands,
lymph nodes and pancreas. As these tissues facilitate the regulation of human hormone levels, they are
important for longꢀterm health and should be carefully avoided during surgical resection in surrounding
areas.
After in vivo studies, we found compounds that exhibited high SBR in targeted tissues (high is
designated as a ratio of target signal to surrounding tissue being >2.0). Without surface or subcellular
receptors readily available for the particular native tissues, it has been difficult to infer mechanistic
insight of how these fluorophores localize within their respective tissues. However, we have found
several key structural components that help facilitate structureꢀinherent biodistribution and localization
in important tissues, including the salivary, thyroid, pituitary, adrenal glands, lymph nodes and pancreas.
In surgical resections such as thyroidectomies, it is crucial to remove the entire thyroid gland
while maintaining complete integrity of the parathyroid gland. Selective thyroid gland imaging is
paramount in the ability to avoid partial thyroid resection or accidental parathyroid transection which
can cause several abnormal human conditions such as hypocalcemia.
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The common clinical indications of salivary gland imaging are pain and swelling. Imaging is
useful in identifying the masses of salivary glands and also in differentiating them from the
masses/pathologies of adjacent cervical spaces. In proven case of salivary gland tumors, imaging helps
in delineating the extent of the lesion and invasion of adjacent cervical spaces. Cancerous tissues in this
area can grow quickly and surgical resection of the diseased tissue is needed while sparing excision of
the exocrine salivary glands themselves.
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Toward finding the thyroid and salivary glands targeting by using NIR fluorophores, we
evaluated the imaging capability of these compounds in thyroid and salivary glands. Table 3 shows the
thyroid and salivary glands targeting for the entire set of compounds. The targeting of these two tissues
seemed to parallel each other suggesting a biological connection between these two tissues, though
despite an extensive literature review, an explanation for this remains unclear. Among the entire set of
prepared compounds we found that central halogenation reduced the targeting efficacy into these glands.
Surprisingly, the parent set of compounds (14ꢀ16) all targeted the salivary gland with superior SBR.
From our data, we can see overall that the salivary glands are sensitive to the central halogenation but
remain tolerant to the heterocyclic halogen incorporation. Compounds 20ꢀ25 exhibit the halogen trend
we observed. With the heterocyclic halogen substitution the salivary gland targeting remains high;
however, when the central halogen is incorporated within the same compounds we have a severely
diminished targeted ability (i.e. from compound 20 with SBR of >5 to compounds 21 and 22 with SBR
of 1ꢀ2). Specifically compounds 17ꢀ25 shown in Figures 2 and 3 exemplify this trend. Compounds 17,
20, 23 do not feature halogens at the central carbon of the polymethine chain and exhibit the most
effective thyroid and salivary gland localization. As the halogen incorporation increases, the thyroid and
salivary gland localization is forfeited in deference to nonꢀspecific accumulation and high liver uptake
due to hepatobiliary clearance arising from the increase in hydrophobicity.
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As one of the important endocrine glands, the pituitary gland (dubbed the master gland) is
responsible for controlling the hormonal release of signaling chemicals throughout the body.
Transecting this precious gland during surgery causes lasting side effects from hormone dysregulation
with imbalances perpetuating during the lifetime of the patent. Tumors associated with the pituitary
gland can interfere with signaling the release of many hormones such as thyroidꢀstimulating hormone
which directly influences metabolic activity, energy levels, growth hormones and the nervous system.
Efforts to remove pituitary gland tumors must critically and clearly image the native tissue to prevent
accidental damage. We found excellent pituitary gland targeting with the trifluoromethyl compounds 26ꢀ
28 (Figure 4). The trifluoromethyl substitutions increase the dipole of the compound considerably (Table
2) which we anticipated to elicit a drastic biological response compared to the alternate halogenated
counterparts. Indeed, the vast change in electronic dipole from 1ꢀ4 debye (other compounds) to >7.5
debye in the trifluoromethyl substituted compounds (which also corresponds to an increase in
polarizability) lowered the target recognition to some endocrine tissues; however, the thyroid gland
targeting remained elevated when the central position was halogenꢀfree.
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Lymph nodes are widely distributed and are responsible for immune system function and can
indicate an abnormal physiological condition. For example, an increase in size may indicate leukemia
due to increased trafficking of lymphocytes from the blood into the lymph nodes. Additionally, thorough
examination of the lymph nodes is crucial for the staging of cancer as tumor metastases normally first
localize in sentinel lymph nodes. For surgical avoidance and the frequent necessity to biopsy, a NIR
imaging technique to visualize this tissue is highly important. In the developed set of compounds, we
noticed effective targeting of the lymph node by the methoxy derivatives 31ꢀ33 which is surprising in
the context of the methylene dioxy compounds 36ꢀ38 exhibiting low lymph node targeting. Interestingly
we see an inverse effect of the previous set of compounds where the meso halogenation increases the
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imaging signal as compared to the surrounding tissue in the lymph node with 32 and 33 having a SBR of
>5 as indicated by the +++ in Table 3. Figure 5 shows two successful examples of nodal imaging in the
inguinal and lumbar nodes with compound 33 exhibiting excellent targeting characteristics.
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The adrenal glands, located above the kidney, produce essential and nonessential hormones that
primarily control the body’s metabolism, blood pressure and stress response. In Figure 6, we observed
that the methylene dioxy compounds 36ꢀ38 exhibited adrenal gland targeting characteristics (++, SBR 3ꢀ
5) that also seemed to be tolerant to the addition of mesoꢀhalogens. It seems that the logD values
predominantly govern the localization of these compounds as the 5 compounds (31, 32, 36ꢀ38) with the
lowest logD were the most effective at targeting this tissue with the methylene dioxy compounds 36ꢀ38
prevailing as the best contrast agents for the adrenal gland across the series. This finding is also true in
their pancreas targeting ability as all three compounds within this set exhibit excellent SBR in pancreatic
tissue. Interestingly, the small change from methoxy 31ꢀ33 to the locked ring structure of 36ꢀ38 alters
the targeting ability of the compound (i.e. comparing 33 and 38). This finding also corroborates our
previous results that pancreatic targeting requires low comparative logD values. Indeed the highest
pancreas targeting characteristics are from compounds exhibiting comparatively low logD values (i.e.
compounds 36ꢀ38, logD 2.80ꢀ3.21). Additionally, the central halogenation seems to lower pancreasꢀ
targeting substantially except for the methylene dioxy set of compounds. It is very interesting that
independent halogenation (either central or terminal heterocycles) does not seem to greatly perturb the
pancreatic tissue uptake, except for when the trifluoromethyl group is incorporated. Halogenating both
positions, however, has a detrimental effect on the compoundꢀpancreas localization. For example,
compounds 15, 16, 17, 20, 23 are all effective at pancreas targeting and only have halogens at either the
heterocycles or bridge. Compounds 18, 19, 21, 22, 24, 25 with all sites halogenated exhibit reduced
pancreas uptake. We attribute this effect to an increase in overall hydrophobicity imparted by too many
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halogens on the core structure. Additionally, structures with higher molecular weight in addition to
greater hydrophobic character seem to be poor at pancreatic localization.
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Preliminary cytotoxicity of NIR fluorophores. To confirm cell cytotoxicity of the tissueꢀspecific
fluorophores, NIH/3T3 mouse fibroblast cells were incubated with the selected NIR fluorophores,
o
respectively, for 1 h at 37 C and gently washed with DMEM. Then, the viability of fibroblasts was
assessed by the alamarBlue assay. The assay system incorporates an oxidationꢀreduction (REDOX)
indicator that both fluorescence and changes color in response to chemical reduction of growth medium
resulting from cell growth. As shown in Figure 7, over 75% of the cells were viable when treated with
NIR fluorophores at concentrations of 2 ꢁM and 10 ꢁM. Therefore, notable cell death was not observed
even with the high concentration of the NIR fluorophores. These results confirm that tissueꢀspecific NIR
fluorophores do not show obvious cytotoxic effects.
Conclusions
Toward satisfying the clinical need for native tissue targeted contrast agents in realꢀtime NIR imageꢀ
guided surgery, we present compounds that are effective at imaging the salivary, thyroid, pituitary,
adrenal glands, lymph nodes and pancreas. We are confident that this work will lay the foundation for
development of native tissue contrast agents for clinical use.
Experimental Details
Synthesis of pentamethine cyanine analogs. The chemical reagents used in the synthesis of these
compounds were obtained from Acros Organics, Alfa Aesar and Matrix Scientific. The reactions were
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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
60ꢀ200 ꢁm, 60A, 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 Bruker Avance (400 MHz) spectrometer using DMSOꢀd6 or MeODꢀ
d4 containing tetramethylsilane (TMS) as an internal calibration standard set to 0.0 ppm. UVꢀVis/NIR
absorption spectra were recorded on a Varian Cary 50 spectrophotometer. Highꢀresolution accurate mass
spectra (HRMS) were obtained either at the Georgia State University Mass Spectrometry Facility using
a Waters QꢀTOF micro (ESIꢀQꢀTOF) 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 DeltaꢀPak 5 ꢁM 100A 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. The integral under the curve was determined for both the LC and ELSD spectra and
was used to confirm the purity (>95%) of the synthesized contrast agents.
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Optical and physicochemical property analyses. All optical measurements were performed in various
solvents, including ethanol, dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS, pH 7.4) and at
37oC 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 RFꢀ5301PC spectrofluorometer (350–
1000 nm). For fluorescence quantum yield (QY) measurements, rhodamine 800 in absolute ethanol (QY
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= 28%) was used as a calibration standard, under conditions of matched absorbance at 620 nm. In silico
calculations of physicochemical distribution coefficient (logD at pH 7.4) was calculated using Marvin
and JChem calculator plugins (ChemAxon, Budapest, Hungary). Electrostatic maps were calculated
using Spartan DFT calculations at the B3LYP level.
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Photostability analysis. The photostability experiments were performed to determine the photo
bleaching threshold of the fluorophores. We determined the photobleaching rate by measuring the
decrease in absorbance at the wavelength of maximum absorption over a 48h time period. The light
condition involved a glass cell containing individual contrast agent (0.01 mm in methanol) was affixed
250mm away from a 15W F15T8 broad spectrum bulb being irradiated using a portable lamp at room
temperature. The dark control was also examined to exclude chemical decomposition phenomena. The
absorbance values were measured at various time points and then plotted versus time to obtain the
photostability graphs in light and dark.
LCꢀMS analysis. The purity of all compounds was measured using liquid chromatographyꢀmass
spectrometry (LCꢀMS) on a Waters system consisting of a 1525 binary HPLC pump with a manual
7725i Rheodyne injector, a 996 Photodiode Array (PDA) detector, and a 2475 multiwavelength
fluorescence detector. The column eluent was divided in 2 using a flow splitter (Upchurch Scientific). A
portion of the eluent flowed into an ELSD (Richards Scientific) while the rest flowed into a Micromass
LCT ESIꢀTOF spectrometer (Waters) equipped with a Symmetry (R) C18 (4.6 x 150 mm, 5 ꢁm)
reverseꢀphase HPLC column. For mass spectrometry mobile phase was solvent A = 0.1% formic acid
(FA) in water and solvent B = CH3CN with 95% A for 5 min and a linear gradient from 5% to 40%
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CH3CN (from A to B for 30 min) at a flow rate of 1 mL/min, capillary voltage was ꢀ3317V, and sample
cone voltage was ꢀ50V.
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Cell viability assay. The cell toxicity and proliferation were assessed by alamarBlue® (Thermo
Scientific) assay. The NIH/3T3 fibroblasts were seeded onto 96ꢀwell plates (1 × 104 cells per well). To
test cytotoxicity depending on the fluorophore type and concentration, cells were treated with 2 and 10
ꢁM of each NIR fluorophore (n = 6) for 1 h and cultured at 24 h postꢀtreatment. On each assay time
point, the incubation cell media were replaced with 100 ꢁL of fresh media. 10 ꢁL of alamarBlue solution
was directly added to 100 ꢁL each well and incubated for 4 h at 37 ◦C in a humidified 5% CO2 incubator.
Finally, the 96ꢀwell plates were placed into a microplate reader (Spark 10M, TECAN, Switzerland) for
measuring the absorption intensity at 570 nm and fluorescence intensity at 590 nm. The data were
presented by dividing by dyeꢀuntreated control group. The statistical significance was determined by
oneꢀway analysis of variance (ANOVA).
Animal models and biodistribution. Animals were housed in an AAALACꢀcertified facility and were
studied under the supervision of BIDMC’s IACUC in accordance with the approved institutional
protocol #057ꢀ2014. Male CDꢀ1 mice (20–30 g, 6ꢀ8 weeks, Charles River Laboratories, Wilmington,
MA) were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine intraperitoneally (Webster
Veterinary, Fort Devens, MA). For intraoperative imaging and biodistribution, the imaging system
equipped with a custom filter set (Chroma Technology Corporation, Brattleboro, VT) composed of a
650 ± 22 nm excitation filter, a 680 nm dichroic mirror, and an 710 ± 25 nm emission filter was used at
a fluence rate of 4 mW/cm2, with white light (400ꢀ650 nm) at 40,000 lx. Color and NIR fluorescence
images were acquired simultaneously with custom software at rates up to 15 Hz over a 15 cm diameter
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field of view. The imaging system was positioned at a distance of 18 inches from the surgical field. For
each experiment, camera exposure time and image normalization was held constant.
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Quantitative analysis. At each time point, the fluorescence and background intensity of a region of
interest (ROI) over each tissue was quantified using custom imaging software and ImageJ software
(NIH, Bethesda, MD) version 1.45q. The signalꢀtoꢀbackground ratio (SBR) was calculated as SBR =
fluorescence/background, where background is the signal intensity of neighboring tissues such as
muscle or skin obtained over the period of imaging time. All NIR fluorescence images for a particular
fluorophore were normalized identically for all conditions of an experiment. At least three animals were
analyzed at each time point. Statistical analysis was carried out using the unpaired Student’s tꢀtest or
oneꢀway ANOVA. Results were presented as mean ± s.d. and curve fitting was performed using Prism
version 4.0a software (GraphPad, San Diego, CA).
Synthesis and Characterization of NIR Fluorophores.
To obtain the final dyes 14ꢀ28 and 31ꢀ33 and 36ꢀ38, the precursor salts 6ꢀ10, 30 and 35 (2 mol.
eq.) were added to a clean, dry round bottom flask. The compounds were stirred in 10 mL of acetic
anhydride followed by either sodium acetate or triethyl amine (3 molar equivalents) were added along
with 1 molar equivalent of individual malondialdehyde linker 11ꢀ13. The reaction was allowed to stir at
40ꢀ60oC for 2h. The reactions were monitored closely using regular phase thin layer chromatography
with a mobile phase of DCM/MeOH (99:1) as well as UVꢀVisꢀNIR spectrophotometer in quartz cuvettes
with methanol as a solvent to visualize the absorption band at ~650 nm against that of the starting
materials ~400 nm. Upon completion of the reaction, the mixtures were allowed to cool, and the solvent
was evaporated. The residue was extracted using DCM and was washed with DI water (3 x 70 mL). The
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resulting organic layer was dried under magnesium sulfate, gravity filtered and evaporated to afford
crystals that were washed with diethyl ether (2 x 50 mL) and hexanes (50 mL) to yield crystals that were
dried under vacuum overnight. The pure product was obtained after dissolving in methanol and
precipitating with ether several times or the compounds were isolated using flash column
chromatography and 5% methanol in DCM as the eluting solvent. After purification, the compounds
were obtained in the designated yields and were fully characterized.
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1,3,3-Trimethyl-2-((1E,3E,5E)-5-(1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-3H-
o
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indol-1-ium iodide (14): Yield 64%, MP >260 C ; H NMR (400 MHz, MeODꢀd4) δ: 1.71 (s, 12H),
3.63 (s, 6H), 6.28 (d, J = 16.0 Hz, 2H), 6.65 (t, J = 12.0 Hz, 1H), 7.24 (t, J = 8.0 Hz, 2H), 7.29 (d, J =
8.0 Hz, 2H), 7.39 (t, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 8.25 (t, J = 12.0 Hz, 2H); 13C NMR (100
MHz, MeODꢀd4) δ 27.90, 31.71, 50.50, 104.44, 111.83, 123.31, 126.20, 129.71, 142.56, 144.29, 155.52,
175.28. TOF HRMS m/z [M]+ calculated for [C27H31N2]+ 383.2487, found 383.2474.
2-((1E,3Z,5E)-3-Chloro-5-(1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-
1
3H-indol-1-ium iodide (15): Yield 64%, M.P. 230ꢀ232 °C, H NMR (400 MHz, MeODꢀd4) δ 1.76 (s,
12H), 3.71 (s, 6H), 6.435 (d, J = 12 Hz, 2H), 7.32 (t, J = 8 Hz, 2H), 7.39 (d, J = 8 Hz, 2H), 7.45 (t, J = 8
13
Hz, 2H), 7.54 (d, J = 8 Hz, 2H), 8.345 (d, J = 12 Hz, 2H). C NMR (100 MHz, MeODꢀd4) δ 25.03,
29.53, 48.55, 98.93, 110.02, 121.00, 121.86, 124.55, 127.43, 140.42, 141.67, 146.72, 174.28. TOF
HRMS m/z [M]+ calculated for [C27H30N2Cl]+ 417.2098 found 417.2107.
2-((1E,3Z,5E)-3-Bromo-5-(1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-trimethyl-
o
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3H-indol-1-ium iodide (16): Yield 61%, M.P. 230ꢀ232 C ; H NMR (400 MHz, CDCl3ꢀd1) δ 1.91 (s,
12H), 3.82 (s, 6H), 6.36 (d, J = 8 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H, 7.26ꢀ7.31 (m, 4H), 7.39ꢀ7.44 (m, 4H)
8.93 (d, J = 12 Hz, 2H). 13C NMR (100 MHz, DMSOꢀd6) δ 27.04, 31.98, 49.85, 102.61, 112.19, 115.98,
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122.94, 125.97, 128.93, 141.78, 143.02, 149.64, 175.14. TOF HRMS m/z [M]+ calculated for
[C27H30N2Br]+ 461.1592 found 461.1585.
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5-Fluoro-2-((1E,3E,5E)-5-(5-fluoro-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-
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trimethyl-3H-indol-1-ium iodide (17): Yield 78%, MP >260 oC, HꢀNMR(400 MHz, DMSOꢀd6) δ 1.68
(s, 12H), 3.59 (s, 6H), 6.23 (d, J = 12.0 Hz, 2H), 6.52 (s, 1H), 7.24 (s, 2H), 7.39 (s, 2H), 7.62 (s, 2H),
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8.29 (s, 2H). CꢀNMR (100 MHz, DMSOꢀd6) δ: 27.27, 31.85, 49.54, 103.64, 103.76, 110.82, 111.07,
112.43, 112.49, 112.59, 112.66, 115.15, 125.54, 139.55, 143.60, 143.69, 154.27, 154.29, 159.32, 161.72,
173.64. TOF HRꢀMS ESI m/z [M]+ calculated for [C27H29F2N2]+ 419.2299, found 419.2296.
2-((1E,3Z,5E)-3-Chloro-5-(5-fluoro-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-
fluoro-1,3,3-trimethyl-3H-indol-1-ium (18): Yield 37%, MP >260 oC, 1HꢀNMR(400 MHz, DMSOꢀd6) δ:
1.72 (s, 12H), 3.67 (s, 6H), 6.26 (d, J = 14 Hz, 2H), 7.29,7.31 (t, J = 6.8 Hz, 8.8 Hz, 2H), 7.52ꢀ7.54 (m,
2H), 7.70 (d, J = 6 Hz, 2H), 8.41 (d, J = 14 Hz, 2H). 19F NMR (375 MHz, DMSOꢀ d6) δ: ꢀ116.67 (s, 2F).
TOF HRꢀMS ESI m/z [M]+ calculated for [C27H29F2ClN2]+ 453.1909 found 453.1914.
2-((1E,3Z,5E)-3-Bromo-5-(5-fluoro-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-
fluoro-1,3,3-trimethyl-3H-indol-1-ium (19) Yield 52%, 1HꢀNMR(400 MHz, DMSOꢀd6) δ: 1.72 (s, 12H),
3.67 (s, 6H), 6.26 (d, J = 13.2 Hz, 2H), 7.32 (t, J = 8.8 Hz, 2H) , 7.55ꢀ7.52 (m, 2H), 7.70 (d, J = 7.6, 2H),
8.46 (d, J = 13.6, 2H). 19F NMR (375 MHz, DMSOꢀ d6) δ: ꢀ116.67 (s, 2F). TOF HRꢀMS ESI m/z [M]+
calculated [C27H29F2N2Br]+ was 497.1404 found 497.1394.
5-Chloro-2-((1E,3E,5E)-5-(5-chloro-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-
trimethyl-3H-indol-1-ium iodide (20): Yield 59%, MP >260 oC, 1HꢀNMR(400 MHz, DMSOꢀd6) δ: 1.69
(s, 12H), 3.59 (s, 6H), 6.27 (d, J = 14 Hz, 2H), 6.551 (t, J = 12.4, 12.0 Hz, 1H), 7.39 (d, J = 8.8 Hz, 2H),
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7.46 (d, J = 8.4 Hz, 2H), 7.80 (s, 2H), 8.32 (t, J = 13.2 Hz, 2H). CꢀNMR (100 MHz, DMSOꢀd6) δ:
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27.30, 31.84, 49.51, 104.09, 112.87, 123.29, 126.16, 128.67, 129.50, 142.23, 143.54, 154.72, 173.65.
TOF HRꢀMS ESI m/z [M]+ calculated for [C27H29Cl2N2]+ 451.1708, found 451.1722.
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yl)-1,3,3-trimethyl-3H-indol-1-ium (21): Yield 53%, MP >260 C, HꢀNMR(400 MHz, DMSOꢀd6) δ:
1.72 (s, 12H), 3.67 (s, 6H), 6.26 (d, J = 14 Hz, 2H), 7.52 (m, 4H), 7.89 (s, 2H), 8.43 (d, J = 13.6, 2H).
13CꢀNMR (100 MHz, DMSOꢀd6) δ: 26.81, 32.15, 49.98, 100.72, 113.62, 123.06, 123.49, 128.80, 130.31,
142.02, 143.83, 147.96, 174.97. TOF HRꢀMS ESI m/z [M]+ calculated for [C27H29Cl3N2]+ 485.1318
found 485.1317.
2-((1E,3Z,5E)-3-Bromo-5-(5-chloro-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-
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chloro-1,3,3-trimethyl-3H-indol-1-ium iodide (22) Yield 69%, MP 252ꢀ254 C HꢀNMR(400 MHz,
DMSOꢀd6) δ: 1.73 (s, 12H), 3.67 (s, 6H), 6.28 (d, J = 13.2 Hz, 2H), 7.52 (m, 4H), 7.89 (s, 2H), 8.47 (d,
J = 13.2 Hz, 2H). 13CꢀNMR (100 MHz, DMSOꢀd6) δ: 26.79, 32.16, 50.03, 102.96, 113.65, 116.40,
123.51, 128.82, 130.35, 141.99, 143.83, 149.98, 175.15. TOF HRꢀMS ESI m/z [M]+ calculated
[C27H28BrN2Cl2]+ was 529.0813 found 529.0810.
5-Bromo-2-((1E,3E,5E)-5-(5-bromo-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-1,3,3-
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trimethyl-3H-indol-1-ium iodide (23): Yield 62%, MP >260 C, HꢀNMR(400 MHz, DMSOꢀd6) δ: 1.69
(s, 12H), 3.58 (s, 6H), 6.27 (d, J = 14 Hz, 2H), 6.56 (t, J = 12.4, 12 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H),
7.59 (d, J = 8.4, 2H), 7.93 (s, 2H), 8.33 (t, J = 13.2 Hz, 2H). 13CꢀNMR (100 MHz, DMSOꢀd6) δ 27.25,
31.71, 49.48, 104.07, 113.33, 117.45, 126.09, 131.51, 142.63, 143.85, 154.86, 173.45. TOF HRꢀMS
ESI m/z [M]+ calculated for [C27H29Br2N2]+ 539.0697, found 539.0695.
5-Bromo-2-((1E,3Z,5E)-5-(5-bromo-1,3,3-trimethylindolin-2-ylidene)-3-chloropenta-1,3-dien-1-
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yl)-1,3,3-trimethyl-3H-indol-1-ium (24): Yield 72%, MP >260 C, 1HꢀNMR(400 MHz, DMSOꢀd6) δ:
1.72 (s, 12H), 3.67 (s, 6H), 6.28 (d, J = 12.0 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.63 (d, J = 8.0 Hz, 2H),
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8.01 (s, 2H), 8.43 (d, J = 12.0 Hz, 2H). 13CꢀNMR (100 MHz, DMSOꢀd6) δ: 26.81, 32.17, 49.96, 100.69,
114.05, 118.37, 123.13, 126.26, 131.63, 142.40, 144.11, 147.96, 174.78. TOF HRꢀMS ESI m/z [M]+
calculated for [C27H28Br2N2Cl]+ 573.0308, found 573.0316.
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yl)-1,3,3-trimethyl-3H-indol-1-ium iodide (25): Yield 46%, MP 231ꢀ232 C, HꢀNMR(400 MHz,
DMSOꢀd6) δ: 1.72 (s, 12H), 3.66 (s, 6H), 6.28 (d, J = 13.2 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 6.64 (d, J =
13
8.4 Hz, 2H), 8.01 (s, 2H), 8.48 (d, J = 13.6 Hz, 2H). CꢀNMR (100 MHz, DMSOꢀd6) δ: 26.79, 32.11,
50.03, 102.97, 114.07, 116.47, 118.42, 126.27, 131.65, 142.40, 144.13, 150.03, 175.01. TOF HRꢀMS
ESI m/z [M]+ calculated [C27H28Br3N2]+ was 616.9803, found 616.9814.
1,3,3-Trimethyl-5-(trifluoromethyl)-2-((1E,3E)-5-((E)-1,3,3-trimethyl-5-(trifluoromethyl)indolin-
2-ylidene)penta-1,3-dien-1-yl)-3H-indol-1-ium iodide (26): Yield 55%, MP 218ꢀ221°C, 1H NMR (400
MHz, CDCl3): δ 1.81 (s, 12H), 3.79 (s, 6H), 6.53 (d, J = 13.6 Hz, 2H), 7.11 (t, J = 13.6 Hz, 1H), 7.21(d,
J = 8.4 Hz, 2H), 7.60 (s, 2H), 7.70 (d, J = 7.6 Hz, 2H), 8.02 (t, J = 13.6, 2H). 19FNMR: (375 MHz,
DMSOꢀd6): δ 62.00. TOF HRMS m/z [M]+ calculated for [C29H29N2F6]+ 519.2297, found 519.2393.
2-((1E,3Z)-3-Chloro-5-((E)-1,3,3-trimethyl-5-(trifluoromethyl)indolin-2-ylidene)penta-1,3-dien-
1
1-yl)-1,3,3-trimethyl-5-(trifluoromethyl)-3H-indol-1-ium iodide (27): Yield 71%, MP 226ꢀ228 °C, H
NMR (400 MHz, CDCl3): δ 1.91 (s, 12H), 3.87 (s, 6H), 6.47 (d, J = 13.6 Hz, 2 H), 7.30 (s, 2H), 7.65 (m,
4H), 8.90 (d, J = 10.4 Hz, 2H). 19F NMR (375 MHz, DMSOꢀd6): δ 62.05. TOF HRMS m/z [M]+
calculated for [C29H28N2F6Cl]+ 553.1840, found 553.1996.
2-((1E,3Z)-3-Bromo-5-((E)-1,3,3-trimethyl-5-(trifluoromethyl)indolin-2-ylidene)penta-1,3-dien-
1
1-yl)-1,3,3-trimethyl-5-(trifluoromethyl)-3H-indol-1-ium iodide (28): Yield 71%, MP 226ꢀ228 °C, H
NMR (400 MHz, CDCl3): δ 1.91 (s, 12H), 3.87 (s, 6H), 6.46 (bs, 2H), 7.30 (s, 2H), 7.69 (m, 4H), 8.91
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(d, J =, 7.2 Hz, 2H). F NMR (375 MHz, DMSOꢀd6), δ: 62.01. TOF HRMS m/z [M]+ calculated for
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[C29H28N2F6Br]+ 597.1335, found 597.1597.
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1
5-Methoxy-2,3,3-trimethyl-3H-indole (29): Yield 78%, reddish brown oil; H NMR (400MHz,
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DMSOꢀd6) δ 1.17 (s, 6H), 2.16 (s, 3H), 3.68 (s, 3H), 6.74 (m, 2H), 7.38 (s,1H).
1
5-Methoxy-1,2,3,3-tetramethyl-3H-indol-1-ium iodide (30): H NMR (400 MHz, DMSOꢀd6) δ
1.51 (s, 6H), 2.71 (s, 3H), 3.86 (s, 3H), 3.94 (s, 3H), 7.14 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 7.81 (d, J =
8.0 Hz, 1H).
5-Methoxy-2-((1E,3E,5E)-5-(5-methoxy-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-
1,3,3-trimethyl-3H-indol-1-ium iodide (31): Yield 75%, MP 228ꢀ230 °C, 1H NMR (400 MHz, DMSOꢀ
d6): δ 1.66 (s, 12H), 3.56 (s, 3H), 3.81 (s, 6H), 6.17 (d, J = 12 Hz, 2H), 6.46 (t, J = 12 Hz, 1H), 6.95 (d, J
= 8 Hz, 2H), 7.30ꢀ7.28 (m, 4H), 8.23 (t, J = 12 Hz, 2H). 13C NMR (100 MHz, DMSOꢀd6): δ 26.48,
30.62, 48.45, 55.29, 102.06, 108.43, 111.02, 112.84, 123.73, 135.80, 142.15, 151.94, 156.98, 171.55.
TOF HRMS m/z [M]+ calculated for [C29H35N2O2]+ 443.2699, found 443.2692.
2-((1E,3Z,5E)-3-Chloro-5-(5-methoxy-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-
methoxy-1,3,3-trimethyl-3H-indol-1-ium iodide (32): Yield 80%, MP 235ꢀ237 C, 1H NMR (400 MHz,
o
MeODꢀd4) δ 1.73 (s, 12H), 3.67 (s, 6H), 3.85 (s, 6H), 6.34 (d, J = 12 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H),
7.14 (s, 2H), 7.29 (d, J = 8.0 Hz, 2H), 8.23 (d, J = 12 Hz, 2H). 13C NMR (100 MHz, DMSOꢀd6) δ 27.05,
31.96, 49.88, 56.33, 99.74, 109.36, 112.76, 114.07, 121.81, 136.50, 143.49, 146.18, 158.51, 173.76.
TOF HRMS m/z [M]+ calculated for [C29H34N2O2Cl]+ 477.2309; found 477.2327.
2-((1E,3Z,5E)-3-Bromo-5-(5-methoxy-1,3,3-trimethylindolin-2-ylidene)penta-1,3-dien-1-yl)-5-
o
1
methoxy-1,3,3-trimethyl-3H-indol-1-ium iodide (33): Yield 55%, MP 214ꢀ216 C, H NMR (400 MHz,
DMSOꢀd6): δ 1.69 (s , 12H), 3.63 (s, 6H), 3.81 (s, 6H), 6.18 (d, J = 12 Hz, 2H), 6.97 (d, J = 8.0 Hz, 2H),
7.35 (s, 2H), 7.40 (d, J = 8 Hz, 2H), 8.38 (d, J = 12 Hz, 2H). 13C NMR (100 MHz, DMSOꢀd6): δ 26.20,
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31.16, 49.13, 55.53, 101.20, 108.56, 112.00, 113.28, 114.41, 135.67, 142.68, 147.32, 157.72, 173.09.
TOF HRMS m/z [M]+ calculated for [C29H34N2O2Br]+ 521.1804 found 521.1801.
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5,7,7-Trimethyl-6-((1E,3E,5Z)-5-(5,7,7-trimethyl-5H-[1,3]dioxolo[4,5-f]indol-6(7H)-
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ylidene)penta-1,3-dien-1-yl)-7H-[1,3]dioxolo[4,5-f]indol-5-ium iodide (36): Yield 38%, H NMR (400
MHz, DMSOꢀd6) δ: 1.63 (s, 12H), 3.54 (s, 6H), 6.06 (s, 4H), 6.18 (d, J = 16.0 Hz, 2H), 6.46 (t, J = 16.0
13
Hz, 1H), 7.13 (s, 2H), 7.28 (s, 2H), 8.16 (t, J = 15 Hz, 2H). C NMR (100 MHz, DMSOꢀd6) δ: 27.59,
31.89, 49.29, 94.35, 102.18, 103.40, 104.03, 125.11, 134.93, 137.52, 145.55, 148.02, 152.68, 173.32.
TOF HRMS m/z [M]+ calculated for [C29H31O4N2]+ 471.2278 found 471.2268.
6-((1E,3Z,5E)-3-Chloro-5-(5,7,7-trimethyl-5,7-dihydro-6H-[1,3]dioxolo[4,5-f]indol-6-
ylidene)penta-1,3-dien-1-yl)-5,7,7-trimethyl-7H-[1,3]dioxolo[4,5-f]indol-5-ium (37): Yield 39%, 1H
NMR (400 MHz, DMSOꢀd6) δ: 1.66 (s, 12H), 3.63 (s, 6H), 6.10 (s, 4H), 6.21 (d, J = 13.6 Hz, 2H), 7.29
(s, 2H), 7.38 (s, 2H), 8.30 (d, J = 13.6 Hz, 2H). 13C NMR (100 MHz, DMSOꢀd6) δ: 27.10, 32.20, 49.70,
94.92, 100.10, 102.34, 104.12, 135.49, 137.25, 146.04, 146.12, 148.10, 174.41. TOF HRMS m/z [M]+
calculated for [C29H30N2O4Cl]+ 505.1894 found 505.1871.
6-((1E,3Z,5E)-3-Bromo-5-(5,7,7-trimethyl-5,7-dihydro-6H-[1,3]dioxolo[4,5-f]indol-6-
ylidene)penta-1,3-dien-1-yl)-5,7,7-trimethyl-7H-[1,3]dioxolo[4,5-f]indol-5-ium (38): Yield 27%, 1H
NMR (400 MHz, DMSOꢀd6) δ: 1.66 (s, 12H), 3.62 (s, 6H), 6.10 (s, 4H), 6.21 (d, J = 13.6 Hz, 2H), 7.29
(s, 2H), 7.39 (s, 2H), 8.36 (d, J = 13.6 Hz, 2H). 13C NMR (100 MHz, DMSOꢀd6) δ: 27.08, 32.20, 49.76,
94.94, 102.34, 104.14, 113.95, 135.50, 137.22, 146.16, 146.16, 148.02, 174.59. TOF HRMS m/z [M]+
calculated for [C29H30N2O4Br]+ 549.1389 found 549.1368.
Supporting Information
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The Supporting Information including instrumentation, general methods, synthetic procedures, and
NMR and MS spectra is available free of charge on the ACS Publications website at pubs.acs.org.
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Abbreviations: NIR, nearꢀinfrared; LogD, distribution coefficient; SBR, signalꢀtoꢀnoise ratio;
DMEM, Dulbecco's Modified Eagle's Medium; ANOVA, analysis of variance.
Acknowledgments
This study was supported by a Georgia State University dissertation grant to E.A.O., a Brains and
Behavior grant to M.H., a Health Innovation Grant and the NIH/NIBIB grant #R01ꢀEBꢀ011523
(H.S.C.). EAO was supported through a preꢀdoctoral fellowship from the Center for Diagnostics and
Therapeutics (CDT). HH was financially supported by Chonnam National University (2015). Thank you
Andrew Levitz and Nicholas Bruschi for helping with the synthesis.
*Corresponding Authors
Maged Henary, Ph.D.
100 Piedmont Ave. #315
Atlanta, GA 30303
Office: 404ꢀ413ꢀ5566; Fax: 404ꢀ413ꢀ5505
Email: mhenary1@gsu.edu
Hak Soo Choi, Ph.D.
BIDMC, Room SLꢀ418
330 Brookline Avenue
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Boston, MA 02215
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Office: 617ꢀ667ꢀ6024; Fax: 617ꢀ667ꢀ0214
Email: hchoi@bidmc.harvard.edu
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Tables and Figures
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Table 1. Optical properties of 700 nm emitting NIR fluorophores. All measurments were performed in
dimethyl sulfoxide (DMSO), ethanol (EtOH), phosphateꢀbuffered saline (PBS), and fetal bovine serum
o
(FBS), pH = 7.4 at 37 C. Abbreviations: λabs, wavelength of maximum absorbance; λem, wavelength of
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maximum emission; QY, quantum yield; and MB, molecular brightness.
Extinction Coefficient ( , Mꢀ1cmꢀ1)
λabs (nm)
DMSO EtOH PBS FBS DMSO
λem (nm)
EtOH FBS EtOH FBS EtOH FBS EtOH
Stokes Shift
QY ( , %)
MB
x
)
ID
EtOH PBS FBS
FBS
31.0 81,161 57,195
212,500 248,200 218,800 184,500
235,300 207,800 171,000 168,800
257,330 253,200 204,400 211,700
647
645
645
645
646
642
652
653
650
653
655
643
642
644
641
671
672
669
691
689
687
645
644
641
641
643
641
649
652
648
650
653
641
638
640
638
668
671
669
690
690
687
640
642
638
638
638
635
645
647
643
653
660
635
636
638
635
658
658
655
680
678
675
649
638
634
660
659
657
663
658
658
658
664
665
670
667
657
656
656
653
696
694
689
705
716
715
662
665
660
662
660
658
669
666
663
671
671
663
654
655
651
696
692
688
716
712
716
15
15
16
22
15
17
19
14
17
20
14
16
18
16
15
28
23
20
15
26
28
13
27
26
13
22
14
9
32.7
29.2
30.1
31.6
16.5
16.3
41.5
18.3
15.4
42.4
20.8
15.5
58.7
20.5
21.1
9.6
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
31
32
33
36
37
28.8
43.6
60,677 48,614
76,213 92,301
649 216,700 225,800 196,500 164,400
638 210,900 233,700 193,400 193,400
644 173,200 210,500 170,900 145,800
660 183,400 204,200 174,200 136,000
658 192,800 207,800 140,200 136,000
655 233,100 250,600 185,000 153,700
662 181,200 174,500 144,800 141,100
648 140,200 189,600 121,500 119,700
644 169,000 169,600 166,900 159,600
35.2 71,352 58,868
15.3 38,561 29,590
11.9 34,312 17,350
57.5 84,743 78,200
28.5 38,027 38,760
17.4 38,592 26,744
57.0 73,988 80,427
23.0 39,437 27,531
11.8 26,288 18,833
89.2 26,532 27,295
47.4 23,555 9,243
29.0 10,487 2,436
11.9 19,680 16,070
8
12
9
23
21
6
648
649
644
41,200
72,000 114,900 56,300
32,400 49,700 19,600
45,200
28,700
30,600
19,500
8,400
6
7
674 152,300 205,000 133,600 135,000
676 148,100 210,000 135,900 162,000
22
16
12
18
19
25
3.9
9.7
9.7
6.5
4.2
3.1
8,190 15,710
7,218 10,570
676
94,200 154,000 82,100 109,000
85,900
5.1
698 107,200 108,100 89,200
4.7
5,081
4,698
4,808
5,584
4,948
3,875
693 147,200 156,600 127,100 117,800
691 154,900 165,800 135,700 125,000
3.0
2.9
41 38
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment