Boronic acid derived salicylidenehydrazone complexes for wash-free fluorescence imaging of cellular organelles

Article abstract of DOI:10.1016/j.dyepig.2017.10.027

Wash-free fluorescence imaging process has intrinsic nature of monitoring biochemical process in real time fashion and enable us to get more insight on living organism. In this contribution, it was proved that boronic acid derived salicylidenehydrazone complexes (BSs) are suitable candidates for wash-free fluorescence labels. Through pre-installation of organelles' targeting group morpholine and triphenylphosphonium (TPP⁺) on phenyboronic acid and then assembly it with salicylidenehydrazone ligand by dehydration and dative N-B bond, Lyso-BS and Mito-BS were acquired, which were shown to selectively stain lysosomes and mitochondria with circumventing tedious wash-off procedures in Hela cells respectively, without inducing any appreciable cytotoxicity. Our findings indicate that BSs are potential candidates as wash-free fluorescent stains.

Full text of DOI:10.1016/j.dyepig.2017.10.027

Accepted Manuscript
Boronic acid derived salicylidenehydrazone complexes for wash-free fluorescence
imaging of cellular organelles
Boyu Zhang, Gang Feng, Shichao Wang, Xuanjun Zhang
PII:
S0143-7208(17)31875-2
10.1016/j.dyepig.2017.10.027
DYPI 6325
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 5 September 2017
Revised Date: 14 October 2017
Accepted Date: 15 October 2017
Please cite this article as: Zhang B, Feng G, Wang S, Zhang X, Boronic acid derived
salicylidenehydrazone complexes for wash-free fluorescence imaging of cellular organelles, Dyes and
Pigments (2017), doi: 10.1016/j.dyepig.2017.10.027.
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Graphical Abstracts

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Boronic Acid Derived Salicylidenehydrazone Complexes for
Wash-free Fluorescence Imaging of Cellular Organelles
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Boyu Zhang, Gang Feng, Shichao Wang, Xuanjun Zhang*
Faculty of Health Sciences, University of Macau, Macau SAR, China.
Email: xuanjunzhang@umac.mo
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Abstract: Wash-free fluorescence imaging process has intrinsic nature of monitoring
biochemical process in real time fashion and enable us to get more insight on living organism. In
this contribution, it was proved that boronic acid derived salicylidenehydrazone complexes (BSs)
are suitable candidates for wash-free fluorescence labels. Through pre-installation of organelles’
targeting group morpholine and triphenylphosphonium (TPP+) on phenyboronic acid and then
assembly it with salicylidenehydrazone ligand by dehydration and dative N-B bond, Lyso-BS and
Mito-BS were acquired, which were shown to selectively stain lysosomes and mitochondria with
circumventing tedious wash-off procedures in Hela cells respectively, without inducing any
appreciable cytotoxicity. Our findings indicate that BSs are potential candidates as wash-free
fluorescent stains.
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Keywords: Wash-free; Boronic acid; Salicylidenehydrazone; Lysosomes: Mitochondria;
Fluorescence imaging
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1. Introduction
Owning to its non-invasion, technical simplicity, multiple signals, and high sensitivity,
fluorescence imaging technology have become a powerful tool for interrogating the biomolecular
and biochemical process, stretching across applications from fundamental biological research to
clinical diagnostics.[1] From practical point of view, a fluorescent image with high-quality must
possess large signal-to-noise (S/N) ratio, which requires both an enhanced fluorescent signal at a
region of interest and minimized background fluorescence.[2] A washing protocol step is
indispensable to protein,[3, 4] cell,[5] bacteria[6, 7] and tissue[8] imaging for alleviating the
background signals from the fluorescent labels unbounded to region of interest.[9] However, the
rigorous, repeating and time-consuming washing-out process disturbs the continuous observation
of targets and inevitably delays the acquisition of microscopic data.[10, 11] In addition, the
washing-out steps alter the environment of specimen and might induce thermal stress and
apoptosis, which make it difficult to monitor the subcellular organelles's changes in real time,[12]
also make fluorescent quantification assay unreliable due to the quantitative loss of specimen in
course of washing[9]. Therefore, it is highly desirable to develop new wash-free imaging method.
Its essential requirement is the fluorophores that could light up or turn on with special biochemical
events or internal stimuli, but keep its dark state otherwise, especially in water medium.
Up to date, several strategies have been exploited for flourishing the arsenal of wash-free labels.
Fluorogenic dyes with emitter-quencher pairs are especially common for creating activatable
fluorescent labels through mechanisms of photoinduced electron transfer, internal charge transfer
or energy transfer.[13] Depending on quenching efficiency, parts of activatable fluorescent labels
have a wash-free feature but many others need a wash-out process to realize imaging or
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monitoring of biological processes with good sensitivity and reliability.[14] Fluorescent
environment-sensitive dyes are an another more promising strategy for developing wash-free
labels, due to its fluorescent quenching characteristic in water.[15] The dyes with free
intramolecular rotational bonds in water would disrupt its conjugated planarity and relax the
excited energy through non-radiative channels.[16] As it is restricted to a confined
microenvironment, its fluorescence will be restored.[17-19] Based on this fundamental concept,
many wash-free nuclear stains are developed and widely employed in cell life cycle analysis, flow
cytometry and also as counterstains in fluorescence microscopy,[20] such as DAPI and Hoechst
33342, representatives of Indole and benzimide dyes. Sparked by the phenomenon of
aggregation-induced emission (AIE)[21], wash-free AIE fluorogenic dyes was developed from
anchoring hydrophilic motifs onto parent AIEgens. Dissimilar to nuclear stains possessing
well-defined binding modes for light-up, the wash-free AIEgens normally requires reasonable
extent of accumulation inside of cells to arouse AIE. As a large class of environment-sensitive
dyes, push-pull dyes undergoing intramolecular charge transfer (ICT) creates a highly dipolar
excited state, which relaxes through interaction with the dipoles of solvent and thus its emission
shifts to longer wavelengths in more polar solvents. Most of push-pull dyes are quenched in polar
protic solvents, providing an avenue of designing new wash-free dyes.[22] Flavones conjugated
with targeting group have been exploited for wash-free fluorescence imaging,[23] further for
targeting of mitochondrial[24] and endoplasmic reticulum[5]. Although the "scaffold approach",
where one of the reactants contains a chromophore, have been successful in many cases of
preparing chromogenic structures, a new and promising “chromophore approach”[25] have been
developed during the past years, where the multicomponent reaction generates a chromophore
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from non-fluorescent materials.[26]
In this contribution, we present the synthesis and application of two new wash-free fluorogenic
dyes through chromophore approach by two step: 1) pre-installation of organelles targeting groups
on phenylboronic acid first, morpholine and triphenylphosphonium (TPP+),[27] for lysosomes and
mitochondria respectively, 2) followed by involving it in construction of a modular fluorogenic
dyes boronic acid salicylidenehydrazone (BSs) based on a previous work[28] While Lip-BS was
reported as stain for lipid droplets,[29] it is shown that Lyso-BS and Mito-BS could rapidly locate
lysosomes and mitochondria with high Pearson’s colocalization coefficient (Scheme 1).
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2. Experimental
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2.1 Synthesis. (Z)-2-(((E)-4-(diethylamino)-2-hydroxybenzylidene)hydrazono)-2-phenylacetic
acid (Ligand). Adopting a literature procedure with some reasonable modification,[29]
hydrazonoe (2.5 mmol, 490 mg) was completely dissolved in 5 mL water firstly and 20 mL
methanol was added. After shaking moderately, this solution was eluted through a columned
packed with Amberlyst@ 15 (5 g wet resin exchanged with 10 mL methanol for three times) and
dropped into a methanolic solution of SA (2.5 mmol, 483 mg in 5 mL methanol). The ultimate
solution was stirred for 2 h at room temperature and then incubation at 4
in refrigerator
overnight. The slurry was filtered and washed with cold methanol (5 mL) three times, and
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afterwards collected as orange powder: yield 65%; H NMR (400 MHz, CDCl3) δ 8.16 (s, 1H),
8.09 – 8.01 (m, 2H), 7.71 (d, J = 2.3 Hz, 1H), 7.43 (td, J = 7.0, 5.8, 3.0 Hz, 3H), 7.19 (d, J = 9.2
Hz, 1H), 6.36 (dd, J = 9.2, 2.3 Hz, 1H), 3.59 (q, J = 7.1 Hz, 4H), 1.31 (t, J = 7.1 Hz, 6H). 13C
NMR (101 MHz, DMSO-d6) δ 167.3, 164.9, 161.9, 159.5, 152.4, 134.7, 131.9, 131.7, 129.6,
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127.3, 106.5, 104.9, 97.3, 44.5, 13.0. HRMS-ESI calcd for C19H22N3O3 ([M+H]+), 340.1661;
found, 340.1703.
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(4-(morpholinomethyl)phenyl)boronic acid (LB). A mixture of BPBA (2.33mmol, 500 mg),
morpholine (4.65 mmol, 405 mg) and K2CO3 (9.31 mmol, 1.29 g) in 10 mL CH3CN was stirred at
room temperature for 24 h. After completion of the reaction, the solid K2CO3 was filtered out and
filtrate was concentrated. The residue was dissolved in DCM (10 mL), washed with brine (3 ×10
mL) until neutrality, and then the orange extracts was dried over anhydrous Na2SO4 and
concentrated in vacuo to give white solid. Because of the intrinsic tendency to exist as mixtures of
oligomeric anhydrides, it was directly used for next step without purification.
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(4-boronobenzyl)triphenylphosphonium bromide (MB). A solution containing BPBA (5.2
mmol, 1.12 g) and PPh3 (5.72 mmol, 1.5 g) in 160 mL MeCN was refluxed for 12 h. After the
completion of reaction, the solvent was removed in vacuo and the residue was triturated with
diethyl ether (3 × 10 mL) and collected as white precipitate: yield 95%; 1H NMR (400 MHz,
DMSO-d6) δ 7.88 – 7.78 (m, 3H), 7.71 – 7.51 (m, 17H), 6.90 (dd, J = 8.1, 2.5 Hz, 2H), 4.98 (d, J =
15.4 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 135.6, 135.6, 134.9, 134.8, 134.6, 134.5, 130.6,
130.5, 130.4, 130.4, 118.7, 117.9, 29.0. HRMS-ESI calcd for C25H23BO2P ([M-Br]+), 397.1523;
found, 397.1613.
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General procedures for the synthesis of Lip-BS, Lyso-BS and Mito-BS. The derivate of
phenylboronic acid (0.3 mmol) was dissolved in CH3CN (3 mL), toward which the ligand (0.33
mmol, 112 mg) was added. The resulting solution was refluxed until the termination of reaction.
And then, the solution was concentrated and the residue was triturated with cold methanol for
washing away minor impurities.
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Compound Lip-BS. Orange solid; Yield 95%; 1H NMR (400 MHz, CDCl3) δ 8.21 (s, 1H), 7.96 –
7.88 (m, 2H), 7.41 – 7.27 (m, 5H), 7.18 – 7.08 (m, 4H), 6.29 (dd, J = 9.1, 2.4 Hz, 1H), 6.13 (d, J =
2.3 Hz, 1H), 3.37 (ddt, J = 16.6, 14.8, 7.4 Hz, 4H), 1.16 (t, J = 7.2 Hz, 6H). 13C NMR (101 MHz,
CDCl3) δ 161.6, 157.3, 155.8, 154.0, 153.6, 134.6, 132.7, 130.8, 130.8, 129.5, 128.2, 127.8, 127.6,
107.7, 106.4, 98.7, 45.4, 12.8. HRMS-ESI calcd for C25H25BN3O3 ([M+H]+), 426.1989; found,
426.1873.
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Compound Lyso-BS. Orange solid; Yield 85%; H NMR (400 MHz, Chloroform-d) δ 8.31 (s,
1H), 8.06 – 7.99 (m, 2H), 7.51 – 7.30 (m, 5H), 7.21 (dd, J = 26.9, 8.4 Hz, 3H), 6.39 (dd, J = 9.2,
2.4 Hz, 1H), 6.24 (d, J = 2.3 Hz, 1H), 3.68 (t, J = 4.6 Hz, 4H), 3.58 – 3.38 (m, 6H), 2.42 (t, J = 4.6
Hz, 4H), 1.27 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 161.6, 157.3, 155.9, 155.5, 154.0,
153.5, 134.5, 132.7, 132.3, 130.8, 129.4, 128.2, 114.6, 107.6, 106.4, 98.7, 45.4, 12.7. HRMS-ESI
calcd for C30H34BN4O4 ([M+H]+), 525.2673; found, 525.2405. Melting point 230-232 °C.
Elemental analysis calcd (%) for C30H33BN4O4: C 68.71, H 6.34, N 10.68, found (%): C 68.51, H
6.50, N 10.54.
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Compound Mito-BS. Orange solid; Yield 93%; Melting point 195-197 °C. H NMR (400 MHz,
CDCl3) δ 8.06 – 7.99 (m, 2H), 7.74 – 7.40 (m, 19H), 7.24 (d, J = 9.2 Hz, 1H), 7.14 (d, J = 7.5 Hz,
2H), 6.89 (dd, J = 8.0, 2.6 Hz, 2H), 6.41 (dd, J = 9.2, 2.4 Hz, 1H), 6.19 (d, J = 2.2 Hz, 1H), 5.17
(dt, J = 35.4, 14.7 Hz, 2H), 3.49 (ddq, J = 18.2, 14.8, 7.4 Hz, 4H), 1.31 – 1.19 (m, 6H). 13C NMR
(101 MHz, CDCl3) δ 157.6, 155.9, 153.6, 134.9, 134.9, 134.8, 134.3, 134.2, 132.8, 131.4, 131.4,
130.9, 130.6, 130.6, 130.1, 130.0, 129.4, 128.3, 118.3, 117.4, 108.2, 106.6, 98.6, 45.5, 31.2, 12.8.
HRMS-ESI calcd for C44H40BN3O3P ([M-Br]+), 700.2900; found, 700.2411. Melting point
195-197 °C. Elemental analysis calcd (%) for C44H40BBrN3O3P: C 67.71, H 5.17, N 5.38, found
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(%): C 67.81, H 5.20, N 5.36.
2.2 Fluorescence Quantum Yield. The fluorescence quantum yields (QYs) were quantified using
Fluorescein as the standard (Φf = 0.89, in 0.1 M NaOH).[30] The QYs can be calculated through
adopting the following equation:
Φs = Φr(Arns2Fs)/(Asnr2Fr)
(1)
where the subscripts s and r denote the sample and the standard (Fluorescein), respectively, Φ is
the quantum yield, F is the integrated emission intensity, A is the absorbance, and n is the
refractive index.
2.3 Cell Culture and Imaging. Hela cells, kind gift of the Faculty of Health Sciences (University
of Macau), were cultured and propagated in serum-containing RPMI 1640 medium supplemented
with 10% (v/v) fetal bovine serum (FBS), 1% penicillin – streptomycin solution with incubation in
a humidified atmosphere containing 5% CO2 at 37
according to the manufacturer’s
specifications. It was seeded at a density of 1× 104 cells/cm2. One or two days prior to imaging,
the cells were passaged and plated in phenol red-free medium Confocal Dish, and allowed to grow
to 50-70% confluence and washed with cell culture medium twice. To achieve nuclear staining,
cells were incubated with 1 µM Hoechst at 37
for 10 min and washed with pre-warmed cell
culture medium solution. For the Lyso-BS and LysoTracker Red (LysoTracker® Red DND-99)
co-staining, a solution of LysoTracker Red (100-150 nM) and Lyso-BS (1 µM) in 1 mL of medium
and 0.05 mL of DMSO was added to pre-washed cells and incubated at 37 for less than 1 min.
For the Mito-BS and MitoTracker Red (MitoTracker® Red CMXRos), the optimum working
concentration of Mito-BS and MitoTracker red are 3 µM and 250 nM respectively. The cell
imaging was obtained on a Carl Zeiss Confocal LSM710 microscope. To alleviate the energy
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transfer artifacts, two lasers were used, the 488-nanometer argon-ion laser line was employed to
excite Lyso-BS and Mito-BS, whereas LysoTracker Red and MitoTracker Red were excited with
the 543-nanometer helium neon laser line. The blue channel filter was as follows: excitation at 365
nm, beam splitter FT at 395 nm, and emission at 445/50 nm. The green channel filter was as
follows: excitation at 450−490 nm, beam splitter FT at 510 nm, and emission at 515−565 nm. The
red channel filter was as follows: excitation at 587/25 nm, beam splitter FT at 605 nm, and
emission at 647/70 nm.
We chose two lases for multiple labeling experiments for alleviating the energy transfer artifacts,
the Lyso-BS was excited at 488 nm using argon laser, whereas LysoTracker® Red was excited at
543 nm using HeNe laser. Filter sets, including bandpass barrier, emission filter and beam splitter
were also used for diminishing bleed-through. In addition, it should be noted that multitrack line
scanning is an excellent method to generate very nice images.
2.4 Cytotoxicity Assay. The cytotoxicity of Lyso-BS and Mito-BS against Hela cells was studied
using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell-viability assay.
HeLa cells were seeded into a 24-well plate at a density of 7 × 103 cells in each well, maintained
overnight in RPMI-1640 medium containing 10% FBS at 37
with a 5% CO2 humidified
atmosphere. And then, the cells were treated with various concentration of Lyso-BS, Mito-BS,
LysoTracker red or MitoTracker red (0, 2, 5, 10 µM) for 24 h. After pouring out the medium, 100
µL of freshly prepared MTT (5 mg/mL in PBS，Beyotime Biotechnology) was added to each well
and incubated for 4 h. After removing the MTT medium solution, the cells were lysed by adding
100 µL of DMSO. The plate was gently shaken for 5 min, and then the absorbance of purple
formazan at 570 nm was monitored using a PerkinElmer Victor X3 Microplate Reader.
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3. Results and discussion
3.1 Synthesis
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Ligand was synthesized from the reaction of 4-diethylaminosalicylaldehyde (SA) with
hydrazine, produced from phenylglyoxylic acid.[29] Hydrazone solution eluted
through a column packed with Amberlyst@ 15 prior to dropping into methanolic
solution of SA for cation exchange. Solid product was filtered from
methanolic-aqueous solution and given in high purity and moderate yield. To
demonstrate the advantages of modular chromophore approach, LB and MB were
synthesized from BPBA using a facile nucleophilic substitution reaction of boronated
alkyl bromide precursors with triphenylphosphine or morpholine (Scheme 2). With
three derivatives of phenylboronic acids in hands, it assembled with ligand in CH3CN
and afforded NO2 - type boron complexes, in which the dative bond B-N furnished an
extra molecular stability.[31, 32]
3.2 Optical properties
With the aim of developing wash-free fluorogenic dyes in mind, the optical properties
of Lip-BS, Lyso-BS and Mito-BS were first tested in 20 mM HEPES solution,
revealing λmax at 400 nm for all of three dyes (Fig. 1a-c). Gratifyingly, any fluorescent
signals weren’t detected in HEPES solution (Fig. 1d-f), which is essential prerequisite
to wash-free dyes. In polar protic solvent CH3OH, the adsorption spectra of all three
showed a dual band adsorption trait, where the high-energy band from 310 nm to 430
nm originated from π-π* transition of local aromatic chromophore (red parts in
Scheme 1), and the low-energy band in the range of 430 – 540 nm attributed to the
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ICT absorption between the donor (diethylamino group) and the acceptor
(oxadiazaborinine ring), manifesting that the ICT effect of BS dyes evanesced
completely in aqueous solution and partially reinstated in CH3OH.[33] The
fluorescence peek also appeared at λmax 540 nm albeit with low QY (< 1%, in line
with literature[29]), measured out using fluorescein as reference. In polar aprotic
solvent DMSO and THF, the absorption peak at 400 nm entirely disappeared and only
one peak at 470 nm was in existence, which presented that energy absorption of BS
completely changed from local chromophore absorption to π-π* transition of ICT
state.[34] The QYs of Lip-BS, Lyso-BS and Mito-BS were 7.8 %, 11.8% and 7.8% in
DMSO, whereas significantly enhanced to 37.6%, 46.5% and 26.0% in THF
respectively. In addition, the fluorescence emission maxima were located at 550 nm in
DMSO and 520 nm in THF. The bathochromic shift of the emission, discerned from
the polar DMSO (ET (30) = 45), further supports partial involvement of ICT in the
excited state, as has been suggested recently by quantum–chemical calculations of
BSAHY dyes.[35] These make a viewpoint become more and more apparent, which is
hydrogen bonds between protic solvent and carbonyl group open π-π* transition of
local aromatic chromophore and close the ICT emission channel. Thereafter, the
optical properties of Lip-BS, Lyso-BS and Mito-BS were tested in apolar solevent,
toluene and CHCl3, in which the Uv-vis absorption spectra were similar as in THF as
shown in Fig. 1. The blue shift of emission maxima was 5 nm in toluene as compared
with in CHCl3, indicating the less influence of solvent polarity as in apolar solvent.
The QYs were 40%, 65% and 36% in toluene, and 62%, 68% and 46% in CHCl3 for
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Lip-BS, Lyso-BS and Mito-BS respectively. Together our results and the observations
on recent reports,[28, 29] it demonstrates that the electronic properties of the boronic
acid in BSs have only minor influence on the wavelength of emission, whereas
influence on the QYs to a certain extent.
3.3 Cytotoxicity Tests
A comparative study on cytotoxicities of BSs with commercial tracker was conducted
prior to its application for cell imaging through MTT array to assess the tolerance of
BSs to their working concentrations. Hela cells as biological models were incubated
with 0, 2, 5, or 10 µM Lyso-BS, Mito-BS, and LysoTracker Red, MitoTracker red for
24 h (Fig. 2). The results indicate Lyso-BS and Mito-BS were shown to exhibit low
cytotoxicity, reaching high levels of cell viability (>95 %), even incubation with high
concentration (10 µM). On the contrary, commercial MitoTracker Red shown higher
cytotoxicity to Hela cells, only about 25% of which were viable at 10 µM of working
concentration, 60% of cell viable for LysoTracker.[36] These results indicate Lyso-BS
and Mito-BS have additional advantages in biocompatibility, inheriting it from
boronic acids which were used in medicinal chemistry and chemical biology
widely.[37]
3.4 Intracellular localization
To certify that attachment of targeting group to BSs endowed BSs with staining of
specific organelles, HeLa cells were incubated with Lyso-BS and LysoTracker red
together, or Mito-BS and MitoTracker red. Hoechst 33342 was employed for nucleus
staining,[38] in order to confirm cell viability throughout the experiment. It has been
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proved that the Lip-BS have intrinsic tendency of staining lipid droplets, but its
wash-free feature hasn’t been emphasized.[29] In our experiments, the nucleus
staining was arranged prior to other organelles staining for the convenient observation
of wash-free staining process. The optimum working concentration of LysoTracker
red was in the range of 100 nM-150 nM in our experiments. Higher working
concentration of LysoTracker red could cause artifacts from overloading. For avoiding
“alkalizing effect”[39] on the lysosomes and facilitating direct observation, the
incubated HeLa cells were quickly scanned under microscopy after incubation with
LysoTracker red and Lyso-BS (1 µM) for 1 min. As shown on Fig. 3, punctuated
staining patterns were snapped in both green channel and red channel, which match
very well as merged image shown (Fig. 3e and Fig. S1, ESI†). It’s well-known that
the morpholine group with a lower pKa value can target lysosomes that contain a lot
of acid hydrolases in the pH range from 4.5 to 5.5.[40] The hydrophobic pockets of
proteins in lysosomes could provide accommodations for Lyso-BS, thus lighting it up.
To estimate the degree of colocalization of Lyso-BS and LysoTracker red, a
quantitative analysis of confocal images was performed. The quantification of the
degree of colocalization between two channels were achieved through calculation of
Pearson’s correlation coefficient (POC), which was high up to 0.93 for Lyso-BS and
LysoTracker. In the beginning stage of apoptosis, lysosomes were considered as
“suicide bags”[41] to participate in cell death pathways. It was found that the
LysoTracker red spread through the whole cell with Hoechst 33342, whereas the
Lyso-BS were detained in lysosomes (Fig. S2). It’s demonstrated that introducing
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morpholine group into BSs dye dramatically alter the final subcellular fate of BSs
from lipid droplets to lysosomes and that Lyso-BS might have higher affinity with
lysosomes than commercial LysoTracker.
Subsequently, the subcellular fate of Mito-BS was looked into through colocalization
of Mito-BS with commercial MitoTracker red. TPP+ cations were substantially
utilized to deliver the probes, antioxidants, and pharmacophores to mitochondria.[27]
The negative membrane potential of the mitochondrial inner membrane enables
Mito-BS with TPP+ cations to accumulate in the mitochondrial matrix against their
concentration gradient. The hydrophobic membrane environment might trigger the
light-up of Mito-BS. Since the dyes was relatively non-fluorescent in an aqueous
environment, the observed fluorescence was assumed to arise from its interaction with
cellular lipids or hydrophobic pockets of proteins. Unlike the isolated punctuate
lysosomes pattern, mitochondria exist as a dynamic and punctate network in the
perinuclear region, with an overall filamentous morphology and dispersing throughout
the cell (Fig. 4). The experiment revealed that Mito-BS specifically localized
mitochondria with a high Pearson’s coefficient of approximately 0.91 (Fig. 4f). Taken
together, all the results illustrate that BSs are promising candidates for wash-free
imaging subcellular organelles.
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4. Conclusions
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In conclusion, this study bespeaks that BSs have intrinsic wash-free fluorescence property. Polar
protic solvent, especially water, engender the extinction of ICT effect of BSs, giving rise to its
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non-fluorescent characteristic in aqueous solution. Through pre-installation morpholine and TPP+
on phenyboronic acid and then assembly it with salicylidenehydrazone ligand, the resulting
Lyso-BS and Mito-BS was shown to selectively stain lysosomes and mitochondria in Hela cells
respectively, without inducing any appreciable cytotoxicity. All above features make the BSs a
potentially useful fluorophore platform (e.g. handy and multi-assembly synthesizing approach) for
the wider application.
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Acknowledgements
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This work was supported by the Macao Science and Technology Development Fund under Grant
No.: 052/2015/A2, 082/2016/A2; the University of Macau research grants under grant No.:
MYRG2016-00058-FHS, MYRG2017-00066-FHS, and SRG2015-00007-FHS.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at
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References
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Scheme 1 Pre-installation of organelles targeting groups on phenylboronic acid for constructing labels of
specific cellular organelles
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Scheme 2 Synthetic routine of Lip-BS, Lyso-BS and Mito-BS
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Fig. 1 The absorption spectra (a-c) and the fluorescence spectra (d-f) of Lip-BS (a, d), Lyso-BS (b, e) and
Mito-BS (c, f). The corresponding color (g) and fluorescence photograph (h) of Lip (1, 4, 7, 10, 13, 16), Lyso
(2, 5, 8, 11, 14, 17) and Mito (3, 6, 9, 12, 15, 18) in toluene, CHCl3, THF, DMSO, CH3OH and 20 mM
HEPES Buffer solution. Concentration: 5 uM
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Fig. 2 Cytotoxicity experiments with Lyso-BS, Mito-BS, LysoTracker and MitoTracker at various
concentrations in HeLa cells (0, 2, 5, and 10 µM) for 24 h.
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Fig. 3 Intracellular distribution of Lyso-BS (1 µM) compared to LysoTracker Red (100 nM). HeLa cells were
imaged on Carl Zeiss Confocal LSM710 using 40 × oil immersion objective lens. Panel (a) shows image of
differential interference contrast (DIC), each panel (b-e) shows a confocal micrograph obtained at a different
fluorescence channel of Hoechst 33342 (b), Lyso-BS (c), LysoTracker Red (d), overlay of the three fluorescence
images (e) and intensity correlation plot of Lyso-BS (horizontal axis) and LysoTracker red (vertical axis) (f).
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Fig. 4 Intracellular distribution of Mito-BS (3 µM) compared to LysoTracker Red (250 nM). HeLa cells were
imaged on Carl Zeiss Confocal LSM710 using 40 × oil immersion objective lens. Definition of panels (a-f) were
same as Fig. 3.
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Highlights
ò The wash-free feature of boronic acid derived salicylidenehydrazone complexes
(BSs) were reported.
ò Pre-installation of organelles targeting groups on phenylboronic acid for
constructing labels of specific cellular organelles.
ò The targeting groups of specific cellular organelles have dominant influence on
final subcellular fate of BSs