A Fluorescent Probe for Stimulated Emission Depletion Super-Resolution Imaging of Vicinal-Dithiol-Proteins on Mitochondrial Membrane

Article abstract of DOI:10.1021/acs.bioconjchem.8b00128

Realizing the significant roles of vicinal-dithiol proteins (VDPs) in maintaining the cellular redox homeostasis and their implication in many diseases, we synthesized a smart arsenate based fluorescent probe 1 which can preferentially target the mitochon

Full text of DOI:10.1021/acs.bioconjchem.8b00128

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A Fluorescent Probe for STED Super-Resolution Imaging of
Vicinal-Dithiol-Proteins (VDPs) on Mitochondrial Membrane
Zhigang Yang, Dong Hoon Kang, Hoyeon Lee, Jinwoo Shin, wei yan, Bhowmira Rathore, Hye-
Ri Kim, Seo Jin Kim, Hardev Singh, Liwei Liu, Junle Qu, Chulhun Kang, and Jong Seung Kim
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00128 • Publication Date (Web): 23 Mar 2018
Downloaded from http://pubs.acs.org on March 25, 2018
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Bioconjugate Chemistry
A Fluorescent Probe for STED SuperꢀResolution Imaging of Vicinalꢀ
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DithiolꢀProteins (VDPs) on Mitochondrial Membrane
†
,‡,§
¶,§
#,§
‡,§
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†
Zhigang Yang,
Dong Hoon Kang, Hoyeon Lee, Jinwoo Shin, Wei Yan, Bhowmira Rathore,
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HyeꢀRi Kim, Seo Jin Kim, Hardev Singh, Liwei Liu, Junle Qu, * Chulhun Kang, * and Jong Seung
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Kim *
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Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong
Province, College of Optoelectronic Engineering, Shenzhen University, Shenzhen, Guangdong 518060,
China.
‡
Department of Chemistry, Korea University, Seoul 02841, Korea.
¶
Asan Medical Center, College of Medicine, University of Ulsan, Seoul 138ꢀ736, Korea.
#
The School of EastꢀWest Medical Science, Kyung Hee University, Yongin, Gyeonggiꢀdo 17104, Korea.
§
These authors contributed equally to this work.
Eꢀmail: jlqu@szu.edu.cn (J. Qu); kangch@khu.ac.kr (C. Kang); jongskim@korea.ac.kr (J. S. Kim)
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Abstract
Realizing the significant roles of vicinalꢀdithiol proteins (VDPs) in maintaining the cellular redox
homeostasis and their implication in many diseases, we synthesized a smart arsenate based fluorescent
probe 1 which can preferentially target the mitochondrial membraneꢀbound vicinal dithiol proteins
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(VDPs), especially voltageꢀdependent anion channel (VDAC2). The probe targetability was
demonstrated by ‘‘in vitro’’ studies such as coꢀlocalization, stimulated emission depletion (STED) superꢀ
resolution imaging, proteomic MS/MS analysis and by western blot analysis. The probe represents a
rare example of fluorescence labeling of mitochondrial membraneꢀbound VDPs and can provide a new
way to construct VDPsꢀspecific fluorescent probes to gain deeper understanding of their roles in
mitochondrialꢀrelated disorders.
Introduction
Vicinal dithiol proteins (VDPs) are proteins where two cysteine residues are proximal in their primary
structures, for example, CXXC, and the residues may act as molecular switch depending on the cellular
redox potential through formation of the corresponding disulfide bonds. The VDPs have received
significant attention because of its fundamental roles in various biological processes including protein
folding and stabilization of its native structure, maintaining cellular redox homeostasis and regulation of
1ꢀ5
mitochondrial functions. Moreover, VDPs are also recognized to be implicated in many human
diseases such as cancer, diabetes, platelet function, human immunodeficiency virus type 1 (HIVꢀ1),
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neurodegeneration and so forth.
Mitochondria have abundant VDPs, which are estimated as 5–15% of the total protein thiols and are
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presumed to play a significant role in mitochondrial antioxidant defences and in redox signalling.
Taking advantages of arsenicals such as 1, 3, 2ꢀdithiarsenolane to selectively bind to the vicinal dithiols
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14ꢀ15
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in proteins,
a arsenicalꢀbased fluorescent probe was developed to label VDPs in situ in live cells.
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Interestingly, the probe seems to prefer the mitochondrial VDPs even without any mitochondrial
targeting unit such as a triphenylphosphonium group which may be attributed to relatively high VDP
contents.
Among the mitochondrial VDPs, the membrane VDPs deserve much attention based on the following
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two considerations: i) the significant portion of mitochondrial VDPs is found on membrane, and ii) the
onꢀoff switching of permeability transition pore complex in mitochondrial membrane is susceptible to
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alter redox environments and further to trigger cell apoptosis.
Likely, mitochondrial membraneꢀ
bound VDPs may be involved in pathogenic events in the organelle. In this regard, development of a
fluorescent probe targeting mitochondrial membraneꢀbound VDP's would provide valuable information
for understanding the role of mitochondrial redox balance in various mitochondriaꢀrelated human
diseases, which has not been attempted so far.
Accordingly, we successfully synthesized a fluorescent probe 1 in which a BODIPY based
fluorophore, on one end, is conjugated to triphenylphosphonium group (a typical mitochondrial
targeting unit) via an alkyl spacer which provides suitable lipophilic character for membrane
localization, and on other end it was conjugated to phenylarsenicate (a recognition site for VDPs)
particularly for imaging and labelling the VDP's on mitochondrial membranes.
Results and discussion
22
The BODIPY probe 1 was prepared according to the protocols reported in our previous work with
some modifications. The complete synthetic scheme and the synthetic details are described in Scheme 1.
Briefly, 4ꢀhydroxyphenylꢀtetramethyl BODIPY (6) was obtained from 4ꢀhydroxybenzaldehyde (7) in
20% yield. Then, alkylation of 6 with 1,6ꢀdibormohexane to produce 5, followed by VilsmeierꢀHacck
reaction resulted in the formation of aldehyde derivative 4. Compound 3 was obtained by refluxing
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mixture of 4 and triphenylphosphine in toluene for 12 hours and finally condensed with arsenical 8 and
nonꢀarsenical derivative through a baseꢀcatalysed reaction to yield target compound 1 and reference
compound 2 in moderate yields, respectively.
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Scheme 1. Synthetic route of target compound 1 and reference compound 2.
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As shown in Figure 1, probe 1 in methanol exhibits absorption and emission maxima at 513 and 534
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nm, respectively. The spectra for reference compound 2 are almost identical to those of 1, indicating that
the organoarsenic moiety does not affect the BODIPY’s spectral behaviours (Figure S1). In addition, the
emission of 1 was insensitive to solvent polarity as it displayed negligible shift in its emission maxima
in different solvents (Figure S2). Moreover, as suggested by confocal fluorescence images, cells
incubated with probe 1 displayed intense green fluorescence intensity and it was anticipated that
fluorescence signals intensified as the time of incubation increases. These results collectively indicate
that probe 1 showed efficient labelling properties towards VDP's and fluorescence intensity is directly
proportional to degree of VDP's modification by organoarsenic moiety of probe 1.
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Chemicalprobes
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fluorescence (red in b) and excitation (blue in b) spectra of 1 (1.0 ꢁM) in methanol; The emission
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spectrum was taken upon excitation at 534 nm and the excitation spectrum was obtained with emission
at 514 nm.
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In order to demonstrate whether 1 can selectively react with vicinal dithiol motifs of proteins, the
reduced and oxidized form of bovine serum albumin (rBSA and oBSA) were prepared by treating it with
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,4ꢀdithiothreitol (DTT) and hydrogen peroxide (H O ), respectively. Then, BSAs were allowed to
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react with probe 1 and their products were analyzed using SDSꢀPAGE. The gel electrophoretic profiles
of the reaction mixtures of BSAs with probe 1 are shown in Fig. 2a, where the protein bands were
visualized using CBB G250 staining or by following the probe 1 emission. As shown in Fig. 2a,
although the similar intensities on the CBBꢀstained image were observed for both BSAs, single
fluorescent band was found only on the rBSAꢀloaded lane. Considering that a strong detergent (SDS)
was applied to the experimental condition for the gel electrophoresis, the emission on the gel is not due
to any nonꢀcovalent complex between the probe and proteins, but due to covalent bonding of probe 1
with rBSA, presumably through its reaction with rBSA’s vicinal dithiol groups.
To verify that probe 1 can covalently modify the cellular VDPs in live cells, HeLa cells were
incubated with 1 or 2 for 1 hour and the images were recorded using confocal laser fluorescence
microscopy (Figures 2b, S3, S4). The confocal images of both 1 and 2ꢀtreated HeLa cells showed strong
green signals after 1 hꢀincubation as shown in left panel of Figure 2b. However, after a sequential
washing steps with PBS/50% ethanol (1:1 v/v)/formaldehyde solution, only the 1ꢀtreated cells displayed
persistent fluorescence intensity (Figures 2b and S3), while the intensity from 2ꢀtreated cells was
dramatically reduced, attributing to the nonꢀcovalent interaction between 2 and the cell matrix. This
suggests that the fluorescence of 1ꢀtreated cells is mostly aided by covalent labelling of the cellular
macromolecules, presumably the vicinal dithiol proteins. Quantization of the fluorescence intensities
(Figures 2c, S3) further validates this conclusion. Further, we also investigated the effect of
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phenylarsinoxide (PAO), a wellꢀknown modifier of VDPs on the 1ꢀlavelling of the cells. The intensity
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of the confocal images of 1ꢀtreated cells in the presence of PAO displayed gradually decrease in a doseꢀ
dependent manner (Figure S4), implying that the modification by 1 is directed to VDPs.
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Figure 2. SDSꢀPAGE of rBSA and oBSA after reaction with 1 and fluorescent labelling of 1 and 2 (1.0
M) for 1 hour (37 °C, 5% CO ); Fluorescent and CBB G250 stained images were taken from a SDSꢀ
ꢁ
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PAGE gel; (a) Protein size markers are shown on M lane. The amount of BSAs on the panel (a) was 1
ꢁg per lane and the fluorescent images were obtained using a fluorescence scanner (Typhoon FLA 9400,
GE), excited at 488 nm, collected at 500ꢀ540 nm; (b) Fluorescent images of HeLa cells were obtained
before and after 1×PBS washing followed by ethanol (1:1 ratio) and three rounds of 4% formaldehyde
solution; (c) Histogram of relative fluorescence intensity (R. F. I.) per cell was represented using image
J program. Results represent the mean (± SEM) of three independent experiments (n = 3). The statistical
signification was marked as *** for p < 0.001, compared with each of the fluorescence intensity before
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washing.
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Identification of the intracellular location of 1 would be a reasonable step for further investigation of
mitochondrial VDPs which was performed with fluorescence coꢀlocalization experiments using various
organelle markers (i.e., Mitoꢀ, Lysoꢀ, and ERꢀtrackers). As seen in Figure 3, the fluorescent image of 1ꢀ
treated cell is well overlapped with that of Mitoꢀtracker as compared to Lysoꢀ and ERꢀtrackers with
Pearson’s correlation coefficient values are 0.89, 0.41, and 0.51 for the Mitoꢀ, Lysoꢀ, and ERꢀtrackers
respectively. The preferential mitochondrial localization is attributed to its triphenylphosphonium group,
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a wellꢀknown mitochondrion guiding unit due to the positivelyꢀcharged bulky hydrophobic moiety.
Taken together, these results demonstrated that 1 is localized into the mitochondria and covalently
modify the macroꢀmolecules, most likely the reduced forms of its vicinal dithiol proteins.
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Figure 3. Coꢀlocalization images of 1 (1.0 ꢁM) in HeLa cells coꢀtreated with organelle staining dye
MitoꢀTracker Red (0.1 ꢁM) or LysoꢀTracker Red (0.1 mM) or ERꢀTracker Red) (0.2 ꢁM)]. Scale bar
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Further, to evaluate whether the fluorescent labelling by 1 is sensitive to oxidative stress of
mitochondria, confocal fluorescence images were recorded in the presence of oligomeric Aβ1ꢀ42, a wellꢀ
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known mitochondrion damaging agent.
As shown in Figure 4, the Aβ1ꢀ42 treatment induced the
dramatically increased ROS production in mitochondria, which were visualized by increased red signals
of MitoSOX, a mitochondrial superoxide indicator. This result indicates that the Aβ1ꢀ42 treatment indeed
provokes ROS generation in mitochondria to the cells. The subsequent addition of 1 to the oligomeric
Aβ1ꢀ42ꢀtreated cells showed significantly reduced fluorescence intensity as compared to that without
Aβ1ꢀ42 treatment. However, it is interesting that under similar conditions the change in fluorescent
intensity of MitoꢀTracker was negligible (Figure S5), indicating that the cells maintain the mitochondria
membrane potentials yet. Nonetheless, the dramatic reduction of 1’s emission may be attributed to the
increased oxidized forms of mitochondrial VDPs upon Aβ1ꢀ42 treatment; thereby implicates that probe 1
is highly sensitive to the reduced state of mitochondrial VDPs.
(a)
(b)
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Figure 4. Confocal microscopic images of HeLa cells coꢀlabelled with oligomeric Aβ1ꢀ42 (a
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mitochondrial damaging agent) and MitoSOX (red emissive mitochondrial ROS indicator) as function
of incubation time. Cells were seeded at 35 mm confocal dish. Cells were pretreated with oligomeric
Aβ1ꢀ42 (10.0 ꢁM) for 12 h in incubator (37 °C, 5% CO ). After each time of incubation, cells were coꢀ
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labeled with MitoSOX (2.0 ꢁM) in 1×PBS for 20 min at 37 °C in dark. The fluorescent confocal images
were collected using an excitation wavelength of 488 nm and a longꢀpath 530 nm emission filter and
compared with oligomeric Aβ1ꢀ42 untreated control cells. Scale bar indicate 50 ꢁm. Histogram of relative
fluorescence intensity (R. F. I.) per cell was represented using image J program. Results represent the
mean (± SEM) of five independent experiments (n = 5). The statistical signification was marked as ***
for p < 0.001.
In order to further confirm the exact localization of 1 in mitochondria, we used time gated STED
(stimulated emission depletion) super resolution imaging microscope to observe the fine structure of
mitochondria using fluorescent imaging. First, it is significant to test STED depletion ability of 1 in live
cells. HeLa cells were incubated with 1 (2 ꢁM) for 30 minutes and then upon excitation at 514 nm (1%
of excitation Laser power), different STED laser powers at 592 nm were used to test the stimulated
depletion effect. It was demonstrated that 10% of STED laser power is enough to acquire marked
depletion in fluorescence imaging (Figure S6).
The STED super resolution imaging showed much clear cellular imaging with enhanced imaging
resolution of ca. 170 and 84 nm at 10% and 40% of STED Laser power, respectively (Figures S7 and
S8). As shown in Figure 5, compared to the confocal imaging (FWHM > 250 nm in Figure 5A, 5E
Black line), STED CW laser at 10% of laser power provided high resolution image (Figure 5B). After a
series of image optimization steps as shown in Figure 5C, a fine fluorescence images of mitochondria
with ca. 90 nm resolution was obtained (Figure 5D). The image shows tubular appearance with spotted
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Bioconjugate Chemistry
labelling which is anticipated for modification of clustered proteins on membrane rather than
mitochondria matrix proteins which would appear as continuous features. Likewise, this result suggests
1
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9
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1
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that 1 can specifically labelled the VDPs localizing on the mitochondrial membrane in HeLa cells.
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Figure 5. (A) Confocal fluorescence of selected mitochondria; (B) STED super resolution image of
mitochondria recorded with 10% energy power of STED laser; (CꢀD) STED super resolution images
recorded with 10% (C) and 40% (D) of STED laser power at 592 nm after switching on the time gate to
eliminate the background fluorescence with lifetime less than 0.5 ns; (E) Line traces through the single
mitochondrial segment show the markedly improved resolving power of STED; excitation wavelength
was 514 nm with 1% of Laser power and depleted Laser wavelength was 592 nm with 10% (B, C) and
40% (D) of energy power of CW Laser source. These optical properties were measured on a Leica SP8
fluorescent imaging system.
To identify the 1ꢀmodified proteins in the cells, a proteomic mass spectral (MS) analysis was carried
out for the fluorescently labelled band of SDSꢀPAGE of mitochondrial fractionation. After incubation
with 1, HeLa cells were fractionated into three subcellular compartments (cytosol, mitochondria and
nuclei) according to the protocol provided by the supplier. The purities of the obtained fractions were
verified by SDSꢀPAGEꢀbased Western blot analysis using organelleꢀselective proteins markers (Figure
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S9). The gel for three fractions was subjected to fluorescence imaging and CBBꢀstaining (Figure 6a). As
anticipated from the confocal microscopic study, the fluorescent intensity of the lanes showed strong for
the mitochondria fraction. Moreover, surprisingly, there is a major band on the fluorescence image of
the gel and its intensity was diminished upon oligomeric Aβ1ꢀ42 treatment. This result implies that the
fluorescent image of the cells by 1 is mostly contributed by modification of single VDP in mitochondrial
membrane and the VDP’s redox state seems to be involved in mitotoxicity by Aβ1ꢀ42. Therefore, the
peptides collected from the fluorescent band on SDSꢀPAGE gel were applied to MS/MS analysis. The
analysis suggests 12 protein names as Voltageꢀdependent anion channel (VDAC2) (Figure S9).
Considering the proteins; molecular weight, location, and roles in mitochondria, the best candidate for
the bands in Figure 6 would be VDAC2 where Figure S9 shows any significant change in its total
expression level (Figures 6b, S9). These results suggest that the vicinal thiols in VDAC2 behaves as a
1
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33ꢀ37
molecular sensor for redox environment in mitochondria.
(a)
(b)
Cyto
Mito
Nuc
Mito
Mito
AsꢁTP:
Mr (kDa)
ꢁ
+
ꢁ
+
ꢁ
+
ꢁ
+
AsꢁTP:
ꢁ
+
Mr (kDa)
1
80
40
180
140
1
100
100
IB: VDAC2
7
6
5
0
75
60
4
3
2
5
5
5
45
35
25
VDAC2
CBB
F.L.
Figure 6. Identification of 1ꢀmodified proteins in mitochondrial fraction of HeLa cells; (a) HeLa cells
were incubated with 2 ꢁM of AsꢀTP for 1 hour; three subcellular fractions from HeLa cells were
separated on a 10% SDSꢀPAGE gel and the fluorescence image of gel was taken by Typhoon 9400
variable mode imager with 100 ꢁm resolutions (Amersham Bioscience/GE Healthcare). After
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Bioconjugate Chemistry
fluorescence imaging, the gel was CBBꢀstained and merged with the corresponding fluorescence image
for excision of protein band with the same position of electrophoretic mobility on SDSꢀPAGE gel; Cyto,
cytosol; Mito, mitochondria; Nuc, nuclei; (b) Mitochondrial fractions from HeLa cells were
immunoblotted using an antiꢀVDAC2 antibody. Shown is a representative of three independent
experiments.
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Conclusion
A phenylarsenicateꢀbased fluorescent probe 1 was developed for highly selective and robust staining of
VDPs on mitochondrial membrane. The probe can predominantly bind to vicinal dithiol proteins in
mitochondria by covalent interactions between the arsenicate moiety and the reduced dithiols in proteins.
STED super resolution imaging of live HeLa cells using the phenylarsenicateꢀbased fluorescent probe 1
provided clear fluorescent profiles of mitochondrial membrane. Furthermore, probe 1 was proven to
preferentially target the VDAC2 on the mitochondrial membrane via covalent binding of its arsenicate
moiety to the vicinal dithiols, which was verified by different approaches, e.g. the comparison of SDSꢀ
PAGE of rBSA and oBSA, colocalization imaging, proteomic MS/MS analysis and Western blot
analysis. Releasing the important roles played by mitochondrial VDPs as mentioned above, we believe
that our system will have significant contribution to understand various diseases resulted from the
malfunctions of VDAC2.
Experimental section
Synthesis of 4ꢀaminophenylarsenoxide (10). Compound 10 was prepared according to the literature
procedure. pꢀArsanilic acid (11) (22 g, 100 mmol) was dissolved in methanol (100 mL) and heated to
reflux. Phenylhydrazine (23 mL, 220 mmol) was titrated dropwise to above mixture in 30 minutes.
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o
When N ꢀproduction ceases, refluxing was continued for 1 h. The mixture was condensed at 80 C to
2
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
2
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3
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5
5
5
5
5
5
5
6
exclude the solvent, then washed with water (100 mL) and an aqueous NaOH solution (0.2 M, 60 mL)
and finally with diethyl ether (2×150 mL). The aqueous solution was treated with aqueous NH Cl
4
o
solution (5 M, 80 mL) suspended overnight at 0 C. Precipitates were collected through a Büchner funnel
filter, washed by iceꢀwater (25 mL×2) and dried over KOH, which gave 10 as white powder (6.5 g, 26%
0
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2
3
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7
8
9
0
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8
9
0
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7
8
9
0
yield).
Synthesis of 2ꢀpꢀaminophenylꢀ1,3,2ꢀdithiarsenolane (9) To a solution of 10 (2.6 g, 13 mmol) in dry
ethanol (25 mL), ethanedithiol (1.5 mL, 20 mmol) was added dropwise and heated to reflux for 20
minutes with constant stirring. The solution was chilled in dry ice/acetone, coꢀdistilled with toluene (106
mL) and concentrated. A white crystal was recrystallized from ethanol yielding 1.8 g (34% yield) of 9.
1
H NMR (DMSOꢀd , 400 MHz): δ 3.13ꢀ3.36 (m, 4H), 5.40 (s, NꢀH, 2H), 6.56 (d, 2H, J = 8.8 Hz), 7.27
6
(d, 2H, J = 4.8 Hz) ppm.
Synthesis of compound 8. In 50 ml round bottom flask, 2ꢀcyanoacetic acid (0.86 g, 10 mmol), EDCI (1
g, 7 mmol), DMAP (0.12 g, 1mmol) and DIPEA (0.5 mL) were added in DMF (20 mL) and the mixture
was stirred for few minutes at room temperature. Then, amine 9 (1.3 g, 5 mmol) dissolved in DMF was
added dropwise to above solution and stirring was continue overnight at room temperature. The reaction
mixture was poured into saline solution (70 mL) and extracted with DCM (100 mL), dried over
anhydrous Na SO . After evaporation of the DCM, the product was purified by column chromatography
2
4
1
to provide product 8 (1.3 g, yield 80%). H NMR (DMSOꢀd , 400 MHz): δ 3.11 (t, CH , 2H, J = 6.0 Hz),
6
2
3
.28 (t, CH , 2H, J = 6.0 Hz), 3.84 (s, CH , 2H), 7.50ꢀ7.60 (dd, ArH, 4H, J = 8.0 Hz), 7.89 (s, NH, 1H);
2 2
1
3
C NMR (DMSOꢀd , 100 MHz): δ 31.40, 36.49, 42.09, 49.41, 116.38, 119.63, 132.15, 138.85, 161.81
6
ppm.
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Bioconjugate Chemistry
Synthesis of compound 6. 4ꢀHydroxybenzaldehyde (3.0 g, 24 mmol) and 2, 4ꢀdimethylpyrrole (5 mL)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
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4
4
4
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5
5
5
6
o
were mixed in 10 mL freshly dried DCM followed by addition of 0.1 mL TFA at 0 C under Arꢀ
atmosphere. After stirring the resulting mixture overnight, DDQ (2.8 g) was added and stirred
continuously for one hour more. Finally, BF ·OEt (3 mL) and TEA (6 mL) were added into the reaction
3
2
mixture with stirring for additional 5 hours. Upon the completion of reaction, the raw product was
0
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2
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7
8
9
0
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9
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3
4
5
6
7
8
9
0
separated by silica gel chromatography (hexaneꢀDCM as eluent phase) to give the product of 6 (1.8 g,
2
1%).
Synthesis of compound 5. Compound 6 (1.4 g, 4 mmol), 1, 6ꢀdibromobexane (2.0 g, 8 mmol) and
K CO (0.6 g, 4 mmol) were stirred in 15 mL dry DMF at 50 °C overnight. The product was separated
2
3
1
through silica gel chromatography (DCMꢀmethanol) to give 5 (1.6 g, 80%). H NMR (CDCl , 400
3
MHz): δ 1.38 (s, CH , 6H), 1.48 (m, 4H, CH ), 1.78 (m, 2H, CH ), 1.82 (m, 2H, CH ), 2.49 (s, 6H, CH ),
3
2
2
2
3
3.38 (t, 2H, CH , J = 4.0 Hz), 3.95 (t, 2H, CH , J = 4.0 Hz), 5.92 (s, 6H, CH ), 6.93 (d, 2H, ArH, J = 8.0
2
2
3
1
3
Hz), 7.05 (d, 2H, ArH, J = 8.0 Hz); C NMR (CDCl , 100 MHz): δ 14.76, 14.81, 25.52, 28.17, 29.29,
3
3
2.87, 34.09, 68.02, 115.26, 121.33, 126.89, 129.30, 132.02, 142.22, 143.38, 155.28, 159.83 ppm.
Synthesis of compound 4. POCl (1.54 g, 10 mmol) was dropped into dry DMF (6 mL) at 0 °C, over a
3
period of 30 minutes. Then, 5 (1.6 g, 3 mmol) dissolved in 3 mL dry DMF was added into above
solution, and the obtained mixture was heated at 60 °C for 5 hours. After cooling down to room
temperature, the mixture was poured into iceꢀwater (100 mL), and then neutralized with NaHCO . The
3
crude product was extracted with DCM and purified through silica gel chromatography (DCMꢀmethanol
1
mixture solvent) to give 4 (1.2 g, 75.5%). H NMR (CDCl , 400 MHz): δ 1.49 (s, CH , 3H), 1.55 (m, 4H,
3
3
CH ), 1.72 (s, 3H, CH ), 1.92 (m, 4H, CH ), 2.61 (s, 3H, CH ), 2.82 (s, 3H, CH ), 3.45 (t, 2H, CH , J =
2
3
2
3
3
2
4
.0 Hz), 4.03 (t, 2H, CH , J = 4.0 Hz), 6.15 (s, 1H, CH ), 7.03 (d, 2H, ArH, J = 8.0 Hz), 7.16 (d, 2H,
2
3
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1
3
ArH, J = 8.0 Hz); 10.01 (s, 1H, CHO); C NMR (CDCl , 100 MHz): δ 12.00, 15.34, 25.57, 26.88,
3
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
5
6
2
8.10, 29.25, 32.68, 33.99, 45.23, 68.14, 115.39, 124.07, 126.13, 129.39, 134.72, 143.06, 144.05,
147.54, 148.23, 156.56, 160.22, 161.61, 186.17 ppm.
Synthesis of compound 3. Compound 4 (1.1 g, 2 mmol) was placed in toluene (20 mL) together with
triphenylphosphine (1 g, 3.8 mmol) and the obtained reaction solution was heated at 80 °C for 8 hours.
After cooling down to ambient temperature, mixture was diluted with diethyl ether (150 mL) to
precipitate the crude product which was filteredꢀoff and washed with excess diethyl ether to get pure
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
product 3 (1 g, 70%). H NMR (CDCl , 400 MHz): δ 1.21 (bp, CH , 2H), 1.26 (bp, 2H, CH ), 1.48 (s,
3
2
2
3H, CH ), 1.52ꢀ1.54 (m, 2H, CH ), 1.53 (s, 3H, CH ), 1.69ꢀ1.72 (m, 2H, CH ), 2.60 (s, 3H, CH ), 2.80
3
2
3
2
3
(s, 3H, CH ), 3.80 (t, 2H, CH , J = 6.0 Hz), 4.01 (t, 2H, CH , J = 6.0 Hz), 6.16 (s, 1H, ArH), 7.02 (d, 2H,
3 2 2
ArH, J = 8.0 Hz), 7.13 (d, 2H, ArH, J = 8.0 Hz), 7.72ꢀ7.88 (m, 15 H, ArH, J = 8.0 Hz), 9.98 (s, 1H,
1
3
CHO); C NMR (CDCl , 100 MHz): δ 11.94, 13.27, 15.36, 22.84, 25.95, 27.73, 29.03, 29.84, 30.24,
3
6
1
8.13, 84.97, 115.60, 118.01, 118.86, 124.14, 126.27, 130.41, 130.65, 133.76, 142.94, 147.78, 156.09,
60.23, 161.65, 186.14 ppm.
Synthesis of compound 2. Compound 3 (0.72 g, 1 mmol), 2ꢀcyanoactylanaline (0.32 g, 2 mmol) and
piperazine (0.17 g, 2 mmol) were mixed in 20 mL DMF/methanol solvent (1/1 v/v). The obtained
mixture was stirred at room temperature overnight. Then, the reaction mixture was poured into dry
diethyl ether (200 mL) to form precipitates which were further purified on silica gel column
1
chromatography (DCMꢀmethanol) to obtain final product 2. H NMR (CDCl , 500 MHz): δ 9.98 (s, 1H),
3
8
.00 (s, 1H), 7.84 – 7.76 (m, 15H), 7.70 – 7.66 (m, 10H), 7.12 (d, J = 8.43 Hz, 2H), 7.00 (d, J = 8.55 Hz,
2H), 6.13 (s, 1H), 4.09 (t, J = 6.59 Hz, 2H), 3.97 (t, J = 6.26 Hz, 2H), 2.79 (s, 2H), 2.59 (s, 2H), 2.25 –
2
.21 (m, 2H), 2.02 – 1.96 (m, 2H), 1.79 – 1.46 (m, 20H) ppm.
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Bioconjugate Chemistry
Synthesis of probe 1. Compound 3 (0.72 g, 1 mmol), 8 (0.48, 1.5 mmol) and piperazine (0.17 g, 2
mmol) were mixed in 20 mL DMF/methanol solvent (1/1, v/v). The obtained mixture was stirred at
room temperature for overnight. Then the reaction mixture was poured into dry diethyl ether (200 mL),
to form precipitates which were further purified on silica gel column chromatography (DCMꢀmethanol)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
to obtain final product 1 (0.5 g, 50%). H NMR (CDCl , 400 MHz): δ 1.44 (s, CH , 3H), 1.47 (s, 3H,
3
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
CH ), 1.50 (t, 4H, CH , J = 4.0 Hz), 1.73 (t, 4H, CH , J = 4.0 Hz), 2.56 (s, 3H, CH ), 2.63 (s, 3H, CH ),
3
2
2
3
3
3
1
7
.12ꢀ3.15 (m, 2H, CH ), 3.31ꢀ3.34 (m, 2H, CH ), 3.73ꢀ3.75 (m, 2H, CH ), 3.96 (bp, 2H, CH ), 6.09 (s,
2 2 2 2
H, ArH), 6.96 (d, 2H, ArH, J = 8.0 Hz), 7.10 (d, 2H, ArH, J = 8.0 Hz), 7.55 (d, 2H, ArH, J = 8.0 Hz),
1
3
.67 (m, 8H, ArH), 7.73 (m, 9H, ArH), 7.80 (s, 1H, CH), 7.83 (s, 1H, NH) ppm; C NMR (CDCl , 100
3
MHz): δ 14.87, 15.40, 22.50, 22.88, 25.89, 29.07, 36.71, 42.02, 53.42, 68.12, 115.67, 118.00, 118.92,
20.49, 123.98, 129.21, 130.77, 135.90, 138.57, 140.53, 147.51, 153.43, 160.18, 162.76 ppm;
1
+
+
+
Calculated [M] , C H AsBF N O PS , molecular weight: m/z Fw = 1021.27; Mass spectra ESI ,
5
5
54
2
4
2
2
found peak, 1021.50.
UV/Vis absorption and fluorescence spectroscopy. Stock solutions of 1 (1 mM) was prepared in
DMSO solvent. UV/Vis absorption spectra of 1 were measured using a Perkin 3000 spectrophotometer
(PE, USA), and fluorescent emission were recorded on a Horiba spectrofluorometer (Horiba) equipped
with a xenon lamp, and regular PMT and an In/Ga/As detector for near infrared (window II)
measurement.
Formation of reduced BSA (rBSA) and oxidized BSA (oBSA). rBSA was obtained by treating
solution of BSA with 1.0 mM DTT overnight at 4 °C and oBSA was obtained by treating BSA with 100
ꢁM H O for 10 min at 25 °C. These samples were diluted 50 times with distilled water and then the
2
2
proteins were recovered by precipitation in 50% acetone for 2 h at ꢀ20 °C.
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1
2
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8
9
1
1
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5
6
Formation of oligomeric Aβ1ꢀ42. Peptide (Beckham) with a concentration of 1 mg/mL was obtained by
dissolving in 100% HFIP (1, 1, 1, 3, 3, 3ꢀhexafluoroꢀ2ꢀpropanol) and incubated at room temperature for
3
days with vortex at moderate speed. This was sonicated for 10 min while the tube is held in the
upright position. And then sample was dried under a gentle nitrogen gas stream and vial was capped
immediately. Aβ peptide stock sol. (50 ꢁg) was suspended in 13 ꢁL DMSO and 50 ꢁL of DMEM was
0
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added, mixed and incubated for 2 h (5% CO , 37 °C). This sample has 63 ꢁL total volume and 100 ꢁM
2
βꢀamyloid in oligomeric form.
Cell culture. HeLa cells were cultured at moderate density in DMEM medium. All culturing media
were supplemented with penicillin (100 units/mL), 10% (v/v) FBS (WelGene) and streptomycin (100
mg/mL) under 5% CO atmosphere and 95% humidity at 37 °C.
2
Confocal microscopy. 24 hours before imaging on a microscope, the cells were seeded on cover glass
bottom dish (SPL Lifesciences Co., Ltd.) which was incubated in a humidified atmosphere containing
o
5
% (v/v) CO at 37 C. Cell images were obtained using confocal laser scanning microscopy (Leica SP8,
2
Leica, Germany). Other information is available in the figure captions.
SDSꢀPAGE and fluorescence image of gels. The selectivity of compound 1 towards proteins or cells
was verified by 10% SDSꢀPAGE. Samples were treated with 1 in deꢀionized water (DW) or DMEM at
o
5
% CO and 95% humidity at 37 C for 1 hour. After labeling, the samples were precipitated with 50%
2
o
(
v/v) acetone for 2 hours at ꢀ20 C and then mixed with SDSꢀPAGE loading buffer with tris (2ꢀ
carboxyethyl) phosphine (TCEP) and the electrophoresis was started immediately. The gel was imaged
by a fluorescent scanner (Typhoon FLA 9400, GE) upon 488 nm excitation with a band path filter
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ranging from 500 nm to 540 nm. The same gel was stained via Coomassie brilliant blue (CBB) G250
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after the fluorescent image was obtained.
Mass spectrometry. The trypsin digests (5.0 ꢁL) were prepared in buffer A (H O/acetonitrile/HCO H;
2
2
TM
9
5:5:0.2, v/v/v) and then injected into an analytical column (3.0 ꢁm particle size, Atlantis dC18,
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Waters) equipped with C reversedꢀphase of 75 ꢁm i.d. × 150 mm with an integrated electrospray
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TM
ionization Silica Tip . The peptides were desalted in a line before separation through a trap column
TM
(0.35 × 50 mm, OPTIꢀPAK
C , Waters) and rinsed by a linear gradient of buffer Bof 5ꢀ80%
18
ꢀ1
(
ACN/water/formic acid; 95 : 5 : 0.2, v/v/v) for 2 hours. A split/splitless inlet speed of 200 nL. Min was
set and the capillary voltage (3 kV) was employed to the HPLC mobile phase prior to spraying. The
mass spectrometer was set up to measure scan cycles consisting of one MS scan followed by MS/MS
scans of the most abundant ions in each scan in 10 seconds. The obtained spectra were automatically
analyzed through Proteo Lynx Global Server 3.0.2 (Waters, Manchester, UK) and then matched against
amino acid sequences in human Swiss Prot database (version 57.8). The search parameters were shown
as follows: Dalton tolerance (0.2) for peptide and fragments; digested with trypsin enzyme was up to
one missed cleavage allowed, deꢀamidation (NQ), propionamidation, oxidation (M) or
cabamidomethylation (C) were set as a variable modification. Results were recorded using probabilityꢀ
based Mowse score (Protein score is ꢀ10 × log (p), where p is the probability that the obtained match is a
random event. Protein with scores greater than 20 is counted to be significant (p < 0.05)).
Western blotting. The same volume of subcellular fraction was mixed with buffer solution of 5×
Laemmle and then boiled for 5 min. The obtained mixtures were separated on 10% SDS (SDSꢀPAGE)
and then transferred to nitrocellulose membranes which were incubated with a blocking buffer
composed of 5% nonfat milk or 1% BSA in Trisꢀbuffered saline with 0.5% Tweenꢀ20) for 1 hour with
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shaking at room temperature. The blots were cultured with the primary antibodies in the blocking buffer
solution at 4 °C for overnight. The specific immune reactive proteins were observed by Tanon gel
Imaging/Analysis System (Tanonꢀ5220S, China).
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STED super resolution imaging. All superꢀresolution experiments were achieved on a customꢀbuilt
STED microscope system. For excitation section, a superꢀcontinuum laser power with pulse rate of 80
MHz was applied. The excitation wavelength of 514 nm and its fluorescence intensity was adjusted by
an acoustoꢀoptical tunable filter (AOTF). A continuousꢀwave of 592 nm (MPB Communications Inc.,
Montreal, Canada) as the depletion beam (STED beam) was used on this system. Both laser beams pass
through an optical fiber with polarizationꢀmaintain and were focused into a scanner. The STED beam
was induced through an easy STED phase plate (BꢀHalle, Berlin, Germany) on aback focal plane of
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STED objective (HCX PL APO CS 100×/1.40 oil, Leica Microsystems). The fluorescence of backꢀ
2
propagating sample was collected by the same objective and passed through the detection path by a
direction beam splitter, and then refined by a pinhole with diameter of 151.6 ꢁm (~ 1 Airy unit). The
fluorescence emission ranging from 520 to 560 nm was collected on the hybrid detector. Images were
obtained via timeꢀgated detection of the gate opening upon excitation and the STED laser passing
through. Acquisition of images was generally accomplished with multiple lines and frame
accumulations. Here a typical acquisition set was carried out accumulate four lines and two frames
totally with a pixel dwell time (2.4 ꢁs), the field view of images was set at 26.53 ꢁm × 26.53 ꢁm with
1024 × 1024 pixels and each pixel size was 6 nm.
Acknowledgments
This research was supported by the National Natural Science Foundation of China (No. 21406125),the
National Basic Research Program of China (No. 2015CB352005); Guangdong Natural Science
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Bioconjugate Chemistry
Foundation Innovation Team (No. 2014A030312008); Hong Kong, Macao and Taiwan cooperation
innovation platform & major projects of international cooperation in Colleges and Universities in
Guangdong Province (No. 2015KGJHZ002); and the Ministry of Science, ICT & Future Planning
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(MSIP) of National Research Foundation of Korea (No. 2009ꢀ0081566, JSK), (No.
2
015R1A5A1037656, C.K.) and (No. 2014R1A6A3A04058006, DHK).
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Associated Content
Supporting information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
1
0.1021/acs.bioconjchem.xx
Materials for the synthesis, Supporting absorbance and emission spectra, Supporting confocal
and superꢀresolution cell images, Western blot analysis of organelle fractions and NMR spectra
PDF)
(
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Table of Content
Realizing the significant roles of vicinalꢀdithiol proteins (VDPs) in maintaining the cellular redox
homeostasis and their implication in many diseases, a smart arsenate based fluorescent probe 1 was
designed which can preferentially target the mitochondrial membraneꢀbound VDPs, especially
Voltageꢀdependent anion channel (VDAC2) as demonstrated by ‘‘in vitro’’ studies such as coꢀ
localization, stimulated emission depletion (STED) superꢀresolution imaging, proteomic MS/MS
analysis and by western blot analysis. As far we know, our probe represents a rare example of
fluorescence labelling the mitochondrial membraneꢀbound VDPs. We believe that these findings can
provide a new way to construct VDPsꢀspecific fluorescent probes in order to gain deeper
understanding of their roles in mitochondrialꢀrelated disorders.
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
27
ACS Paragon Plus Environment