Fluorescence phenomena in nerve-labeling styryl-type dyes

Article abstract of DOI:10.1016/j.jphotochem.2015.05.033

Several classes of diversely substituted styryl type dyes have been synthesized with the goal of extending their expected fluorescent properties as much toward red as possible given the constraint that they maintain drug-like properties and retain high affinity binding to their biological target. We report on the synthesis, optical properties of a series of styryl dyes (d1-d14), and the anomalous photophysical behavior of several of these donor-acceptor pairs separated by long conjugated π-systems (d7-d10). We further describe an unusual dual emission behavior with two distinct ground state conformers which could be individually excited to locally excited (LE) and twisted intramolecular charge transfer (TICT) excited state in push-pull dye systems (d7, d9 and d10). Additionally, unexpected emission behavior in dye systems d7 and d8 wherein the amino-derivative d7 displayed a dual emission in polar medium while the N,N-dimethyl derivative d8 and other methylated derivatives d12-d14 showed only LE emission but did not show any TICT emission. Based on photophysical and nerve binding studies, we down selected compounds that exhibited the most robust fluorescent staining of nerve tissue sections. These dyes (d7, d9, and d10) were subsequently selected for in vivo fluorescence imaging studies in rodents using the small animal multispectral imaging instrument and the dual-mode laparoscopic instrument developed in-house.

Full text of DOI:10.1016/j.jphotochem.2015.05.033

Accepted Manuscript
Title: Fluorescence Phenomena in Nerve-Labeling
Styryl-Type Dyes
Author: Tiberiu M. Siclovan Rong Zhang Victoria Cotero
Anshika Bajaj Dmitry V. Dylov Siavash Yazdanfar Randall
Carter Cristina A.Tan Hehir Arunkumar Natarajan
PII:
S1010-6030(15)00198-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2015.05.033
JPC 9921
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
25-11-2014
20-5-2015
25-5-2015
Please cite this article as: Tiberiu M.Siclovan, Rong Zhang, Victoria Cotero,
Anshika Bajaj, Dmitry V.Dylov, Siavash Yazdanfar, Randall Carter, Cristina
A.Tan Hehir, Arunkumar Natarajan, Fluorescence Phenomena in Nerve-Labeling
Styryl-Type Dyes, Journal of Photochemistry and Photobiology A: Chemistry
http://dx.doi.org/10.1016/j.jphotochem.2015.05.033
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Fluorescence Phenomena in Nerve-Labeling Styryl-Type Dyes
Tiberiu M. Siclovan, Rong Zhang, Victoria Cotero, Anshika Bajaj, Dmitry V.
Dylov, Siavash Yazdanfar, Randall Carter, Cristina A. Tan Hehir, Arunkumar
Natarajan*natararu@ge.com
GE Global Research, One Research Circle, Niskayuna NY 12309
*Corresponding author. Tel.: 518-387-4772
Graphical Abstract
Highlights
Synthesized and investigated photophysical properties of several substituted styryl type dyes
d1-d15.
Three dyes d7, d9 and d10 showed good fluorescent properties and maintained drug-like
binding property.
Observed an unusual dual emission behavior with two distinct ground state conformers that
could individually be excited.
Observed an anomalous emission behavior on dye systems d7 and d8 the amino- derivative d7
showed a dual emission characteristic in the polar medium, while the N,N-dimethyl derivative
d8 did not.
Interestingly, when compound d7 was injected into a mouse and the dye bound to nerves were
excited by either 405 nm or 447 nm laser, a wavelength dependent emission described above
was observed similar to fluorescence in DMSO.
in-vivo fluorescence imaging studies in rodents using small animal multispectral imaging
instrument and the dual-mode laparoscopic instrument developed in-house.
1

Abstract
Several classes of diversely substituted styryl type dyes have been synthesized with the goal of
extending their expected fluorescent properties as much towards red as possible given the
constraint that they maintain drug-like properties and retain high affinity binding to their
biological target. We report on the synthesis, optical properties of a series of styryl dyes (d1-
d14), and the anomalous photophysical behavior of several of these Donor-Acceptor pairs
separated by long conjugated -systems (d7-d10). We further describe an unusual dual
emission behavior with two distinct ground state conformers which could be individually
excited to locally excited (LE) and twisted intramolecular charge transfer (TICT) excited state
in push-pull dye systems (d7, d9 and d10). Additionally, unexpected emission behavior in dye
systems d7 and d8 wherein the amino- derivative d7 displayed a dual emission in polar
medium while the N,N-dimethyl derivative d8 and other methylated derivatives d12-d14
showed only LE emission but did not show any TICT emission. Based on photophysical and
nerve binding studies, we down selected compounds that exhibited the most robust fluorescent
staining of nerve tissue sections. These dyes (d7, d9, and d10) were subsequently selected for
in-vivo fluorescence imaging studies in rodents using the small animal multispectral imaging
instrument and the dual-mode laparoscopic instrument developed in-house.
Keywords
Styryl dyes, TICT emission, nerve labeling, dyes, laparoscopy, in vivo and ex vixo imaging,
fluorescence imaging of nerves.
Introduction
Nerves are often difficult to visualize during surgery, due to their intricacy and anatomical
variations in their location.1 As a result, unintended nerve injury has been recognized as an
important cause of morbidity associated with several lifesaving surgical procedures such as
prostatectomy,2,3 thyroidectomy,4,5 rhytidectomy,6 and breast cancer surgery.7 The need for
guidance during surgery has been recognized for about a decade, beginning with the evaluation
of techniques employing pre-operative CT and MR imaging.8-11 However, real time decision
2

making has directed intraoperative identification efforts towards fluorescence imaging,12
including approaches relying on visualizing the nerves based on contrast agents designed to
elicit selective nerve fluorescence during surgery, reviewed recently.13
To facilitate clinical use, the ideal in vivo nerve contrast agent must be designed to
cross the tight junctions of the blood nerve barrier, a crucial requirement for systemic
injection. Thus, in addition to being fluorescent, the contrast agent should show high
selectivity to a nerve target as well as exhibit properties of small molecules. The targeting
moiety has to be inherently fluorescent because conjugating the targeting component to a
fluorescent dye would significantly increase its molecular weight beyond the desirable range.
The styryl dye, BMB, (1,4-bis(p-aminostyryl)-2-methoxybenzene), developed to be a
PET tracer for imaging myelin absorbs in the near-ultraviolet range and emits in the blue
region.14 While it bound with high affinity to myelin extracts from brain, BMB suffered from
poor aqueous solubility. Moreover, intraoperative imaging at wavelengths optimal for BMB
produced high background in non-target tissue because tissue autofluorescence was high in
this region. Using BMB as a starting point, we sought to explore whether chemical
modifications applied to bis-styryl dyes15,16 would allow us to elaborate them into dyes having
optical and biological properties suitable for their use in an intraoperative setting.
We have since reported on bis-styryl dyes (d1, d7 and d9) for selectively targeting
myelin basic protein (MBP), a major component of nerves,17-19 along with compact optical
imaging instruments for open and minimal access surgeries.20,21 Following intravenous
injection, these dyes visualized central and peripheral nerves in vivo with high contrast relative
to the surrounding muscle tissue, despite absorbing and emitting in the visible region. The
affinity for MBP was maintained provided certain structural features were conserved.22 We
have shown that both the absorption and the emission maxima can be red-shifted significantly
by employing through- system conjugated donor-acceptor (D-A) group, producing dyes with
large Stokes shifts, thus minimizing the impact of autofluorescence in the visible region.
Herein, we report the screening and optical characterization of a series of molecules
(d1-d14) with the aim of creating dyes with red-shifted fluorescence, large Stokes shifts and
high fluorescence yields while maintaining binding to nerves and improved aqueous solubility.
Furthermore, we elaborate on the unusual photophysical properties of our previously reported
3

fluorophores, which were observed for both in-vitro (in organic solvent) and in-vivo (in mouse
nerves).
MATERIALS AND METHODS
Synthesis of dyes: The dyes shown in Table 1 were synthesized in step-wise procedures as
illustrated in the Schemes below. Details of the synthetic methodology can be found in the
Supplemental Material. Compounds were purified by medium performance liquid
chromatography on silica gel (ISCO Companion with RediSep Gold silica columns). Those
selected for advancement into in vivo imaging studies were further purified by high
performance liquid chromatography (HPLC). For normal phase HPLC, a Shimadzu LC-8A
equipped with a SIL-10AP autosampler, an SPD-M20A diode array detector and an FRC-10 A
fraction collector were used, together with a Waters Sunfire preparative silica OBD column, 5
m, either 19x150 mm or 50x100 mm, and a hexanes/ ethyl acetate gradient. For reverse phase
HPLC, a GE AKTA Avant system was used, with a Waters XTerra PrepMS C18 column, 5
m, 30x100 mm and water - 0.1% formic acid/ acetonitrile/ 0.1% formic acid gradient.
Bis-styryl dyes having three ring structures similar to that of d7 (vide infra) have been
synthesized following the methodology previously reported.18 Essentially, this consists of a
tandem Heck coupling of the appropriately functionalized aminostyrene with the
correspondingly substituted bromo arylaldehyde, followed by a Horner-Wittig olefination with
the appropriately substituted benzyldiethyl phosphonate (Scheme 1).
Compounds d5 and d8 were prepared via reductive amination of d1 and d7,
respectively. Compound d11 was prepared as illustrated in Scheme 2.
Compounds d12 and d14, containing multiple electron withdrawing groups were
prepared via a Knoevenagel condensation of the corresponding styryl aldehyde with the active
methylene compounds, as described previously23 and illustrated in Scheme 3 for compound
d14 and detailed in the experimental sections for compounds d12-d14.
4

The styryl compounds d2-d4 have been prepared via straightforward transformations
16
depicted in Scheme 4. Compound d2 has been previously reported and has been included
here for comparison within the polyene series. Updated reviews of the general methodologies
described above have been published recently.24-26
Characterization of nerve labeling dyes: Methods for spectroscopic and physicochemical
characterizations were as previously described.18 Briefly, absorbance spectra were taken using a
Lambda 20 UV/Vis spectrometer (Perkin Elmer, Waltham, MA). The wavelength of maximum
absorbance was then used as the excitation wavelength for the collection of the fluorescence
emission spectra on a steady state fluorimeter (Photon Technology International, Birmingham,
NJ). A 10 mM stock solution of each dye in dimethylsulfoxide (DMSO) was used in the
preparation of 10 µM solutions of the dye in DMSO, unless indicated otherwise.
Ex vivo and in-vivo imaging: Ex vivo nerve tissue staining on each of the dye was performed as
described.17 A buffer only control (no dye) was performed using exactly the same procedure to
determine autofluorescence under identical imaging settings.
In vivo nerve imaging studies on selected dyes were described previously.18,27 All
procedures were approved by the Institutional Animal Care and Use Committee at GE Global
Research. Briefly, CD- 1 mice ranging in body weight from 25 to 30 g, and Sprague-Dawley
rats ranging in body weight from 250 to 300 g, were purchased from Charles River
Laboratories (Wilmington, MA). On the day of the experiment, mice and rats were
anesthetized using 2%– 4% isofluorane and given a single tail vein injection each of 50 mg/kg
of d7 (GE3082), 8 mg/kg of d9 (GE3111), or 12.2 mg/kg of d10 (GE3126). The animals were
then returned to the home cage until the designated time-point for imaging (3 h post-injection
for d7 and d9; 1 h post-injection for d10). The nerves were exposed and multispectral imaging
was initially performed using a fluorescence stereomicroscope (SteREO Lumar V12, Carl
Zeiss Inc., Thornwood, NY) equipped with a multispectral imaging camera (Nuance, CRI,
Woburn, MA). Imaging using our custom laparoscopic instrument was as described.27
Imaging of control animals (with no dye injected) was performed under identical conditions.
5

Spectral measurements of tissue fluorescence were done using a spectroscopic probe.
The probe consisted of illumination (405 nm laser, which was the same as the laparoscopic
instrument illumination, or a 447 nm laser) coupled to a fiber, a premium-grade reflection
ferrule (Ocean Optics, 6 illumination fibers, 1 600-micron read fiber, 3-inch long ferrule), a
collimator with a holder for excitation rejection filter, and a spectrometer (Ocean Optics,
USB2000+UV-VIS). The 405 nm excitation spectra were measured with BLP01-405R-25
rejection filter, while the 447 nm excitation spectrum was measured with LP03-458RU long-
pass filter.
RESULTS & DISCUSSION
We investigated the absorption and emission properties of dyes d1-d14 in non-polar (toluene) and
polar (DMSO, MeOH) solvents. The dyes d1-d14, shown in Table 1, can be classified into two
categories, where dyes d1-d5 have conjugated amine based electron donating “push-push” end
groups, while d6-d14 are based on electron-donating and electron-withdrawing “push-pull” end
groups in conjugation. The purpose of screening these individual categories was to see which one
of these gave the longest wavelength emission and highest fluorescence yields while maintaining
nerve binding.
Since we had previously shown that BMB (d1) can bind and visualize nerves in vivo,19 we
utilized this molecule as a starting point to develop dyes with improved optical properties. The
absorbance and fluorescence emission maxima in DMSO for each synthesized dye are shown in
Table 1. Compounds d2 and d3 lacking a central arene group (Scheme 4) displayed a
hypsochromic shift in the absorption and emission while retaining high emission intensity as
compared to d1. Compound d4, in which the central arene was preserved while incorporating an
extra alkene on each side of the arene, showed a reasonable bathochromic shift of the emission
compared to d1, however its fluorescence intensity was reduced dramatically (fluorescence
emission intensity: d1=106,800 vs. d4=200). Alkylation of the amine groups (d5) helped in
further bathochromic shift of absorption and emission with respect to d1 while maintaining
sufficient fluorescent intensity.
In the push-pull dye system category, d6 was used as a model compound which displayed
solvent dependent behavior; In the push-pull dye systems category, d6 was used as a model
6

compound which displayed solvent dependent behavior; absorption and emission maxima of 368
nm and 432 nm respectively in non-polar solvents (toluene). In polar solvent (DMSO), d6
displayed an absorption maximum at 385 nm and emission maximum at 522 nm which we assign
to a Twisted Intramolecular Charge Transfer (TICT) state .28 Extending the conjugation of this
system by adding an arene middle ring (d7) resulted in a bathochromic shift of ~30 nm in
absorption and a dual emission in DMSO at 491 nm and 621 nm. The solvent dependent behavior
of d7 relative to the analogous non push-pull system (d1) is shown in Figure 1.
N-methylated dye system d8 resulted in a slight (5 nm) hypsochromic shift of the
absorption with a maximum at 412 nm and a modest 27 nm shift in emission, with respect to the
short wavelength emission of the unalkylated amine dye system d7. To our surprise this system
failed to show dual emission in polar solvents. Replacing the electron withdrawing cyano group
with other electron withdrawing groups -SO2Me, -SO2-piperazine (d9 and d10) showed similar
absorption trends and values and large Stokes shifts, with emission maxima around 620 nm.
Replacing the electron donating system –NH2 by a hydroxy (-OH) group in d11 resulted in a
hypsochromic shift in absorption and emission with respect to d6. Similar to compounds d7, d9
and d10 also showed dual a emission in polar solvents.. Using the indoline or N,N-dimethyl amino
groups as electron donating groups with a variety of electron withdrawing moieties such as
dicyanovinyl, methylsulfonyl, dicyanopyranyl (d12-d14), resulted in bathochromic shifts in
absorption (467, 419, and 482nm, respectively), and reasonable Stokes shifts albeit low emission
yields.
To identify the most promising nerve labeling dyes, a qualitative ex vivo tissue staining
assay was performed using each of the dye with detectable fluorescence. Figure 2 shows
examples of microscopic images of fluorescently labeled rat nerve tissue sections. Here,
compounds d7, d9, and d10 exhibited the most robust fluorescent staining of nerve tissue
sections. These dyes were subsequently selected for in vivo fluorescence imaging studies in
rodents- using (1) the small animal multispectral imaging instrument shown in Figure 3; and
(2) the dual-mode laparoscopic instrument developed in-house shown in Figure 4.28
Figure 3 shows examples of multispectral imaging in mice. In general, the nerves
appeared red-orange and the surrounding muscle tissue was dark. Because the dyes are
7

lipophilic, there was some non-specific partitioning to adipose tissue which appeared as
yellow-green fluorescence under the multispectral camera.
Figure 4 shows the dual-mode laparoscopic imaging of a rat injected with d7. Note the
thin layer of fascia obscuring the nerve under white light imaging, but not under fluorescence
visualization. More detailed in vivo dosing and kinetics results for these three dyes were
described elsewhere (d717,19,28; d918; manuscript submitted for d10;).
We have also developed a spectroscopic probe for exact spectral measurements of
tissue fluorescence in-situ. The probe allows precise local measurement to be taken at a tissue
of interest at a high-resolution (1 nm) and relatively small integration times, typically 1-2 sec
for nerve, 0.5 sec for adipose tissue, and 2-5 sec for muscle. This spectral probe used the same
illumination as the dual-mode laparoscopic instrument (405 nm excitation). Figure 5 shows
spectral measurements of the nerve, adipose tissue, and muscle in a dissected mouse treated
with of d10. The probe was pointed at the relevant tissue inside the mouse. The shape and
intensity of the spectrum for nerve, adipose tissue, and muscle was different from one another.
The emission spectrum of the nerve was reminiscent of the dual emission wavelength observed
in DMSO (Table 1),
Our approach to improving the photophysical properties from our initial model system
BMB (d1) focused on increasing conjugation of the push-push based dye system, and
investigating the effect of a series of push-pull based Donor-Acceptor (D-A) groups,
conjugated across a variable length -system, followed by down-selection and biological
screening. Of particular interest was the number of connecting double bonds, the number of
aromatic rings, and the strength of the acceptor group, including the effect of multiple acceptor
groups. Additionally, we were presented with the challenge of having to determine what
molecular characteristics were necessary to retain in order to maintain the binding affinity to
our target of interest - myelin basic protein.17 In order to obtain reasonable solubility in the
three ring system and attenuate lipophilicity we incorporated a methoxy- group in the middle
aryl ring.29
8

We sought to understand the influence of conjugation length in this structural space by
extending the -system via the addition of double bonds, an aim most readily achieved, from a
synthetic point of view, in symmetrical D--D systems. Extending the conjugation of the
simple 2-ring diaminostilbene d2 by one double bond provided the expected, modest
bathochromic (red) shift to both absorption and fluorescence emission. The same
transformation applied to the 3-ring BMB system (compound d1) led to compound d4, in
which the absorbance displayed a slight hypsochromic (blue) shift and the fluorescence was
essentially suppressed. In agreement with previous reports on diene chromophores30,31 we are
compelled to conclude that extended alkene systems such as d4, despite the addition of only
one double bond on each side of a rigidifying middle ring, possess too flexible a backbone.
Thus the fluorescence quantum yields and lifetimes in these systems are dependent on
mechanisms that compete with non-radiative decay which do not lead to photoisomerization
and might not involve large amplitude torsional motions. Through NMR and LC-MS analysis
we established that isomerization to cis-products does not occur upon irradiation of our lead
candidate dyes, d7, d9 and d10. Similarly, in the trans-diphenylbutadiene system, Saltiel et.
al30 have observed radiationless decay pathways that do not lead to photoisomerization
resulting in the reduction of fluorescence quantum yields and lifetimes in contrast to the
corresponding trans-stilbene derivatives.
Electronic Donor-Acceptor (push-pull) D--A systems, where D = -NH2, -N(CH3)2, -
OH and A= –CN, -NO2 etc., have been studied extensively for their tendency to exhibit large
stokes shifts and a solvent dependent dual fluorescence behavior.32 These observations have
been explained using a three-state model as opposed to a conventional two-state model as seen
in Figure 6, adapted from the work reported by Lapouyade and coworkers33,34 wherein LE*
represents a highly polar, locally excited fluorescent state with planar geometry; A* represents
a highly polar, TICT fluorescent state with a twisted group, and P* represents a weakly polar,
non-fluorescent funnel to the ground state with twisting about the stilbene double bond. The
dynamic transformation process between LE* and A* has been hypothesized to go through an
energy barrier that has been shown to be sensitive to the polarity of solvents and the
concentration of the dye.33
9

We sought to exploit this effect in the bis-styryl system. Substitution of an electron-
withdrawing cyano moiety for one of the amines in the two-ring stilbene (d2) led to a large
stokes shift as a result of accessing the ICT (A*) state.35 Applying this same substitution to
the three-ring BMB system (comparison of compounds d7 vs. d1) led to a modest 9 nm
bathochromic shift in the absorbance, a primary emission band at 491nm attributed to the LE*
state and an additional long wavelength emission attributed to the A* ICT state at 621 nm.
Substitution with alternative electron withdrawing groups methylsulfonyl or sulfonamide,
introduced to improve pharmacokinetics and pharmacology properties, (compounds d9 and
d10) resulted in similar shifts in absorption and emission. The long wavelength emission is
only observed in polar solvents such as DMSO, Figure 7A, and providing further evidence in
support of formation of an ICT state.
Alkylation of the amine in the 3-ring, D--D BMB system (d1) led to compound d5
with a 7 nm bathochromic shift in absorption and a 16 nm shift in emission, as might be
expected; however, the emission intensity was significantly reduced. Similar N-alkylation in
the push-pull D--A system (d8 & d12) led to complete suppression of the long wavelength
emission. This observation is contrary to the behavior reported in the well-studied DMABN
system in which aminobenzonitrile displayed emission only from the LE* state and N,N-
dimethyl aminobenzonitrile displayed a long wavelength emission from the A* (ICT) state, in
turn attributed to twisting about the aniline bond.35 Gruen & Gorner also reported lack of dual
emission in the 2-ring N,N-dimethylamino-4-cyanostilbene.36 We hypothesize that the
dissimilar emission behavior in the alkylated vs. non-alkylated 3-ring system may be due to a
combination of twisting about the stilbene bonds and the presence of an electron-donating
methoxy substituent in the central ring though more extensive photophysical studies are
required to explain the behavior of this unique system
Further investigation of the four systems (d7-d10) uncovered an additional
phenomenon which is unprecedented, at least in the case of the three-ring, push-pull stilbene
systems. Compound d7 showed a dual emission in a polar solvent (DMSO) similar to that of 4-
amino-4’-cyanostilbene (ACS).37 However, to our surprise, this dual emission displayed two
distinct excitation bands at 380 and 420 nm respectively for the 491 nm (LE) and 621 nm (CT)
emissions, yet only a single excitation and emission in a non-polar solvent (Figure 7). The
wavelength dependent emission of compound d7 is further illustrated by exciting from 300
10

nm-500 nm in 5 nm intervals and collecting the corresponding emission spectra in DMSO
(Figure 8). The observed solvent dependence and large Stokes shift associated with the long-
wavelength emission strongly support the assignment of dual emission peaks at 491 and 621
nm to LE and CT states respectively. The wavelength dependence and distinct excitation
bands would suggest the excitation of two different species in polar media. We hypothesize
that these may represent ground-state conformers that differ through twisting about one of the
stilbene single-bonds and that one of these conformers has an arrangement that facilitates
formation of the ICT state.
Recently, Atsbeha et al.38 and Catalan et al.39 reported that a similar trend was seen for
DMABN in polar solvents. Catalan hypothesized that a dipolar solvent forms species with
charge transfer properties in its ground electronic state and such forms can be directly excited
to a TICT structure. However the anomalous behavior with the systems under our study stems
from the fact that while the amino derivative d7 showed dual emission in a polar medium, the
N,N-dimethyl derivative d8 did not display the expected long-wavelength TICT emission.
Compound d8 showed a single LE emission maximum at 460 nm in non-polar medium
(toluene) and a single emission peak maximum at 500 nm in polar medium (DMSO) as shown
in Figure 7. Unlike DMABN where twisting of N,N-dialkyl group is responsible for TICT
emission (Scheme 5), the push pull stilbene based system is known to undergo aniline moiety
twisting which could also thought be responsible for dual emission characteristics.40 Unlike 4-
cyano-4’-amino stilbene systems in which twisting about the aniline bond represents the only
degree of rotational freedom (Scheme 5), multiple ground state conformers that may exist in
the three ring stilbene-type systems as shown in Scheme 6.
Thus, it was not surprising that compound d7, with a less bulky amino group, could
still show a dual emission behavior in a polar solvent, as the dual emission in the stilbene type
system does not depend on amine bond twisting. However, one would have expected the
corresponding N,N-dimethyl system d8 to still show at least a similar dual emission behavior,
even if not a more pronounced effect. We believe that the methoxy- substitution has an
important role to play for the dual emission behavior of d7 and absence of the same in d8.
11

Attempts to cross check if the methoxy- substituent is indeed responsible for the above
emission behavior in d7 and d8 by making the parent tri-ring stilbene system without the
methoxy- susbstituent was not pursued because this molecule was shown to be insoluble in
polar solvents such as MeOH and DMSO.41 Recently Singh et al.42 reported a similar
observation in the 1,2-diarylethene system having a linear acceptor group such as –CN on one
of the phenyl rings and two electron donor groups such as –OMe on the other phenyl group,
giving systems capable of solvent polarity-dependent dual fluorescence. The short wavelength
fluorescence band was assigned to LE state, which is electronically delocalized with a planar
geometry. The long wavelength fluorescence band has been attributed to a TICT state having a
dipolar character. The authors reported that they couldn’t determine the exact site of excited
state bond twist leading to the formation of TICT species.
There is literature precedence for molecular structures that possess N,N-dimethyl
substituted push-pull dyes which show no TICT emission. Since double bond rotation
transcis is excluded in these systems, we hypothesized that the deactivation from the
singlet-excited state is mainly achieved through an internal conversion mechanism in polar
solvents. Fromherz, Heilmann and others reported that N,N-dimethyl substituted hemicyanine
undergoes internal twisting and a yields a twisted internal charge transfer (TICT) state on
photoexcitation [Scheme 7].43,44 However, no dual emission was observed in this molecule
(HC-3) and the authors hypothesized a non-emissive TICT state. Due to multiple bond
rotations producing rotamers in hemicyanine dyes, the ICT states are deactivated. Strehmel
and co-workers performed quantum mechanical calculations on various TICT conformations
of hemicyanine dyes, and ruled out the possibility that deactivation of TICT state is due to
twisting of diamino group, since this charge transfer state is approx. 1.5 eV higher in energy
than that of the Frank–Condon state.45 The authors attributed twisting of single bonds adjacent
to the central double bond to the formation of CT states comparable in energy to that of the FC
state and causing the singlet-excited state twisting of the aniline ring to become barrierless in
polar
solvents.
Fromherz
also
studied
two
other
dye
systems,
(dimethy1amino)methylazafluorenium with
a
rigid fluorene group [HC-3] and
[(dimethylamino)phenyl]methylpyridinium [HC-2] with a freely rotatable biphenyl frame.43
The quantum yield of the more rigid dye is around 50%, while the quantum yield of the
twistable homolog is about 0.05% in solvents of high polarity and fluidity. The difference in
12

the fluorescence behavior is attributed to the formation of a TICT state with efficient
radiationless deactivation.
We investigated additional systems to verify that the emission behavior of compound
d7 and d8 was neither a single anomaly nor an artifact. Compounds d9 and d10 are similar to
Compound d7 but the electron accepting –cyano group replaced by another electron accepting
groups methylsulfonyl and piperazinylsulfonyl respectively. Compound d9 and d10 still
showed similar LE and TICT dual emissions similar to that of d7 and exhibited two ground
state conformers that could be individually excited. Dual emission of compound d9 in DMSO
obtained using excitation wavelength 380 nm is shown in Figure 9.
A phenol replacing the amino group proved to be a much poorer donating group
(compound d11) even under pH conditions which ensured complete deprotonation to the
respective phenoxide resulting in hypsochromic shift by ~20 nm and reduced stokes shift
compared to d7. Locking the tertiary amine moiety in a planar conformation in the indoline
system (compounds d13-d14) resulted in different effects depending on the system it was
implemented. Compound d13 showed reasonable red-shifted absorption characteristics and
modest fluorescence. Increasing the strength of the acceptor group by using
dicyanomethylene-pyranone, as in compound d14, resulted in a significant bathochromic shift
in absorbance ( = 63 nm in compound d14 vs. compound d13) accompanied by the same
large fluorescence shift but had very poor emission.
Interestingly, when compound d7 was injected into a mouse and the dye bound to
nerves were excited by either 405 nm or 447 nm laser, the wavelength dependent emission
described above was observed (Figure 10). Excitation at longer wavelength (447 nm) showed a
more pronounced red-shifted second peak. There were subtle differences between the in vivo
(in mouse nerve) and in vitro (in DMSO solution) spectra of d7, which was expected because
one medium is in solution while the other is in rigid tissue environment.
Conclusion:
13

We have successfully screened a series of styryl- and distyryl dye systems (d1-d14) and
identified three promising dyes (d7, d9 and d10) that exhibit long wavelength emission, large
Stokes shifts and reasonable fluorescence intensities while maintaining adequate solubility and
high affinity to myelin. We observed an unusual wavelength-dependent dual emission
behavior in which two distinct ground state conformers could individually be excited to favor
emission from the locally excited (LE) or twisted intramolecular charge transfer (TICT)
excited states in push-pull dye systems d7, d9 and d10. Additionally, we observed an
anomalous emission behavior of dye systems d7and d8 under our study conditions, wherein
the amino- derivative d7 showed dual emission characteristic in polar medium, and the N,N-
dimethyl derivative d8 and the other N-alkylated derivatives d12-d14 did not show any TICT
emission at all in either ground state or excited state. We envision that results presented here
will elicit other research groups to elaborate on these photophysical observations.
The in vivo fluorescence imaging studies in rodents injected with selected dyes showed
that visualization of nerves against a dark surrounding muscle tissue was possible. Because of
their lipophilicity, the dyes also showed some non-specific uptake in adipose tissue, which
appeared a different color than that of the nerves under multispectral imaging. These nerves
were detectable while surrounded by fascia under fluorescence guidance.
Despite operating in the visible spectrum, we demonstrated that intraoperative
fluorescence imaging in this optical window is a viable solution under certain conditions. For
example, the large Stokes shift helps to minimize the impact of autofluorescence, similar to the
approach taken in near-infrared imaging. Our future work on this class of dyes includes the
development of derivatives that are less lipophilic, while maintaining their ability to cross the
blood-nerve-barrier, in conjunction with applying novel strategies for differentiating the
fluorescence of nerves from that of adipose tissue.
ACKNOWLEDGMENTS: We thank Bruce Johnson, Eric Williams, Jorge Ramos,
Joseph Greene, Nicole LaPlante, Daniel Gray, and Kenneth Fish for technical assistance. This
work is supported by NIH grant EB022872.
SUPPLEMENTAL MATERIAL
14

See Supplemental Material for detailed synthetic procedures for dyes d1-d11 and the
corresponding characterization of the compounds using NMR and other relevant spectroscopic
analysis.
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18

Tiberiu Siclovan obtained his early
education in chemical engineering from
Timisoara Polytechnic, Romania. In
1992 he moved to the United States,
and in 1996 received his Ph.D. in
organic chemistry from Iowa State
University under the direction of
professor George A. Kraus. That same
year he accepted a position at GE
Global Research, where he is currently
a senior scientist. His research interests
include various aspects of biomedical
and imaging technologies.
Rong Zhang received her B.S in
chemistry from Nanjing University in
China and her MS in organic chemistry
from Worcester Polytechnic Institute.
Prior to joining GE Global Research in
2005, she was a medicinal chemist at
Abbott Laboratories. At GE Global
Research, Rong is a lead chemist working
on developing technologies and agents to
enable diagnostic medical imaging.
Victoria Cotero received her B.S in
chemistry from Fairleigh Dickinson
University and her Ph.D. in
pharmacology and human physiology
from New Jersey Medical School, now
Rutgers Medical School, in 2009. She
then served as a postdoctoral fellow in
neuroendocrinology/behavioral
neuroscience at SUNY-Albany. At GE
Global Research, Victoria is a lead
biologist working on medical
technologies for imaging and preclinical
applications.
19

Anshika Bajaj obtained a B.S. in Pharmacy and M.S.
in Biological sciences from BITS, Pilani, India in
2000, after which she moved to the United States to
pursue a Ph.D. in Biochemistry from the University
of Rochester. In 2006 she started her post-doctoral
work at Albany Medical College and focused on
studying signaling mechanisms involved in
angiogenesis. She joined GE Global Research in 2010
where she contributed to and led biology tasks on
multiple neuro, cell therapy, and immunotherapy
projects. In 2015 she joined Immune Design a clinical
stage immunotherapy company where she is
responsible for in vitro characterization of lenti-
Dmitry V. Dylov received his B.S.and
M.S. degree in applied physics and
mathematics from the Moscow Institute of
Physics and Technology and Ph.D. from
Princeton University in 2010. His
research interests include theoretical
aspects of imaging, spectroscopy and
biophotonics.
Siavash Yazdanfar received his BS from
Boston University and MS and PhD from
Case Western Reserve University in
biomedical engineering. Prior to joining
GE in 2005, he was a postdoctoral fellow
at the Massachusetts Institute of
Technology. He has published 38 peer
reviewed journal articles, 5 book chapters,
and 22 issued US patents, primarily in the
area of biomedical optical imaging and
instrumentation.
20

Randall L. Carter received his B.S. in
chemistry from the Pennsylvania State
University and Ph.D. from Yale
University in 1993. He served as an NIH
post-doctoral fellow and visiting assistant
professor at Duke University before
joining GE Global Research in 1996. His
is currently managing the Translational
Sciences Laboratory with a research focus
on the development of technologies and
agents to enable diagnostic medical
imaging.
Cristina Tan Hehir received her
bachelor’s degree in chemistry from the
University of the Philippines Diliman.
She obtained her Ph.D. in
Chemistry/Biochemistry from Boston
College and was a visiting associate at the
Laboratory of Cell Biology at
NHLBI/NIH. At GE Global Research,
Tina is a senior scientist and project leader
working on technologies for fluorescence
guided surgery and development of
imaging probes for neuroscience. She is
also an NIH panel reviewer for Clinical
Molecular Imaging Probe Development.
21

Arunkumar Natarajan obtained his B.S. degree in 1997 from Loyola College, Chennai,
India and obtained his M.S. from the Indian Institute of Technology (IIT), Mumbai, India in
1999. Then he moved to the United States, and in 2005 obtained his Ph.D. from Tulane
University. He continued his postdoctoral studies at UCLA from 2005-07. He joined GE
Global Research in 2008, where he currently holds a lead scientist position. He has over 42
peer reviewed publications including 7 book chapters and 24-filed and 15-issued US patents.
His scientific interests include photochemistry, polymers and materials chemistry for various
technology applications including biological imaging, holography and additive manufacturing.
Figure captions
Scheme 1. Synthesis of D-A bis-styryl dyes d7-d10 and their analogs d7: R1=R2=H, R3=CN;
d8: R1=R2=Me, R3=CN; d9: R1=R2=H, R3=MeSO2; d10: R1=R2=H, R3=N’-(2-hydroxyethyl)-
N-piperazinylsulfonyl.
Scheme 2. Synthesis of compound d11
Scheme 3. Synthesis of dyes bearing multiple electron-withdrawing groups
Scheme 4. Synthesis of styryl and polyene dyes d2-d4
Scheme 5: Access of TICT state in polar medium via Intramolecular N,N-dimethyl- group
twisting of DMABN in polar medium and anilino- group twisting in push-pull stilbenes.
Scheme 6: Possibility of multiple ground state conformations (rotamers) due to single bond
rotations in push-pull tri ring systems.
Scheme 7: Dependence of bond rotations on TICT emission resulting in non-Fluorescent
TICT (HC-1); weakly fluorescent (HC-2) and strongly fluorescent (HC-3).
22

Figure 1: Overlay of emission of d1 and d7 in Toluene (A) and in DMSO (B)
Figure 2: Qualitative ex vivo nerve labeling screening of several fluorophores.10 M of each
dye was incubated with rat nerve tissue sections to assess their ability to stain the myelin
bundles by fluorescence microscopy. The control tissue was incubated with buffer only. Scale
bar ~50 m.
Figure 3: In vivo multispectral imaging of mice injected with d7 (GE3082), d9 (GE3111), or
d10 (GE3126). The control animal received injection buffer only, and imaged under the same
conditions as the dye-treated mice. Arrows are pointing to the trigeminal and optic nerves (d7)
and to the sciatic nerve (d9, d10). Scale bar ~ 1 mm.
Figure 4: Simultaneous dual-mode (white light and fluorescence laparoscopic imaging of the
thoracic cavity of a rat injected with d7 (GE3082). The white arrows are pointing to the
phrenic nerve, and yellow arrows are pointing to a layer of fascia surrounding the nerve. Scale
bar ~ 1 mm.
Figure 5: Fluorescence emission spectra of nerve, adipose tissue, and muscle of a mouse
injected with d10 (GE3126).
Figure 6: Interpretation of photophysical behavior of push-pull stilbenes (Left) Conventional
Two-State and (Right) Proposed Three-State Kinetic Scheme.
Figure 7: Excitation and emission spectra of compounds d7 (A) and d8 (B). d7 shows two
distinct excitation peaks corresponding to LE and TICT state emission in DMSO, while and
compound d8 shows only one excitation and emission corresponding to the LE state.
Figure 8: Wavelength dependent dual-emission spectrum of compound d7 (GE3082) showing
the presence of two distinct conformers in the ground state with different population levels.
Figure 9: A. Dual-emission seen in compound d9. Emission obtained using excitation
wavelength-380 nm. B. Wavelength dependent dual-emission spectrum of compound d10
showing presence of two distinct conformers in the ground state with different population.
Figure 10: Wavelength dependent emission spectrum of compound d7 (GE3082) in the sciatic
nerve of a mouse injected with d7.
Tables
Table 1. Styryl and bis-styryl fluorophores in solution

Emc, nm
Absb, nm
Namea
Chemical Structure
(Intensity)d
495
d1
(BMB)a
408
362
(106,800)
414
d2
(246,900)
23

448
383
400
d3
d4
(84,300)
575
(200)
511
415
d5
d6
(93,300)
522
385
417
(2,800)
491, 621*
(1,700; 2,520*)
d7
(GE3082)a
518
412
412
d8
(264)
480, 624*
(2,250; 4,700*)
d9
(GE3111)a
510, 618*
(2,100; 3,970*)
d10
(GE3126)a
414
396
532
d11
(12,640)
24

582
467
419
d12
d13
(180)
533
(2,900)
649
482
d14
(150)
aThe d-numbering scheme for each dye is used for simplicity. For previously published
fluorophores, the original designations are shown in parenthesis.
bAbsorbance maximum wavelength measured in DMSO
cEmission maximum wavelength measured in DMSO, the relative fluorescence intensity at the
maximum wavelength shown in parenthesis
d Relative emission intensity values at Em RFU. For dual emissions, the emission intensities in
parentheses correspond to the Em shown, respectively.
* Dual emission (TICT) in polar solvents.
Fig. 1
25

Fig. 2
26

Fig. 3
Fig. 4
27

Fig. 5
Fig. 6
28

A
d7-excitation spectrum
corresponding to CT-
650nm emission
d7-excitation spectrum
corresponding to LE-
470nm emission
d7-dual emission in
DMSO
350
450
550
650
Wavelength (nm)
B
Fig. 7
29