Near-Infrared Fluorescent Rosol Dye Tailored toward Lymphatic Mapping Applications

Article abstract of DOI:10.1021/acs.analchem.8b05709

An optical molecular imaging contrast agent that is tailored toward lymphatic mapping techniques implementing near-infrared (NIR) fluorescence image-guided navigation in the planning and surgical treatment of cancers would significantly aid in enabling the real-time visualization of the potential metastatic tumor-draining lymph node(s) for their needed surgical biopsy and/or removal, thereby ensuring unmissed disease to prevent recurrence and improve patient survival rates. Here, the development of the first NIR fluorescent rosol dye (THQ-Rosol) tailored to overcome the limitations arising from the suboptimal properties of the generic molecular fluorescent dyes commonly used for such applications is described. In developing THQ-Rosol, we prepared a progressive series of torsionally restrictive N-substituted non-NIR fluorescent rosol dyes based on density functional theory (DFT) calculations, wherein we discerned high correlations amongst their calculated energetics, modeled N-C3′ torsion angles, and evaluated properties. We leveraged these strong relationships to rationally design THQ-Rosol, wherein DFT calculations inspired an innovative approach and synthetic strategy to afford an uncharged xanthene core-based scaffold/molecular platform with an aptly elevated pK_a value alongside NIR fluorescence emission (ca.700-900 nm). THQ-Rosol exhibited 710 nm NIR fluorescence emission, a 160 nm Stokes shift, robust photostability, and an aptly elevated pK_a value (5.85) for affording pH-insensitivity and optimal contrast upon designed use. We demonstrated the efficacy of THQ-Rosol for lymphatic mapping with in vitro and in vivo studies, wherein it revealed timely tumor drainage and afforded definitive lymph node visualization upon its administration and accumulation. THQ-Rosol serves as a proof-of-concept for the effective tailoring of an uncharged xanthene core-based scaffold/molecular platform toward a specific imaging application using rational design.

Full text of DOI:10.1021/acs.analchem.8b05709

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Article
A NIR Fluorescent Rosol Dye Tailored
toward Lymphatic Mapping Applications
Kenneth S. Hettie, Jessica Lee Klockow, Timothy Edward Glass, and Frederick T. Chin
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05709 • Publication Date (Web): 23 Jan 2019
Downloaded from http://pubs.acs.org on January 26, 2019
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Analytical Chemistry
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A NIR Fluorescent Rosol Dye Tailored toward Lymphatic Mapping
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Kenneth S. Hettie1‡, Jessica L. Klockow1‡, Timothy E. Glass2, Frederick T. Chin1*
1Molecular Imaging Program at Stanford (MIPS), Department of Radiology, Stanford University School of Medicine,
Stanford, California 94305
2Department of Chemistry, University of Missouri, Columbia, MO 65211
ABSTRACT: An optical molecular imaging contrast agent that is tailored toward lymphatic mapping techniques implementing
near-infrared (NIR) fluorescence image-guided navigation in the planning and surgical treatment of cancers would significantly aid
in enabling the real-time visualization of the potential metastatic tumor-draining lymph node(s) for needed surgical biopsy and/or
removal, thereby ensuring unmissed disease to prevent recurrence and improve survival rates. Here, the development of the first NIR
fluorescent rosol dye (THQ-Rosol) tailored to overcome limitations arising from the suboptimal properties of the generic molecular
fluorescent dyes commonly used for such applications is described. In developing THQ-Rosol, we prepared a progressive series of
torsionally-restrictive N-substituted non-NIR fluorescent rosol dyes based on DFT calculations, wherein we discerned high
correlations amongst their calculated energetics, modeled N-C3’ torsion angles, and evaluated properties. We leveraged these strong
relationships to rationally design THQ-Rosol, wherein DFT calculations inspired an innovative approach and synthetic strategy to
afford an uncharged xanthene core-based scaffold/molecular platform an aptly-elevated pKa value alongside NIR fluorescence
emission (c.a. 700-900 nm). THQ-Rosol exhibited 710 nm NIR fluorescence emission, a 160 nm Stokes shift, robust photostability,
and an aptly-elevated pKa value (5.85) for affording pH-insensitivity and optimal contrast upon designed use. We demonstrated the
efficacy of THQ-Rosol for lymphatic mapping with in vitro and in vivo studies, wherein it revealed timely tumor drainage and
afforded definitive lymph node visualization upon administration and accumulation. THQ-Rosol serves as a proof-of-concept for the
effective tailoring of an uncharged xanthene core-based scaffold/molecular platform toward a specific imaging application using
rational design.
The favorable prognosis and successful surgical treatment of
many cancer types hinge on the accurate preoperative outlining
(demarcation) and intraoperative assessment (surgical biopsy),
delineation, and surgical removal (resection) of the malignant
tumor tissue and potential metastatic tumor-draining lymph
node(s) from visually-indistinguishable surrounding normal
tissue.1–3 The use of an intratumorally-injected contrast agent
that can drain into and accumulate within the sentinel and its
downstream lymph node(s) assists in improving patient survival
rates by enabling their visualization in real-time for their needed
preoperative demarcation, surgical biopsy, delineation, and
potential subsequent complete resection.4,5 Optical molecular
imaging contrast agents explored for use in lymphatic mapping
applications that implement fluorescence imaging techniques
such as fluorescence-guided surgery (FGS) currently involves a
limited set of commonly-used generic molecular fluorescent
dyes that primarily include fluorescein, methylene blue (MB),
and indocyanine green (ICG) (Figure 1A and Table S1).6–10 As
repurposed molecules, these generic molecular fluorescent dyes
were neither originally designed nor intended for use toward
lymphatic fluorescence imaging applications.11–13 As such,
fluorescein, MB, and ICG possess inherent photophysical and
physical properties that hamper their efficacy toward
facilitating the real-time visualization of tumor-draining
lymphatics for all stages of their needed pre- and intraoperative
treatment, and thereby prevent the accurate identification,
surgical biopsy, and complete resection of the potential
metastatic tumor-draining lymph nodes that are crucial for
ensuring unmissed disease in an effort to prevent recurrence and
improve patient survival rates.14–16 In particular, these
molecular fluorescent dyes collectively suffer from (i)
exhibiting visible wavelength fluorescence emission (ca. 390-
700 nm) which provide limited penetration depths in tissue, (ii)
displaying a small Stokes shift (ca. < 40 nm) which reduces
detection sensitivity due to promoting an elevated
instrumentation source background, (iii) maintaining a charge
which hinders their prompt tumor diffusion and timely drainage
into the lymphatics following their administration, (iv)
displaying a pH-sensitive fluorescence or submaximal response
within tumor-draining lymphatic pH levels (pH 6.8-7.4), and/or
(v) demonstrating poor photostability.4,17 The development of a
molecular fluorescent dye designed to overcome these
shortcomings could prove invaluable in the lymphatic mapping
of applicable cancer types. Accordingly,
a molecular
fluorescent dye fashioned for this purpose would exhibit
fluorescence emission in the near-infrared (NIR) optical
imaging window (ca. 700-900 nm), a large Stokes shift (ca. >
80 nm), a pH-insensitive (steady) and maximal fluorescence
response within the aforementioned pH levels, robust
photostability, excellent water-solubility, and be uncharged, all
of which would collectively enhance its efficacy toward
affording the real-time visualization of tumor-draining
lymphatics.4
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Page 2 of 9
The rhodol molecular platform is a topologically-equivalent
hybrid of the fluorescein and rhodamine molecular platforms
and serves as an enhanced base structure for the development
of fluorescence imaging constructs due to consisting of, in part,
an uncharged underlying xanthene core-based scaffold that
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A)
B)
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2
pendant
benzene
O
X = N,O
XR
COO
COO
COO
8'
H
1
O
1'
4'
N
N
2'
3'
7'
6'
scaffold
RX
O
O
O
O
O
NR3R4
R2R1N
O
O
O
O
R2R1N
5'
pH-Induced Spirolactone
Fluorescein
(3',6'-dihydroxyxanthene)
Rhodamine
(3',6'-diaminoxanthene)
Rhodol
THQ-Rosol
(Non-fluorescent)
(3'-amino-6'-hydroxyxanthene) (2',3'-diamino-6'-hydroxyxanthene)
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UV
VISIBLE
NIR
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Fluorescence Emission
200 nm
800 nm
Figure 1. Progressive exocyclic heteroatom modifications to the xanthene core structure of topologically-equivalent molecular fluorescent
dyes that paved the way for the development of the first NIR fluorescent rosol dye, THQ-Rosol. A) General molecular platform of
fluorescein-, rhodamine-, and rhodol-based fluorescent dyes, all of which consist of a charged non-NIR fluorescent xanthene core-based
scaffold having a pendant benzene moiety with a charged 2-carboxylate group (-COO-) that promotes pH-induced spirolactonization and
affords their scaffolds a pH-sensitive fluorescence response at more elevated pH levels than it otherwise demonstrates when without it.18–20
B) Molecular platform of rationally-designed THQ-Rosol, which consists of i) an uncharged NIR fluorescent scaffold that evolves from the
non-NIR scaffold that also underlies the rhodol molecular platform and ii) a pendant benzene moiety without a charged 2-carboxylate group
to afford an inherently uncharged molecular platform, preclude pH-induced spirolactonization, and afford the scaffold a steady maximal NIR
fluorescence response at more elevated pH levels than it would otherwise demonstrate when a charged 2-carboxylate group is present.
affords favorable physical and photophysical properties for
bioimaging applications, which are much unlike its parent
molecular congeners whose charged underlying xanthene core-
based scaffolds afford photobleaching, limited cell
permeability, and/or permanent intracellular retention (Figure
1A).21–24 All else aside, the rhodol molecular platform is yet
similar to the fluorescein and rhodamine molecular platforms in
that it too consists of a pendant benzene moiety with a charged
2-carboxylate group (-COO-), which imparts an increased pKa
value of ~1.5 pH units to its respective underlying scaffold
(Figure 2A).21,25,26 As such, this physical effect affords its
scaffold a pKa value of ~6.3.21,27 The resultant pKa value affords
and an aptly-elevated pKa value of ~5.8-6.0 to better situate its
off-on fluorescence intensity-pH profile would provide the
rosol molecular platform with optimum viability for deep-tissue
fluorescence imaging of tumor-draining lymphatics by being
tailored to concurrently afford a steady maximal NIR
fluorescence response and optimal contrast, as it could provide
minimal overlapping background fluorescence response if it
demonstrated any appreciable residual retention within tumor
tissue shortly after being administered.
Only scant information involving modifications to the
photophysical and/or physical profile of the xanthene core-
based scaffold when underlying the rosol molecular platform is
currently available, presumably due to the numerous constraints
of the conventional synthetic strategy that allow for its direct
preparation (Figure 3A). However, the literature is replete with
examples having made use of the limited number of standard
approaches for altering the pKa value and/or fluorescence
emission wavelength of the xanthene core-based scaffolds that
underlie the fluorescein, rhodamine, and rhodol molecular
platforms, namely site-specific halogenation, π-system
elongation, and endocyclic heteroatom substitution.
Unfortunately, none of these approaches i) allow for the direct
preparation of an uncharged xanthene core-based
scaffold/molecular platform that provides fluorescence
emission beyond 700 nm and/or ii) provide a means for directly
modifying the xanthene core-based scaffold when as an
integrated component of these molecular platforms such that it
elevates its pKa value. Moreover, as these approaches largely
derive from generalized empirical bases rather than rational
design, their use permits only crude and unpredictable
adjustments to the fluorescence emission wavelength and/or
pKa value of their xanthene core-based scaffolds. Taken
together, both a new approach and synthetic strategy for directly
modifying and allowing for the direct preparation, respectively,
of an uncharged xanthene core-based scaffold/molecular
platform such that it affords a NIR fluorescence emission
wavelength and/or an elevated pKa value that each derive from
a quantitative basis could significantly advance both the
efficient development and the efficacy of fluorescent rosol dyes
toward specific imaging applications.
the rhodol molecular platform
a pH-sensitive and/or
submaximal fluorescence response upon its use in
environments having a pH level of less than ~7.4, such as those
of tumor-draining lymphatics (pH 6.8-7.4) (Figure 2B). With its
identical underlying xanthene core-based scaffold to that which
underlies the rhodol molecular platform, but having a pendant
benzene moiety without a charged 2-carboxylate group, the
rosol molecular platform is well-suited for use in such types of
environments because its cross-platform scaffold demonstrates
a
pH-insensitive and maximal fluorescence response
throughout those lower pH levels due to maintaining a lower
pKa value of ~4.8.25,28 As an uncharged small-molecule that is
water-soluble, the rosol molecular platform could readily lend
itself to unrestricted cell permeability and limited intracellular
retention. However, its non-NIR fluorescent scaffold and lower
pKa value could not afford both the NIR fluorescence emission
and contrast levels that are needed for its use in the deep-tissue
fluorescence imaging of tumor-draining lymphatics, as non-
NIR fluorescence emission wavelengths provide only topical
penetration depths and its current pKa value of ~4.8 would
provide suboptimal contrast between the significant
overlapping background fluorescence response (ca. 50%) it
would exhibit between if it demonstrated appreciable residual
retention within tumor tissue after being administered (e.g., in
acidic subcellular compartments) and after having drained into
the lymphatics (Figure 2C).29 Tuning the physical and
photophysical properties such that its xanthene core-based
scaffold affords both a NIR fluorescence emission wavelength
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Analytical Chemistry
Herein, we describe the design, synthesis, and evaluation of
the first NIR fluorescent rosol dye (THQ-Rosol), which serves
as a proof-of-concept for the effective tailoring of an uncharged
xanthene core-based scaffold/molecular platform toward
lymphatic mapping applications using rational design (Figure
1B). As part of our efforts in efficiently developing THQ-
Rosol, we designed a small progressive series of rotationally-
constrained
provided the energy of the highest occupied and lowest
unoccupied molecular orbital (EHOMO and ELUMO) values of each
N-substituted fluorescent rosol scaffold, wherein the versatility
of our synthetic strategy also allowed for the preparation of the
non-NIR fluorescent rosol dyes as designed (Figure 3B). In
discerning high correlations amongst their calculated
energetics, modeled N-C3’ torsion angles, and evaluated
properties, we determined that fluorescent rosol dyes can be
designed in a quantitative manner with the fluorescence
emission wavelength and pKa value of their scaffolds
predetermined and/or finely tuned simply based on
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A)
H
O
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2. HNR1R2
R2R1N
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PG-O
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B)
R2
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R2
H
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2. resorcinol
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R1
Cl
Ph
OMe
N
O
R1
Z
R1
Z
PG = protecting group X = H, C, or N Z = H or C R1, R2 = alkyl
Figure 3. Generalized synthetic strategies that allow for the direct
preparation of fluorescent rosol dyes. A) Conventional synthetic
strategy that affords only non-NIR (3’-amino-substituted)
fluorescent rosol dyes of limited diversity. B) Newly-established
synthetic strategy that affords non-NIR and NIR (2’,3’-diamino-
substituted) fluorescent rosol dyes of broad diversity.
their calculated and modeled values. We leveraged those strong
relationships to rationally design THQ-Rosol, wherein DFT
calculations inspired an innovative approach and synthetic
strategy for extending the fluorescence emission wavelength
and elevating the pKa value and of an uncharged xanthene core-
based scaffold such that it would provide the first NIR
fluorescent rosol dye (2’,3’-diamino-substituted) with both a
finely-tuned NIR fluorescence emission wavelength and an
aptly-elevated pKa value that fittingly situates its off-on
fluorescence intensity-pH profile to provide optimal contrast
for deep-tissue fluorescence imaging of tumor-draining
lymphatics. High correlations between the similarly calculated,
modeled, and evaluated properties of THQ-Rosol further
bolstered the utility of computationally-efficient DFT
calculations and molecular modeling in serving as a simple
model that can afford a priori the determination and fine-tuning
of the fluorescence emission wavelength and pKa value,
respectively, for the efficient development and efficacy of both
non-NIR and NIR fluorescent rosol dyes toward specific
imaging applications. We performed in vitro and in vivo
comparative studies with THQ-Rosol to assess its efficacy in
affording timely tumor drainage and definitive lymph node
visualization with excellent contrast upon its administration and
accumulation, respectively. In all, the development of THQ-
Rosol both represents a rationally-designed optical molecular
imaging contrast agent based on the rosol molecular platform
that is well-suited for lymphatic mapping applications and
provides a compelling means to fashion non-NIR and NIR
fluorescent rosol dyes toward specific imaging applications that
could prove effective for other similar fluorescent molecular
platforms having a xanthene core-based scaffold.
Figure 2. Molecular platform of 9’-phenyl-3’-hydroxyfluorone-,
rosamine-, and rosol-based fluorescent dyes, their analogues
having a pendant benzene moiety with a charged 2-carboxylate
group (-COO-), each respective initial pKa value, and the off-on
fluorescence intensity-pH profile of the molecular platforms having
a 3’-amino-6’-hydroxyxanthene-based scaffold and the maximum
contrast level they would afford upon use toward visualizing
tumor-draining lymphatics. A) The presence of the charged 2-
carboxylate group imparts an increased pKa value to their
respective underlying xanthene core-based scaffold. B) The off-on
fluorescence intensity-pH profiles of a generic rosol (X) and rhodol
(Z) dye with a pKa value of ~4.8 and ~6.3, respectively, and a
proposed rosol dye (Y) designed with a pKa value of 5.8-6.0. The
dashed red lines circumscribe the pH level range that a fluorescent
molecular dye having a pKa value within them would afford
optimal contrast upon use in lymphatic mapping applications. The
circle identifies the pKa value. C) Only the proposed rosol dye (Y)
designed to have a pKa value of 5.8-6.0 would afford a steady
maximal response and optimal contrast. A solid or striped intensity
ratio bar indicates that the dye would be either pH-insensitive or
pH-sensitive when within tumor-draining lymphatic pH levels (pH
6.8-7.4), respectively.
N-substituted non-NIR fluorescent rosol dyes (3’-amino-
substituted) based on density functional theory (DFT)
calculations with increasingly torsionally-restrictive alkyl
substituents appended to the exocyclic N-heteroatom of their
underlying xanthene core-based scaffold in order to examine
the relationships amongst their calculated energetics, modeled
N-C3’ torsion angles, experimentally-determined pKa values,
and measured photophysical properties. DFT calculations
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Analytical Chemistry
state energies. Many starting geometries were used to ensure the
Page 4 of 9
Experimental Section
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optimized structure corresponds to a global minimum.
DFT Calculations. DFT calculations using the hybrid
exchange-correlation function B3LYP with the 6-31G(d) basis
set as implemented in Gaussian 09W Rev. A.02 were performed
on the truncated N-substituted xanthene core-based scaffold
underlying each fluorescent rosol dye to best represent the
electronic contributions influencing the ground and excited
Chemicals and Reagents. Unless noted otherwise, all
chemicals were obtained from Aldrich, Fisher, TCI America,
Alfa Aesar, or Combi-Blocks and used without further
purification. Flash chromatography was performed with 32-63
mm silica gel.
7
Table 1. Calculated, Modeled, and Measured Properties of Non-NIR Fluorescent Rosol Dyes and THQ-Rosol.
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Me-Rosol
Et-Rosol
Pip-Rosol
Jul-Rosol
THQ-Rosol
Ph
Ph
Ph
Ph
Ph
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N
Compound
N
O
O
N
O
O
N
O
O
N
O
O
N
O
O
ELUMO (hartrees)a
EHOMO (hartrees)a
-0.07500
-0.18814
0.11314
-
-0.07520
-0.18811
0.11291
-
-0.07795
-0.19086
0.11291
-
-0.07281
-0.18333
0.11052
-
-0.06856
-0.17245
0.10389
12.21
10.34
550
E (hartrees)
Torsion angle N-C2’ ()b
Torsion angle N-C3’ ()b
abs (nm)c
em (nm)d
18.57
520
14.86
524
12.01
525
7.45
540


557
558
563
574
710
Stokes shift (nm)
Photostability (I/I0)e
 (M-1cm-1)
37
34
38
34
160
0.99
0.98
0.97
0.98
0.99
46,136
42,629
42,460
0.048 ± 0.001
2.0
30,420
29,382
f
0.182 ± 0.007
0.098 ± 0.001
0.414 ± 0.025
0.029 ± 0.001
0.85
fl
Brightness (M-1cm-1)
8.4
4.2
13
pKag
4.84
4.97
4.95
5.76
5.85
aDFT calculations were performed on the truncated scaffolds of each modeled fluorescent rosol dye to obtain their respective ELUMO and
EHOMO values. bThe modeled torsion angle is the offset from planarity (in degrees) of the alkyl substituent that is appended to the C2’- or
C3’-nitrogen atom in relation to their respective xanthene core-based scaffold. cMeasured maximum absorption wavelength of the
fluorescent rosol dye (20 µM) in aqueous buffer (50 mM phosphate, 150 mM NaCl, pH 7.4). dMeasured maximum emission wavelength of
the fluorescent rosol dye (20 µM) upon exciting it at its respective abs in aqueous buffer (50 mM phosphate, 150 mM NaCl, pH 7.4). eI0 is
the measured initial fluorescence intensity of the fluorescent rosol dye and I is its measured fluorescence intensity after 30 min of continuous
photoirradiation. ffl is the measured fluorescence quantum yield. gpKa values were determined using 20 M of the fluorescent rosol dye in
aqueous buffer (50 mM phosphate, 150 mM NaCl, pH 7.4). Error in fl and pKa values are ±5% based on triplicate measurements.
Spectroscopy. NMR spectra were obtained on an Agilent
400-MR NMR Spectrometer. All HRMS analyses were
completed using positive-ion mode electrospray ionization with
an Apollo II ion source on a Bruker 12 Tesla Apex Q FTICR-
MS. Absorption spectra were recorded using an Agilent 8453
UV-visible Spectrophotometer at 25C. Steady-state
fluorescence spectroscopy was performed on a Horiba Jobin
nm long-pass emission filter for THQ-Rosol and a 710-760 nm
band-pass excitation filter and 800 nm long-pass emission filter
for ICG.
Results and Discussion
To efficiently develop an optical molecular imaging contrast
agent that is tailored toward lymphatic mapping applications
based on the rosol molecular platform using a quantitative basis,
we initially designed an appropriate panel of non-NIR
fluorescent rosol dyes with the aid of DFT calculations to
systematically examine the relationships amongst the calculated
energetics and modeled N-C3’ torsion angles of their
underlying xanthene core-based scaffolds and accompanying
evaluated spectroscopic, photophysical, and physical
properties. Although subsequently discussed, we present the
relevant results of THQ-Rosol that we later developed and
evaluated next to those of the non-NIR fluorescent rosol dyes
for easy comparison.
Yvon,
Inc.
PTI
Quantamaster
400
(QM-400)
spectrofluorometer.
In Vivo Imaging. All experimental procedures involving
animals were approved by the Stanford University Institutional
Animal Care and Use Committee (IACUC). For tumor models,
cells (0.5 × 106 cells; 150 μL of serum-free media) were
subcutaneously injected on the back of female nu/nu mice (age
17-18 weeks; Charles River Laboratories) and allowed to grow
for 6 weeks. Tumor size was monitored by calipers and
bioluminescence imaging. Animals were anesthetized with 2%
isoflurane in O2 and kept on a heating pad during imaging.
Imaging was performed on a CRi, Inc. MaestroTM spectral
fluorescent imager with a 503-555 nm excitation filter and 580
DFT Calculations and Molecular Modeling. Calculated
molecular orbital energy values helped guide the design and
selection of an appropriate panel of non-NIR fluorescent rosol
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Analytical Chemistry
dyes. We selected the non-NIR fluorescent rosol dyes having a
small as those of fluorescein, MB, and ICG. Despite the non-
NIR fluorescent rosol dyes revealing a general trend of
decreasing molar absorptivity values for Me-Rosol, Et-Rosol,
Pip-Rosol, and Jul-Rosol ranging from 46,136 M-1cm-1 to
30,420 M-1cm-1, respectively, all of these non-NIR fluorescent
rosol dyes provided excellent photostability by essentially
maintaining their initial fluorescence emission intensity when
under continuous photoirradiation. Although Me-Rosol, Et-
Rosol, and Pip-Rosol revealed a weak general trend of both
decreasing fluorescence quantum yield and brightness values
ranging from 0.182 to 0.048 and 8.4 M-1cm-1 to 2.0 M-1cm-1,
respectively, Jul-Rosol displayed superior fluorescence
quantum yield and brightness value of 0.414 and 13 M-1cm-1,
respectively, due to the reduced nonradiative decay afforded by
its annulated torsionally-restrictive alkyl substituents appended
to the exocyclic N-heteroatom of its underlying xanthene core-
based scaffold. The non-NIR fluorescent rosol dyes revealed a
general trend of increasing experimentally-determined pKa
values ranging from 4.84 to 5.76
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dimethylamino (Me), diethylamino (Et), piperidino (Pip), and
julolidino (Jul) moiety that we denote as Me-Rosol, Et-Rosol,
Pip-Rosol, and Jul-Rosol, respectively, which provided a
general trend of decreasing molecular orbital energy gap (E)
values ranging from 0.11314 to 0.11052 hartrees via an
accompanying relatively weak general trend of increasing
EHOMO values ranging from -0.18814 to -0.18333 hartrees
(Table 1). Their modeled N-C3’ torsion angle () revealed a
general trend of increasingly torsionally-restrictive alkyl
substituents appended to the exocyclic N-heteroatom of the
underlying xanthene core-based scaffold which accompanied
their general trend of decreasing molecular orbital energy gap
values. Accordingly, Jul-Rosol maintained the smallest
modeled N-C3’ torsion angle (7.45) and molecular orbital
energy gap value (0.11052 hartrees) amongst the designed non-
NIR fluorescent rosol dyes.
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Scheme 1. Synthesis of Non-NIR Fluorescent Rosol Dyes
and THQ-Rosol.a
Ph
Ph
A)
O
a
b
R2N
OMe
R2N
OH
R2N
O
O
1
2
Me-, Et-, Pip-, & Jul-Rosol
B)
Ph
Ph
N
N
N
N
N
O
c
d
OMe
OH
N
O
O
3
4
THQ-Rosol
aReagents and conditions: (a) 1, benzoyl chloride, AlCl3, DCM,
0C  RT; BBr3, DCM, 0C  RT; Me-2 (89%), Et-2 (92%), Pip-
2 (94%), Jul-2 (27%); (b) 2, resorcinol, MeSO3H, 100C, 3 hr, Me-
Rosol (69%), Et-Rosol (8%), Pip-Rosol (25%), Jul-Rosol (4%);
(c) 3, benzoyl chloride, AlCl3, DCM, 0C  RT, 2 hr, N2, 85%; (d)
4, resorcinol, MeSO3H, 90C, overnight, 49%.
Synthesis. Non-NIR fluorescent rosol dyes were also
prepared according to our newly-established synthetic strategy
as shown in Scheme 1A. Compounds 1 were acylated under
Friedel-Crafts conditions, which ultimately provided
Figure 4. Linear regression analyses and statistical measure
between select set of parameters of the non-NIR fluorescent rosol
dyes without and with inclusion of relevant values of THQ-Rosol.
Correlation plots interrelating the calculated molecular orbital
energy gap (E) value to the A) maximum fluorescence emission
wavelength (λem) and B) pKa value of the evaluated fluorescent
rosol dyes. Correlation plots interrelating the modeled N-C3’
torsion angle to the C) maximum fluorescence emission
wavelength (λem) and D) pKa value of the evaluated fluorescent
rosol dyes. The r2 value is the accompanying coefficient of
determination of the aforementioned analyzed set of parameters of
the evaluated non-NIR fluorescent rosol dyes without and with (**)
THQ-Rosol. Yellow highlights that the correlation was maintained
between the set of parameters upon inclusion of the relevant values
of THQ-Rosol.
compounds
demethylating
2
in high yield by way of additionally
their acylated methoxy-intermediates
(byproducts not shown) for the purpose of preserving the initial
starting materials. Intermolecular condensation of compounds 2
with resorcinol under acidic conditions and heat gave Me-
Rosol, Et-Rosol, Pip-Rosol, and Jul-Rosol ranging from
moderate to low yields. Presumably, low-volume solution (1
mL) stirring difficulties and progressive efforts in isolating each
non-NIR rosol dye upon work-up and purification account for
the varied reaction yields.
Evaluated Spectroscopic, Photophysical, and Physical
Properties. The measured spectroscopic, photophysical, and
experimentally-determined physical properties of the prepared
non-NIR fluorescent rosol dyes were obtained using standard
techniques/methods and are also provided in Table 1. The non-
NIR fluorescent rosol dyes revealed general trends of increasing
measured maximum absorption wavelength ranging from 520
nm to 540 nm alongside increasing measured maximum
emission wavelength ranging from 557 nm to 574 nm for Me-
Rosol, Et-Rosol, Pip-Rosol, and Jul-Rosol, respectively. As
such, all of these non-NIR fluorescent rosol dyes displayed a
small Stokes shift ranging from 34 nm to 38 nm, which are as
for Me-Rosol, Et-Rosol, Pip-Rosol, and Jul-Rosol,
respectively, due to the progressive decrease in their N-C3’
torsion angle that results from the steric hindrance their
increasingly torsionally-restrictive alkyl substituents afford
them.
Linear Regression Analyses. The calculated energetics and
the modeled N-C3’ torsion angle of each non-NIR fluorescent
rosol dye were separately plotted against their evaluated
photophysical and physical properties, which allowed for
statistical measure of a coefficient of determination (r2) from
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each correlation plot (Figures 4A-D). We discerned high
Page 6 of 9
rosol dyes to rationally design THQ-Rosol, wherein we
extrapolated from them that achieving an appropriately small
molecular orbital energy gap value and modeled torsion angle
could potentially impart a finely-tuned NIR fluorescence
emission wavelength and an aptly-elevated pKa value to the
rosol molecular platform such that it fittingly situates its off-on
fluorescence intensity-pH profile to be optimal for deep-tissue
fluorescence imaging of tumor-draining lymphatics. The 2’,3’-
diamino-substituted xanthene core-based scaffold in
conjunction
correlations (r2 ≥ 0.64) amongst both their calculated molecular
orbital energy gap values and modeled N-C3’ torsion angles
when each was interrelated to their measured maximum
emission wavelengths and their respective experimentally-
determined pKa values, wherein we obtained an r2 value of 0.91,
0.99, 0.90, and 0.77 from their linear regression analyses,
respectively. As such, the calculated molecular orbital energy
gap value and modeled N-C3’ torsion angle provided a
quantitative means for designing THQ-Rosol.
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THQ-Rosol. We leveraged the strong relationships that we
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derived from the correlation plots of the non-NIR fluorescent
Figure 5. Measured photophysical and experimentally-determined physical properties of the non-NIR fluorescent rosol dyes and THQ-
Rosol. A) Fluorescence emission profile of THQ-Rosol accentuating i) the effect of its lower calculated molecular orbital energy gap value
and ii) the difference in its maximum fluorescence emission wavelength when compared to those of the non-NIR fluorescent rosol dyes. B)
Photostability of the generic molecular fluorescent dyes commonly used for lymphatic mapping applications, non-NIR fluorescent rosol
dyes, and THQ-Rosol. C) Experimentally-determined off-on fluorescence intensity pH-profile of a non-NIR fluorescent rosol dye, THQ-
Rosol, and its proposed analogue having a pendant benzene moiety with a charged 2-carboxylate group (-COO-), all of which accentuate that
by minimizing the N-C3’ torsion angle of a 3’-amino-6’-hydroxyxanthene-based scaffold as an integrated component of a molecular platform
having a pendant benzene moiety without a charged 2-carboxylate group a pKa value can be modulated such that it could afford a pH-
insensitive and maximal fluorescence response. The green-shaded region identifies tumor-draining lymphatics pH levels (pH 6.8-7.4).
with ethyl substituents appended to its annulated and
adjacently-situated exocyclic N-heteroatoms concurrently
afforded its rosol-based molecular platform the optimal
combination of i) an appropriate molecular orbital gap value of
0.10389 hartrees via a concomitant increased EHOMO value (-
0.17245 hartrees) and a decreased ELUMO value (-0.06856
hartrees) and ii) the smallest modeled N-C2’ and N-C3’ torsion
angles (12.21 and 10.34, respectively) amongst additionally-
explored N-substituted THQ-Rosol analogues having
appended alkyl substituents with either less or more steric bulk
(Table 1 and Figure S1). As DFT calculations inspired an
innovative approach of designing a rosol molecular platform
Intermolecular condensation of intermediate 4 with resorcinol
under acidic conditions provided THQ-Rosol in moderate
yield.
The measured spectroscopic, photophysical, and
experimentally-determined physical properties of THQ-Rosol
were similarly obtained using traditional techniques/methods
and are provided in Table 1. THQ-Rosol maintained the
general trend we observed for the non-NIR fluorescent rosol
dyes by revealing a maximum absorption wavelength of 550 nm
and a noteworthy measured maximum emission wavelength of
710 nm, which is a difference of ≥ 136 nm in the measured
maximum emission wavelength when compared to the prepared
non-NIR fluorescent rosol dyes Me-Rosol, Et-Rosol, Pip-
Rosol, and Jul-Rosol, which can be attributed to its lower
molecular orbital energy gap value (Figure 5A). As such, THQ-
Rosol represents the first uncharged xanthene core-based
scaffold/molecular platform that provides NIR fluorescence
emission beyond 700 nm under aqueous conditions (i.e.,
without any appreciable organic co-solvent also in the media).
Accordingly, THQ-Rosol displayed a remarkable Stokes shift
of 160 nm, and thereby would better accommodate high
detection sensitivity by promoting minimal instrumentation
source background upon use when compared to those of
fluorescein, MB, and ICG. Moreover, THQ-Rosol also
displayed robust photostability via maintaining over 99% of its
original fluorescence emission intensity when under continuous
photoirradiation and is superior to that of the only generic
molecular fluorescent dye commonly used towards lymphatic
fluorescence imaging applications having NIR fluorescence
emission (i.e., ICG) (Figure 5B). Notably, ICG demonstrated
with
a 2’,3’-diamino-substituted xanthene-based scaffold
alongside appending appropriate torsionally-restrictive
substituents to its adjacently-situated exocyclic N-heteroatoms,
further development of THQ-Rosol required we establish a
new synthetic strategy that could allow for its direct preparation
because current efforts leading to molecular platforms that have
a 3’-amino-6’-hydroxyxanthene-based scaffold either 1) afford
a rhodol molecular platform or 2) cannot afford a rosol
molecular platform having
a
2’,3’-diamino-substituted
xanthene-core based scaffold (Figure 3A). As such, we
established a synthetic strategy that would allow for directly
preparing a NIR fluorescent rosol dye that has a 2’,3’-diamino-
substituted xanthene core-based scaffold (Figure 3B). THQ-
Rosol was prepared in two steps from known starting materials
according to Scheme 1B.30–32 Compound 3 was concurrently
acylated and demethylated under Friedel-Crafts conditions to
give compound 4 in high yield with marginal formation of its
acylated methoxy-intermediate (byproduct not shown).
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rapid and significant photobleaching by maintaining less than
1
64% of its original fluorescence emission intensity under
identical conditions. In addition, we found that its aptly-
elevated pKa value of 5.85 fittingly situates its off-on
fluorescence intensity-pH profile such that it could afford
minimal overlapping background fluorescence response
between if it demonstrated appreciable residual retention within
tumor tissue after being administered and having drained into
the lymphatics, whereupon THQ-Rosol would then afford a
pH-insensitive maximal fluorescence response (Figure 5C). As
shown from the comparable r2 values of 0.97 and 0.70, its
molecular orbital energy gap value and small N-C3’ torsion
angle of THQ-Rosol primarily accounted for its pronounced
fluorescence emission of 710 nm and its experimentally-
determined pKa value of 5.85, respectively (Figures 4A and
4D). THQ-Rosol also demonstrated excellent stability to
standard bioanalytes under their normal biological and elevated
concentrations by maintaining its initial NIR fluorescence
response (Figure S10). Lastly, based on its absorption-based
linearity profile, we determined that THQ-Rosol maintains an
apparent solubility of ~15 mg/L (40 µM), which could prove to
be effective for clinical applications contingent on the outcomes
of additional studies capable of determining its
pharmacokinetic, pharmacodynamic, and dose-escalation
profile (Figure S11). Given all of its favorable properties, we
next assessed its efficacy to afford the real-time visualization of
tumor-draining lymphatics by evaluating THQ-Rosol in vitro
and in vivo.
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Figure 6. In vivo efficacy of THQ-Rosol toward lymphatic
mapping applications. A) Representative images of THQ-Rosol
following its administration to a GBM39 xenograft mouse model
at select time points. B) The accompanying normalized
fluorescence tumor-to-background ratio of THQ-Rosol (λem2 = 710
nm) compared to ICG (λem1 = 800-900 nm) at select time points
(each 50 µL, 25 µM). Accumulation of THQ-Rosol in the axillary
lymph node C) pre- and D) post-administration. TOI = time of
injection. Dashed white circle circumscribes the axillary lymph
node region.
Cellular Analyses. We evaluated THQ-Rosol for
maintaining cellular viability using Calcein-AM as a live-cell
stain, wherein the cells maintained their viability across a range
of applicable concentrations up to 25 µM (Figure S12). We
adopted an appropriate protocol upon applying THQ-Rosol to
a cancer cell line and used confocal fluorescence microscopy to
assess its ability to i) translocate across the cell membrane, ii)
not demonstrate permanent intracellular retention, and iii)
afford only a minimal fluorescence response if so. As such, we
gleaned from performing subsequent washing steps, the need
for an elevated PMT detector gain level, and its punctate
staining pattern we observed, that its comparable pKa value
allowed THQ-Rosol to i) demonstrate transient intracellular
retention in acidic subcellular compartments, and ii) display a
minimal fluorescence response when within them (Figure S13).
We validated its transient equilibrium-driven lysosomal
localization by performing colocalization studies using a
standard lysosomal labeling dye alongside THQ-Rosol,
wherein we used two different pairs of appropriate excitation
and emission channels with different PMT detector gain levels
to visualize their fluorescence responses (Figures S13A and
S13B). Analysis of the puncta from the images of each
respective dye when overlaid provided a Pearson coefficient (r)
of 0.70, which strongly supports that THQ-Rosol localizes to
lysosomes (Figure S13C).
In Vivo Imaging. We mimicked the standard use of a generic
molecular fluorescent dye toward lymphatic mapping
applications by administering THQ-Rosol to a xenograft tumor
model, wherein we assessed its efficacy to afford prompt
diffusion and timely drainage from within a tumor following its
administration (Figure 6A). In evaluating its fluorescence
tumor-to-background ratio at select time points, we determined
that THQ-Rosol outperforms ICG under identical conditions
(Figure 6B). Accordingly, we observed that ~90% of THQ-
Rosol drained from the tumor in 2 hours post-administration
following its prompt diffusion throughout the tumor upon its
administration, whereas we observed a 5-fold increase in the
fluorescence tumor-to-background ratio of ICG over the first 2
hours post-administration due to its delayed diffusion and only
~60% of ICG drained from the tumor within the subsequent 2
hours. We also observed that only ~5% of THQ-Rosol
remained in the tumor tissue throughout the course of 6 hours,
whereas over ~40% and ~10% of ICG remained in the tumor
tissue throughout the course of 4 hours and 6 hours,
respectively. Presumably, the significant difference in the post-
administration diffusion and drainage profiles between THQ-
Rosol and ICG can be attributed to their uncharged and charged
physical properties, respectively, as well as its well-situated
pKa value that fosters its transient intracellular retention. As
such, these properties could further aid in the visualization of
the lymphatics by also affording a minimal overlapping
background fluorescence response. Accordingly, we assessed
its ability to accumulate within an axillary lymph node, wherein
it demonstrated a strong fluorescence response within the
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Vahrmeijer, A. L.; Hutteman, M.; van der Vorst, J. R.; van
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de Velde, C. J. H.; Frangioni, J. V. Image-Guided Cancer Surgery
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We described the development of the first NIR fluorescent
rosol dye (THQ-Rosol) tailored with optimal properties for
overcoming the limitations arising from those of the generic
molecular fluorescent dyes commonly-used for lymphatic
mapping applications. We utilized efficient DFT calculations to
facilitate the design of both non-NIR and NIR fluorescent rosol
dyes, wherein we leveraged the strong relationships that we
discerned between select calculated, modeled, and evaluated
properties of the former dyes to rationally design an uncharged
xanthene core-based scaffold as an integrated component of a
rosol molecular platform such that it would have a finely-tuned
NIR fluorescence emission wavelength (710 nm) and a pKa
value (5.85) that fittingly situates its off-on fluorescence
intensity-pH profile to provide optimal contrast for the deep-
tissue fluorescence imaging of tumor-draining lymphatics. In
doing so, we established a simple model to fashion a priori non-
NIR and NIR fluorescent rosol dyes with specific properties.
We demonstrated that THQ-Rosol outperforms ICG by
affording timely tumor drainage and definitive lymph node
visualization with excellent contrast due to its design. As such,
THQ-Rosol could significantly aid in the visualization of the
potential metastatic tumor-draining lymph node(s) for their pre-
and intraoperative treatment.
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ASSOCIATED CONTENT
Supporting Information
Molecular modeling and calculations, H and 13C NMR spectra,
1
UV/vis and fluorescence spectra, pH titrations, cell assays, and
animal study protocols. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* chinf@stanford.edu
Author Contributions
‡These authors contributed equally.
ACKNOWLEDGMENT
We thank Tim Doyle (Stanford Small Animal Imaging Facility)
and Kitty Lee (Stanford Cell Science Imaging Facility) for
technical assistance. The project was supported, in part, by the
National Center for Research Resources: S10 OD010580; the
National Science Foundation: CHE-1112194 (TEG); the
Department of Energy: DE-SC0008397 (FTC); The Ben and
Catherine Ivy Foundation (FTC); and the National Cancer Institute:
R21 CA205564 (FTC), F32 CA213620 (KSH), and T32 CA118681
(JLK).
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Carboxaldehyde. Dyes Pigm. 2009, 1 (82), 84–89.
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