A General Strategy for Through-Bond Energy Transfer Fluorescence Probes Combining Intramolecular Charge Transfer: A Silyl Ether System for Endogenous Peroxynitrite Sensing

Article abstract of DOI:10.1002/chem.201903880

A general strategy is reported for developing through-bond energy transfer (TBET) fluorescence probes by combining intramolecular charge transfer (ICT). The strategy uses a coplanar donor-π-bridge-acceptor system (SiOPh-PyOH) without spirolactam. The off-on switch of TBET and ICT is controlled by coplanar structure changes in the sensing process instead of spirolactam ring-opening in traditional TBET probes. DFT calculations showed that the energy and charge transfers from SiOPh to PyOH are prohibited. Since the SiOPh has no fluorescence, the probe SiOPh-PyOH shows fluorescence properties similar to that of pyrene. After sensing ONOO^?, the silyl ether is removed and the probe changes into ^?OPh-PyO^?. Electron-donating ICT from OPh to PyO^? induces a large redshift of emission to 594 nm (179 nm shift). TBET from OPh to PyO^? ensures the probe exhibits a large pseudo-Stokes shift of 213 nm. Furthermore, the probe was successfully used in endogenous ONOO^? detection. This study offers a new strategy for the construction of TBET probes emitting in the red region without spirolactam ring-opening, a new ONOO^? sensing system using silyl ether as a reaction site, and a method for the deprotection of silyl ethers with ONOOH under mild conditions.

Full text of DOI:10.1002/chem.201903880

A Journal of
Accepted Article
Title: A General Strategy for Through-bond Energy Transfer
Fluorescence Probes Combining Intramolecular Charge Transfer:
a Silyl Ether System for Endogenous Peroxynitrite Sensing
Authors: Gonghao Lu, Yuxin Guo, Jiezhen Zhuo, Xue Li, Haijun Chi,
and Zhiqiang Zhang
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201903880
Link to VoR: http://dx.doi.org/10.1002/chem.201903880
Supported by

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A General Strategy for Through-bond Energy Transfer
Fluorescence Probes Combining Intramolecular Charge Transfer:
a Silyl Ether System for Endogenous Peroxynitrite Sensing
[
a]
[a]
[a]
[b]
[a]
[a]
Gonghao Lu,* Yuxin Guo, Jiezhen Zhuo, Xue Li,* Haijun Chi and Zhiqiang Zhang
3
+
Abstract: A general strategy is reported for developing through-
bond energy transfer (TBET) fluorescence probes combing
intramolecular charge transfer (ICT). The strategy uses a coplanar
donor-π-bridge-acceptor system (SiOPh-PyOH) without spirolactam.
The off–on switch of TBET and ICT is controlled by the coplanar
structure changes in sensing process instead of spirolactam ring-
opening in traditional TBET probes. DFT calculations showed the
energy and charge transfers from SiOPh to PyOH are prohibited.
Since the SiOPh has no fluorescence, the probe SiOPh-PyOH
shows the similar fluorescence properties with pyrene. After sensing
However, one of them is a quenching probe for Fe in red
25
region, and the other only shows a small PSS and emission
3+
26
shift for Al in green region. Therefore, the TBET fluorescence
probe is largely undocumented for fluorophores without
spirolactam.
Since TBET is based on excitation energy transfer from
donor to acceptor, a good TBET system requires some specific
conditions for smooth energy transfer, in other words, the energy
levels of the donor must match those of the acceptor. Through
analyzing results in literatures, we found that almost all of the
reported TBET probes achieved the energy level matching by
the structure changes of spirolactam ring-opening (Figure 1b).
The highest occupied molecular orbital (HOMO) and lowest
unoccupied molecular orbital (LUMO) energy levels of donors
are higher than those of receptors and the energy gaps of
donors are slightly larger than those of receptors, which
effectively achieve energy transfer from donors to acceptors. For
example, the density functional theory (DFT) calculations for the
probe in reference 4 confirmed this energy matching (Figure 1c).
The HOMO is mainly located in coumarin-oxadiazole part
−
−
ONOO , the silyl ether is removed and the probe changes into OPh-
−
−
−
PyO . Electron-donating ICT from OPh to PyO makes the probe
possess a large red-shifted emission to 594 nm (179 nm shift). TBET
−
−
from OPh to PyO ensures the probe a large Pseudo-Stokes shift of
13 nm. Further, the probe was successfully used in endogenous
2
−
ONOO detection. This study offers
a new strategy for the
construction of TBET probes emitting in red region without
−
spirolactam ring-opening, a new ONOO sensing system using silyl
ether as a reaction site and a method for the deprotection of silyl
ethers by ONOOH in mild conditions.
(
(
donor) while the LUMO is mainly located in rhodamine core
acceptor). The HOMO and LUMO energy levels of the donor
Introduction
are higher than thos of the receptor and the energy gap of the
donor (3.5318 eV) is slightly larger than that of the acceptor
(
2.8093 eV), which makes energy transfer effective from donor
In a through-bond energy transfer (TBET) system, the energy
donor and acceptor are linked with electronically conjugated
bonds and the energy transfer occurs through the conjugated
to acceptor. However, for these probes using spirolactam ring-
opening, the conjugation is poor because the plane of the donor
is almost perpendicular to that of the acceptor based on the
configuration optimization (Figure 1d), which makes it impossible
for the intramolecular charge transfer (ICT) from donor to
acceptor. That is the reason why these TBET probes exhibited
almost same maximum emission wavelength (λem) with that of
rhodamine core itself, nearly without any red-shifts. From
another perspective, probes using spirolactam ring-opening are
actually more like fluorescence resonance energy transfer
strategy instead of TBET mechanism.
1
bonds. Thus, there is no requirement of substantial spectral
2
overlap between the donor emission and acceptor absorbance,
leading to a large Pseudo-Stokes shifts (PSS) and emission shift,
which favorable to avoid self-quenching and fluorescence
3
detection errors because of excitation back-scattering effects.
Therefore, TBET has been widely reported as a strategy for
ratiometric fluorescence probes with large PSS and emission
4-26
shifts.
Although numerous ratiometric fluorescence probes
have been implemented with TBET mechanism, these probes
are predominantly based on spirolactam ring-opening to sensing
In addition to the structure change caused by the
spirolactam ring-opening, it is conceivable that the sensing
reaction on the reaction site of a donor-π-bridge-acceptor
system may give rise to the energy level changes, which is
possible to achieve the energy matching between the donor and
acceptor, leading to the occurrence of TBET (Figure 2a).
Furthermore, since the donor and acceptor are coplanar and
have good conjugation, ICT effect from donor to acceptor
occurs simultaneously when TBET occurs, which leads to a red-
shifted emmison (Figure 2b). Thus, we can achieve a ratiometric
TBET probe based on a single fluorophore. This distinguishing
feature makes TBET probes possess the advantages of desired
flexibility in the choices of fluorophores instead of traditional
TBET probes focusing on rhodamine or fluorescein spirolactam
structures as well as large PSS and large emission shifts.
2+
4-13
2+ 14-16
ions or molecules (Figure 1a), such as Hg ions,
Cu ,
picric acid and so on. To the
best of our knowledge, only two TBET probes were recently
2+ 17
2+ 18
4+ 19
–
20-23
24
Pd , Sn , Ce , OCl,
3+
3+ 25,26
reported based on non-spirolactam for Fe
and Al .
[
[
a]
b]
Dr. G. Lu, Y. Guo, J. Zhuo, H. Chi, Prof. Dr. Z. Zhang
School of Chemical Engineering
University of Science and Technology Liaoning (USTL)
85 Qianshan Zhong Road, Anshan, Liaoning,114051 (P. R. China)
1
E-mail: ghlu@ustl.edu.cn
Dr. X. Li
School of Material and Metallurgy
University of Science and Technology Liaoning
Supporting information for this article is given via a link at the end of
the document.
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a
b
TBET off
X
TBET on
NEt2
NEt2
Poor
Non-conjugated
linker
conjugated
linker
After sensing
O
Acceptor
O
After sensing Donor
LUMO
Donor
LUMO
N
O
O
Donor
NH
Non
fluorescent
precursor
Donor
LUMO
HOMO
NEt2
Non
NEt2
Acceptor
HOMO
HOMO
fluorescent
precursor
d
Acceptor
TBET on
X
ICT off
or weak
Donor
Figure 1. Traditional strategy for TBET probe designs. (a) Traditional TBET probes based on spirolactam ring-opening. (b) Energy matching for traditional TBET
probes. DFT calculations for (c) energy matching and (d) vertical planes of TBET probe in reference 4.
a
TBET off
TBET on
b
X
Reaction
site
Reaction
site
TBET on
Conjugated
linker
Conjugated
linker
X
ICT off
Acceptor
or
Donor
LUMO
Acceptor
Donor1
ICT on
Acceptor1
Donor1
Acceptor
or
LUMO
HOMO
LUMO
HOMO
ICT on
After sensing
LUMO
HOMO
Acceptor1
HOMO
Figure 2. Donor-π-bridge-acceptor system for TBET probe designs. (a) Energy matching for new strategy in TBET probe designs combining ICT. (b) Configuration
optimization for new strategy.
2
7
‒
In our previous work, a donor-π-bridge-acceptor system
was designed and the phenylboronate as a reaction site exert a
mechanism of TBET and ICT process using the chemical
transformation from phenylboronate to phenol, resulting from the
strong oxidation of peroxynitrite. However, the probe using
a new tool for the design of ONOO probe in biological detection
and a new method for the deprotection of silyl ether in organic
synthesis. The precise DFT calculations showed that the most
‒
‒
matchable energy between two donors is OPh-PyO , instead of
‒
27
OPh-PyOH in previous report. The experimental results
‒
‒
‒
oxidation strategy is easily interfered by ClO . More importantly,
proved that OPh-PyO is a good TBET system with large PSS
and emission shift. In a word, a precise energy level matching is
a good strategy for TBET probe designs combining ICT.
the relations between energy and charge transfer efficiencis and
energy matching were not elucidated clearly. These limited the
application of this TBET design platform in other fluorophores. In
silyloxyphenyl group was connected with
hydroxypyrene or silyloxypyrene structure through
conjugated 1,2-dimethylenehydrazine linker to give two donor-π-
bridge-acceptor probes (SiOPh-PyOH and SiOPh-PyOSi)
Scheme 1). Phenoxy silyl ether was selected as a reaction site
to change the energy levels of the donors as it is frequently used
as a protective group for sensing fluorine ions (F ).
CHO
OH
NH2
Si O
CHO
N
this work,
a
a
well
NH
2
NH
OH
Reflux
2
•H
2
O
3
OH
a
C
2
H
5
C
2
H
5
OH
1
2
Reflux
O
O
Si
Si
3SiCl
(
(CH
3
)
N
N
N
N
Imidazole
CH Cl
0°C - rt
(H
3C)3
SiO
HO
SiOPh–PyOH
2
2
−
28, 29
SiOPh–PyOSi
Surprisingly, the selectivities of two probes showed that SiOPh-
‒
PyOH exhibited high sensitivity and selectivity to ONOO instead
Scheme 1. Synthetic routes for SiOPh-PyOH and SiOPh-PyOSi.
−
of F by silyl deprotection. To the best of our knowledge, this is
‒
the first time that silyl ether is cleaved by ONOO , which provide
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Results and Discussion
intensity of probe SiOPh-PyOH at 354 nm decreased with the
simultaneous appearance of two new red-shifted absorption
bands centered at 381 nm and 506 nm (Figure 4a).
Fluorescence titrations with ONOO (0 – 5.5 equiv.) showed that
the emission of SiOPh-PyOH at 415 nm decreased distinctly,
Syntheses of Donor-π-bridge-acceptor Probes and
Derivatives. SiOPh-PyOH was synthesized in three simple
steps (Scheme 1). The starting 1-hydroxypyrene-2-carbaldehyde
−
(
1) was synthesized according to the literature (Synthesis details
while the emission band at 594 nm underwent a noticeable
increment simultaneously (Figure 4b). The F-F of the emission
0
30,31
in Scheme S1).
The hydroxyl group on pyrene of SiOPh-
PyOH can be further silylated to give probe SiOPh-PyOSi.
Similar procedures gave derivatives HOPh-PyOH, MeOPh-
PyOH and HOPh-PyOMe (Synthesis details in Scheme S2).
intensities at 594 nm linearly correlated with the concentration of
−
ONOO (2.5 – 4.5 equiv.) with a correlation coefficient of
2
R =0.95 and the limit of detection was calculated to be 36.2 nM
based on S/N=3 (Inset in Figure 4b). It is worthy to note that
both absorption and fluorescence titrations show low linear
changes with the concentration of ONOO , especially in low
concentrations (0 – 2 equiv.) (Insets in Figure 4), and the reason
will be discussed in mechanism part.
Spectrometric Responses of the Probes. The UV–Vis
absorption spectra were first studied to confirm the responses of
SiOPh-PyOH to different RNS/ROS and other common anions
−
(Figure 3a and S1). DMF-PBS (7:3, v/v, pH = 7.4) was selected
as the suitable solvent. Free probe SiOPh-PyOH showed an
intensive absorption band at 354 nm, which is the pyrene
absorption. Upon the addition of ONOO (5.5 equiv.), the band
Selectivity and Effect of pH. Competitive experiments of
SiOPh-PyOH showed that the fluorescence intensity (I594)
changed little in the presence of other ROS/RNS and anions
(Figure S6). These results indicated that probe SiOPh-PyOH
showed an excellent selectivity toward ONOO over other
ROS/RNS. In addition, the effect of pH on the fluorescence
−
broadening and red-shift were observed bacause of the
favorable ICT from phenolate to pyrene, accompanied with a
color change from pale yellow to pink, while no obvious changes
occurred when other ROS/RNS and common anions at a higher
concentration (25 equiv.) were added (Figure S2). Compared
with the analogues 2 and p-hydroxybenzaldehyde, the peaks at
−
response was also tested and probe SiOPh-PyOH exhibited
−
stable responses to ONOO in the wide range of pH 3.5 − 11.5
381 nm and 506 nm were assigned to the phenolate and pyrene
(Figure S7). Thus, probe SiOPh-PyOH can work at the
moiety, respectively. The absorption spectra for probe SiOPh-
PyOSi showed that this probe had no response to all detected
RNS/ROS and common anions at the detected concentration
physiological pH range.
a) 0.6
−
Probe and H
2
2−
O
2
, ∙OH, O
2
−
, NO∙, TBHP
1
−
−
−
(25 equiv.) (Figure S3a).
2 2 2 4
TBO∙, O , S , NO , F , Pj , H PO ,
2− 2− − 3− − −
0.5
HPO
CH COO
3
4 4 4 4
, SO , Cl , PO , Br , HSO
−
−
Probe Probe
Probe
The response of probe SiOPh-PyOH to ONOO was further
p-Hydroxy-
benzaldehyde
Probe
+
-
+
0
.4
.3
.2
.1
+
-
confirmed by fluorescent spectra in DMF-PBS buffer (Figure 3b
and S4). Blue fluorescence at 415 nm was observed for free
probe SiOPh-PyOH. Although some anions partially quenched
ONOO ClO
-
+
OH
0
p-Hydroxy-
benzaldehyde
−
-
the fluorescence of SiOPh-PyOH at 415 nm, only ONOO nearly
ONOO
0
completely quenched the fluorescence at 415 nm and promoted
a dramatic enhancement of the emission at 594 nm and an
orange-red fluorescence was gradually strengthened under UV
light (Figure S5). Emission shift and PSS are 179 nm and 213
nm, respectively. Probe SiOPh-PyOH produced a fluorescence
1
-
-
1 + OH
ClO
0
0.0
3
00 350 400 450 500 550 600 650 700
Wavelength / nm
“
OFF-ON” at 594 nm up to 21.3 times in a low concentration of
b)600
−
ONOO (5.5 equiv.). In contrast, no obvious changes in emission
intensities at 594 nm occurred when the probe was incubated
with other ROS/RNS and other common anions at a higher
concentration (25 equiv.), even ClO , which easily interferes the
ONOO sensing in many reports.
-
λ
= 506, ONOO
ex
5
00
Probe Probe
-
λ
= 381, ONOO
ex
−
Probe
+
+
400
-
-
ONOO
ClO
−
32-34
When the probe after
λex= 365, ONOO-
−
300 Probe and H2O2
−
sensing ONOO was excited at 365 or 381 nm (phenolate) or
, ∙OH, O
2
, NO∙,
1
2−
−
−
−
TBHP, TBO∙, O
2
, S , NO
, SO
2
, F , Pj ,
506 nm (pyrene moiety), respectively, the λem remained constant
−
−
2−
2−
−
00 H2
PO
4
, I , HPO
4
4
, Cl ,
2
3
−
−
−
−
at 594 nm and only the fluorescence intensities slightly varied,
indicating that the emission property of the probe is excitation
independent. This suggests that the energy transfer from
phenolate to pyrene ring is efficient and smooth. The fluorescent
spectra for probe SiOPh-PyOSi further confirmed that SiOPh-
PyOSi showed no response to all detected RNS/ROS and
anions at the detected concentration (25 equiv.) (Figure S3b).
PO
4
4 3
, Br , HSO , CH COO
-
ClO
100
0
4
00
450
500
550
600
650
700
750
Wavelength / nm
Figure 3. Spectra of probe SiOPh-PyOH (10 μM) with various anions. (a) UV-
Vis spectra. (b) Fluorescence emission spectra (λex = 381 nm). ONOO is 55
μM and other anions are 250 μM in DMF-PBS (7:3, v/v, pH=7.4). Insets
showing the color changes before and after the addition of ONOO .
−
To further understand the properties of SiOPh-PyOH in the
−
−
presence of ONOO , the titration experiments were conducted in
DMF-PBS (7:3, v/v, pH = 7.4) (Figure 4). With increasing
−
conentration of ONOO from 0 to 5.5 equiv., the absorbance
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a) 0.6
−
1
.6
ethers. For probe SiOPh-PyOH, when excessive F or bases
1
.4 Y = 0.0411X - 0.626
(
70 equiv.) were added, similar fluorescence emissions with
ONOO (4 equiv.) were observed at about 610 nm (Figure S10a),
indicating that the Si-O bond is cleaved by ONOO to give the
same sensing product with F or bases. In contrast, probe
SiOPh-PyOSi exhibited high stability to ONOO even at higher
concentrations up to 2.5 times (10 equiv.), while equal amounts
of F or bases, as the traditional Si-O bond cleavage reagents,
still cleaved the Si-O bonds of SiOPh-PyOSi to give similar
fluorescence emissions at 610 nm (Figure S10b). In a word,
ONOO cleaves the Si-O bond in SiOPh-PyOH but not the Si-O
bonds in SiOPh-PyOSi. These results indicate the Si-O bond in
SiOPh-PyOH is not directly cleaved by ONOO and the hydroxy
group on pyrene ring should play a decisive role on the Si-O
bond cleavage. Therefore, we tried to add two molar equivalents
of phenol into the SiOPh-PyOSi solution, the surprising result
1.2
2
R = 0.984
0
.5
.4
.3
.2
.1
.0
1
.0
.8
.6
.4
.2
.0
−
0
−
0
0
0
−
0
0
−
0
0
10
20
30
40
50
60
-
[ONOO / μM
]
0
−
0
−
0
3
00 350 400 450 500 550 600 650 700
Wavelength / nm
−
b) 300
3
00
Y = 12.7798X - 346.3624
2
R
00
= 0.9478
2
−
200
100
0
showed that ONOO cleaved the Si-O bonds of SiOPh-PyOSi in
the presence of phenol.
0
10
20
30
40
50
60
-
Confirmation of Fluorescent Structure. In order to confirm the
fluorescent structures, fluorescent titration experiments for
donor-π-bridge-acceptor derivatives to base were carried out for
[ONOO ] / μM
100
−
comparison with probe SiOPh-PyOH to ONOO in pure DMF
0
(Figure S11). To reduce the protonation effects, the polar aprotic
400
450
500
550
600
650
solvent (DMF) and an organic base (TBAOH in THF) were used.
Wavelength / nm
−
For SiOPh-PyOH (Figure S11a), when ONOO was added, λem
−
originally appeared at 625 nm in low concentrations of ONOO .
Figure 4. Spectrometric titrations of probe SiOPh-PyOH upon addition of
ONOO . (a) UV-Vis spectra. Inset showing a plot of changes of A381/A354 for
absorption titration. (b) Fluorescence emission spectra (λex = 381 nm). Inset
0
showing a plot of changes of F – F for fluorescence titration. Probe SiOPh-
−
−
With increasing the concentration of ONOO , λem occurred a
blue-shift to 609 nm. These results indicate that there must be
an obvious change in fluorescent structure in the process of
−
PyOH is 10 μM and ONOO is 0 – 55 μM in DMF-PBS (7:3, v/v, pH=7.4).
−
SiOPh-PyOH sensing ONOO . HOPh-PyOH showed the similar
Considering that the silyloxyphenyl group is a well-known
emission maximums and a blue-shift (from 622 nm to 608 nm)
with SiOPh-PyOH when TBAOH was added (Figure S11b),
indicating that the fluorescent change of SiOPh-PyOH sensing
−
reactive site for the detection of F , our original aim was to
−
design a probe for sensing F . The practical results made us
−
−
further investigate the activity of the probe to F (Figure S8). The
ONOO should result from the formations of aryloxides. To
−
fluorescence titration experiments of the probe to F were
further confirm which aryloxide is the fluorescent intermediate in
sensing process, the fluorescence properties of MeOPh-PyOH
and HOPh-PyOMe with a stable methyl protecting group were
studied. Upon the addition of TBAOH, MeOPh-PyOH exhibited a
carried out in pure DMF and DMF-PBS (7:3, v/v, pH = 7.4). The
−
results showed that organic F (tetrabutylammonium fluoride,
TBAF) with a concentration up to 17.5 times (70 equiv.) induced
−
−
the similar fluorescence intensity with ONOO in pure DMF,
weak fluorescence enhancement at 621 nm for MeOPh-PyO
−
−
while inorganic F (NaF) in DMF or organic F (TBAF) in
aqueous DMF showed no fluorescence responses at 594 nm.
Based on the DFT calculations for SiOPh-PyOH and SiOPh-
(Figure S11c), while HOPh-PyOMe showed only a slight red-
shift in the blue region and no emission in the red region (Figure
−
−
S11d). Since MeOPh-PyO instead of OPh-PyOMe emits in the
−
PyO , pyrene ring is the actual electron-donor. The strong
red region, accordingly, the fluorescent structure with the
−
−
electron-donating pyrene structure can reduce the positive
charge on the silicon atom through the conjugated structure,
thus largely reduce the activity of the nucleophilic reaction that
emission at 625 nm should be SiOPh-PyO instead of OPh-
−
PyOH when probe SiOPh-PyOH senses ONOO . In addition,
fluorescent titrations for MeOPh-PyOH and HOPh-PyOMe
showed that both of them are weak fluorescent systems.
Therefore, the strong fluorescent emissions of SiOPh-PyOH and
−
F attacks silicon atom.
−
Si-O Bond Cleavage by ONOO . To confirm the response
−
−
−
HOPy-PhOH should be attributed to the formation of OPh-PyO .
reaction of SiOPh-PyOH to ONOO , HRMS measurements were
−
carried out for SiOPh-PyOH before/after sensing ONOO
Sensing Mechanism. Since the conjugate acid (ONOOH) of
−
(
Figure S9). For the sample after sensing, a peak at 363.1107
ONOO is a weak acid and the hydroxy on pyrene has a certain
−
−
m/z corresponding to aryloxides OPh-PyOH or HOPh-PyO was
acidity just as phenol, we think that the initial step of the
−
observed, indicating the Si-O bond breaking. Then, in order to
response mechanism is the equilibrium of ONOO with the
−
−
investigate how ONOO cleaves Si-O bond, the fluorescence
hydroxy on pyrene, which give SiOPh-PyO and ONOOH
properties of SiOPh-PyOH were compared with those of SiOPh-
(
Scheme 1). ONOOH is highly reactive and can decompose into
−
−
+
−
35,36
+
PyOSi by adding ONOO , F and bases (NaOH and tetrabutyl-
nitronium ion (NO
eletrophilicity and OH has nucleophilicity, meanwhile, Si-O bond
2
−
) and hydroxyl ion ( OH).
2
NO has strong
−
ammonium hydroxide, TBAOH) in pure DMF (Figure S10), as F
and bases are effective reagents for the deprotections of silyl
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Chemistry - A European Journal
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+
is a strong polar covalent bond (partial charge: -0.6488 for O
ONOOH
NO
2
OH
Si
O
+
and 0.9082 for Si in SiOPh-PyOH by DFT). Therefore, NO
takes an electrophilic attack on oxygen and OH simultaneously
2
N
O
−
N
ONOO–
ONOOH
O
Si
N
OH
N
takes a nucleophilic attack on silicon, leading to the Si-O bond
−
HOPy–PhOSi
–
breaking to give an intermediate O
2
NOPh-PyO , which is further
OPy–PhOSi
−
−
−
hydrolyzed to OPh-PyO by ONOO . In low concentrations of
ONOO , there are several possible equilibriums between the
Si OH
−
−
−
−
−
aryloxides ( OPh-PyO , OPh-PyOH and HOPh-PyO ) in
sensing system, and these aryloxides have slightly different
absorptions and largely different fluorescence properties, which
make the linearities of the titration curves low (Insets in Figure 4).
ONO
2
O
N
N
–
OPy–PhONO
2
ONOO–
O
DFT Calculation. To verify our original hypothesis for the new
+
NO
2
NO3
O
design strategy of TBET probe, DFT calculations were carried
O
−
out for probe SiOPh-PyOH, possible structures ( OPh-PyOH,
N
N
N
H
3
O
N
−
−
−
SiOPh-PyO and OPh-PyO ) and their fragment structures
OH
–
H3
–
–
O
OPy–PhO
(Figure S12).
–
HOPy–PhO
Weak fluorescence
For probe SiOPh-PyOH, fragment structure SiOPh has
Excessive ONOO–
– H
H
3
O
3
O
N
lower HOMO and higher LUMO energy level than PyOH, thus,
SiOPh has much larger energy gap than PyOH (Figure S12a).
The energy and charge cannot spontaneously transfers from
SiOPh with a low energy level to PyOH with a high energy level.
Therefore, both TBET and ICT from donor SiOPh to donor
PyOH are turn-off at silyl protection. In addition, it appears that
the HOMO of SiOPh-PyOH is mainly located in pyrene ring in
character and the LUMO distributes throughout the conjugated
structure. The energy gap of SiOPh-PyOH is only slightly less
than that of PyOH because of conjugation effect. These results
indicate that the pyrene ring in SiOPh-PyOH is an actual donor
and SiOPh-PyOH should have a similar or slightly red-shifted
emission compared with PyOH. Therefore, the probe SiOPh-
PyOH should give an emission (400 − 450 nm).
OH
O
O
N
N
N
–
Excessive ONOO
O
–
–
–
OPy–PhOH
OPy–PhO
Weak fluorescence
Strong fluorescence
−
Scheme 1. Proposed response mechanism of SiOPh-PyOH sensing ONOO .
−
In contrast, the energy gap of OPh structure is a little higher
but close to that of PyO and the HOMO and LUMO energy
levels of OPh are also a little higher but close to these of PyO ,
which makes that the energy and charge transfer can smoothly
'flow' from OPh to PyO (Figure 12d). Furthermore, for OPh-
PyO structure, the HOMOs are mainly delocalized onto
−
−
−
−
−
−
−
phenolate but through the whole molecule, while the LUMOs are
mainly distributed on pyrene ring but through the whole molecule.
These results indicate that there is good conjugation between
−
When the probe SiOPh-PyOH changes into SiOPh-PyO by
deprotonation, the capacity of pyrene ring as an electron
donating group is further enhanced, which makes the LUMO
distribution on SiOPh increase (Figure 12b). Although the
energy gap of SiOPh-PyO is greatly reduced to 2.2436 eV,
which corresponds to an emissions of about 600 ~ 650 nm, the
fluorescence intensity largely decreases because of less LUMO
distribution on PyO structure. Therefore, SiOPh-PyO should
emit weak fluorescence at 600 ~ 650 nm, which was confirmed
by the results of MeOPh-PyOH with added a base.
−
−
−
OPh and PyO and the phenolate OPh is an electron donor to
−
−
−
PyO , which is beneficial to ICT from OPh to PyO . Therefore,
−
−
−
−
OPh-PyO should be a better TBET system than OPh-PyOH
and SiOPh-PyO . The favorable electron conjugations greatly
reduce the energy gaps of OPh-PyO to 2.4485 eV, which
−
−
−
−
−
corresponds to an emission at about 580 ~ 620 nm.
Next, energy levels, absorption transitions and oscillator
strengths (f) were calculated for probe SiOPh-PyOH and
possible structures in the sensing process (Figure S13). As
shown in Figure S13a, two allowed transitions in SiOPh-PyOH
If silyl ether of SiOPh-PyOH was directly cleaved to give
OPh-PyOH (Figure 12c), phenolate would be an electron donor.
The energy gap of phenolate OPh is reduced to 3.9737 eV, a
little higher but close to the energy gap of acceptor 5 (3.4458
eV). However, the HOMO and LUMO energy levels of OPh are
much higher than those of PyOH. Therefore, the conjugation
between OPh and PyOH should be very poor, and the electron-
donating effect from OPh to PyOH should very weak, which
makes the emission of fluorophore PyOH red-shifted little.
Further, since the HOMO energy level of donor OPh is higher
than the LUMO energy level of acceptor PyOH, photo induced
electron transfer effect from OPh to PyOH greatly reduces the
fluorescence of PyOH. Therefore, OPh-PyOH is a very poor
fluorescent structure with a slight red shift, which is similar to the
−
−
are S
0
→ S
2
(3.4744 eV) and S
0
→ S
4
(3.5752 eV) while the
→ S , f = 0.1297).
transition from HOMO to LUMO is very low (S
0
1
−
The HOMO and LUMO+1 are mainly located in the pyrene part,
while the HOMO-1, LUMO and the LUMO+2 are distributed
through the whole molecule. Therefore, S → S and S → S
0 4 0 2
can be assigned to the absorption bands of the pyrene core and
the whole conjugated structure, respectively. However, those
allowed transitions have almost the same calculated energy
gaps (3.4744 and 3.5752 eV). These theoretical results are in
good accordance with the experimental data that SiOPh-PyOH
shows no obvious change in the absorption and fluorescence
spectra of the pyrene moiety.
−
−
−
−
−
results of HOPh-PyOMe with added a base.
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Chemistry - A European Journal
10.1002/chem.201903880
FULL PAPER
‒
37,38
After deprotonation, two allowed transitions of SiOPh-PyO
γ (IFN-γ) and lipopolysaccharide (LPS).
Thus, we further
0 1 0 7
are S → S (2.0220 eV) and S → S (3.3413 eV) (Figure S13b). tested the ability of SiOPh-PyOH for imaging the endogenous
−
The energy gaps should correspond to the red and blue
fluorescence emission, respectively. Although the transitions
ONOO in the cells. When the cells were stimulated with IFN-
γ/LPS and then treated with SiOPh-PyOH, dramatic
a
from HOMO (S
0
→ S
1
, f = 0.6694) and HOMO-1 (S
0
→ S
7
, f =
fluorescence enhancement was also observed in red channel
0
.7169) to LUMO are highly allowed, the LUMO is mainly
(Figure 5b), indicating the SiOPh-PyOH is capable of imaging
the stimulation-induced ONOO .
−
located in SiOPh moiety instead of pyrene moiety, which makes
the fluorescence intensities weak in both the red and blue
regions. These week fluorescent properties were proved by
HOPy-PhOMe when the base was added (Figure S11c).
‒
Unlike SiOPh-PyO , the transition from HOMO to LUMO (S
0
‒
→
1
S , f = 0.0636, 1.8678 eV) for OPh-PyOH is completely
forbidden (Figure S13c). This shows that there is no
fluorescence emission in the red region. On the other hand, for
the pyrene fluorophore, the transitions from HOMO-2 to LUMO
0 6 0 8
(S → S , f = 0.1421) and LUMO+1 (S → S , f = 0.4948) are
‒
lowly allowed, which indicates OPh-PyOH a weak fluorescence
structure in blue region. These fluorescent properties were also
proved by MeOPy-PhOH when the base was added (Figure
S11d).
Figure 5. Fluorescence images of RAW264.7 murine macrophages under
different conditions. (a) Cells were treated with SiOPh-PyOH (10 μM, 30 min).
(b) Cells were stimulated with LPS (1 μg/mL) and IFN-γ (50 ng/mL) for 6 h,
and then treated with SiOPh-PyOH (10 μM, 30 min).
‒
‒
For structure OPh-PyO , the most allowed transition is S
(2.2492 eV), which is the transition from HOMO to LUMO
Figure S13d). Moreover, the HOMO is mainly located in the Conclusions
pyrene part, while the LUMO is mainly located in phenolate
moiety. Therefore, this S → S transition is assigned to the
0
→
(
S
1
0
1
In summary, this paper describes (1) a general design strategy
for a TBET probe with high energy and electron transfer
efficiencies, (2) a mechanism of silyl ether cleaved by ONOOH,
(3) a TBET fluorescence probe designed based on only one
absorption band from phenolate moiety to pyrene core. These
theoretical results are in good accordance with the experimental
data. Collectively, these results confirm that OPh-PyO is a
‒
‒
−
TBET system.
fluorophore for endogenous ONOO with high selectivity, large
PSS and large emission shift. Probe HOPy-PhOSi consists of a
silyloxyphenyl group and a pyrene fluorophore. Probe HOPy-
PhOSi shows the blue emission (415 nm) of pyrene ring without
Cellular imaging experiments. SiOPh-PyOH showed the
excellent sensing ability to recognize ONOO in a physiological
solution. We further explored its potential use for bioimaging in
human breast cancer cells (MCF-7) (Figure S14 and S15).
When MCF-7 cells were incubated with SiOPh-PyOH for 30 min,
strong blue fluorescence (Figure S14b) and no red fluorescence
−
−
−
−
ONOO , while OPy-PhO shows the significantly red-shifted
absorption and organe-red emission (594 nm), resulting from the
Si−O bond cleavage. The results proved that ONOOH is an
effective deprotection reagent for silyl ethers. To the best of our
(
Figure S14c) were observed. When the stained cells were then
−
knowledge, this is the first system using silyl ether for ONOO
−
incubated with ONOO for 10 min. The strong blue fluorescence
darkened (Figure S14d) and the red fluorescence brightened
−
sensing instead of F . Probe HOPy-PhOSi exhibits high
selectivity to ONOO , large PSS (213 nm) and large emission
−
(Figure S14e). These results indicated that probe SiOPh-PyOH
shift (179 nm), and has been successfully used in endogenous
was cell membrance permeable and suitable for imaging
−
ONOO detection. We believe this probe design strategy will be
−
ONOO in the living cells. When MCF-7 cells with SiOPh-PyOH
widely applicable for the construction of TBET probes emitting in
(
10 μM) were incubated with increasing concentrations of
−
red region, ONOO sensing and deprotection of silyl ether by
−
ONOO , the clear fluorescence imagings were obtained at the
concentrations more than 20 μM (Figure S15). In addition, the
cytotoxicity of SiOPh-PyOH was evaluated in MCF-7 cells. Little
toxicity on cell viability was observed after incubation with
SiOPh-PyOH (10 − 30 μM) for 4 h (Figure S16), which
demonstrated that SiOPh-PyOH could be used for potential
clinical applications in living organisms.
ONOOH.
Experimental Section
Syntheses of Probes and Derivatives. Detailed descriptions of the
syntheses can be found in the Supporting information.
General Procedure for Spectral Measurement. All measurements
were carried out at room temperature. The stock solution of probe
SiOPh-PyOH (0.5 mM/L) was prepared in DMF. Generation of ROS and
common anions (20 mM/L) were prepared using distilled water by
Subsequently, the capability of SiOPh-PyOH for imaging
endogenous ONOO was evaluated in RAW264.7 murine
macrophages (Figure 5). When the cells were incubated with
SiOPh-PyOH, only blue fluorescence was observed (Figure 5a),
suggesting that SiOPh-PyOH may be stable in the cells. It is
−
39
according to the literature. For optical measurements, probe SiOPh-
PyOH was diluted to 10 μM in DMF-PBS buffer (pH = 7.4), and 3.0 mL of
diluted solution was placed in a quartz cell of 1 cm optical path length. All
spectroscopic experiments were carried out at room temperature. The
known
that
RAW264.7
murine
macrophages
could
−
endogenously produce ONOO upon stimulation with interferon-
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Chemistry - A European Journal
10.1002/chem.201903880
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Entry for the Table of Contents (Please choose one layout)
FULL PAPER
Emission
15 nm
381 nm
+
Emission
594 nm
4
ONOOH
NO2
OH
TBET on
Gonghao Lu,* Yuxin Guo, Jiezhen Zhuo,
Xue Li,* Haijun Chi and Zhiqiang Zhang
TBET off
X
Si
O
3
81 nm
O
O
Si
N
N
N
N
N
ONOO–
N
OH
O
O
Page No. – Page No.
X
ICT on
Weak fluorescence
¯
ICT on
ICT off
–
¯
OPy PhO
A General Strategy for Through-bond
Energy Transfer Fluorescence Probes
Combining Intramolecular Charge
Transfer: a Silyl Ether System for
Endogenous Peroxynitrite Sensing
HOPy–PhOSi
A probe provides characteristics of (1) a general design strategy for a TBET probe,
2) a mechanism of silyl ether cleaved by ONOOH, and (3) a TBET fluorescence
probe designed based on only one fluorophore for endogenous ONOO . The probe
shows high selectivity, large Pseudo-Stokes shift (213 nm) and large emission shift
(
−
(179 nm shift).
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