Spiroboronate Si-rhodamine as a near-infrared probe for imaging lysosomes based on the reversible ring-opening process

Article abstract of DOI:10.1039/c5cc02496j

A cyclic boronate structure was incorporated into Si-rhodamine to design a pH-activatable near-infrared (NIR) probe based on the reversible ring-opening process triggered by H⁺. The probe showed general suitability of specific labelling of acid

Full text of DOI:10.1039/c5cc02496j

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Spiroboronate Si-rhodamine as a near-infrared probe for imaging
lysosomes based on reversible ring-opening process
Weiwei Zhu,ab‡ Xiaoyun Chai,b‡ Baogang Wang,b Yan Zou,b Ting Wang,*b Qingguo Meng,a and
Qiuye Wub
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A cyclic boronate structure was incorporated into Si-rhodamine to
design pH-activatable near-infrared (NIR) probe based on
a
reversible ring-opening process triggered by H+. The probe showed
general suitability of specific labelling acidic lysosomes and
tracking their pH changes in living cells.
Intracellular pH plays vital roles in a variety of critical cellular
functions in cells and specific subcellular organelles.1 In lysosomes,
an acidic environment can serve to activate the enzyme, facilitating
the degradation of proteins in cellular metabolism. Meanwhile,
abnormal pH values in lysosomes often associate with cellular
dysfunction.2 Accordingly, labelling acidic organelles and tracking
their pH changes are crucial for studying physiological and
pathological processes. Owing to the high sensitivity and selectivity,
fluorescent probes have become indispensable for monitoring pH in
vivo.3 In particular, design of NIR (650-900 nm) pH-activatable
fluorescent probes4 is especially preferred because NIR light
minimizes photodamage and avoids the influence of
autofluorescence.5 So far, several NIR fluorescent pH probes have
been developed and most of them are based on cyanine, in which
the fluorescence behavior is controlled by the photoinduced
Scheme 1 Chemical structures of spiroboronate derivatives (R-B
and SiR-B) and the reversible transformation between boronate
and boronic acid .
Si-rhodamine (SiR) recently has become rather popular in the
field of bioimaging because of its excellent NIR photophysical
properties and biocompatible characteristics.6 Particularly,
spirocyclization of SiR is an attractive strategy for designing NIR
fluorescent probes, as the spirocyclic SiR is colorless and
nonfluorescent, whereas opening the corresponding spiroring gives
rise to a blue color change and strong NIR fluorescence. Following
the design principle, some SiR-based fluorescent probes have been
8
exploited for imaging Hg2+ 7
,
ClO− and proteins of interest in living
cells.9 Although rhodamine-based pH-activatable probes have been
well developed from spirolactam,3b, 10 similar approach have yet not
successfully achieved in SiR because of the unsatisfied
spirocyclization equilibrium of spirolactam within proper pH range.
Thus, new spirocyclization of SiR with ring-opening process that can
be precisely controlled by H+ arose our interests.
electron
transfer
(PeT)
process
through
the
protonation/deprotonation of their amine-containing side chains.
Although widely used, these NIR pH probes have some inherent
deficiencies for bioimaging: firstly, in most cases cyanine-based
probes easily suffer from photobleaching; additionally, sometimes
the PeT process has poor efficiency, resulting in relatively high and
nonspecific background signals; lastly, only a few of them work in
the pH range of 5-6 and can be used for studying acidic organelles
in vivo.4b, 4e Therefore, the development of new photostable NIR
fluorescent pH probes with high signal/noise ratio and alternative
H+-responsive mechanism is of great significance and practical
value.
As a promising recognition group, boronic acid has attracted
considerable interest owing to their unique binding and reactive
abilities. A variety of boronic acid and boronate-based fluorescent
probes have been developed for detecting biologically relevant
species.11 Boronic acid participates in such equilibria: in neutral or
mildly basic environment, it binds with the hydroxyl groups
containing substances and forms corresponding boronate which can
be reversibly hydrolyzed to boronic acid in acid condition. The
reversible transformation between boronate and boronic acid
under external stimuli of H+ offers a new clue to design boron-
containing spirocyclic derivatives based on rhodamine and SiR
(Scheme 1). Herein we designed and synthesized a SiR-based
spirocyclic derivative SiR-B, in which a boronic acid group was
introduced to replace the carboxyl group at 2’ position. Meanwhile,
its rhodamine analogue R-B was also prepared as a comparison. To
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the best of our knowledge, this is the first example of the
The probe SiR-B formed colorless solution in most aprotic
spiroboronate ring in rhodamine and SiR systems. Probes SiR-B and organic solvents, but underwent a reversible blue color change
R-B both displayed the reversible H+-triggered ring-opening process after adding AcOH or Et3N (Fig. S1, ESI†). Correspondingly, a bright
of the corresponding spirocylic structures accompanied by pink color was observed in initial probe R-B solution and similar H+-
remarkable chromogenic and fluorogenic changes. Furthermore, dependent color response was observed. These phenomena
probe SiR-B showed abrupt fluorescence change in the pH range implied that the boron-containing groups in both probes SiR-B and
from 7.4 to 5.0, demonstrating ideal suitability for specific labelling R-B are sensitive to H+, and the reversible color changes is likely to
acidic lysosomes and tracking their pH changes in living cells.
be induced by the reversible ring-opening process of the
As presented in Scheme 2, probe SiR-B was synthesized by corresponding rings. To fully explore the H+-responsive mechanism,
addition of a lithiated benzene moiety bearing a protected boronic we compared the NMR spectral changes of probe SiR-B upon
8
acid group to Si-containing xanthone (SiX),7,
followed by addition of H+ (Fig. 1). In DMSO-d6, a singlet resonating at δ = 9.26
deprotection in hydrochloric acid (HCl) solution. In the protection of ppm in 1H NMR could be assigned to the B-OH and the signal
boronic acid group, N-butyldiethanolamine was chosen as
resonating at δ = 88 ppm in 13C NMR corresponds to the quaternary
a
protective reagent because of its tolerance to lithiation and carbon atom at position 9 (C9).13 These characteristic peaks
effective deprotection.12 However, a similar synthetic approach has indicated that probe SiR-B existed predominantly in its spirocyclic
failed in synthesizing probe R-B. R-B was obtained from iodine- form in aprotic organic solvents. After adding H+, the B-OH signal
substituted rosamine via coupling reaction and subsequent dismissed, suggesting the interaction between spiroboronate and
deprotection reaction. The structures of probes SiR-B and R-B were H+. Meanwhile, no such signal of the quaternary carbon at similar
confirmed by high-resolution mass spectrometry and NMR region was observed. The significant upfield shift of the C9 signal
spectroscopy.
directly confirmed the ring-opening of the spiroboronate in probe
SiR-B by formation of boronic acid.
The pH-dependent spectral properties of probes SiR-B and R-B
were further evaluated in phosphate buffer solution (containing 1%
DMSO). As shown in Fig. S2 (ESI†), free SiR-B was colorless and
almost nonfluorescent at physiological pH of 7.4 as a result of the
intermolecular spirocyclization. When the pH decreased from 7.4 to
5.0, the solution changed to blue color with appearing and
dramatically increasing of an intense band at 664 nm.
Concomitantly, the fluorescence at 680 nm was significantly
enhanced with a 20-fold intensity increase within the studied pH
range duo to the ring-opening process of spiroboronate. At pH 5.5,
probe SiR-B existed in the ring-opening form with maximum
Scheme 2 Synthesis of probes SiR-B (i and ii) and R-B (iii and iv).
Conditions: i) N-butyldiethanolamine, PhMe, 50 oC; ii) n-BuLi,
THF,−78 oC, then compound SiX, THF, −78 oC to r.t., then 6M HCl,
r.t.; iii) Bis(pinacolato)diboron, PdCl2(dppf)2, KOAc, DMSO, 80 oC; iv)
BBr3, CH2Cl2, 0 oC.
1
fluorescence, possessing an extinction coefficient of 105000 M−
1
cm− and a fluorescence quantum yield of 0.31 (Table 1). The
fluorescence pH titration curve of probe SiR-B yielded pKcycl
(equilibrium constant of intramolecular spirocyclization) of 6.2,
Table 1 Photochemical Properties of Probes SiR-B and R-B
d
Probes
SiR-Ba
R-Bb
λabs/nm
651
λem/nm
667
ε/M−1 cm−1
Φc
pKcycl
105000
0.31 6.2
0.33 9.6
555
576
101000
aMeasured in phosphate buffer (pH = 5.5) containing 1% DMSO. b
Measured in phosphate buffer (pH = 7.4) containing 1% DMSO.
cCy5 (Φ = 0.27, PBS) and Rhodamine B (Φ = 0.31, water) were
used as the references for the fluorescence quantum yields of
probes SiR-B and R-B, respectively. dCalculated from fluorescence
pH titration.
Fig. 1 (a) and (b) 1H and 13C NMR spectral changes of probe SiR-B
upon addition of 1.5 equiv. of CF3COOD (black lines: before adding
CF3COOD; blue lines: after adding CF3COOD). (c) Proposed H+-
responsive mechanism based on the ring-opening process of
spiroboronate in SiR-B.
Fig. 2 Absorbance and fluorescence pH titration curves of probes
SiR-B (blue, λabs = 651 nm, λex = 620 nm, λem = 667 nm) and R-B
(red, λabs = 555 nm, λex = 520 nm, λem = 576 nm).
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suggesting that probe SiR-B may serve as an appropriate NIR pH
probe for imaging acidic pH in vivo. By sharp contrast, probe R-B
1
solution displayed a pink color (ε = 101000 M− cm−1) and strong
fluorescence (Φ = 0.33) at pH 7.4, suggesting that probe R-B
preferred the ring-opening form at physiological pH. As the pH
increased to 12.0, the intermolecular spirocyclization of boronic
acid resulted in the immediately fading of the absorption and
fluorescence intensity (Fig. S3, ESI†). The pKcycl value of probe R-B
was 9.6, which was much higher than that of probe SiR-B. The
comparison of two pH-activatable probes revealed that they shared
similar pH-dependent ring-opening process of corresponding
spiroboronate accompanied with chromogenic and fluorogenic
changes. The intriguing difference of intramolecular spirocyclization
Fig. 3 Fluorescence images of HepG2 (left) and PC12 (right) cells co-
stained with 5.0 ꢀM SiR-B and 1 ꢀM LysoTracker Green DND-26. (a)
Fluorescence microscopic images (red channel) from probe SiR-B.
(b) Fluorescence microscopic images (green channel) from
LysoTracker Green DND-26. (c) Bright-field microscopic images. (d)
Merged fluorescence microscopic images of green and red
channels.
behavior was likely to attribute to the different stability of spiroring
9
as suggested by us and other research groups.7,
Strong
electrophilicity of the SiR fluorophore stabilized the spirocyclic
structure, thereby shifting the pKcycl value to acidic pH region. The
exceptional photophysical characteristics and H+-responsive
behavior of probe SiR-B ensure that it is a promising candidate for
imaging pH in vivo, especially labelling and tracking acidic lysosomes
which can reach pH value as low as ∼5.
The properties of probe SiR-B were scrupulously evaluated
(Fig. S4-8, ESI†). Reversibility is highly required for tracking pH
changes in living cells. After five cycles, no noticeable changes in the
emission were observed in SiR-B. Meanwhile, the color of probe
SiR-B changed repeatedly between colorless and blue. The
selectivity of probe SiR-B to H+ over biologically relevant species at
pH 7.4 and pH 5.5 was evaluated as well. High concentrations of
ions, such as K+, Na+, Ca2+, Mg2+, Cu2+, Zn2+, Fe3+ and F− did not cause
any significant fluorescence intensity changes. Other biologically
important species, including amino acids and carbohydrates, caused
no visible effect on the fluorescence signals. Low concentration of
H2O2 has little influence on the emission, although very high
concentration of H2O2 quenched the fluorescence of probe SiR-B at
pH = 5.5. Like other SiRs, probe SiR-B was highly stable at pH of 7.4
and 5.5, showing remarkably resistance to photobleaching after
irradiation.14 Additionally, MTT assays revealed that the cell
viabilities of HepG2 cells were not seriously affected by incubation
Fig. 4 Fluorescence images of probe 5.0 ꢀM SiR-B in HepG2 cells
stimulated with chloroquine. (a) and (b) Images of the stained cells
before chloroquine stimulation; (c) and (d) Images of cells in
exposed to 100 ꢀM chloroquine for 2 min.
locating group was attributed to the H+-triggered ring-opening
process with appropriate pKcycl that matched well with pH of the
acidic lysosomes. The sensitive H+-response behavior offered
enhanced lysosome-specific NIR fluorescence imaging with minimal
background fluorescence. Furthermore, we examined the feasibility
of applying our probe for tracking the pH changes in lysosomes (Fig.
4). Obvious quenching of the fluorescence signal in the area of the
lysosomes was observed after treating with chloroquine, which is
commonly used in inducing an increase in the lysosomal pH. The
results suggested that probe SiR-B could be used for tracking pH
changes in lysosomes.
with 1∼10 ꢀM probe SiR-B for 24 h.
As stated above, the pH-activatable probe SiR-B possesses
excellent NIR photophysical properties with high molar extinction
coefficient and high fluorescence quantum yield. In addition, it
meets the requirements for imaging pH in vivo, such as reversibility,
promising specificity, high photostability and low bio-toxicity. To
evaluate the feasibility of imaging pH in living cells with probe SiR-B,
it was employed to stain HepG2 and PC12 cells. Confocal
fluorescence microscopy results showed that probe SiR-B
accumulated into specific subcellular organelles, giving red
fluorescence spots dispersed in cells. Co-staining with commercially
available lysosome-specific probe LysoTracker Green DND-26 was
performed to further confirm the subcellular location (Fig. 3). The
yellow merged images indicated that the staining of probe SiR-B fit
well with that of LysoTracker Green DND-26 (Fig. S9, ESI†). These
results thus demonstrated that the probe SiR-B possessed great cell
membrane permeability and general suitability of specific labelling
acidic lysosomes in living cells. The efficient lysosome-targeting
location of SiR-B without intentionally functionalized lysosome-
In conclusion, we incorporated cyclic boronate structure into
rhodamine and SiR systems and successfully synthesized both
spiroboronate rhodamine (R-B) and Si-rhodamine (SiR-B). The
controlled spirocyclization of boronate in SiR-B generated a pH-
activatable NIR probe based on reversible ring-opening process
triggered by H+. We proved that probe SiR-B existed as a closed
spirocyclic structure in aqueous solution at physiological pH 7.4,
whereas converting the spirocyclic structure to the strongly
fluorescent open form at pH ∼5. Taking advantage of the applicative
H+-responsive
behavior
and
exceptional
biocompatible
characteristics, probe SiR-B showed the general suitability of
specific labelling acidic lysosomes and tracking their pH changes.
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14 Y. Koide, Y. Urano, K. Hanaoka, T. Terai, and T. Nagano, ACS
Chem. Biol. 2011, 6, 600-608
These results remind us that spirocyclization of SiR is a powerful
design strategy for designing NIR fluorescent probes in which the
equilibrium between the spirocyclic closed form (colorless and
nonfluorescent) and the open form (strongly fluorescent) can be
precisely controlled to facilitate various applications.
.
This work has been supported by the National Natural Science
Foundation of China (No. 21205135). We also sincerely appreciate
Xiaoyan Cui for her useful discussion and suggestion.
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