A General Descriptor Δ e Enables the Quantitative Development of Luminescent Materials Based on Photoinduced Electron Transfer

Article abstract of DOI:10.1021/jacs.0c01473

Photoinduced electron transfer (PET) is one of the most important mechanisms for developing fluorescent probes and biosensors. Quantitative prediction of the quantum yields of these probes and sensors is crucial to accelerate the rational development of n

Full text of DOI:10.1021/jacs.0c01473

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Article
A General Descriptor #E Enables the Quantitative Development of
Luminescent Materials based on Photoinduced Electron Transfer
Weijie Chi, Jie Chen, Wenjuan Liu, Chao Wang, Qingkai Qi, Qinglong Qiao, Tee Meng Tan,
Kangming Xiong, Xiao Liu, Keegan Kang, Young-Tae Chang, Zhaochao Xu, and Xiaogang Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c01473 • Publication Date (Web): 17 Mar 2020
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A General Descriptor ΔE Enables the Quantitative Development of
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Luminescent Materials based on Photoinduced Electron Transfer
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Weijie Chi,†,# Jie Chen,‡,# Wenjuan Liu,‡,# Chao Wang,†, ‡ Qingkai Qi,‡ Qinglong Qiao,‡ Tee Meng Tan,†
Kangming Xiong,‡ Xiao Liu,§ Keegan Kang,† Young-Tae Chang,§,* Zhaochao Xu,‡,* Xiaogang Liu†,*
† Fluorescence Research Group, Singapore University of Technology and Design, 8 Somapah Road 487372, Singapore.
‡ CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese
Academy of Sciences 457 Zhongshan Road, Dalian 116023, China
§ Department of Chemistry, POSTECH & Center for Self-assembly and Complexity, IBS, Pohang 37673, Republic of Korea
ABSTRACT: Photo-induced electron transfer (PET) is one of the most important mechanism for developing fluorescent probes
and biosensors. Quantitative prediction of the quantum yields of these probes and sensors is crucial to accelerate the rational
development of novel PET-based functional materials. Herein, we developed a general descriptor (Δ
yield of PET probes, with a threshold value of ~0.6 eV. When Δ < ~0.6 eV, the quantum yield is low (mostly <2%) due to the
substantial activation of PET in polar environments; when Δ > ~0.6 eV, the quantum yield is high because of the inhibition of PET.
This simple yet effective descriptor is applicable to a wide range of fluorophores, such as BODIPY, fluorescein, rhodamine, and Si-
Rhodamine. This Δ descriptor not only enables us to establish new applications for existing PET probes but also quantitatively
design novel PET-based fluorophores for wash-free bioimaging and AIEgen development.
E) for predicting the quantum
E
E
E
(Figure 1a), by directly linking the receptor to the fluorophore (i.e.,
via a single C-C bond), such as in a series of PET probes developed
by Nagano and Urano groups.15
INTRODUCTION
Photoinduced electron transfer (PET)1-2 has been frequently
employed for constructing numerous fluorescent probes to detect
various species that are of considerable biological importance.3-10
Modulating PET formations induces significant changes in the
quantum yields of these probes, thus affording a convenient route to
monitor analytes or environmental changes with high sensitivity,
vivid visibility, and good spatiotemporal resolution. Accordingly, the
accurate prediction of quantum yields represents one of the key
requirements in the rational development of novel PET probes.
Nevertheless, few simple and effective methods exist to
quantitatively predict quantum yields,11-12 especially when chemists
venture into new fluorophore structural spaces in search for novel
PET probes with tailored selectivity and sensitivity. To expedite the
molecular engineering of such probes and expand their utility, it is
imperative to formulate a fast and reliable approach to predict the
quantum yields of PET probes, based on a deep understanding of
their structure-property relationships.
Figure 1. Schematic illustration of (a) PET-based fluorescent probes of
the ‘fluorophore-spacer-receptor’ and ‘fluorophore-receptor’ formats;
(b) the change of fluorescence intensity for a PET probe.
Significant efforts have been devoted to formulating equations for
predicting the quantum yield of PET probes. The change in Gibbs
free energy (ΔG), as described by the Rehm-Weller Equation
Molecular structures of PET probes typically employ the
‘fluorophore–spacer–receptor’ format, as summarized by de Silva
and co-workers (Figure 1a).13-14 In the PET ON state, the quantum
yield of the fluorophore (ΦPET-ON) is extremely low, because the fast
PET rate substantially quenches fluorescence. In the PET OFF state,
the fluorophore emits bright fluorescence (high quantum yield;
ΦPET-OFF) as the PET process is inhibited upon the binding of the
analyte at the receptor site (Figure 1b). A low ΦPET-ON value is critical
to ensure a large turn-on or turn-off ratio (approximately ΦPET-
proposed in 1968,16 is an important descriptor to determine the
quantum yields of PET probes (including both ‘fluorophore–
spacer–receptor’ and ‘fluorophore–receptor’ formats), by assessing
the thermodynamic feasibility of PET. In general, a negative Δ
suggests an inferior quantum yield due to the activation of PET.
However, a more negative Δ does not always suggest a lower
G
G
quantum yield, i.e., due to the presence of the Marcus inverted
region.17-18 Moreover, the PET rate needs to kinetically compete
with the fluorescence rate. Although the kinetics of PET could be
quantified via Marcus equation in principle,19-20 it is often
/
OFF
ΦPET-ON), which quantifies the sensitivity of the PET probe. It is
of note that the “spacer” in a PET probe could also be removed
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challenging to accurately quantify many parameters (such as
electronic coupling matrix elements) of this equation.21
demand a thorough revision to the existing MO representation of
the PET process.
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Importantly, for PET probes with the ‘fluorophore–spacer–
receptor’ configuration, de Silva et al. firstly proposed that the
relative energy levels of the frontier molecular orbitals (FMO) in a
fluorophore and a receptor fragment can rationalize PET ON (low
quantum yield) or OFF (high quantum yield) switching.22 For
example, d-PET is on only if the energy level of the highest occupied
molecular orbital (HOMO) of the receptor fragment is higher than
that of the fluorophore (Figure 2a); a-PET is feasible only if the
lowest occupied molecular orbital (LUMO) of receptor is more
stable than that of the fluorophore (Figure 2a), where the receptor
functions as a fluorescence quencher. These ground-breaking works
have generated profound implications for the rational design of PET
probes.5, 9, 13, 23-24 Nevertheless, it is worth highlighting that the MO
representation indicates that the “apparent” change of electronic
energy of FMO must be negative to turn on PET and quench
The existing MO representation of PET involves the HOMOs
and/or LUMOs of both the fluorophore and receptor moieties,
where the latter serves as a quencher. The driving force for the PET
process is energetically described as ΔE = EHOMO, fluorophore – EHOMO,
quencher for a-PET (Figure 2a) and ΔE = ELUMO, quencher – ELUMO, fluorophore
for d-PET (Figure 2a). When the fluorophore and quencher
moieties are present in one molecule and computationally modeled
together, the FMOs are typically HOMO-1, HOMO, LUMO, and
LUMO+1 of this molecule. In such a case, the driving force for PET
can be calculated by mapping these frontier molecular orbitals to
either the fluorophore or the receptor moiety according to the
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electron density distribution, followed by calculating ΔE. The
smaller value between EHOMO, fluorophore – EHOMO, quencher and ELUMO, quencher
– ELUMO, fluorophore should be taken into consideration, to reflect the
energetic preference of a-PET or d-PET. A negative Δ
deemed necessary for activating PET.
E is often
fluorescence (ΔE < 0). This indication, however, is not consistent
with several experimental observations for PET probes with the
‘fluorophore–receptor’ format.25
We collected the molecular structures and quantum yields of 83
meso-phenyl substituted BODIPY dyes from existing literature
(Table S1), and calculated the energy levels of the FMOs of all these
Herein, by investigating ~140 meso-phenyl-substituted
fluorescent dyes with the ‘fluorophore–receptor’ configuration, we
discovered and rationalized a general descriptor (ΔE) for semi-
quantitatively predicting the quantum yield of PET probes, with a
counterintuitive threshold value of ~0.6 eV. Our data showed that
when ΔE < ~0.6 eV, the quantum yields of the fluorophores were
molecules using M062X/Def2SVP level of theory to derive Δ
(Figures S3 – S85). The results revealed an intriguing correlation
between the quantum yields of these BODIPY dyes and their Δ
values: when Δ > ~0.6 eV, the quantum yields of these
fluorophores are high (i.e., >10% for all such compounds, and
50% for 90% of these compounds); in contrast, when Δ < ~0.6 eV,
the quantum yields of these fluorophores in polar solvents are
generally low (i.e., < 10% for all such compounds and < 2% for
E
E
E
Φ
Φ >
low (i.e., mostly <2%) in polar solvents, in contrast, when Δ
eV, the quantum yields of the fluorophores were high in all medium
(i.e., mostly >20%). We further demonstrated that the Δ threshold
E
> ~0.6
E
E
Φ
Φ
of ~0.6 eV could accurately predict fluorescence quantum yields in a
wide range of popular fluorophores, such as BODIPY, fluorescein,
rhodamine, and Si-rhodamine dyes. The descriptor ΔE successfully
enabled us to quantitatively design PET fluorescent probes for
various bioimaging applications (such as wash-free bioimaging of
lipid droplets and mitochondria), and accurately predict AIEgens.
We believe that the descriptor ΔE will serve as a highly useful
indicator for the efficient development of numerous PET-based
luminescent materials.
80% of these compounds; Figures 2b and S86). It is worth
emphasizing that these dyes employ rigid BODIPY fluorophores,
which quantum yields are typically close to 1. That is, other
fluorescence quenching mechanisms such as twisted intramolecular
charge transfer (TICT) and formation of triplet excited states,27-30
are mostly not applicable. Hence, the low quantum yields have been
attributed to PET-induced fluorescence quenching. Our results
suggested that Δ
low/high quantum yields of PET-based fluorophores.
The threshold of Δ = ~0.6 eV is apparently inconsistent with the
existing MO representation of PET (which suggests Δ = 0). In
particular, the MO representation seems to indicate that PET in the
region of 0 < Δ < ~0.6 eV is energetically not feasible. However,
many fluorophores with Δ < ~0.6 eV demonstrated low quantum
E of ~0.6 eV is a useful descriptor to predict the
E
RESULTS AND DISCUSSIONS
E
Correlation between Δ
BODIPY dyes. Our initial investigations started with
E
and quantum yields of PET-based
-NH2-Ph-
p
E
BDP (B4; Figure S1), which quantum yield is very low in polar
solvents (0.3% in methanol).26 Previous reports suggested that a fast
PET process quenched the fluorescence of such BODIPY dyes.
Unfortunately, many of these studies did not include convincing
experimental data to validate the PET process, such as observations
of radical ions using time-resolved spectroscopic techniques.
Moreover, to our surprise, our DFT/TD-DFT calculations show
that B4 does not possess any quenching orbitals sitting in between
the HOMO and LUMO of the BODIPY fragment [Figures S1 and
S2; See the “computational methods” section in the Supporting
Information (SI)], suggesting the absence of both a-PET and d-PET
according to the widely used MO representation of PET. These
results clearly cannot explain the observed low quantum yields in B4.
It is worth emphasizing again that a similar disagreement has also
been noted in the fluorescein derivative.25 These inconsistencies
E
yields, suggesting that the “exception” in B4 is not an anomaly.
Employing a range-separated functional ωB97XD, we obtained
similar computational results (Figure S87 and Table S2). It is
important to note that this ΔE threshold is applicable only when a
PET probe is the ‘fluorophore-receptor’ type and the quencher is
close to the fluorophore (Figure S88).
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Figure 2. (a) Schematic illustration of the PET process upon the photoexcitation of a fluorophore, including both the donor-PET (d-PET)
and acceptor-PET (a-PET). (b) Correlation between the experimental quantum yields and calculated Δ
theory) of meso-phenyl BODIPY derivatives in polar solvents. (c) Schematic illustration of the State-crossing from a Locally-Excited to an
Electron-Transfer state (SLEET) model, and calculated excitation/de-excitation energy (as well as oscillator strength ) of B4 in methanol;
the inset shows the molecular structure of B4; note that values are not drawn in scale for clarify. (d) Optimized molecular structures of B4
E (at the M062X/Def2SVP level of
f
θ
in the ground and excited states, as well as the corresponding electron and hole distributions in methanol. VES and AES denoted vertically
excited state and adiabatic excited state, respectively.
A SLEET model to rationalize the correlation between ΔE and
quantum yields. To further understand the nature of the
fluorescence quenching phenomenon in these BODIPY molecules,
we performed detailed excited-state calculations of B4 at
M062X/TZVP level of theory in methanol. Our calculations show
that the photoexcitation in B4 resulted in a locally-excited (LE) state
in B4 can be rationalized by this State-crossing from a Locally-
Excited to an Electron-Transfer state (SLEET) model.
A key difference between the popular MO representation of PET
and the SLEET model concerns the treatment of the exciton binding
energy in the excited states (especially in the ET state), which
accounts for the difference between the “electronic” gap and the
“optical” gap. The MO representation reflects the “electronic” gap,
and the SLEET model considers the “optical” gap. The “optical” gap
follows more closely with the photo-excitation and de-excitation
processes. Consequently, the SLEET model is able to better explain
experimental observations, including the “not feasible” PET region
with a large oscillator strength (f = 0.62) in the Franck-Condon state
(or the vertically excited state; Figure 2c), since both the photo-
induced hole and electron are mainly located at the BODIPY
scaffold (Figure 2d). However, upon geometry relaxation, a non-
emissive electron-transfer (ET) state takes over the LE state and
becomes the S1 state in excited-state potential energy surfaces
(PESs). The formation of this ET state is accompanied by the
rotation of the meso-phenyl ring to a perpendicular alignment with
respect to the BODIPY scaffold, which leads to a complete charge
separation between the substituted meso-phenyl ring and the
with 0 < ΔE < ~0.6 eV.
BODIPY scaffold and a negligible oscillator strength (
f = 0) (Figures
2c and d). Accordingly, the PET formation and low quantum yield
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Figure 3. (a) Chemical structures and calculated ΔE values (in methanol) of B1—B6. (b) Experimental quantum yields of B1—B6 in various
solvents, including n-hexane (HEX), dichloromethane (DCM), ethyl acetate (ETAC), ethanol (EtOH), acetonitrile (MeCN), methanol
(MeOH), and dimethyl sulfoxide (DMSO). (c) Viscosity responses of fluorescence intensities of B4—B6, in ethanol/glycerol mixtures with
varied volume ratios. (d) Transient absorption spectra of B1—B6 in ethanol, at different time delays. (e) Time evolutions, the best-fit
functions, and the derived lifetime constants of the transient absorption signals at 500.9 nm and 560.7 nm for B5 in ethanol.
Experimental validations of the PET mechanism. Next, we
synthesized and characterized B1–B6 (Figure 3a) in order to
validate the PET mechanism (see the section of “Chemical synthesis
of BODIPY derivatives” and Figures S89 – S94 of the SI). The UV—
Vis absorption and fluorescence spectra, together with the
fluorescence quantum yields of B1–B6 were measured in various
solvents with different polarities. The peak UV-Vis absorption
wavelengths (496–502 nm) and emission wavelengths (506–514
nm) of B1–B6 exhibited a slight shift (Figures S104 – S115) when
the electron-donating groups at the phenyl ring were changed. The
slight shift stemmed from a weak electronic coupling between the
BODIPY scaffold and the meso-substituent groups (Table S3 and
Our calculations also showed that the state-crossing to the non-
emissive ET state is accompanied by the rotation of the substituted
meso-phenyl ring in BODIPYs. Accordingly, restricting such
rotations (i.e., in high-viscosity solvents) should recover the
fluorescence. Hence, we measured the viscosity dependence of the
emission intensities in B4–B6. As we increased solvent viscosity by
raising the volume ratio of glycerol in the ethanol/glycerol mixture,
we noticed a considerable enhancement of fluorescence intensities
in B4–B6, by 7—11 times (Figures 3c). These results are in good
agreement with our theoretical calculations.
Radicals are usually short-lived but key species in PET
processes.24, 31 We next performed transient absorption spectroscopy
(TAS) measurements on B4–B6 to establish the generation of
radicals. TAS of B1-B3 were collected as well for comparison. After
photo-excitation, B1–B3 only showed an overlapped ground-state
bleaching (GSB) and stimulated emission (SE) band around 500
nm (Figure 3d), as the Stokes shifts of BODIPY dyes are small.
These results indicated one dominant conformation in the excited
state (i.e., the LE state). In stark contrast, in addition to this GSB/SE
band, B4–B6 exhibited one additional excited-state absorption
(ESA) band at 530–600 nm (Figure 3d). Moreover, the decay
lifetimes of the GSB/SE and the ESA are significantly different, i.e.,
Figure S116). Importantly, the quantum yields of B1–B3 (with Δ
0.6 eV) remain high in all tested solvents (Table S4 and Figure 3b).
In contrast, B4–B6 (with Δ < 0.6 eV) are highly emissive only in
low-polarity solvents ( = 0.404, 0.314, and 0.279 in hexane,
respectively). They become almost nonfluorescent in polar solvents
= 0.001, 0.001, and 0.004 in ethanol, respectively). According to
E >
E
Φ
(
Φ
the SLEET model, the polarity dependence of quantum yields is
because the highly polar ET state becomes increasingly stable as
solvent polarity increases (due to enhanced electrostatic
interactions) and the state-crossing to the non-emissive ET state
could be substantially activated only in high-polarity solvents.
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fluorescence only from lipid droplets (Figure 4b and Figure S118),
301 ps for GSB/SE and 68 ps for ESA in B5, respectively (Figure 3e).
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These distinct spectra and lifetime suggest the presence of two
excited-state conformations in B4–B6. Moreover, previous reports
have shown that the transient absorption peaks of BODIPY radicals
located at approximately 550 – 580 nm.32-34 We thus attribute the
observed ESA to the ET state of BODIPY dyes, as the ET state
corresponds to the formation of radicals.
which leads to a perfect overlap with emission signals of LD 540.
The background emissions outsides of LDs are extremely low,
although we did not perform any washing steps. The excellent wash-
free LD bioimaging utilities of B4–B6 further allows us to monitor
the dynamics of LDs. For example, we noticed the merge of two
small LDs into a big one at the time of 24 sec and 42 sec, respectively,
with the assistance of B4 (Figure 4c and Figure S119).
The solvent and viscosity dependence of the fluorescence
intensity in B4–B6, as well as their transient absorption spectra, are
entirely consistent with our calculations and provided unambiguous
evidence on the occurrence of PET in these compounds. These
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observations also suggest that ΔE with a threshold value of ~0.6 eV
could serve as a simple yet effective descriptor to predict substantial
occurrences of PET in polar solvents and the resulted low quantum
yields in PET probes.
Application of the descriptor
experiments have confirmed the reliability of descriptor Δ
predicting low/high quantum yields (with a threshold value of Δ
~0.6 eV). We expect that Δ
ΔE
. Both calculations and
E
E
in
=
E
will not only guide the quantitative
design of PET-based fluorescence turn-on/off materials but also
help to expand new applications for existing PET probes. As the
polarity of the environment plays a significant role in controlling
quantum yields of PET fluorescent probes,35 we envisaged that PET-
based luminescent materials could find a broad range of
applications, such as fluorescence turn-on labeling of non-polar
organelles, and the development of novel AIEgens (since molecular
aggregation in polar solvents effectively reduces the local polarity of
the AIEgens).36-37
Wash-free bioimaging of lipid droplets in live cells. Lipid
droplets (LDs) are dynamic organelles that are involved in lipid
storage and metabolism of cells. LDs play critical roles in many
physiological processes,38 and dysfunctions of LDs are related to
many diseases, such as fatty liver, diabetes, obesity, cancer, and
atherosclerosis.39 Studying the dynamics of LD distributions in live
cells are thus critical for gaining biological insights on these crucial
cellular organelles.
Figure 4. (a) Co-staining of HeLa cells using B3 (1 μM) and LD 540 (1
μM); green channel, B3; red channel, LD 540; yellow channel, the
merged image. (b) Co-staining of HeLa cells using
B4
(1 μM) and
LD
540 (1 μM); green channel, B4; red channel, LD 540; yellow channel,
the merged image. (c) Lipid droplet dynamics of HeLa cells, as revealed
by B4. The merge of two small lipid droplets into a big one was observed
at 24 sec and 42 sec, respectively.
We hypothesized that B4–B6 could effectively stain LDs, due to
preferential solvation in lipids than in aqueous solution. Moreover,
because LDs provide a highly viscous (~50 cP; similar to the
viscosity of the ethanol-glycerol mixture with a volume ratio of
43%:57%; Figure S117)40 and non-polar environment, we would
expect that B4–B6 emit bright fluorescence in LDs due to the
inhibition of PET. Besides, as the quantum yields of B4–B6 are
almost zero in high-polarity solvents, the background emissions
from B4–B6 in cytoplasm should be negligible even if a small
amount of these compounds are present. These reasons encouraged
us to perform wash-free bioimaging of LDs using B4–B6. As a
comparison, we also stained cells using B3, whose quantum yields
are high in all tested solvents. Consequently, relatively high
background emissions from cytoplasm are expected.
Overall, guided by the descriptor ΔE, we demonstrate the
excellent wash-free bioimaging utilities of PET-based probes in live-
cell bioimaging, by utilizing the environmental dependence of PET.
Development of PET-based AIEgens. We next explored the
applications of the PET mechanism in developing AIEgens. To date,
luminescent materials with aggregation-induced emission (AIE)
characteristics have found a broad range of applications, such as
biosensors, photodynamic therapy agents, organic light-emitting
41-44
diodes, and wave-guides.36,
Formulating new mechanisms to
design AIEgens is crucial to accelerate the development of functional
AIE materials and applications.37, 41, 45-46
Based on the descriptor ΔE, we reason that B5 and B6 are
Indeed, B3 could penetrate cell membranes and stain live cells
(Figure 4a). Colocalization experiments of this probe with LD 540,
a commercially available LD imaging agent, demonstrates that B3
emits bright fluorescence from lipid droplets (Figure 4a). However,
this compound also generates noticeable emissions from the
cytoplasm. The overall clarity of LDs is thus suboptimal. On the
contrary, after staining live cells, PET probes B4–B6 emit bright
potentially AIEgens: owing to the polarity dependence of PET, these
probes are non-emissive in highly polar solvents. However, in
parallel with molecular aggregation, the immediate local
environment of these fluorophores becomes non-polar and large-
amplitude intramolecular rotations are inhibited,36 thus disabling the
PET process and turning on emissions. Moreover, the meso
-
substitutes and the BODIPY scaffold of B5 and B6 are twisted with
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respect to each other, endowing these compounds highly steric explored the possibility of applying descriptor Δ
Page 6 of 17
to fluorescein,
E
structures, which help to minimize π-π stacking in the aggregates or
the solid-state. Finally, the alkyl-substitution to the amino group in
B5 and B6 would further suppress potential intermolecular
hydrogen bonding induced fluorescence quenching. These factors
could collectively make B5 and B6 excellent AIEgens.
rhodamine and Si-rhodamine derivatives.47-49 These dyes are the
main-stream fluorophores and are heavily utilized in bioimaging,
biosensing, and medical diagnostic applications.50-51 Moreover, their
meso-phenyl substituents have been modified to suit different
applications based on the PET mechanism, similar to those of
BODIPYs.
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To our delight, our computational results showed that ΔE of ~0.6
eV could also serve as a useful threshold in evaluating the tendency
of PET and semiquantitatively predicting the quantum yield of
fluorescein, rhodamine and Si-rhodamine derivatives (Tables S5
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and S6, Figures S123 – S179): Δ
process and low quantum yields (
and
significantly suppresses PET process and affords high quantum
yields (i.e., >10% for all but two such compounds, and > 20%
for 80% of these compounds) (Figures 6a and S180). These results
show that the descriptor Δ is generalizable to many chemical
E
Φ
< ~0.6 eV leads to a fast PET
< 10% for all such compounds
Φ
< 2% for 80% of these compounds); ΔE > ~0.6 eV
Φ
Φ
E
families when a quencher (receptor) is attached at a proximity to the
fluorophore.
It is also interesting to note that in the PET OFF region, the
quantum yields of those fluorophores are relatively lower than those
of BODIPY-based fluorophores. This is because other mechanisms
(such as TICT formations and hydrogen bond interactions) are still
50
applicable in the fluorescence quenching of rhodamines,27,
although PET is largely inhibited when Δ
Quantitative designs of PET-based rhodamine dyes. To further
verify the prediction power of the descriptor Δ , we designed three
E > ~0.6 eV.
E
new rhodamine dyes (M1, M2, and M3 in Figure 6b). These
compounds were locked in the “open-ring” structures, to avoid
interference from spiro-cyclization reactions in conventional
rhodamine dyes. Our quantum chemical calculations showed that
Δ
E
in M1 (1.024 eV) and M2 (1.012 eV) were significantly larger
Figure 5. Fluorescence spectra of B5 (a) and B6 (b) in dioxane/water
mixtures with different water fractions (fw). The inset of (a) and (b)
shows the changes of peak intensities of fluorescence spectra and the
photographs of B5 and B6 in pure dioxane and powder state under UV
irradiations, respectively.
than 0.6 eV, while Δ in M3 (0.522 eV) was less than 0.6 eV in
E
methanol (Figures 6c, S181 – S183). Moreover, the HOMO and
HOMO-1 of M3 were located on the xanthene and meso-phenyl
substituted groups, respectively (Figures 6d and S183). Hence, we
predicted that the quantum yields of M1 and M2 should be high
(>20%), and that of M3 should be poor in polar solvents (as a result
of substantial PET quenching).
We thus measured the UV-Vis absorption and emission spectra of
B5 and B6 in ethanol/water (Figures S120 – S122) and
dioxane/water mixtures (Figure 5). In pure dioxane or ethanol, B5
and B6 emitted almost no fluorescence. However, as the volume
fraction (fw) of water increases, the poor solubility of B5 and B6 in
aqueous solution led to the formation of molecular aggregates.
These aggregates greatly enhanced the emission intensities of B5
and B6 by up to 24 and 28 times, respectively. To further confirm
the AIE characteristics of these compounds, we also measured the
solid-state emissions of B5 and B6 powder. Indeed, bright solid-state
luminescence with a quantum yield of 10% and 12 % was observed
for B5 and B6, respectively (Figure 5). The quantum yields in the
solid powder are ~100 times as high as that in polar solvents.
Next, we synthesized and characterized M1, M2, and M3 (see the
section of “Chemical synthesis of rhodamine derivatives” and
Figures S95 – S103 of the SI). The UV—Vis absorption (Figures
S184 –S186) and fluorescence spectra (Figures S187 – S189), and
the fluorescence quantum yields (Figure 6e; Table S7) of M1, M2,
and M3 were measured in various solvents with different polarities.
As expected, the quantum yields of M1 and M2 are high (30% and
26% in methanol, respectively). In contrast, the quantum yield of
M3 is only 1% in methanol.
These results show that the descriptor ΔE is highly effective in
semi-quantitatively predicting the PET tendency and the resulting
quantum yields in PET-based fluorophores. It is also worth
Overall, as predicted by the descriptor ΔE, our results show that
modulating PET formation serves as an effective mechanism for the
rational development of AIEgens.
mentioning that obtaining Δ
calculations and are highly computationally efficient. Δ
E
only requires ground-state
is thus very
E
Generalization of the descriptor Δ
Motivated by the success of the descriptor Δ
predicting quantum yields in BODIPY fluorophores, we then
E
to other dye scaffolds.
useful for the quantitative design of PET-based luminescent
materials, especially in large-scale computational screening.
E
in rationalizing and
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Figure 6. (a) Correlation between the experimental quantum yields and calculated Δ
phenyl rhodamine, Si-rhodamine and fluorescein derivatives in polar solvents, (b) designing three new rhodamine dyes (M1 – M3), (c) the
calculated Δ of M1 – M3, and prediction of PET status in ethanol, (d) calculated distributions and energies of FMO for M3 in ethanol, (e)
quantum yields of M1, M2, and M3 in various solvents DCM, ETAC, EtOH, MeCN, MeOH, and DMSO, (f) co-stained live HeLa cells
using M3 (3 μM; orange channel) and Hoechst 33342 (a nucleus stain; 3 μM; blue channel) and the merged image.
E (at the M062X/Def2SVP level of theory) of meso-
E
Wash-free bioimaging of mitochondria using PET-based
rhodamine dyes. Finally, we explored the wash-free bioimaging
utilities of PET-based compound M3. This compound is weakly
emissive in aqueous solution but highly emissive in DCM (due to
the activation and disablement of PET, respectively). We expected
that this compound remained dark in the cytoplasm. However, given
that M3 carries a positive charge, we foresaw that this compound
could anchor onto mitochondria, which has a negative membrane
potential.52-53 Besides, since the membrane of mitochondria is non-
polar, bright emissions from M3 may be restored in mitochondria,
thus realizing wash-free bioimaging.
calculation in a highly efficient manner. When Δ
“ON” and the quantum yield of the fluorophores in polar solvents is
low (i.e., mostly <2%). In contrast, when Δ > ~0.6 eV, PET is
“OFF” and the quantum yield of the fluorophore is high (i.e.,
mostly >20%). As a proof of concept, we accurately predicted and
rationally developed wash-free fluorescent stains of lipid droplets
and mitochondria, as well as AIEgens, by modulating PET
formations. We expect that the descriptor ΔE would serve as a simple
and effective indicator for guiding the development of abundant
PET-based luminescent materials.
E < ~0.6 eV, PET is
E
ASSOCIATED CONTENT
We thus co-stained live HeLa cells using M3 (3 μM) and Hoechst
33342 (a nucleus stain; 3 μM). Notably, M3 successfully penetrated
the cellular membrane and accumulated in mitochondria (Figure
6f). The localization of M3 in mitochondria was subsequently
validated via co-staining with Cy5 (Figure S190). Compound M3
and Hoechst 33342 collectively enabled the multi-color bioimaging
of mitochondria and nucleus in a wash-free manner. We also tested
the bioimaging performance of M1 and M2 for comparison.
Unfortunately, M2 exhibits poor cell permeability. While M1
successfully entered cells and stained mitochondria (Figure S191),
it also led to substantial background emissions and poor bioimaging
quality. Overall, these results demonstrate the excellent wash-free
bioimaging utilities of PET probes.
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
AUTHOR INFORMATION
Corresponding Author
* E-mail: xiaogang_liu@sutd.edu.sg
* E-mail: zcxu@dicp.ac.cn
* E-mail: ytchang@postech.ac.kr
Author Contributions
# These authors contributed equally.
Notes
The authors declare no competing financial interest.
CONCLUSIONS
In summary, we discovered and rationalized a general descriptor Δ
to semi-quantitatively predict quantum yields of BODIPY,
fluorescein, O/Si-rhodamines with the ‘fluorophore-receptor’
E
ACKNOWLEDGMENT
This work is supported by Singapore University of Technology and
Design (SUTD) and the SUTD-MIT International Design Centre
(IDC) [T1SRCI17126, IDG31800104], National Natural Science
format. The value of ΔE can be obtained via a simple ground-state
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17.
Goldsmith, Z. K.; Soudackov, Alexander. V.; Hammes-Schiffer,
Foundation of China (21878286, 21908216), Dalian Institute of
Chemical Physics (DMTO201603, TMSR201601) and Institute for
Basic Science (IBS, Korea) [IBS-R077-A1]. The authors would like to
acknowledge the use of the computing service of SUTD-MIT IDC and
National Super-computing Center (Singapore).
S. Theoretical Analysis of the Inverted Region in Photoinduced Proton-
Coupled Electron Transfer. Faraday Discuss. 2019, 216, 363-378.
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Concerted Proton-Electron Transfer Reactions in the Marcus Inverted
Region. Science 2019, 364, 471-475.
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Parada, G. A.; Goldsmith, Z. K.; Kolmar, S.; Pettersson Rimgard,
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Figure 1. Schematic illustration of (a) PET-based fluorescent probes of the ‘fluorophore-spacer-receptor’ and
‘fluorophore-receptor’ formats; (b) the change of fluorescence intensity for a PET probe.
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Figure 2. (a) Schematic illustration of the PET process upon the photoexcitation of a fluorophore, including
both the donor-PET (d-PET) and acceptor-PET (a-PET). (b) Correlation between the experimental quantum
yields and calculated ΔE (at the M062X/Def2SVP level of theory) of meso-phenyl BODIPY derivatives in polar
solvents. (c) Schematic illustration of the State-crossing from a Locally-Excited to an Electron-Transfer state
(SLEET) model, and calculated excitation/de-excitation energy (as well as oscillator strength f) of B4 in
methanol; the inset shows the molecular structure of B4; note that θ values are not drawn in scale for
clarify. (d) Optimized molecular structures of B4 in the ground and excited states, as well as the
corresponding electron and hole distributions in methanol. VES and AES denoted vertically excited state and
adiabatic excited state, respectively.
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Figure 3. (a) Chemical structures and calculated ΔE values (in methanol) of B1—B6. (b) Experimental
quantum yields of B1—B6 in various solvents, including n-hexane (HEX), dichloromethane (DCM), ethyl
acetate (ETAC), ethanol (EtOH), acetonitrile (MeCN), methanol (MeOH), and dimethyl sulfoxide (DMSO). (c)
Viscosity responses of fluorescence intensities of B4—B6, in ethanol/glycerol mixtures with varied volume
ratios. (d) Transient absorption spectra of B1—B6 in ethanol, at different time delays. (e) Time evolutions,
the best-fit functions, and the derived lifetime constants of the transient absorption signals at 500.9 nm and
560.7 nm for B5 in ethanol.
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Figure 4. (a) Co-staining of HeLa cells using B3 (1 μM) and LD 540 (1 μM); green channel, B3; red channel,
LD 540; yellow channel, the merged image. (b) Co-staining of HeLa cells using B4 (1 μM) and LD 540 (1
μM); green channel, B4; red channel, LD 540; yellow channel, the merged image. (c) Lipid droplet dynamics
of HeLa cells, as revealed by B4. The merge of two small lipid droplets into a big one was observed at 24 sec
and 42 sec, respectively.
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Figure 5. Fluorescence spectra of B5 (a) and B6 (b) in dioxane/water mixtures with different water fractions
(fw). The inset of (a) and (b) shows the changes of peak intensities of fluorescence spectra and the
photographs of B5 and B6 in pure dioxane and powder state under UV irradiations, respectively.
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Figure 6. (a) Correlation between the experimental quantum yields and calculated ΔE (at the
M062X/Def2SVP level of theory) of meso-phenyl rhodamine, Si-rhodamine and fluorescein derivatives in
polar solvents, (b) designing three new rhodamine dyes (M1 – M3), (c) the calculated ΔE of M1 – M3, and
prediction of PET status in ethanol, (d) calculated distributions and energies of FMO for M3 in ethanol, (e)
quantum yields of M1, M2, and M3 in various solvents DCM, ETAC, EtOH, MeCN, MeOH, and DMSO, (f) co-
stained live HeLa cells using M3 (3 μM; orange channel) and Hoechst 33342 (a nucleus stain; 3 μM; blue
channel) and the merged image.
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