Dual-Self-Restricted GFP Chromophore Analogues with Significantly Enhanced Emission

Article abstract of DOI:10.1021/acs.jpcb.9b11329

The tremendous gap of fluorescence emission of synthetic green fluorescent protein (GFP) chromophore to the protein itself makes it impossible to use for applications in molecular and cellular imaging. Here, we developed an efficient methodology to enhance the photoluminescence response of synthetic GFP chromophore analogues by constructing dual-self-restricted chromophores. Single self-restricted chromophores were first generated with 2,5-dimethoxy substitution on the aromatic ring, which were further modified with phenyl or 2,5-dimethoxy phenyl to form dual-self-restricted chromophores. These two chromophores showed an obvious solvatofluorochromic color palette across blue to yellow with a maximum emission Stokes shift of 95 nm and dramatically enhanced fluorescence emission in various aprotic solvents, especially in hexane, where the QY reached around 0.6. Importantly, in acetonitrile and dimethyl sulfoxide, the fluorescence QYs of both chromophores were over 0.22, which were the highest reported so far in high polar organic solvents. Meanwhile, the fluorescence lifetimes also improved obviously with the maximum of around 4.5 ns. Theoretical calculations revealed a more favorable Mülliken atomic charge translocation over the double-bond bridge and illustrated much higher energy barriers for the Z/E photoisomerization together with larger bond orders compared with the GFP core chromophore, p-HBDI. Our work significantly improved the fluorescence emission of synthetic GFP chromophore analogues in polar solvents while reserved the multicolor emitting function, providing a solid molecular motif for engineering high-performance fluorescent probes.

Full text of DOI:10.1021/acs.jpcb.9b11329

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B: Liquids, Chemical and Dynamical Processes in Solution, Spectroscopy in Solution
Dual-Self-Restricted GFP Chromophore
Analogues with Significantly Enhanced Emission
Hongping Deng, Chunyang Yu, Deyue Yan, and Xinyuan Zhu
J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b11329 • Publication Date (Web): 13 Jan 2020
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Dual-Self-Restricted GFP Chromophore Analogues
with Significantly Enhanced Emission
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Hongping Deng, Chunyang Yu,* Deyue Yan,* and Xinyuan Zhu*
School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix
Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China.
Fax: +86-21-54741297.
To whom correspondence should be addressed:
C.Y. - chunyangyu@sjtu.edu.cn
D.Y. - dyyan@sjtu.edu.cn
X.Z. - xyzhu@sjtu.edu.cn
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ABSTRACT
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The tremendous gap of fluorescence emission of synthetic green fluorescent protein (GFP)
chromophore to the protein itself makes it impossible for applications in molecular and cellular
imaging. Here, we developed an efficient methodology to enhance the photoluminescence
response of synthetic GFP chromophore analogues by constructing dual-self-restricted
chromophores. Single self-restricted chromophores were firstly generated with 2,5-dimethoxy
substitution on the aromatic ring, which were further modified with phenyl or 2,5-dimethoxy
phenyl to form dual-self-restricted chromophores. This two chromophores showed obvious
solvatofluorochromic color palette across blue to yellow with maximum emission Stokes shift of
95 nm, and dramatically enhanced fluorescence emission in various aprotic solvents, especially in
hexane, where the QY reached around 0.6. Importantly, in acetonitrile and dimethyl sulfoxide, the
fluorescence QYs of both chromophores were over 0.22, which were the highest reported so far in
high polar organic solvents. Meanwhile, the fluorescence lifetimes also improved obviously with
the maximum of around 4.5 ns. Theoretical calculations revealed a more favorable Mülliken
atomic charge translocation over the double-bond bridge and illustrated much higher energy
barriers for the Z/E photoisomerization together with larger bond orders compared to the GFP core
chromophore, p-HBDI. Our work significantly improved the fluorescence emission of synthetic
GFP chromophore analogues in polar solvents while reserved the multicolor emitting function,
providing a solid molecular motif for engineering high-performance fluorescent probes.
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KEYWORDS: Fluorescent dyes, aggregation-induced emission, fluorescent proteins, green
fluorescent protein chromophore, photoisomerization.
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INTRODUCTION
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The genetically encoded green fluorescent protein (GFP) has provided a powerful tool for labeling
and tracing biological targets in biochemistry and cell biology.1-4 The discovery and development
of fluorescent family have achieved simultaneous multicolor imaging of a variety of biomolecules,
and deep tissue or live animal imaging with red to near-infrared proteins.5-9 However, the
fluorescence quantum yields (QYs) dropped dramatically over four orders of magnitude for
denatured proteins.10 It is beneficial to understand protein photophysics, develop new fluorescent
probes and find potential applications by investigating chemically synthesized GFP chromophore
and its analogues.11,12 The loss of fluorescence for these chromophores significantly hindered this
process, owing to an ultrafast internal conversion due to free molecular motions of the carbon-
carbon bonds.13-17
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A variety of methodologies have been applied to increase the QYs of these chromophores by
inhibiting the nonradiative pathways over past years.18,19 Physical encapsulation with different
hosts, such as proteins,20,21 ribonucleic acids,22,23 amphiphilic macromolecules,24 porous
scaffolds,25 has greatly improved the corresponding fluorescence response, but significantly
increased the overall probe size, making it difficult for further molecular labeling. Chemical
locking with covalent bonds or metal chelation,26-29 results in very high fluorescence QYs
comparable to commercial dyes, but loses multicolor emitting function, which is important for
constructing responsive probes. Similar loss of multicolor emission happens for hydrogen-bond
locking chromophores, which generally shows low QYs in different fluid solvents.30,31
Meanwhile, although tremendous efforts have been made to make highly emissive unlocked GFP
chromophore analogues, there is very limited progress in enhancing the emission, especially in
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high polar solvents.18,19 The most widely utilized strategy was to increase the molecular
conjugation length at the aromatic or azalactone ring, but the efficacy was still limited due to
efficient molecular motions.32-34 Meta-amine GFP-like chromophores were synthesized and
reported to have QYs over 0.4 in hexane, but dropped below 0.1 in polar aprotic solvent,
acetonitrile.35 Further modification with a push-pull substituent on the phenyl ring, the QY of the
corresponding chromophore increased to 0.6 in hexane, but still was less than 0.1 in acetonitrile.36
Recently, pyridinium analogues of GFP chromophore were reported to have very good QYs in
water, but still very low in polar organic solvents, which might be due to the poor solubility in
water.37 We have disclosed an unusual enhancement of fluorescence emission for 2,5-dialkoxy-
substituted GFP-like chromophores with QYs over 0.1 in polar solvents, such as acetonitrile,
dimethylformamide and dimethyl sulfoxide, due to the formation of a self-restricted effect.38 It
would be very interesting to investigate the impact of dual-self-restricted effects on the
fluorescence response of the chromophores.
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Here, we designed and synthesized two dual-self-restricted GFP-like chromophores by the
standard Erlenmeyer azalactone methodology. The fluorescence response in various solvents were
studied, showing significant emission enhancement in all tested solvents, especial unexpected
improvement in high polar solvents. Theoretical calculations further revealed the molecular
mechanism due to increased rotation energy barrier along the carbon-carbon double bond.
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RESULTS AND DISCUSSION
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Synthetic GFP-like chromophores are non-emissive owing to the ultrafast internal conversion
along the carbon-carbon bonds.13-15 Theoretically, an efficient methodology to inhibit the carbon-
carbon molecular motions would adopt locking strategy at both the aromatic and azalactone rings.
Thus, single non-covalent “locking” with self-restricted effect was designed at the aromatic ring,
while a second restriction was applied to the azalactone ring via phenyl or 2,5-dimethoxy-phenyl
substitution, resulting in dual-self-restricted chromophores (Scheme 1a). Considering the
synthetic liability and chemical reactivity, these two compounds, 4-(2,5-methoxybenzylidene)-2-
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phenyloxazol-5(4H)-one
(MBDPO)
and
4-(2,5-methoxybenzylidene)-2-(2,5-
methoxyphenyloxazol)-5(4H)-one (MBDMPO), were prepared into oxazolone analogues via the
standard Erlenmeyer synthesis39,40 with relatively good yields, which included the cyclization
reaction between benzaldehyde and benzoylglycine derivatives. For MBDMPO, the
benzoylglycine derivative was prepared via a two-step process, which contained an amidation
reaction between 2,5-dimethoxybenzoic acid and glycine methyl ester hydrochloride, and a
hydrolysis reaction of the corresponding ester product. The synthetic procedure was given in
Scheme 1b and the molecular structures of the two chromophores were shown together with that
of GFP core chromophore, p-HBDI. The chemical structures of MBDPO and MBDMPO were
fully characterized, and detailed synthetic procedures and characterizations were provided in the
Supporting Information.
The optical properties of the two chromophores were firstly tested in ethyl acetate (EA, a good
solvent). As shown in Figure 1, the absorption spectra of MBDMPO and MBDPO displayed two
major peaks at around 340 nm and 425 nm respectively ascribing to electron transitions from π-π*
and n-π*.41 The substitution with 2,5-dimethoy group for MBDMPO weakened the electron
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transition from π-π*, but strengthened that from n-π*, making the second absorption peak red-
shifted compared to that of MBDPO. Generally, GFP-like chromophores are very dim in fluid
solvents due to free rotation along either the single or double bond, resulting in significant
fluorescence quenching.18,19 On the contrary, the inhibition of either the single or double bond
would lead to the increase of fluorescence emission. For the two chromophores, an important
question was whether they could display obvious fluorescence in fluid solvent at room
temperature. Apparently, bright green fluorescence was observed for both MBDMPO and
MBDPO in EA with emission peaks at 509 nm and 516 nm, respectively. These emission peaks
red-shifted more than 80 nm compared to that of p-HBDI due to self-restricted effect and increased
conjugation at the heterocyclic ring. Moreover, the fluorescence quantum yields and lifetimes of
MBDMPO and MBDPO were also tested in EA to determine the degree of fluorescence emission
enhancement (Table S1, Table S2). The QYs of MBDMPO and MBDPO were about 0.36 and
0.34 with fluorescence lifetimes of around 3.1 ns and 3.9 ns, respectively, which improved
significantly compared to the core chromophore (p-HBDI) and other unlocked GFPc analogs,18,19
suggesting the effective inhibition of carbon-carbon molecular motions.
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To prove the generosity of the enhanced emission, the solvent dependence of fluorescence
emission for MBDMPO and MBDPO was further investigated in different solvents. As given in
Figure 2, the absorption spectra for both chromophores showed minor changes with increasing
solvent polarity from Hex to MeOH,42 displaying obvious two-peak absorptions for π-π* and n-π*
transition. One feature was the redshift of the absorption for n-π* transition along with increasing
polarity, which was still less than 15 nm in all solvents tested. The other feature was the enhanced
absorption intensity for π-π* transition compared to that of n-π* transition in MeOH owing to the
formation of strong solvent-solute hydrogen bonding.35,43 Meanwhile, intense fluorescence
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emission could be observed from hexane (Hex) to methanol (MeOH) for both MBDPO and
MBDMPO, proving the existence of the emission enhancement for the two chromophores.
Specifically, the emission spectra of MBDMPO had only one emission peak, which further
redshifted from 478 nm to 552 nm with distensible half band width from Hex to MeOH, displaying
obvious solvatofluorochromism feature.35-37 Meanwhile, the fluorescence QYs and lifetimes of
MBDMPO in these solvents were also tested to tell the degree of emission enhancement. In Hex,
the QY of MBDMPO reached around 0.6, which was unexpectedly high for unlocked GFP-like
chromophores.18,19 With the increase of solvent polarity, the emission QY decreased to 0.28 in
DMSO and further decreased to 0.05 in MeOH due to strong solvent-solute hydrogen-bonding
effect.35,43 The solvent-solute H-bonding effect was proposed by previously published
literatures,14,35 where the H-bond donor (methanol) and the imidazolinone group formed a type of
H-bonded intermediate, inducing a new nonradiative decay channel responsible for fluorescence
loss. The predominant H-bonded intermediate was through solvent hydrogen binding to the
carbonyl oxygen, other than the nitrogen of the imidazolinone group. The lifetimes of MBDMPO
were at about 2.4 ns to 3.2 ns from Hex to DMSO, but also decreased to 0.9 ns in MeOH (Table
S1, Figure S1). Likely, the solvent dependence of MBDPO followed the same phenomenon as
that of MBDMPO. From Hex to MeOH, the emission peak of MBDPO also redshifted to 555 nm
with a maximum Stokes shift of about 93 nm. The emission response of MBDPO was greatly
enhanced with fluorescence QYs between 0.41 and 0.22, and lifetimes between 2.6 ns and 4.5 ns
from Hex to DMSO, which decreased to 0.06 and 1.8 ns in MeOH (Table S2, Figure S2).
Importantly, the fluorescence QYs of both chromophores were over 0.22 in high polar solvents,
acetonitrile and dimethyl sulfoxide, which were the highest reported for unlocked GFP-like
chromophores to our knowledge in polar organic solvents. In short, the fluorescence performance
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of these two chromophores enhanced greatly in a variety of solvents, reflecting efficient inhibition
of carbon-carbon bond molecular motions.
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To investigate the influence of solvent-solute hydrogen bonding on fluorescence quenching, the
titration of MBDMPO and MBDPO in acetonitrile (ACN) with methanol was conducted with
increasing amount of hydrogen donor. As shown in Figure 3, for both chromophores, the
fluorescence intensity decreased gradually with the increasing content of MeOH in ACN from 0
to 250 μL, demonstrating the fluorescence quenching effect due to strong solvent-solute hydrogen
bonding.35,36 At the same time, the decrease of emission intensity for MBDMPO was much greater
than that of MBDPO, which proved that the solvent-solute hydrogen bonding was stronger for
MBDMPO. Fitting hydrogen donor amount (volume) with emission intensity, the fluorescence
quenching followed into an exponential decay manner for both chromophores. Meanwhile, the
effect of deuterated solvent on the fluorescence emission of MBDMPO and MBDPO was also
measured. The addition of MeOD in MeOH increased the emission intensity for both
chromophores, which further revealed the fluorescence quenching of hydrogen bonding.38 And,
the emission enhancement for MBDMPO was more significant than that of MBDPO under the
same condition, which also illustrated the stronger solvent-solute hydrogen bonding for MBDMPO
compared to MBDPO.
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A phenomenon induced by restriction of intramolecular rotation (RIR) was defined as aggregation-
induced emission (AIE), where the typical carbon-carbon single bond molecular motions were
greatly inhibited in aggregated state.44,45 It is interesting to see if these two chromophores could
have strong emission in both fluid solvents and aggregated state. Since both chromophores have
bright fluorescence in fluid solvents, the linear-relationship between chromophore concentration
and fluorescence emission was measured to make sure there was no aggregation due to high
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concentration of chromophores.46 Figure S3 proved that there was a good linear relationship for
MBDPO and MBDMPO between chromophore concentration and emission intensity when the
concentration was less than 10 μM in acetonitrile (a good solvent). A MeOH/water mixed solvent
system was selected to explore the emission response in aggregated state because the QYs of both
chromophores were the lowest in methanol among tested solvents. In the cases of AIE
fluorophores, the emission intensity would increase slowly until exceeding a certain amount of
bad solvent (usually water) in the mixed solvent.44 As shown in Figure 4, the fluorescence
intensity decreased hugely under 50% water content in the mixed solvents for both chromophores,
which most likely was due to solvatochromic effect. Under higher water content, the emission
intensity recovered and enhanced to a maximum of about 2.3-fold increase compared to that in
MeOH, but it was still not able to compete with QYs in aprotic solvents. The enhanced emission
was ascribed to aggregation-induced emission,44,45 where the formation of molecular aggregates
greatly inhibited the free molecular motions of the corresponding chromophores, leading to the
increment of emission intensity. Meanwhile, there was a general redshift phenomena of the
emission peaks with a maximum Stokes shift of about 20 nm, which might be due to solvent-solute
interaction or chromophore aggregation.35,47 Under 90% water content, the emission intensity
decreased for MBDMPO but increased for MBDPO with similar molecular structure. The
introduction of two methoxy groups decreased the overall coplanar conformation for MBDMPO,
leading to the formation of less ordered aggregates under higher water content.47 Together, these
results might suggest that the dual-self-restricted strategy might be able to produce highly emissive
GFP-like chromophores in both solutions and aggregates with molecular tailoring.
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To reveal the mechanism for improved fluorescence response, theoretical calculations were
employed to compare the difference of the three chromophores (Scheme 1a). The optimum
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geometries of p-HBDI, MBDMPO and MBDPO molecules calculated from density functional
theory (DFT) calculations (in gas phase) are provided in Figure 5.48 We can find along with the
rotation around the double bond between the imidazolinone heterocycle and the aromatic ring, p-
HBDI and MBDPO have two configurations, while MBDMPO has four configurations. In the
meanwhile, the relative energies of the most stable structures for these molecules are also
calculated. Specifically, p-HBDI 2 relative to p-HBDI 1 is 2.70 kcal/mol; MBDPO 2 relative to
MBDPO 1 is 3.50 kcal/mol; MBDMPO A2 relative to MBDMPO A1 is 4.13 kcal/mol; MBDMPO
B2 relative to MBDMPO B1 is 3.45 kcal/mol. It should be noted that each Z-E isomerization path
corresponds to one radiationless decay channel in the excited state. So, the increment of
isomerization barrier in the excited state is beneficial for the improvement of the fluorescence
performance.
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Also, the electron density distribution for these compounds were further analysed.49,50 As given
in Figure S4 and S5, the HOMO of MBDPO is delocalized over the aromatic and the azalactone
heterocycle, and the phenyl ring has less contribution. Meanwhile, the HOMO-1, LUMO and
LUMO+1 are delocalized over the whole molecule, which means the electron will transfer along
the double bond between the aromatic and the azalactone heterocycle upon excitation. In contrast,
the HOMO-1 of MBDMPO is localized over the two phenyl rings and the azalactone heterocycle
has less contribution. The HOMO, LUMO and LUMO+1 are the out-of-phase and in-phase
combination of three localized π bonds, and all of them are delocalized over the whole molecule.
However, we can find that the introduction of methoxy group increases the conjugation of
molecule MBDMPO. Thus, relative to molecule MBDPO, a red shift of n-π* absorption for
MBDMPO has been observed both in experiments and theoretical calculations (Figure S6).
Moreover, the obvious solvatofluorochromism feature is observed for all the optimized structures
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of both MBDPO and MBDMPO (Figure S7), which is also in accordance with experimental
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Meanwhile, previous works have shown that the translocation of the negative charge from the
aromatic ring to the azalactone heterocycle favours to restrict the motion around the exocyclic
double bond and inhibit this radiationless channel.38,51,52 As shown in Figure 6, the negative
charges are mainly located on aromatic ring for MBDMPO and MBDPO molecule at S0 state, and
then it has an obviously charge translocation from aromatic ring to azalactone heterocycle upon
excitation, which will be favorable to inhibit the radiationless decay channel around the exocyclic
double bond. Meanwhile, the charge transfer (S1-S0) for heterocycle upon excitation is very
similar for three chromophores, but there is a major difference for the aromatic ring (Table S3).
The aromatic ring charge transfer (S1-S0) order for the three chromophores is MBDMPO >
MBDPO > p-HBDI, which means a bigger part of charge is transferred to the double bond with
the same order as MBDMPO > MBDPO > p-HBDI. The more electrons transfer to the double
bond bridge, the better Z/E photoisomerization inhibition. Thus, the calculated results are agreed
well with the experimental data that the fluorescence emission response follows the order as
MBDMPO > MBDPO > > p-HBDI.
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Moreover, the stability of the twisted state relative to the Z-isomer state for p-HBDI, MBDPO and
MBDMPO has been examined by time-dependent density function theory (TDDFT)
calculations.14,36,53 As shown in Figure 7a, the size of the stability of twisted state is in the order
of p-HBDI (-6.40 kcal/mol) > MBDMPO A (7.13 kcal/mol) > MBDMPO B (7.74 kcal/mol) >
MBDPO (8.30 kcal/mol). Although the order of the relative stability of twisted state is not the
same as the order of the fluorescence performance, the higher relative energy for MBDPO and
MBDMPO than p-HBDI indeed support its fluorescence enhancement. The substituent electronic
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effect on the isomerization process is more complicated than the relative energies of the Z-isomer
and twisted states.36 To further have a comparison between MBDPO and MBDMPO, a Mayer
bond analysis was performed.54-56 As shown in Figure 7b, the C=C bond order of MBDPO 1 and
MBDMPO A1 are 1.8120 and 1.9578, respectively, which is much higher than p-HBDI (1.5840).
As we know, the higher bond order is closely related to the higher rotation energy barriers. That is
to say, the introduction of the methoxy group further increases the conjugation of C=C bond in
molecule MBDMPO. Consequently, molecule MBDMPO has a relatively better fluorescence
performance than molecule MBDPO.
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It is a big puzzle that synthetic GFP-like chromophores generally are non-emissive in solution
while remain their excellent optical properties in intact proteins.4-6,18 In this work, we have
developed emission significantly enhanced dual-self-restricted chromophores, which were highly
emissive in polar solvents and showed variable emission color palette from blue to yellow due to
the self-restricted effect and conjugated effect. Moreover, the mechanism for the enhanced
emission response was illustrated via theoretical calculations of the Mülliken atomic charge
translocation, the rotation energy barrier and the Mayer bond order, showing increased energy
barriers for Z/E photoisomerization. The dual-self-restricted chromophores were designed to be
functionalizable at multiple positions via different chemical synthesis approaches. With enhanced
fluorescence response of variable emission color palette, these chromophores are suitable for the
development of functional fluorescent probes for molecular and cellular applications.
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Scheme 1. Design and synthesis of two dual-self-restricted GFP-like chromophores. (a) Molecular
structures of the two chromophores with the GFP core chromophore, p-HBDI, showing efficient
molecular motions along the carbon-carbon bonds. (b) Synthetic routes towards the dual-self-
restricted chromophores, MBDMPO and MBDPO, via the standard Erlenmeyer synthesis.
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Figure 1. The optical properties of the two chromophores in good solvent, ethyl acetate (EA).
Normalized absorption (blue) and fluorescence emission (green) spectra of MBDMPO (a) and
MBDPO (b) in EA. Both chromophores showed enhanced bright green fluorescence. Emission
spectra were excited at maximum of absorption.
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Figure 2. The effect of solvent polarity on the optical properties of the two chromophores.
Normalized absorption (a, c) and fluorescence emission (b, d) spectra of MBDMPO (a, b) and
MBDPO (c, d) in different solvents. The fluorescence quantum yield (e) and lifetime (f) of
MBDMPO and MBDPO in different solvents, showing significantly increased QYs, especially in
high polar solvents. The emission spectra were excited using the maximum absorption.
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Figure 3. The effect of solvent-solute hydrogen bonding on the optical properties of the two
chromophores. The fluorescence emission of MBDMPO (a) and MBDPO (b) in acetonitrile with
increasing methanol (MeOH) from 0 μL to 250 μL. The exponential fitting of emission intensity
to methanol volume for MBDMPO (c) and MBDPO (d) with equations and R2 values. The
normalized fluorescence emission of MBDMPO (e) and MBDPO (f) in MeOH/MeOD mixed
solvents. The ratio of MeOD in MeOH was given.
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Figure 4. The effect of aggregation on the optical properties of the two chromophores. The
fluorescence emission (a, b) and its fold change of fluorescence intensity together with the change
of emission peak (c, d) for MBDMPO (a, c) and MBDPO (b, d) in mixed solvents of MeOH/H2O
with increasing water content in percentage. The emission spectra were excited using the
maximum absorption wavelengths in methanol.
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Figure 5. The optimized structures of three chromophores in gas phase obtained from DFT
calculations to obtain the most stable configuration of each chromophore. Different molecular
configurations for p-HBDI (a), MBDMPO (b) and MBDPO (c) with Z-configuration (1) or E-
configuration (2) were presented, while MBDMPO showed four configurations.
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Figure 6. The Mülliken atomic charge variation of aromatic ring (A) and imidazolinone
heterocycle (B) upon excitation (S0-S1) for p-HBDI 1, MBDPO 1 and MBDMPO A1 from DFT
calculations. Chromophores with the most stable configuration were selected to calculate the
charge transfer. The atomic charge of the bridge carbon is not included in the sum.
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Figure 7. Theoretical simulations revealed much higher Z/E photoisomerization barriers of the
two chromophores. (a) The TDDFT calculated relative energies (kcal/mol) of the Z* (the reference
state), the twisted intermediate, and E* states of MBDMPO, MBDPO and p-HBDI. (b) The Mayer
bond order analysis of molecule MBDMPO A1, MBDPO 1 and p-HBDI from DFT calculations.
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ASSOCIATED CONTENT
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Supporting Information. Synthesis, characterizations, time-resolved fluorescence and emission
spectra (Figure S1 to Figure S3), optical parameters (Table S1, Table S2) and theoretical
calculations data (Figure S4 to Figure S7, Table S3).
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AUTHOR INFORMATION
Corresponding Authors
*E-mail: chunyangyu@sjtu.edu.cn (C.Y.).
*E-mail: dyyan@sjtu.edu.cn (D.Y.)
*E-mail: xyzhu@sjtu.edu.cn (X.Z.).
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We acknowledge financial support from the National Basic Research Program of China
(2015CB931801) and the National Natural Science Foundation of China (51473093, 21404070).
ABBREVIATIONS
Hex: n-hexane; EA: ethyl acetate; ACN: acetonitrile; DMSO: dimethyl sulfoxide; MeOH:
methanol.
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