BODIPYâCoumarin Conjugate as an Endoplasmic Reticulum

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A BODIPY-coumarin conjugate as an endoplasmic reticulum membrane fluidity sensor and its application to ER stress models Hoyeon Lee, Zhigang Yang, Youngjin Wi, Tae Woo Kim, Peter Verwilst, Yun Hak Lee, Ga-in Han, Chulhun Kang, and Jong Seung Kim Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00508 • Publication Date (Web): 20 Nov 2015 Downloaded from http://pubs.acs.org on November 23, 2015

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Bioconjugate Chemistry

A BODIPY-coumarin conjugate as an endoplasmic reticulum membrane fluidity sensor and its application to ER stress models Hoyeon Lee,†§ Zhigang Yang,‡§ Youngjin Wi,† Tae Woo Kim,† Peter Verwilst,‡ Yun Hak Lee,‡ Ga-in Han,† Chulhun Kang,†* and Jong Seung Kim‡* †

The School of East-West Medical Science, Kyung Hee University, Yongin 446-701, Korea

‡

Department of Chemistry, Korea University, Seoul 136-701, Korea.

§

The authors contributed equally.

E-mail: [email protected] (C. Kang); [email protected] (J.S.Kim) Abstract An endoplasmic reticulum (ER) membrane-selective chemosensor composed of BODIPY and coumarin moieties and a long alkyl chain (n-C18) was synthesized. The emission ratio of BODIPY to coumarin depends on the solution viscosity. The probe is localized to the ER membrane and was applied to reveal the reduced ER membrane fluidity under ER stress conditions. Introduction Membrane fluidity is an important factor in many metabolic pathways, e.g. the electron transfer chain reactions in mitochondrial membranes,1, 2 endoplasmic reticulum (ER) protein trafficking,3-8 cell signaling via the plasma membrane,9-11 and drug delivery.12 Its modulation is largely controlled by the lipid composition in the membrane system. In particular, the extent of saturation of fatty acids is a risk factor in metabolic syndrome where the saturated fatty acids cause ER stress involving the unfolded protein response,13-19 increase in pro-apoptotic C/EBP homologous protein expression,14 disruption of ER homeostasis, and even cell death17. Whereas the supplementation of saturated fatty acids in the culture media induced a high degree of incorporation into the cellular phospholipid pool,20-22 their effect on ER membrane fluidity is rarely accessed due to the lack of ER membrane-selective fluidity chemosensors. Polarity- and viscosity-responsive chemosensors are preferentially adopted for investigation of biomembrane fluidity. Nile red is a well-known membrane polarity sensor which has been used for investigations of the cholesterol content of membranes,23 as well as enabling the discrimination of the types of lipids, e.g. oleic acid vs. cholesterol.24 Recently, new bimodal fluorescent molecular rotors composed of two different fluorophores have been introduced to measure viscosity changes in damaged mitochondria25 and ER viscosity reduction, possibly of the ER membrane, following

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tunicamycin treatment.26 Relative to single fluorophore-based sensors, these bimodal sensors have a clear advantage in that the ratio of two fluorophore’s emissions depends solely on the environment where the probe is located at, independent of the probe’s concentration.27,28 However, whether the probes are located at the lumens or the membranes of the organelles were unclear, thus the locations corresponding to the observed viscosity changes are hitherto uncertain. In this context, an ER membrane-selective viscosity biosensor (1) is herein introduced, composed of BODIPY and coumarin moieties to report the viscosity as the ratio of their fluorescent emissions, with a long alkyl chain (n-C18) as an ER membrane guiding group.29-31 The chemical structures used in this study are shown in Scheme 1. Here biosensor (1) is applied to monitor alterations in the ER membrane fluidity in HeLa cells by the supplementation of a saturated fatty acid (palmitic acid, PA), tunicamycin, or brefeldin A. Results and discussion With the aim of analyzing ER membrane fluidity, fluorescent sensor 1 and the reference molecules 2–4 were synthesized (Scheme 1). The preparation of compounds 3, 4 and related intermediates have been outlined in our previous work and we direct the reader to that work for detailed synthetic procedures for these reference molecules.25, 32-34 5 was used to synthesize target compounds through a reaction with phosgene and the corresponding amines to yield target compounds 1 and 2 (Scheme 1). All 1H NMR, 13C NMR, and mass analysis data for the new probe molecule and its intermediates can be found in the supporting information. (Figures S6 – S10). OH

H N

OH i)

ii)

NH 2

O

O N

O

O

9

8

3 iii)

O

O

iv)

H N

O

N F BN+ F

O Br

7

CHO

6

4

v) H N

O

N H

10 H N

H N

n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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NH

O O

O

vi)

N

O

O

N N

5

+B

F

N

F

1 n = 16 2 n=1


BN+

F

F

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Scheme 1. Synthetic route for target compounds and structures of the reference compounds, i) ethyl iodide/K2CO3, DMF, 60°C, 2 h, 75%; ii) ethylacetoacetate/ZnCl2, ethanol, reflux, 12 h, 77 %; iii) NBS, DMF, r.t., 41%; iv) 4-formylphenylboronic acid/Pd(PPh3)4/K2CO3/DME, reflux, 12 h, 36 %; v) pyrrole/TFA, DCM, r.t., 1 h, then DDQ, DCM, r.t., 1 h, then BF3-Et2O, Et3N, DCM, r.t., 1 h, 10 %; vi) phosgene/DIPEA, DCM, r.t., 1 h, then n-octadecylamine/n-propylamine, DCM, r.t., 6 h, 11 % (for 1).

To study the spectral properties of 1, absorption and emission spectra were measured in methylene chloride. As shown in Fig. 1A, Probe 1 showed two absorption maxima (λabs: 350 and 500 nm) and two emission maxima (λem: 439 and 518 nm). The emissions attribute mainly to those of its coumarin and BODIPY moieties, respectively. Indeed, the excitation spectrum of 1 at 439 nm emission mostly overlaps with the absorption spectrum of 3 (Fig. 1B). Although the peak at 500 nm in the excitation spectrum at 518 nm emission overlaps with the absorption spectrum of 4, the peak around 350 nm is slightly red-shifted compared to the corresponding absorption maximum of 4 (Fig. 1C), possibly due to a partial contribution by the coumarin moiety due to an energy transfer process through the bond between the two fluorophores.

Figure 1. Spectroscopic properties of 1, (A) Normalized absorption and emission spectra of 1 in methylene chloride. (B) Normalized excitation of 1 at 439 nm and absorption spectrum of 3. (C) Normalized excitation spectrum of 1 at 520 nm and absorption spectrum of 4. All compounds were used at a concentration of 1.0 µM, in methylene chloride.

To investigate how the emission ratio of the coumarin and BODIPY moieties from 1 respond to changes in local viscosity and polarity, the fluorescent spectra were recorded in mixed solvents using methanol-glycerol and methanol-1,4-dioxane systems, respectively. As shown in Fig. 2A, with the gradual increase in glycerol concentration from 0 to 70%, the emissions at 439 and 518 nm were enhanced by 5-and 7-fold, respectively. The plot of the fluorescence intensity ratio of 1 (I518/I439) vs. solution viscosity (η) revealed a globally positive correlation (Fig. 2B). The plot of log ratio (I518/I439) versus log η (the inset in Fig. 2B) is roughly fitted by a straight line with a slope of 0.50±0.10, which agreement with the literature data.35 By contrast, the ratio was independent of solvent polarity (ɛ; methanol-1,4-dioxane in Fig. 2D). Therefore, we concluded that compound 1 was sensitive to the viscosity of its environment, and that the effect of the polarity on the ratio was relatively small.



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Figure 2. Fluorescence spectra depend on viscosity and polarity. Changes in the fluorescent spectra (A) of 1 (1.0 µM) were obtained by varying the solution viscosity using different glycerol concentrations (% [v/v]) in a methanol-glycerol system. (B) The relationship between the I518/I439 ratio and solution viscosity. (C) Fluorescence spectra of 1 (1.0 µM) were measured across various solution polarities by altering 1,4-dioxane concentration (% [w/w]) in a methanol-1,4-dioxane system. (D) Fluorescence intensity ratios for BODIPY (I518)/coumarin (I439) were determined in solvents of varying polarity. An excitation wavelength of 350 nm (slit 5/5) was used for all spectra.

Prior to the application of compound 1 in biological systems, its fluorescence was measured in PBS and was compared to that in methanol (Fig S1). Whereas a strong emission with double maxima (439 and 518 nm) was observed in methanol upon excitation at 350 nm, in the case of the aqueous solution however, the emission disappeared with a weak and broad emission at ca. 600 nm, an observation probably caused by the self-aggregation of 1 in PBS (Fig. S1A). When the compound was diluted down to 0.125 µM, the emissions at 439 and 518 nm still remained quenched (Fig. S1B). However, compound 2, which has a shorter alkyl chain, shows strong emission in methanol as well as in PBS (Fig. S1C). These results indicate that compound 1 has a strong emission only in the organic phase and is virtually insoluble in aqueous environments, resulting in a dramatically quenched emission through self-aggregation caused by its long alkyl chain (n-C18). Self-aggregation as the main source of fluorescence quenching was further confirmed by studying the fluorescent changes upon the addition of increasing amounts of SDS, an ionic surfactant, to an aqueous solution of 1 (Fig. S2). When no SDS was added, virtually no fluorescence can be observed. At 1.25% of SDS, a marked peak at ca. 560 nm was observed, attributed to an aggregation emission peak. With the successive addition of SDS, the peak gradually disappeared, restoring a fluorescence spectrum exhibiting all the features also observed in methanolic solutions, observations fully consistent with the self-aggregation of 1 in aqueous solutions. Thereby, taking advantages of compound 1‘s organic-phase selective emission, it was reasonably anticipated that the biosensor would act as a membrane selective probe a cellular environment.


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The biological application of compound 1 requires an efficient cellular delivery system as direct supplementation the self-aggregated insoluble probe to the cell culture would otherwise resulting in poor reproducibility and low cellular uptake. Cyclodextrins (CDs) were selected as a possible delivery system since they have been used to disperse hydrophobic drugs in aqueous solutions and to facilitate the drugs’ import into cells.36,37 As shown in Fig 3A, when β-CD was incubated with 1 in PBS, a sharp emission peak at 518 nm with a weak peak around 450 nm upon excitation at 350 nm was observed. As the disaggregation of the probe by CDs in aqueous environments is evidenced by the recovery of the BODIPY fluorescence, the subsequent experiments were performed with direct excitation of the BODIPY fluorophore at 488 nm. When α-, β-, methyl β-, or γ-CDs were incubated with 1 in PBS, only β-CD showed a sharp emission peak at 518 nm and furthermore, upon gradually increasing the β-CD concentration to 4 mM (Fig. 3C) the fluorescence intensity of 1 at 518 nm dramatically increased. Moreover, the cell viability of HeLa cells was not influenced by the presence of 1 (1.0 µM) and β-CD (4.0 mM; Fig. S3). Thus, β-CD would be used as a carrier for the probe’s intracellular delivery system with low toxicity in aqueous media.

Figure 3. Assessing the fluorescence of the CD/sensor complex. Fluorescent spectra of 1 (1.0 µM) were obtained (A) with or without β-CD (4 mM), (B) with various CDs (4 mM), and (C) with various concentrations of β-CD. All samples were tested after 24 h incubation in 1× PBS. Excitation wavelengths were (A) 350 nm (slit 5/5), and both (B) and (C) 488 nm (slit 3/5).

To further substantiate the capacity of β-CD to improve probe delivery into living cells, confocal laser microscopy experiments were performed using HeLa cells. As shown in Fig. 4A, in the presence of β-CD, a brighter fluorescent signal was detected compared to that in the absence of a solubilizing agent, which confirms β-CD’s usefulness as a delivery agent for hydrophobic insoluble probes to cells. The virtual absence of fluorescent signals before the addition of β-CD (the top left panel of figure 4A) demonstrates that no significant level of autofluorescence, a potential source of interference, was observed. Also, the intracellular localization of compound 1 was further determined by its colocalization with Mito-, ER-, and Lyso-Trackers (Fig. 4B). The merged images showed an overlap of the fluorescence from 1 with that of ER-tracker, indicating that the probe locates in the ER. However, the short alkyl tailed analogue (2) is not selectively localized in any of the organelles (Fig S5). Since 1 emits little in aqueous environment due to its insolubility and shows strong fluorescence only in non-aqueous environments, the intense images in the figures are hypothesised to be



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contributed by the probe molecules residing at non-aqueous regions of the ER, most likely the ER membrane. Taken together, these data suggest that the dye molecule is able to dynamically enter and exit the β-CD cavity, as this type of interaction operates under equilibrium conditions. When the complex of β-CD and BODIPY enters cells, the BODIPY will reside inside the cavity of β-CD in the aqueous cytosol, while it will transfer into the membrane system when the complex approaches intracellular membranes, due to the more hydrophobic nature of the membrane and the long tailed aliphatic anchor of the probe.

Figure 4. (A) Fluorescence of 1 in confocal laser microscopic images depends on β-cyclodextrin concentration. Compound 1 (1.0 µM) was incubated with or without β-CD (4 mM) for 24 h at 4 °C, before being applied to HeLa cells. After 37 °C incubation, confocal images were obtained using 488 nm excitation and a 505 nm longpass emission filter. The scale bars indicate 20 µm. (B) Confocal laser microscopic images of HeLa cells treated with 1 and ER-Tracker Red, MitoTracker Red, or LysoTracker Red. Fluorescence imaging of 1 (1.0 µM) used excitation at 488 nm. Long-path filters of 560, 650, and 585 nm, and excitation wavelengths of 543, 633, and 543 nm were used to obtain fluorescence images of ER-Tracker Red (at 0.2 µM), MitoTracker Red (at 0.1 µM), and LysoTracker Red (at 0.1 µM), respectively. Scale bars indicate 10 µm.

To examine whether compound 1 can monitor biologically relevant changes in the ER membrane fluidity, fluorescence emissions from BODIPY and coumarin moieties of 1 were measured in conjunction with a saturated fatty acid treatment. As shown in Fig. 5, the BODIPY emission largely increased relative to that of the coumarin subunit upon treatment with fatty acids. The emission increase is more profound in the case of the saturated fatty acid (palmitic acid) than that in the case of the unsaturated fatty acid (oleic acid). Taking the results in Fig 2 into account, Fig 5 indicates that the ER membrane fluidity was reduced. Considering the established cytotoxicity of saturated fatty acids,13 this prompts the question whether the reduced ER fluidity might be related with ER stress. Therefore the ER membrane fluidity was characterized by the same method in the presence of other ER stress inducers, namely tunicamycin and brefeldin A.38-41Again, the BODIPY’s fluorescence intensity was increased by both chemicals (Fig. S4). Taken together, these results suggest that the ER membrane fluidity is reduced by ER stress. Considering that ER stress is implicated in many diseases, e.g. Alzheimer’s disease and diabetes42-45 and involves malfunction of cellular protein trafficking,46 our


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findings regarding probe 1 indicate that this biosensor could represent a valuable tool for the investigation of the ER membrane’s role in ER stress.

Figure 5. A) Confocal microscopy images of HeLa cells showing fatty acid-induced ER stress. Cells were incubated with fatty acid-depleted BSA (0.36 mM), palmitic acid (PA; 0.7 mM), or oleic acid (OA; 0.7 mM) for 24 h at 37 °C and then treated with 1 (1.0 µM) and β-CD (4.0 mM) complex for 1 h. Before being added to cells, the complex was incubated for 24 h at room temperature. The upper images were obtained with a 510–600 nm band-pass filter using one-photon excitation at 488 nm, while the lower images were taken with a 400–490 nm band-pass filter using two-photon excitation at 740 nm. Data are represented as means ± SD (n = 4). ***P < 0.001 compared to the control. Scale bars indicate 20 µm. (B) Confocal laser microscopic images of HeLa cells treated with 1 and ER-Tracker Red, MitoTracker Red, or LysoTracker Red. Fluorescence images were obtained using the same conditions given in Fig. 5B, and in the context of ER stress induced by palmitic acid (PA, 0.7 mM). Scale bars indicate 10 µm.

Conclusions In conclusion, an ER membrane-selective biosensor (1), composed of BODIPY and coumarin moieties and a long alkyl chain (n-C18), was synthesized. The emission ratio of two comprising fluorophores (BODIPY/coumarin) depends on the solvent viscosity with a minor interference from the polarity. The sensor mainly localized to the ER membrane in HeLa cells with the aid of β-CD. Our assessment reveals that a reduction of ER membrane fluidity resulted from various ER stress inducers, such as palmitic acid, tunicamycin or brefeldin A. Experimental Procedures



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General methods 1

H and

13

C NMR spectra were collected in CDCl3 (Cambridge Isotope Laboratories, Cambridge,

MA) on a Varian 400 MHz spectrometer. All chemical shifts are reported in ppm value using the peak of TMS as an internal reference. ESI mass spectrometric analyses were carried out using an LC/MS2020 Series (Shimadzu) instrument. Materials and reagents All solvents and reagents used were reagent grade. Silica (230-400 mesh; Merck, Darmstadt, Germany) was used for flash column chromatography for purifications. Water used in all experiments was doubly purified by Milli-Q Systems equipment. Human cervical cancer cell line (HeLa) was purchased from American Type Culture Collection (ATCC) (VA, USA). All reagents for cell culture, ER-Tracker™ Red (BODIPY® TR Glibenclamide), MitoTracker® Deep Red FM, LysoTracker® Red DND-99 were purchased from Invitrogen, Co. (Oregon, USA). Bovine serum albumin (BSA, fraction V, fatty acid free) was purchased from Roche, Ltd. (IN, USA). Palmitic acid, oleic acid, tunicamycin, brefeldin A, thiazolyl blue tetrazolium bromide and other common chemicals were purchased from Sigma-Aldrich, Ltd. (MO, USA). α-, β-, and γ-cyclodextrins, and methyl βcyclodextrin were purchased from Tokyo Chemical Industry Co, Ltd. (JAPAN). Sodium Dodecyl Sulfate (SDS) was purchased from USB, Co. (USA). Glycerin was purchased from DUKSAN Pure Chemical Co, Ltd (KOREA). Methylene chloride, methyl alcohol and 1,4-dioxane were purchased from SAMCHUN Pure Chemical Co, Ltd (KOREA). Procedure for the synthesis of compound 1 3-(ethylamino)phenol

(9)

and

7-(ethylamino)-4-methyl-2H-chromen-2-one

(8):

3-

(ethylamino)phenol (9) and 7-(ethylamino)-4-methyl-2H-chromen-2-one (8) were synthesized following a literature procedure.47 3-bromo-7-(ethylamino)-4-methyl-2H-chromen-2-one (7): To solution of 7-(ethylamino)-4-methyl2H-chromen-2-one (6 g, 30.7 mmol) in DMF (80 mL), NBS (5.47 g, 30.7 mmol) was added slowly. The mixture was stirred during overnight at room temperature. After pouring into brine, and washing, the mixture was extracted with EA. The organic extracts were dried with Na2SO4 and concentrated by rotary evaporation. Purification of solid residue by column chromatography (silica gel; DCM/Ethyl acetate, 20:1, v/v) gave 7 as yellow powder (3.4 g, 41 %). 1H-NMR (400 MHz, CDCl3): 1.30 (t, 3H, CH3, J = 8 Hz), 2.53 (s, 3H, CH3), 3.21 (m, 2H, CH2), 4.25 (s, 1H, NH), 6.42 (d, 1H, ArH, J = 4 Hz),


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6.51 (d, 1H, ArH, J = 8 Hz), 7.38 (d, 1H, ArH, J = 8 Hz). 13C-NMR (100 MHz, CDCl3): 14.60, 19.41, 38.25, 97.70, 106.62, 110.43, 111.08, 126.17, 151.77, 151.84, 154.61, 158.17. 4-(7-(ethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)benzaldehyde (6): A flask with 3-bromo-7(ethylamino)-4-methyl-2H-chromen-2-one (3.4 g, 12.1 mmol), 4-formylphenylboronic acid (2.71 g, 18.15 mmol) and Pd(PPh3)4 (0.70 g, 0.605 mmol) was flushed with nitrogen. After 30 min 200 mL dimethoxymethane and K2CO3 (5.06 g, 36.3 mmol) in 17 mL distilled water were added. The mixture was stirred at 100 °C for overnight. The solvent was removed in vacuo and purified with column chromatography (silica gel; DCM/Ethyl acetate, 20:1, v/v) to give 1.34 g 6 (yield: 36 %) as a yellow solid. 1H-NMR (400 MHz, CDCl3): 1.32 (t, 3H, CH3, J = 8 Hz), 2.25 (s, 3H, CH3), 3.25 (m, 2H, CH2), 4.20 (s, 1H, NH), 6.53 (m, 2H, ArH), 7.47 (m, 3H, ArH), 7.96 (d, 2H, ArH, J = 8 Hz), 10.06 (s, 1H, ArH).

13

C-NMR (100 MHz, CDCl3): 14.47, 16.49, 38.09, 97.68, 110.53, 110.63, 120.37, 126.19,

131.41, 133.51, 141.84, 149.01, 151.57, 155.25, 161.34. 10-(4-(7-(ethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl)-5,5-difluoro-5H-dipyrrolo[1,2c:1',2'-f][1,3,2]diazaborinin-4-ium-5-uide (5): Pyrrol (582 mg, 8.73 mmol) and 4-(7-(ethylamino)-4methyl-2-oxo-2H-chromen-3-yl)benzaldehyde (1.34 g, 4.37 mmol) were dissolved in 70 mL absolute CH2Cl2 under nitrogen atmosphere. One drop of TFA was added and the solution stirred at room temperature until TLC-control showed the complete consumption of the aldehyde. At this point, a solution of 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ, 1.19 g, 9.02 mmol) in 25 mL absolute CH2Cl2 was added, stirring was continued for 1 h followed by the addition of 15 mL of Et3N and 15mL of BF3-Et2O respectively. After stirring for 2 h the reaction mixture was washed with water, dried over Na2SO4 and evaporated to dryness. The residue was purified with column chromatography (silica gel; Hexane/Ethyl acetate, 1:2, v/v) to give 204 mg 5 (yield: 10 %) as a red solid. 1H-NMR (400 MHz, CDCl3): 1.33 (t, 3H, CH3, J = 8 Hz), 2.32 (s, 3H, CH3), 3.26 (m, 2H, CH2), 4.20 (s, 1H, NH), 6.54 (m, 4H, ArH), 7.06 (d, 2H, ArH, J = 4 Hz), 7.49 (t, 3H, ArH, J = 8 Hz), 7.65 (d, 2H, ArH, J = 8 Hz), 7.96 (s, 2H, ArH). 13C-NMR (100 MHz, CDCl3): 14.68, 16.88, 38.31, 97.88, 110.85, 110.87, 118.74, 120.52, 126.43, 130.66, 131.04, 131.97, 133.25, 135.09, 138.38, 144.29, 147.29, 149.27, 151.76, 155.43, 161.81. 10-(4-(7-(1-ethyl-3-octadecylureido)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl)-5,5-difluoro-5Hdipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide

(1):

10-(4-(7-(ethylamino)-4-methyl-2-

oxo-2H-chromen-3-yl)phenyl)-5,5-difluoro-5H-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5uide (100 mg, 0.21 mmol) was dissolved in 40 mL of DCM, phosgene (0.21 mL, 0.42 mmol) was added slowly at ice bath, after stirring for 1 h at room temperature and the solvent was evaporated,



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then added 50 mL of DCM, DIPEA (0.1 mL, 0.63 mmol) and octadecan-1-amine (113 mg, 0.42 mmol) in DCM (25 mL) were added slowly at ice bath, then removed the ice bath, after stirring at room temperature for 6 h, removed the solvent and DIPEA, the residue was purified with column chromatography (silica gel; Hexane/Ethyl acetate, 1:2, v/v) to give 18 mg 1 (yield: 11 %) as a red solid. 1H-NMR (400 MHz, CDCl3): 0.86 (m, 3H, CH3), 1.13 (m, 3H, CH3), 1.25 (m, 30H, CH2), 1.44 (m, 2H, CH2), 2.43 (s, 3H, CH3), 3.21 (m, 2H, CH2), 3.82 (q, 2H, CH2, J = 4 Hz), 4.38 (t, 1H, NH, J = 4 Hz), 6.59 (d, 2H, ArH, J = 4 Hz), 7.03 (d, 2H, ArH, J = 4 Hz), 7.27 (m, 2H, ArH), 7.50 (m, 2H, ArH), 7.69 (m, 2H, ArH), 7.76 (d, 1H, ArH, J = 8 Hz), 7.97 (s, 2H, ArH).

13

C-NMR (100 MHz,

CDCl3): 14.23, 14.35, 17.12, 21.13, 22.91, 27.13, 29.51, 29.57, 29.77, 29.82, 29.91, 30.35, 32.13, 41.22, 44.45, 115.63, 118.90, 118.94, 123.97, 126.20, 126.74, 130.61, 130.78, 131.88, 133.99, 135.04, 137.05, 144.57, 145.81, 146.75, 147.95, 153.71, 156.26, 160.49. MS (ESI): [M] = C46H59BF2N4O3. Cationic mode: [M+H]+ Calculated: 765.47, Found: 765.45; Anionic mode: [M-H]- Calculated: 763.46, Found: 763.35. Solution studies UV/Vis and Fluorescence Spectroscopy. Stock solutions of biosensor 1, 2, 3 and 4 were prepared in DMSO. Absorption spectra were recorded on a V-560 UV/VIS Spectrophotometer (JASCO, Japan), and fluorescence spectra were recorded using an RF-5301 PC spectrofluorometer (Shimadzu) equipped with a xenon lamp. Other information including excitation wavelength is available in the figure captions. Methods for biological evaluation Cell culture and fatty acid treatment. HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM). All media were supplemented with 10% (v/v) FBS (WelGene), penicillin (100 units/mL), and streptomycin (100 μg /ml) under 5% (v/v) CO2 and 95% (v/v) humidity at 37 ℃. The stock solutions of fatty acids were prepared at 100 mM in DMSO. They were diluted with DMEM and the diluted solutions were sonicated for 1 h. Then, the sonicated solutions were mixed with BSA at 2:1 molar ratio in all experiments. Finally, to obtain fatty acid conjugated BSA, the mixtures were constantly stirred for 1 h on a horizontal shaker prior to its addition into the cells. Measurement of cell viability (%). Cell viability assay was performed based on measuring absorption of formazan crystals from 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), formed by intracellular oxidoreductases in the cells. HeLa cells were seeded at 1 × 104


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cells/well in 96-well plates. After proper treatment for the experiments, 10 µl MTT solution (0.5 mg/ml, the final concentration was added to each well and incubated for 2 h at 37 ℃ to allow the cells forming formazan crystals. Subsequently, the remaining solution was removed to leave the cells on the wells and 100 µl DMSO was added into each well to dissolve the crystals. The absorbance of the dissolved crystals from the intact cells was measured at 570 nm using a VICTOR™ X3 Multilabel Plate Reader (Perkin Elmer Inc, USA). To calculate the percentage of dead cells, use negative control of 100% lived cells. Cells were treated nothing and cultured using DMEM with 10% FBS before addition of the MTT.

Cell viability % =

× 100 !

The average of the absorbance values for the negative control was used as a blank value and divided into all other absorbance values and multiplied by 100 to yield the cell viability (%). Confocal microscopy imaging. One day before imaging, the HeLa cells were seeded on Coverglass Bottom Dish (SPL Lifesciences Co., Ltd.) which was incubated in a humidified atmosphere containing 5% (v/v) CO2 at 37 ℃ . Cell images were obtained using confocal laser scanning microscopy (Zeiss LSM 510, Zeiss, Oberko, Germany) and multiphoton microscopy (Leica TCS SP8 MP, Leica, Germany). Other information is available in the figure captions. Acknowledgments This work was supported by NRF (No. 2014R1A2A1A11052325, CK) and CRI (No. 20090081566, JSK) project grants from the National Research Foundation of Korea and a Korea University Grant (PV). References

1.

Chen, J. J. and Yu, B. P. (1994) Alterations in mitochondrial membrane fluidity by lipid peroxidation products. Free Radic. Biol. Med. 17, 411-418.

2.

Colell, A., García-Ruiz, C., Lluis, J.M., Coll, O., Mari, M. and Fernández-Checa, J. C. (2003) Cholesterol impairs the adenine nucleotide translocator-mediated mitochondrial permeability transition through altered membrane fluidity. J. Biol. Chem. 278, 33928-33935.

3.

Oppenheimer, N. and Diamant, H. (2009) Correlated diffusion of membrane proteins and their effect on membrane viscosity. Biophys. J. 96, 3041-3049.

4.

Tortorella, D., Story, C. M., Huppa, J. B., Wiertz, E. J., Jones, T. R., Bacik, I., Bennink, J. R., Yewdell, J. W. and Ploegh, H. L. (1998) Dislocation of Type I Membrane Proteins from the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ER to the Cytosol Is Sensitive to Changes in Redox Potential. J. Cell Biol. 142, 365-376.

5.

Huttner, W. B.and Zimmerberg, J. (2001) Implications of lipid microdomains for membrane curvature, budding and fission. Curr. Opin. Cell Biol. 13, 478-484.

6.

Huijbregts, R.P., Topalof, L. and Bankaitis, V. A. (2000) Lipid metabolism and regulation of membrane trafficking. Traffic 1, 195-202.

7.

Bonifacino, J. S. and Glick, B. S. (2004) The mechanisms of vesicle budding and fusion. Cell 116, 153-166.

8.

Schroeder, F., Gallegos, A. M., Atshaves, B. P., Storey, S. M., McIntosh, A. L., Petrescu, A. D., Huang, H., Starodub, O., Chao, H., Yang, H., et al. (2001) Recent advances in membrane microdomains: rafts, caveolae, and intracellular cholesterol trafficking. Exp. Biol. Med. 226, 873-890.

9.

Deliconstantinos, G., Villiotou, V. and Stavrides. J. C. (1995) Modulation of particulate nitric oxide synthase activity and peroxynitrite synthesis in cholesterol enriched endothelial cell membranes. Biochem. Pharmacol. 49, 1589-1600.

10.

Laude, A. J. and Prior, I. A. (2004) Plasma membrane microdomains: organization, function and trafficking. Mol. Membr. Biol. 21, 193-205.

11.

Goodwin, J. S., Drake, K. R., Remmert, C. L. and Kenworthy, A. K. (2005) Ras Diffusion Is Sensitive to Plasma Membrane Viscosity. Biophys. J. 89, 1398-1410.

12.

Goldstein, D. B. (1984) The effects of drugs on membrane fluidity. Annu. Rev. Pharmacol. 24, 43-64.

13.

Artwohl, M., Lindenmair, A., Roden, M., Waldhäusl, W. K., Freudenthaler, A., Klosner, G., Ilhan, A., Luger, A. and Baumgartner-Parzer, S. M. (2009) Fatty acids induce apoptosis in human smooth muscle cells depending on chain length, saturation, and duration of exposure. Atherosclerosis 202, 351-362.

14.

Pfaffenbach, K. T., Gentile, C. L., Nivala, A. M., Wang, D., Wei, Y. and Pagliassotti, M. J. (2010) Linking endoplasmic reticulum stress to cell death in hepatocytes: roles of C/EBP homologous protein and chemical chaperones in palmitate-mediated cell death. Am. J. Physiol. Endocrinol. Metab. 298, E1027-E1035.

15.

Kim, D. S., Jeong, S. K., Kim, H. R., Kim, D. S., Chae, S.W. and Chae, H. J. (2010) Metformin regulates palmitate-induced apoptosis and ER stress response in HepG2 liver cells. Immunopharmacol. Immunotoxicol. 32, 251-7.

16.

Zhang, Y., Xue, R., Zhang, Z., Yang, X. and Shi, H. (2012) Palmitic and linoleic acids induce ER stress and apoptosis in hepatoma cells. Lipids Health Dis. 11, 1.

17.

Wei, Y., Wang, D., Gentile, C. L. and Pagliassotti, M. J. (2009) Reduced endoplasmic reticulum luminal calcium links saturated fatty acid-mediated endoplasmic reticulum stress


Page 12 of 16

Page 13 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and cell death in liver cells. Mol. Cell Biochem. 331, 31-40.

18.

Wei, Y., Wang, D. and Pagliassotti, M. J. (2007) Saturated fatty acid-mediated endoplasmic reticulum stress and apoptosis are augmented by trans-10, cis-12-conjugated linoleic acid in liver cells. Mol Cell. Biochem. 303, 105-113.

19.

Wei, Y., Wang, D., Topczewski, F. and Pagliassotti, M. J. (2006) Saturated fatty acids induce endoplasmic reticulum stress and apoptosis independently of ceramide in liver cells. Am. J. Physiol. Endocrinol. Metab. 291, E275-E281.

20.

Ariyama, H., Kono, N., Matsuda, S., Inoue, T. and Arai, H. (2010) Decrease in membrane phospholipid unsaturation induces unfolded protein response. J. Biol. Chem. 285, 2202722035.

21.

Leamy, A. K., Egnatchik, R. A., Shiota, M., Ivanova, P. T., Myers, D. S., Brown, H. A. and Young, J. D. (2014) Enhanced synthesis of saturated phospholipids is associated with ER stress and lipotoxicity in palmitate treated hepatic cells. J. Lipid. Res. 55, 1478-1488.

22.

Zambo, V., Simon-Szabo, L., Szelenyi, P., Kereszturi, E., Banhegyi, G. and Csala, M. (2013) Lipotoxicity in the liver. World J. Hepatol. 5, 550-557.

23.

Mukherjee, S., Raghuraman, H. and Chattopadhyay, A. (2007) Membrane localization and dynamics of Nile Red: Effect of cholesterol. BBA-Biomembranes 1768, 59-66.

24.

Diaz, G., Melis, M., Batetta, B., Angius, F. and Falchi, A. M. (2008) Hydrophobic characterization of intracellular lipids in situ by Nile Red red/yellow emission ratio. Micron 39, 819-824.

25.

Yang, Z., He, Y., Lee, J. H., Park, N., Suh, M., Chae, W. S., Cao, J., Peng, X., Jung, H., Kang, C. et al. (2013) A self-calibrating bipartite viscosity sensor for mitochondria. J. Am. Chem. Soc. 135, 9181-9185.

26.

Yang, Z., He, Y., Lee, J. H., Chae, W. S., Ren, W. X., Lee, J. H., Kang, C. and Kim, J. S. (2014) A Nile Red/BODIPY-based bimodal probe sensitive to changes in the micropolarity and microviscosity of the endoplasmic reticulum. Chem. Commun. 50, 11672-11675.

27.

Yuan, L., Lin, W., Xie, Y., Chen, B., and Zhu, S., (2012) Single fluorescent probe responds to H2O2, NO, and H2O2/NO with three different sets of fluorescence signals. J. Am. Chem. Soc. 2012, 134, 1305-1315.

28.

Yuan, L., Lin, W., Zheng, K., and Zhu, S., (2013) FRET-based small-molecule fluorescent probes: Rational Design and Bioimaging Applications. Acc. Chem. Res., 46, 1462-1473.

29.

Kadakol, A., Ghosh, S. S., Sappal, B. S., Sharma, G., Chowdhury, J. R. and Chowdhury, N. R. (2000) Genetic lesions of bilirubin uridine-diphosphoglucuronate glucuronosyltransferase (UGT1A1) causing Crigler-Najjar and Gilbert syndromes: correlation of genotype to phenotype. Hum. Mutat. 16, 297-306.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

30.

Handschin, C. and Meyer, U. A. (2003) Induction of drug metabolism: the role of nuclear receptors. Pharmacol. Rev. 55, 649-673.

31.

Zucker, S. D., Goessling, W., Zeidel, M. L. and Gollan, J. L. (1994) Membrane lipid composition and vesicle size modulate bilirubin intermembrane transfer. Evidence for membrane-directed trafficking of bilirubin in the hepatocyte. J. Biol. Chem. 269, 1926219270.

32.

Kim, H. J and Kim, J. S. (2006) BODIPY appended cone-calix[4]arene: selective fluorescence changes upon Ca2+ binding. Tetrahedron Lett. 47, 7051-7055.

33.

Lee, M. H., Lee, S.W., Kim, S. H., Kang, C. and Kim, J. S. (2009) Nanomolar Hg(II) Detection Using Nile Blue Chemodosimeter in Biological Media. Org. Lett. 11, 2101-2104.

34.

Kim, J. H., Kim, H. J., Kim, S. H., Lee, J. H., Do, J. H., Kim, H. J. and Kim, J. S. (2009) Fluorescent coumarinyldithiane as a selective chemodosimeter for mercury(II) ion in aqueous solution. Tetrahedron Lett. 50, 5958-5961.

35.

Kuimova, M. K., Yahioglu, G., Levitt, J. A., Suhling, K. (2008) Molecular rotor measures viscosity of live cells via fluorescence lifetime imaging. J. Am. Chem. Soc. 130, 6672-6673.

36.

Uekama, K., Hirayama, F. and Irie, T. (1998) Cyclodextrin Drug Carrier Systems. Chem. Rev. 98, 2045-76.

37.

Challa, R., Ahuja, A., Ali, J. and Khar, R. K. (2005) Cyclodextrins in drug delivery: an updated review. AAPS Pharm. Sci. Tech. 6, E329-E357.

38.

Klausner, R. D., Donaldson, J. G. and Lippincott-Schwartz, J. (1992) Brefeldin A: insights into the control of membrane traffic and organelle structure. J. Cell Biol. 116, 1071-1080.

39.

Chan, S. W. and Egan, P. A. (2005) Hepatitis C virus envelope proteins regulate CHOP via induction of the unfolded protein response. FASEB J. 19, 1510-1512.

40.

Helms, J. B. and Rothman, J. E. (1992) Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF. Nature 360, 352-354.

41.

Heifetz, A., Keenan, R. W., Elbein, A. D. (1979) Mechanism of action of tunicamycin on the UDP-GlcNAc: dolichyl-phosphate Glc-NAc-1-phosphate transferase. Biochemistry 18, 2186-2192.

42.

Yoshida, H. (2007) ER stress and diseases. FASEB J. 274, 630-658.

43.

Hanzal-Bayer, M. F. and Hancock, J. F. (2007) Lipid rafts and membrane traffic. FEBS Lett. 581, 2098-2104.

44.

Simons, K. and Toomre, D. (2000) Lipid rafts and signal transduction. Nat. Rev. Mol. Cell


Page 14 of 16

Page 15 of 16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Biol. 1, 31-39.

45.

Tian, T., Harding, A., Inder, K., Plowman, S., Parton, R. G. and Hancock, J. F. (2007) Plasma membrane nanoswitches generate high-fidelity Ras signal transduction. Nat. Cell Biol. 9, 905-914.

46.

Preston, A. M., Gurisik, E., Bartley, C., Laybutt, D. R. and Biden, T. J. (2009) Reduced endoplasmic reticulum (ER)-to-Golgi protein trafficking contributes to ER stress in lipotoxic mouse beta cells by promoting protein overload. Diabetologia 52, 2369-2273.

47.

Lin, Q., Huang, Q., Li, C., Bao, C., Liu, Z., Li, F. and Zhu, L. (2010) Anticancer Drug Release from a Mesoporous Silica Based Nanophotocage Regulated by Either a One- or Two-Photon Process. J. Am. Chem. Soc. 132, 10645-10647.



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BODIPYâCoumarin Conjugate as an Endoplasmic Reticulum

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