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Two-photon probes for Golgi apparatus: Detection of Golgi apparatus in live tissue by two-photon microscopy Ji-Woo Choi, Seung Taek Hong, Mun Seok Kim, Kyu Cheol Paik, Man So Han, and Bong Rae Cho Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00607 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019
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Analytical Chemistry
Two-photon probes for Golgi apparatus: Detection of Golgi apparatus in live tissue by two-photon microscopy Ji-Woo Choi†, Seung Taek Hong†, Mun Seok Kim‡, Kyu Cheol Paik‡, Man So Han‡, and Bong Rae Cho*,‡,§ †KU-KIST
Graduate School of Converging Science and Technology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea ‡ Department
of Chemistry, Daejin University, 1007 Hoguk-ro, Pocheon-si, Gyeonggi-do, 11159, Republic of Korea
§ Department
of Chemistry, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Republic of Korea
ABSTRACT: We have developed blue- and yellow-emitting two-photon probes (BGolgi-blue and PGolgi-yellow) derived from 6-(benzo[d]oxazol-2-yl)-2-naphthalylamine and 2,5-bis(benzo[d]oxazol-2-yl)pyrazine derivatives as the fluorophores and trans-Golgi network peptide (SDYQRL) as the Golgi apparatus targeting moiety. HeLa cells labeled with BGolgi-blue and PGolgi-yellow emitted two-photon excited fluorescence at 462 and 560 nm, respectively, with effective two-photon action cross-section values of 1860 and 1600 10-50 cm4s/photon, respectively. The probes can detect the Golgi apparatus in live cells and deep inside live tissue using two-photon microscopy at widely separated wavelength regions with high selectivity and minimum pH interference, as well as being photostable and having low cytotoxicity.
The Golgi apparatus is an intracellular organelle located near the rough endoplasmic reticulum (ER) and nucleus. It receives proteins and lipids synthesized in the ER, packs them into vesicles, and sends them to other organelles for use in the cell or extracellular space for secretion.1,2 The Golgi apparatus can be detected using fluorescence microscopy after transfecting cells with a fluorescent plasmid Golgi marker, such as green fluorescent protein (GFP), or by labeling the cells with antibody fluorophore conjugates.3–5 However, these methods are either timeconsuming or have cytotoxic effects and instability problems. To circumvent these problems, a few small molecule fluorescent probes derived from 7-nitro-2,1,3benzoxadiazol-4-amine (NBD) and boron-dipyrromethene (BODIPY) as the fluorophores and ceramide and transGolgi network (TGN) peptide (SDYQRL) as the targeting moieties have been developed.6,7 However, the ceramide derivative shows modest selectivity because the targeting is based on rather weak hydrophobic interactions between the probes and lipids in the Golgi apparatus. In addition, the utility of the TGN derivative must be confirmed because the compound has, to date, only been used after checking the purity by thin layer chromatography (TLC) without characterizing the chemical structure. Moreover, all these probes are useful for one-photon microscopy (OPM) and not for two-photon microscopy (TPM). TPM, which utilizes the two near infrared photons for the excitation, is a useful tool for the long term imaging of intact tissue with intrinsically localized emission and minimum interference from background fluorescence.8–13 Recently, a few two-photon (TP) probes that can detect specific targets such as cyclooxygenase-2 (COX-2) and zinc ions in the Golgi apparatus have been reported.14–16
However, there have been no reports of TP probes for the organelle itself. Therefore, we have developed blue- and yellow-emitting TP probes for the Golgi apparatus (BGolgiblue and PGolgi-yellow) derived from 6-(benzo[d]oxazol2-yl)-2-naphthalylamine (B) and 2,5-bis(benzo[d]oxazol-2yl)pyrazine (P) derivatives as the fluorophores and SDYQRL as the Golgi apparatus targeting moiety (Figure 1). We have used B and P from our earlier work because our previous probes, BLT-blue and PLT-yellow, showed emission maxima at 451 and 564 nm, respectively, with effective TP action cross-section (eff) values of 2120 and 2110 10-50 cm4s/photon (GM) in HeLa cells,17 respectively.
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Figure 1. Structures and synthesis of BGolgi-blue, and PGolgiyellow. (a) (i) KOH, EtOH, rt. (ii) N-hydroxysuccinimide, 1ethyl-3-(3-dimethylaminopropyl)-carbodiimide·HCl (EDCI), 4-dimethylaminopyridine (DMAP), CH2Cl2, rt. (b) SDYQRL, Et3N, DMSO, rt.
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Figure 2. NMR spectra (700 MHz) of (a) BGolgi-blue, (b) compound 1, and (c) SDYQRL peptide in DMSO-d6. Most of the aliphatic C-H peaks are excluded for simplicity.
We have used B and P from our earlier work because our previous probes, BLT-blue and PLT-yellow, showed emission maxima at 451 and 564 nm, respectively, with effective TP action cross-section (eff) values of 2120 and 2110 10-50 cm4s/photon (GM) in HeLa cells,17 respectively. In addition, we used SDYQRL from the work of Wirtz with the expectation that it will strongly interact with TGN38 in the Golgi membrane, thereby increasing the selectivity for the Golgi apparatus.18 Herein, we report that BGolgi-blue and PGolgi-yellow are efficient TP probes that can selectively detect the Golgi apparatus at widely separated wavelength regions in live tissue using TPM. EXPERIMENTAL SECTION Synthesis of BGolgi-blue and PGolgi-yellow. The synthesis and structural characterization of BGolgi-blue and PGolgi-yellow are described in the Supporting Information. Spectroscopic measurements. Absorption and emission spectra were obtained as reported. The fluorescence quantum yield (Ф) was measured using coumarin 307 as the reference molecule, according to established methods.19 Solubility. The solubility of BGolgi-blue in PBS buffer was determined by a fluorescence method, while that of PGolgi-yellow in PBS buffer was measured by an absorption method, as previously described.17 Measurements of the TP action cross-section (Фδ) and effective TP action cross-section (Фδeff). The Фδ and Фδeff values for BGolgi-blue and PGolgi-yellow were determined using literature methods (Figure 3).17,20 Briefly,
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In addition, we used SDYQRL from the work of Wirtz with the expectation that it will strongly interact with TGN38 in the Golgi membrane, thereby increasing the selectivity for the Golgi apparatus.18 Herein, we report that BGolgi-blue and PGolgi-yellow are efficient TP probes that can selectively detect the Golgi apparatus at widely separated wavelength regions in live tissue using TPM.
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the Фδ values were determined by comparing the TP excited fluorescence (TPEF) intensities of the probes and that of Rhodamine 6G in methanol, whereas the Фδeff values were determined by comparing the TPEF intensities of the probe-labeled cells with that of 5.0 µM of Rhodamine 6G in methanol under the same imaging conditions.17,20 Cell culture. HeLa (human cervical carcinoma cell line) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco's modified eagle medium (DMEM, WelGene Inc., Gyeongsangbuk-do, Korea) supplemented with heat-inactivated 10% fetal bovine serum (FBS, WelGene), penicillin (100 U/mL), and streptomycin (100 µg/mL). The cells were kept under a humidified atmosphere in an incubator containing 5/95 (v/v) of CO2/air at 37 °C. Two days before imaging, the cells were detached and placed on glass-bottomed dishes (MatTek, Ashland, MA, USA). For labeling, the growth medium was removed and replaced with DMEM without FBS. The cells were then incubated with BGolgi-blue (5 µM) or PGolgiyellow (2 µM) for 30 min at 37 °C, prior to imaging. Tissue preparation. Ex vivo brain Slices were prepared from the hippocampus and hypothalamus of a two-weekold rat (SD, Orient Bio Inc., Gyeonggi-do, Korea) in accordance with the approved protocol of the institutional review board of Korea University. Coronal slices were cut into 400-µm-thick slices by using a vibrating-blade microtome and stored in artificial cerebrospinal fluid (ACSF: 138.6 mM NaCl, 3.5 mM KCl, 21 mM NaHCO3, 0.6 mM NaH2PO4, 9.9 mM D-glucose, 1 mM CaCl2, and 3 mM MgCl2). The slices were incubated with BGolgi-blue (5 µM) or PGolgi-yellow (3 µM) in ACSF bubbled with 95% O2 and 5% CO2 for 30 min at 37 °C. Slices were then washed three times with ACSF and transferred to glass-bottomed dishes (MatTek Corp.). The slices were observed under a spectral confocal multiphoton microscope. OPM imaging. OPM images of BTC-labeled cells were obtained in a wavelength range of 600–650 nm upon excitation at 543 nm using confocal microscopy, as previously reported.14
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Analytical Chemistry TPM imaging. TPM images of probe-labeled cells were obtained over 400–450 nm (Ch1, ABI-Nu, BGolgi-blue) and 550–650 nm (Ch2, PGolgi-yellow) wavelength ranges upon excitation at 750 nm using multiphoton microscopy, as described previously.21 Tissue imaging was performed by the same method except that 50–100 sectional TPM images were obtained in 1.5-µm steps at 120–270 µm depths from the brain section surface along the z-direction to minimize the possibility of image errors being introduced by damage to the surface caused during tissue preparation. Cytotoxicity. The cytotoxicity of BGolgi-blue and PGolgi-yellow was measured using CCK-8 kit (Dojindo, Kumamoto, Japan) following the manufacturer’s protocol. Photostability. The photostability was determined under imaging conditions by monitoring the timedependent decrease in TPEF intensity in HeLa cells labeled with BGolgi-blue and PGolgi-yellow, as described previously.20 RESULTS AND DISCUSSION Synthesis. BGolgi-blue and PGolgi-yellow were synthesized by conjugating B-SIM and Pyr-SIM (see Figure 1) with SDYQRL and obtained in 56% and 40% yields, respectively. The 1H NMR spectra of BGolgi-blue and PGolgi-yellow were complex because the aliphatic C-H protons showed similar chemical shifts and multiple splitting patterns (Figures S15 and S17). In addition, it was difficult to obtain 13C NMR spectra because of the poor solubility of the compounds in dimethyl sulfoxide (DMSO). Except for the aliphatic C-H, CO2H, and N-H proton peaks, all other peaks of BGolgi-blue could be assigned (Figures 2a and S2) and were almost the same as the sum of those of the fluorophore and SDYQRL (Figure 2c). A similar result was observed in the 1H NMR spectrum of PGolgi-yellow (Figure S3). Moreover, the highresolution mass spectrometry (HRMS) results of BGolgiblue and PGolgi-yellow showed molecular weight ion peaks at m/e = 1109.5056 (calcd. 1109.5051) and 1294.5731 (calcd. 1294.5739), respectively (Figures S16 and S18). These results confirm that the fluorophores are attached to SDYQRL in a 1:1 ratio. Spectroscopic properties. The spectroscopic data for BGolgi-blue measured in phosphate buffered saline (PBS, pH 7.4) revealed an absorption maximum (λmax) at 357 nm with a molar extinction coefficient (ε) of 14,700 cm-1M-1 and emission maximum (λfl) at 479 nm with a fluorescence quantum yield () of 0.67 (Table 1 and Figure S4). The water solubility of BGolgi-blue, as determined by the fluorescence method,22 was 7.0 µM, which is sufficient to stain the cells (Figure S5). On the other hand, PGolgiyellow showed λmax at 437 nm (ε = 39,470 cm-1M-1) and emitted little fluorescence in PBS buffer (pH 7.4), presumably because of the efficient intramolecular charge transfer (ICT) from the donors at both ends of the fluorophore to the central pyrazine ring.17 Because of the weak fluorescence, the water solubility of PGolgi-yellow was determined by the absorption method. The solubility was 0.2 μM (Figure S5). Despite the poor water solubility,
there was no problem in staining the cells and tissues with PGolgi-yellow, probably because the equilibrium between the undissolved and dissolved probes rapidly shifted toward the latter as the labeling progressed. When the solvent was changed to a more polar one, the λmax remained nearly the same, whereas the λfl increased gradually (Table 1). The larger bathochromic shift in the λfl (53 vs. 21 nm) for PGolgi-yellow than BGolgi-blue, which was noted after the change in the solvent from 1,4-dioxane to EtOH, can be attributed to the greater stabilization of the charge-transfer excited state of the pyrazine moiety that contains a stronger electron-withdrawing group (Table 1). The TPEF spectra of HeLa cells labeled with BGolgi-blue and PGolgi-yellow are shown at λfl of 462 and 560 nm, respectively (Table 1 and Figure 4). The values are very similar to the λfl measured in EtOH and 1,4dioxane/H2O (200/1), indicating that these solvents can adequately represent the polarity of the intra-organelle environments of BGolgi-blue and PGolgi-yellow, respectively, and that PGolgi-yellow is located in a more hydrophobic environment (Table 1). This outcome can be attributed to the longer structure of fluorophore P, which has a diethylene glycol tail. Although the two probes should be located near the Golgi surface because of the favorable interaction between SDYQRL and TGN38, P is more likely to be embedded in the Golgi membrane than B, providing a more hydrophobic environment for PGolgiyellow. Table 1. Spectroscopic properties. Compo und
BGolgi -blue
PGolgi -yellow
a Solvent (𝑬𝑵 𝑻)
λmax /λflb
c
1,4-dioxane (0.164)
356/436
0.85
28
EtOH (0.654)
361/457
0.94
96
PBS buffer (1.00)
357e/479
0.67
109
HeLa Cell
/462f
1860g
1,4-dioxane (0.164)
442/537
0.62
115
1,4-dioxane/H2O (200/1)
442/561
0.14
65
1,4-dioxane/H2O (40/1)
442/576
0.14
55
EtOH (0.654)
444/590
PBS buffer (1.00)
437h/
HeLa Cell
/560f
1600g
δd
aThe
numbers in the parenthesis are normalized empirical parameters of solvent polarity.23 bOne-photon absorption and emission maxima except otherwise noted. cFluorescence quantum yield. dTwo-photon action cross-section in GM 15%. eMolar extinction coefficients () = 14,700 cm-1M-1. fλfl of TPEF spectra. gEffective two-photon action cross-section values (eff) measured in HeLa cells. h = 39,470 cm-1M-1.
Two-photon brightness. TP action cross-sections (max) were determined in the model solvents by the fluorescence method.17,20 BGolgi-blue and PGolgi-yellow showed good TP properties, having max values of 96 and 65 GM, respectively, at 750 nm (Figure 3 and Table 1). The
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nm (Ch2 and PGolgi-yellow), and 600–700 nm (Ch3 and BTC) (Figure 4). The degree to which the emission intensities of the probes contribute to the emission intensities of the other probes in each channel are as follows: PGolgi-yellow contributes to BGolgi-blue and ABINu in Ch1 by 5% and 17%, respectively; BGolgi-blue and ABI-Nu contribute to PGolgi-yellow in Ch2 by 6% and 16%, respectively; BGolgi-blue contributes to BTC in Ch3 by 5%; and BTC contribute to BGolgi-blue in Ch1 by 2%. Therefore, the emission intensities of the BGolgi-blue/BTC, BGolgiblue/PGolgi-yellow, and ABI-Nu/PGolgi-yellow pairs could be independently determined in the probe-labeled cells. 1.2
20 0
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Figure 3. Two-photon excitation spectra of BGolgi-blue in EtOH and PGolgi-yellow in 1,4-dioxane/H2O (200/1).
Cytotoxicity, photostability, and pH dependency. BGolgi-blue and PGolgi-yellow demonstrated additional advantages of low cytotoxicity, as revealed by Cell Counting Kit-8 (CCK-8) assays (Figure S6). Moreover, the TPEF intensity at a given spot on the probe-labeled HeLa cells showed little decay during continuous irradiation with femtosecond pulses for 1 h, indicating high photostability (Figure S7). Further, the TPEF intensities of these probes measured in the universal buffer remained nearly the same at pH 4–10 (Figure S8). These results confirmed that BGolgi-blue and PGolgi-yellow can be applied for the detection of Golgi apparatus using TPM with minimum artifacts arising from cytotoxicity, photostability, and pH. Detection windows. The detection windows were determined using the one- (BODIPY TR ceramide (BTC)) and two-photon (others) excitation spectra of the HeLa cells labeled with ABI-Nu, a TP probe for the nucleus;21 BGolgi-blue; PGolgi-yellow; and BTC, such that the emission bands from the two probes under comparison are well separated and their emission intensities are similar (Figure 4). The detection windows determined as such were 400–450 nm (Ch1, BGolgi-blue, and ABI-Nu), 550–650
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effective TP action cross-section (eff), a measure of the TP brightness of the bright spots in the TPM images, were determined by comparing the TPEF intensities of the probe-labeled cells with that of Rhodamine 6G under identical imaging conditions.17,20 The eff values of BGolgiblue and PGolgi-yellow were 1860 and 1600 GM, respectively, at 750 nm (Table 1). The effective intravesicular concentrations (ceff) of BGolgi-blue and PGolgi-yellow, calculated by the equation ceff = eff/max,20 were 19 and 25 M, respectively. The values were 56 times higher than those used in the staining medium, a result that can be attributed to the more favorable interactions of the probes with the intravesicular environment than that with the staining medium. Thus, the large max values and favorable interaction of the probes with the intravesicular environment seem to be responsible for the bright TPM images. It should be noted that ceff is a measure of the staining ability of the probe in a cell and will vary depending on the cell line.
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Figure 4. Normalized one- (BTC) and two-photon (others) excited fluorescence spectra of HeLa cells labeled with BTC, ABI-Nu, BGolgi-blue, PGolgi-yellow. The wavelengths for oneand two-photon excitation were 543 and 750 nm, respectively.
Detection of Golgi apparatus in live cells by TPM. We then tested whether BGolgi-blue and PGolgi-yellow can detect the Golgi apparatus in cells. For this purpose, HeLa cells were co-labeled with BGolgi-blue and BTC and the emission intensities were collected at Ch1 and Ch3. Because BTC emitted little TPEF upon TP excitation at 750 nm, the TPM and OPM images were obtained one after another. The two images overlapped well, having a Pearson’s co-localization coefficient (A)24 of 0.77 (Figures 5a–c). In addition, the two TPM images collected at Ch1 and Ch2 of the cells co-labeled with BGolgi-blue and PGolgi-yellow showed a nearly perfect overlap, having an A value of 0.95 (Figures 5d–f). This superior overlap between two TPM images compared to that between TPM and OPM images has been reported previously,17,20 and this is attributed to the following two factors: (i) the two TPM images were obtained simultaneously, whereas the OPM and TPM images were obtained one after the other, and, in this period, the organelles can move, and (ii) the TPM images were obtained in each section and the OPM image was obtained over the entire depth of the cells. Here, again, the importance of using two TP probes for co-localization experiments by TPM is clearly demonstrated. Moreover, the TPM images of HeLa cells labeled with BGolgi-blue and PGolgi-yellow showed bright spots only in the Golgi apparatus (Figures 5a, d, e). In addition, the TPM image of HeLa cells co-labeled with PGolgi-yellow and ABI-Nu
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clearly revealed the Golgi apparatus near the nucleus (Figure S9). In contrast, the OPM images of the cells labeled with BTC show blurred regions in the cytosol in addition to the bright spots in the Golgi apparatus, indicating modest selectivity for the Golgi apparatus (Figure 5b). The superior selectivity of the newly developed probes to that of BTC can be attributed to the stronger intermolecular interactions between SDYQRL, which was employed as the targeting moiety, and TGN38 in the Golgi membrane than the hydrophobic interactions between the ceramide moiety in the BTC and lipids in the Golgi apparatus. These results demonstrate the utility of BGolgiblue and PGolgi-yellow as the TP probes for the Golgi apparatus. (a) BGolgi-blue
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A = 0.77
A = 0.95
Figure 5. (a–c) TPM (a) and OPM (b) images of HeLa cells colabeled with BGolgi-blue and BTC and a merged image (c). (d– f) TPM images (d, e) of HeLa cells co-labeled with BGolgi-blue and PGolgi-yellow and a merged image (f). The images were collected at Ch1 (a, d), Ch2 (e), and Ch3 (b) upon excitation at 543 nm (b) and 750 nm (a, d, e), respectively. The A value is the co-localization coefficient of the red with the green domains. Cells shown are representative images from replicate experiments (n = 5). Scale bar: 30 μm.
Dual-color imaging of apoptosis by TPM. We next tested the ability of PGolgi-yellow to monitor apoptosis, the process of programmed cell death.25–27 Upon apoptotic stimulation, the cells undergo blebbing, nuclear fragmentation, and complete disassembly of the Golgi complex.28,29 To visualize apoptosis, the TPM images of HeLa cells co-labeled with PGolgi-yellow and ABI-Nu were monitored after treating the cells with 1 µM doxorubicin (Dox), an anticancer drug that causes DNA damage, to induce apoptosis.30,31 At t = 0, the TPM image collected at Ch1 and Ch3 clearly revealed a morphologically wellstructured Golgi apparatus close to the nucleus. After 8 h, the nucleus was enlarged and the Golgi apparatus began to be abnormally redistributed to the cytoplasm and membrane. Similar changes were continued until the Golgi apparatus was internalized into the nucleus after 16 h, and the cell membrane was partially disrupted after 24 h. Apoptosis was complete after 48 h, as indicated by the fragmented nucleus, disrupted cell membrane, is assembled Golgi apparatus, and dispersed cell debris (Figures 6a–f). Hence, PGolgi-yellow is clearly capable of monitoring the apoptosis.
(a) t = 0
(b) 4 h
(c) 8 h
(d) 16 h
(e) 24 h
(f) 48 h
Figure 6. Dual-color TPM images of HeLa cells co-labeled with ABI-Nu and PGolgi-yellow. (a–f) The images were obtained 048 h after the treatment of the cells with doxorubicin to induce apoptosis. The images were collected at Ch1 (ABI-Nu) and Ch2 (PGolgi-yellow) upon excitation at 750 nm. Scale bar: 30 µm.
Detection of Golgi apparatus in live tissue by TPM. We further evaluated the utility of BGolgi-blue and PGolgiyellow for tissue imaging. For this experiment, a fresh rat hippocampus section obtained from a two-week-old rat was used. The sectional TPM images of the slice labeled with BGolgi-blue obtained at depths of 120–270 µm revealed the distribution of the Golgi apparatus in the CA3 region along the xy plane throughout its entire depth (Figure 7a). Moreover, the images taken at a higher magnification clearly resolved the Golgi apparatus in the same region (Figures 7e–h). In addition, the dual-color TPM image of the tissue co-labeled with ABI-Nu and PGolgi-yellow showed the distribution of the nuclei and (a)
(b)
100 slices
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Analytical Chemistry
(c)
(e)
(f)
(g)
(h)
(d)
Figure 7. (a,b) Sectional TPM images of a rat hippocampal slice labeled with BGolgi-blue at 120–270 μm (a) and 180 μm (b) depths. (c) Enlarged image of the white dotted box in (b). (d) Dual-color TPM image of a rat hippocampal slice co-labeled with ABI-Nu and PGolgi-yellow at 220 μm depth. (e–g) TPM images of the nucleus (e) and Golgi apparatus (f) in the white dotted box in (d) and merged image (g). (h) Enlarged image of the white dotted box in (g). The images were collected at Ch1 (ABI-Nu, BGolgi-blue) and Ch2 (PGolgi-yellow) upon excitation at 750 nm with magnification at 10× (a, b, d), 100× (c, e–g), and 100× with 2× zoom (h). Scale bars: (d) 100 µm and (c, g) 10 µm.
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Golgi apparatus in each section at 170–270 and 220 µm depths, respectively (Figures S10a and 7d). The images collected in Ch1 and Ch2 with a higher magnification showed the nuclei and Golgi apparatus (Figures 7e, f), and the merged image showed the two organelles in the same section (Figure 7g). When the image in Figure 7g was further magnified, the presence of the Golgi apparatus near the nucleus could be clearly visualized (Figure 7h). These results demonstrate the ability of BGolgi-blue and PGolgiyellow to detect Golgi apparatus in live tissues at depths of 120–270 µm using TPM. CONCLUSIONS To conclude, we have developed blue- and yellow-emitting TP probes for the Golgi apparatus that show large effective TP action cross-sections and can detect the Golgi apparatus in live cells and deep inside live tissue by TPM with high selectivity and minimum interference from pH in a biologically relevant pH range, as well as having minimal cytotoxicity and being photostable. These probes will have applications in bio-medical research. Moreover, the TP fluorophore-peptide conjugate strategy employed in this study will provide a useful guideline for the design of organelle specific TP probes and TP tracers for cancer specific antigens.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details for the synthesis of compounds, spectroscopic characterization (Figures S1–S10), 1H and 13C NMR, and HRMS data (Figures S11–20) (PDF)
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected] ORCID Bong Rae Cho: 0000-0001-8560-5399 Author Contributions J.-W.C. and S.T.H. contributed equally to this work. Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (NRF-2018R1A2B6006029).
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BGolgi-blue PGolgi-yellow
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Golgi apparatus Nucleus
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