Development and characterization of BODIPY-derived tracers for fluorescent labeling of the endoplasmic reticulum

Author Statement

Author

Flip de Jong

Role

Writing - Review & Editing, Investigation , Validation, Formal analysis

Funding acquisition, Writing - Review & Editing, Resources

Writing - Review & Editing, Investigation, Formal analysis

Writing - Review & Editing, Investigation, Validation, Formal analysis

Jan Pokorny

Bella Manshian

Brent Daelemans

Johannes Vandaele

Justyna B. Startek

Stefaan Soenen

Funding acquisition, Writing - Review & Editing, Conceptualization,

Resources

Mark Van der Auweraer

Funding acquisition, Writing - Review & Editing, Conceptualization,

Resources

Wim Dehaen

Funding acquisition, Writing - Review & Editing, Resources

Susana Rocha

Supervision, Writing - Review & Editing, Conceptualization, Methodology

Gaston Silveira-Dorta

Project administration, Supervision, Writing - Original Draft,

Conceptualization, Investigation, Methodology, Validation, Formal

analysis

Development and characterization of BODIPY-derived tracers for fluorescent labeling of the endoplasmic

reticulum

a

b

c,d

e

a

f

Flip de Jong, Jan Pokorny, Bella Manshian, Brent Daelemans, Johannes Vandaele, Justyna B. Startek,

c,d

a

e

*a

*e

Stefaan Soenen, Mark Van der Auweraer, Wim Dehaen, Susana Rocha Gaston Silveira-Dorta

a

Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Leuven Chem&Tech, Celestijnenlaan

2

00F, 3001 Leuven, Belgium

b

Department of Organic Chemistry, Faculty of Science, Palacky University in Olomouc, 17, Listopadu 1192/12,

7

71 46 Olomouc, Czech Republic

c

NanoHealth and Optical Imaging Group, Department of Imaging and Pathology, KU Leuven, B3000 Leuven,

Belgium

d

MoSAIC, KU Leuven, B3000 Leuven, Belgium

e

Molecular Design and Synthesis, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven,

Belgium

f

Department of Cellular and Molecular Medicine, Laboratory of Ion Channel Research, KU Leuven, VIB Center for

Brain & Disease Research, Herestraat 49, Campus Gasthuisberg, O&N1 Box 801, 3000 Leuven, Belgium

E-mail: gaston.silveira@kuleuven.be; susana.rocha@kuleuven.be

Abstract. Visualization of the structure of the Endoplasmic Reticulum (ER) in living cells is important for the

understanding of its function. Here we synthesized a library of BODIPY labeled 8 hydroxyquinoline derivatives

and evaluated their spectroscopic properties, cytotoxicity, and intracellular localization. The compounds were

easily obtained in 34 – 80% yield. Based on the spectral properties and low cytotoxicity, we selected the

quinolin-8-yl pentanoate derivative 17 for in vivo labeling of the ER. Fluorescence staining of the ER was

evaluated by comparison with a commercial ER tracker. The molecules here developed are smaller than the

alternatives commercially available while still presenting a high specificity towards the ER.

1

. Introduction

The endoplasmic reticulum (ER) is a crucial organelle involved in the homeostasis, maintenance, and survival of

1-5

mammalian cells. This is the place where essential steps in the synthesis of proteins take place, namely

6

-7

assembling, folding, post-translational protein modifications, e.g. glycosylation and disulfide bond formation,

and protein secretion. Recently, the ER has attracted the attention of the scientific community due to the ER

stress. This process is associated with a dysregulation in the function of the ER, and it is characterized by an

undesirable accumulation of unfolded/misfolded proteins, resulting in the stimulation of an unfolded protein

8-11

response (UPR), which could induce apoptosis. ER stress can be triggered by multiple stimuli including the

formation of reducing/oxidizing species (redox), calcium dysregulation, glucose deprivation, and viral

1

2-15

infection.

Due to the role of ER stress in various pathological conditions such as strokes, heart and

5,16

neurodegenerative diseases, ischemia, diabetes and cancer, developing new tools to detect and visualize the

effect of different stimuli on the ER structure is of vital importance.

1

7

In the last 30 years, fluorescence microscopy has emerged as a powerful tool to specifically visualize cellular

organelles. Currently, there are three general strategies to fluorescently label the ER: (a)

1

8,19

20-22

immunofluorescence,

molecules (SFMs).

(b) genetically encoded fluorescent tags (GEFTs)

and (c) small fluorescent

2

3-30

Immunostaining of the ER is classically done by targeting the ER-associated protein

3

1

disulfide isomerase (PDI) with a specific antibody (e.g. SelectFX® Alexa Fluor® 488). Despite the high specificity

of immunostaining, it requires permeabilization of the cell membrane, which makes this method incompatible

with live cell imaging. Alternatively, GEFTs can be used to stain the ER in live cells. Commonly, transient

21, 31

overexpression of green-fluorescent protein (GFP) fused to calreticulin or to the KDEL target peptide is used.

However, this also presents some disadvantages such as the alteration of some cellular signaling pathways due

21, 28-30

to the large size of the GFP tag.

Among the commercially available SFMs for fluorescent labeling of the ER, a prominent class is formed by

hybrid 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) molecules. Commercially, there are two BODIPY

based molecules available: ER-Tracker™ Green, 1 Figure 1a and ER-Tracker™ Red 2, Figure 1a. In both

31

compounds, the BODIPY moiety is bonded to glibenclamide to target specifically the ER. Glibenclamide targets

+

32

the sulfonylurea receptors and ATP-sensitive K channels expressed in the ER. While these ER-tracker dyes

offer an alternative to visualize the ER in living conditions, we cannot discard the intrinsic biological effect of the

32

glibencalmide. In addition, the variability in the expression of sulfonylurea receptors in some cell lines may

3

1

result in non-ER labeling.

Therefore, continuing efforts to design new non-invasive small ER probes that lack these disadvantages are

warranted. Since only compounds within a particular range of lipophilic and amphiphatic characteristics can be

2

6

23-27

located in the ER, the number of SFMs that can specifically target the ER is limited.

developing new methodologies to obtain fluorescent dyes for detection of metals inside mammalian cells,

we observed that compound 3 (Figure 1a and 1b) was localized in the ER, making it a potential ER-targeting

Recently, while

33-36

2

+ 37

molecule. Although compound 3 was previously reported as a specific ion chelator for Hg , to the best of our

knowledge there are no studies reporting its intracellular localization. Starting from this meso-8-

hydroxyquinolin-BODIPY, we synthesized a small library of eleven analogues and studied their potential

application for fluorescent labeling of the ER. Here we present the synthesis, photophysical properties,

intracellular localization studies and cytotoxicity of eleven compounds that accumulate preferentially in the ER

of mammalian cells.

(a)

(b)

Figure 1. (a) Structure of hybrid commercially available glibenclamide - BODIPY derivatives 1 and 2 and 3. (b)

Intracellular localization imaging of HeLa cells with 3 (10 µM, λ = 405 nm, collected at 420 - 480) with ER-

ex

tracker red 2 (0.2 µM, λ = 559 nm, collected at 590 – 640 nm), Mitotracker red (1 µM, λ = 635 nm, collected

ex

at 640 – 690 nm) and Lysotracker red (1 µM, λex = 635 nm, collected at 640 – 690 nm). Scale bar 30 µm.

2

. Experimental Section

.1. Materials and instruments

Unless otherwise noted, all organic compounds, inorganic salts, and solvents were analytically pure and used

directly after purchase. NMR spectra were acquired on commercial instruments (Bruker Avance 300 MHz,

Bruker AMX 400 MHz or Bruker Avance II+ 600 MHz) and chemical shifts (δ) are reported in parts per million

1

13

(

ppm), which are referenced to tetramethylsilane ( H) or the internal (NMR) solvent signal ( C). Exact mass

spectra were acquired on a quadrupole orthogonal acceleration time-of-flight mass spectrometer (Synapt G2

−

1

HDMS, Waters, Milford, MA). Samples were infused at 3 µL min and spectra were obtained in positive (or

negative) ionization mode with a resolution of 15 000 FWHM using leucine enkephalin as lock mass. Melting

points (not corrected) were determined using a Reichert Thermovar apparatus. For column chromatography,

7

0–230 mesh silica 60 (E. M. Merck) was used as the stationary phase. Chemicals received from commercial

sources were used without further purification. Dry-reaction solvents were used as received from commercial

sources. Reactions were monitored using thin-layer chromatography (TLC) on aluminum packed percolated Silica

Gel 60 F254 plates. Flash column chromatography was carried out with silica gel 60 (particle size less than 0.020

mm) by using appropriate mixtures of ethyl acetate and hexanes as eluents. Compounds were visualized on the

TLC plates by using UV light. Anhydrous magnesium sulfate was used for drying solutions. Chemical

nomenclature was generated using Chem Bio Draw Ultra 13.0.

2

.2. Synthesis

2

.2.1. 8-(benzyloxy)quinoline-2-carbaldehyde (5).

8

-Hydroxyquinoline-2-carbaldehyde (1.0 g, 5.8 mmol) was dissolved in dry DMF (14 mL) under N . Then

2

anhydrous K CO (1.6 g, 11.6 mmol) and benzyl bromide (1.0 mL, 8.6 mmol) were added, and the reaction was

2

3

heated at 85 °C for 18 h. Afterwards, the mixture was cooled to room temperature, diluted with water and

extracted with DCM (20 mL × 3). The organic layers were dried with MgSO , filtered and the solvent was

4

removed under reduced pressure. The product was purified by flash column chromatography (petroleum

ether/EtOAc = 95 : 5) affording 5 (1.5 g, 90% yield) as a pale yellow solid. The spectroscopic data agree with the

5

0 1

previously reported for the same product. H NMR (400 MHz, CDCl ) δ 10.32 (s, 1H), 8.26 (d, J = 8.4 Hz, 1H),

8

3

1

3

.06 (d, J = 8.4 Hz, 1H), 7.56–7.51 (m, 3H), 7.46–7.23 (m, 3H), 7.15 (d, J = 7.6 Hz, 1H), 5.48 (s, 2H). C NMR (75

MHz, CDCl ) δ 193.9, 155.1, 151.5, 140.3, 137.2, 136.5, 131.4, 129.6, 128.7, 128.0, 127.1, 119.9, 117.8, 111.0,

3

+

7

1.1. HRMS (ESI ): m/z calcd for C H NO [M+H] : 264.1025; found 264.1019.

17 13 2

2

.2.2. N,N-dibenzyl-2-methylquinolin-8-amine.

2

-Methylquinolin-8-amine (3.0 g, 18.0 mmol), was dissolved in dry ACN (20 mL) under N atmosphere. Then

2

anhydrous K CO (7.9 g, 57.0 mmol) and benzyl bromide (6.7 mL, 57.0 mmol) were added, and the reaction was

2

3

heated at 120 °C for 18h. Afterwards, the mixture was cooled to room temperature, diluted with water and

extracted with DCM (30 mL × 3). The organic layers were dried with MgSO , filtered and the solvent was

4

removed under reduced pressure. The product was purified by flash column chromatography (petroleum

1

ether/EtOAc = 90 : 10) affording N,N-dibenzyl-2-methylquinolin-8-amine (5.7 g, 90% yield) as a yellow oil. H

NMR (400 MHz, CDCl ) δ 7.95 (d, J = 8.3 Hz, 1H), 7.41 (d, J = 7.6Hz, 4H), 7.26–7.18 (m, 10H), 6.90 (d, J = 7.3 Hz,

3

13

1

3

H), 4.7 (s, 4H), 2.67 (s, 3H). C NMR (75 MHz, CDCl ) δ 155.9, 146.5, 142.3, 139.4, 136.4, 128.3, 128.0, 127.8,

3

+

26.6, 125.4, 121.4, 119.9, 117.8, 56.6, 25.4. HRMS (ESI ): m/z calcd for C H N [M+H] : 339.1861, found

24

22

2

39.1854.

2

.2.3. 8-(dibenzylamino)quinoline-2-carbaldehyde (6).

N,N-Dibenzyl-2-methylquinolin-8-amine (2.0 g, 5.9 mmol) and selenium dioxide (0.98 g, 8.8 mmol) were

suspended in 1,4-dioxane (25mL) under N atmosphere. Then the reaction was stirred at 80 °C for 18h to give a

2

greenish yellow solution containing a black precipitate of the selenium. Afterwards, the mixture was cooled to

room temperature, diluted with DCM (50 mL) and filtered using a pad of celite. The filter cake was washed with

DCM (3 times) and the organic layer was dried with MgSO

4

, filtered and the solvent was removed under reduced

pressure. The product was purified by flash column chromatography (DCM) affording 6 (1.6 g, 80% yield) as a

1

yellow solid (m.p. = 85–86 °C). H NMR (400 MHz, CDCl

d, J = 8.5 Hz, 1H), 7.33–7.31 (m, 4H), 7.20–7.07 (m, 8H), 6.93 (dd, J = 1.2, 7.8 Hz, 1H), 4.73 (s, 4H). C NMR (75

MHz, CDCl ) δ 139.8, 149.6, 148.4, 141.7, 138.9, 137.5, 131.9, 129.7, 128.6, 128.5, 128.3, 127.8, 127.7, 126.9,

3

) δ 9.75 (d, J = 0.8 Hz, 1H), 8.03 (d, J = 8.1 Hz, 1H), 7.82

1

3

(

3

+

1

18.8, 117.0, 116.8, 56.9. HRMS (ESI ): m/z calcd for C H N O [M+H] : 353.1654; found 353.1646.

24 20 2

2

.2.4. Quinoline-2-carbaldehyde (7).

Following the procedure described for N,N-dibenzyl-2-methylquinolin-8-amine. 2-methylquinoline (5g, 34.9

mmol), selenium dioxide (0.52 g, 5.8 mmol) and 1,4-dioxane (30 mL) were employed. The product was purified

by flash column chromatography (petroleum ether/EtOAc = 90 : 1) affording 7 (1.6 g, 70% yield) as a yellow solid

5

1 1

(

m.p. = 72–73 °C). The spectroscopic data agree with the previous report for the same product. H NMR (400

MHz, CDCl ) δ 10.24 (s, 1H), 8.29 (dd, J = 8.4, 24.1 Hz, 2H), 8.04 (d, J = 8.16 Hz, 1H), 7.91 (d, J = 8.16 Hz, 1H), 7.84

3

13

(

1

t, J = 7.23 Hz, 1H), 7.70 (t, J = 7.70 Hz, 1H). C NMR (75 MHz, CDCl ) δ 193.77, 152.61, 147.95, 137.40, 130.50,

30.44, 130.09, 129.22, 127.88, 117.37. HRMS (ESI ): m/z calcd for C H NO [M+H] : 158.0606; found 158.0605.

10 7

3

+

2

8

.2.5. ((2-Formylquinolin-8-yl)oxy)methyl acetate (9).

-Hydroxyquinoline-2-carbaldehyde (200 mg, 1.2 mmol) was dissolved in dry DMF (10 mL) under N . Then

2

bromomethyl acetate (226 µL, 2.3 mmol) and K CO anhydrous (479.0 mg, 3.4 mmol) were added and the

2 3

reaction was refluxed 18h. Afterwards, the mixture was cooled to room temperature, diluted with water and

extracted with Et O (10 mL × 3). The organic layers were dried with MgSO , filtered and the solvent was

2

4

removed under reduced pressure. Finally, the product was purified by flash column chromatography (petroleum

1

ether/EtOAc = 80 : 20) affording 9 (220 mg, 75% yield) as a yellow oil. H NMR (400 MHz, CDCl ) δ 10.30 (s, 1H),

8

3

13

.30 (d, J = 7.8 Hz, 1H), 8.07 (d, J = 7.8 Hz, 1H), 7.42 (dd, J = 3.02, 6.8 Hz, 1H), 6.15 (s, 2H), 2.14 (s, 3H). C NMR

(

100 MHz, CDCl ) δ 193.6, 168.7, 153.2, 151.9, 140.3, 137.5, 131.4, 129.4, 122.3, 118, 113.9, 86.4, 20.9. HRMS

3

+

ESI ): m/z calcd for C H NO [M+H] : 246,0766; found 246.0770.

13

11

4

General procedure for the synthesis of BODIPY analogs. To a solution of 1H-pyrrole (2,5 eq.) and the

corresponding hydroxyquinoline aldehyde (1 eq.) in dry DCM, trifluoracetic acid (TFA, 0.1 eq.) was added under

N . The reaction mixture was stirred for 3h at room temperature, then a solution of p-chloranil (1 eq.) in dry

2

DCM was added. After stirring 1h at room temperature, Et N (10 eq.) and BF ·OEt (10 eq) were added and

3

2

stirred 30 min at room temperature until a bright-green fluorescence was observed. Then, the mixture was

diluted with water and extracted with DCM 3 times. The organic layer was dried with MgSO , filtrated and the

4

solvent was removed under reduced pressure. Finally, the product was purified by flash column chromatography

using an appropriate solvent mixture of petroleum ether, EtOAc and/or DCM.

2

8

.2.6. 2-(5,5-Difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)quinolin-

-ol (3).

Following the general procedure, 2,4-dimethyl-1H-pyrrole (420

µL, 4.0 mmol), 8-hydroxyquinoline-2-

carbaldehyde (300 mg, 1.73 mmol), TFA (19 L, 0.17 mmol) and dry DCM (75 mL) were employed. Then p-

µ

chloranil (420 mg, 1.71 mmol) was dissolved in dry DCM (60 mL) and added to the mixture. Afterwards, Et N (3

3

mL) and BF ·OEt (3 mL) were added. The product was purified by flash column chromatography (petroleum

3

2

ether/EtOAc = 90 : 10) affording 3 (342 mg, 60% yield) as a green solid (m.p. = 203–204 °C). The spectroscopic

3

7 1

data correspond with the previously reported. H NMR (400 MHz, CDCl ) δ 8.33 (d, J = 8.3 Hz, 1H), 8.03 (br, 1H),

3

1

3

7

.59–7.53 (m, 2H), 7.45–7.42 (m, 1H), 7.28–7.25 (m, 1H), 6.00 (s, 2H), 2.58 (s, 6H), 1.21 (s, 6H). C NMR (75

+

3

MHz, CDCl ) δ 152.5, 137.4, 128.9, 122.5, 121.4, 117.9, 111.1, 14.7, 13.9. HRMS (ESI ): m/z calcd for

+

C

22

H

20BF

2

N

3

O [M+H] : 392.1748; found 392.1742.

2

.2.7.

10-(8-(benzyloxy)quinolin-2-yl)-5,5-difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-

f][1,3,2]diazaborinine (10).

Following the general procedure, 2,4-dimethyl-1H-pyrrole (0.31 mL, 2.85 mmol), 5 (300 mg, 1.14 mmol), TFA (19

µL, 0.17 mmol) and dry DCM (75 mL) were employed. Then p-chloranil (279 mg, 1.14 mmol) was dissolved in dry

DCM (60 mL) and added to the mixture. Afterwards, Et N (3 mL) and BF ·OEt (3 mL) were added. The product

3

2

was purified by flash column chromatography (petroleum ether/EtOAc = 90 : 10) affording 10 (186 mg, 34%

1

yield) as a green solid (m.p. = 105–107 °C). H NMR (400 MHz, CDCl ) δ 8.26 (d, J = 8.35 Hz, 1H), 7.53 (d, J = 8.4

3

Hz, 1H), 7.43 (d, J = 4 Hz, 4H), 7.31 (t, J = 7.3 Hz, 2H), 7.26 (m, 1H), 7.08 (d, J = 5.6 Hz, 1H), 5.98 (s, 2H), 5.51 (s,

1

3

2

1

4

H), 2.57 (s, 6H), 1.27 (s, 6H). C NMR (75 MHz, CDCl ) δ 156.2, 154.5, 142.7, 140.4, 139.3, 137.1, 137.1, 131.7,

3

+

28.8, 128.6, 127.7, 127.6, 126.6, 122.2, 121.1, 119.8, 111.7. HRMS (ESI ): m/z calcd for C H BF N O [M+H] :

2

9

26

2

3

82.2215; found 482.2209.

2

1

.2.8. N,N-dibenzyl-2-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-

0-yl)quinolin-8-amine (11).

Following the general procedure, 2,4-dimethyl-1H-pyrrole (0.14 mL, 1.2 mmol), 6 (200 mg, 0.6 mmol), TFA (10

L, 0.06 mmol), and dry DCM (30 mL) were employed. Then p-chloranil (147.5 mg, 0.6 mmol) was dissolved in

DCM (50 mL) and added to the mixture. Afterwards, Et N (1.4 mL) and BF ·OEt (1.4 mL) were added. The

µ

3

2

product was purified by flash column chromatography (petroleum ether/EtOAc = 90 : 10) affording 11 (139 mg,

1

4

5% yield) as a red solid (m.p. = 180–181 °C). H NMR (400 MHz, CDCl ) δ 8.19 (d, J = 8.4 Hz, 1H) 7.41 (d, J = 8.4

3

Hz, 1H), 7.35–7.34 (m, 2H), 7.22–7.20 (m, 4H), 7.17–7.11 (m, 6H), 7.01–6.99 (m, 1H), 5.96 (s, 2H), 4.71 (s, 4.0 H),

1

3

2

1

.58 (s, 6H), 1.24 (s, 6H). C NMR (75 MHz, CDCl ) δ 155.9, 150.5, 147.3, 142.6, 142.3, 139.7, 138.9, 137.6, 131.5,

3

+

29.1, 128.1, 127.9, 127.6, 126.7, 121.1, 121.0, 119.6, 118.8, 57.1, 14.7, 13.9. HRMS (ESI ): m/z calcd for

C H BF N [M+H] : 571.2845; found 571.2842.

+

36

33

2

4

2

.2.9. 5,5-difluoro-1,3,7,9-tetramethyl-10-(quinolin-2-yl)-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinine

12).

Following the general procedure, 2,4-dimethyl-1H-pyrrole (780 mg, 7.1 mmol), 7 (500 mg, 3.12 mmol), TFA (15

L, 0.31 mmol) and dry DCM (70 mL) were employed. Then p-chloranil (775 mg, 3.12 mmol) was dissolved in

DCM (80 mL) and added to the mixture. Afterwards, Et N (5 mL) and BF ·OEt (5 mL) were added. The product

(

µ

3

2

was purified by flash column chromatography (gradient petroleum ether/EtOAc = 80 : 10 – DCM) affording 12

1

(

397 mg, 34% yield) as an orange solid (m.p. = 306–307 °C). H NMR (400 MHz, CDCl ) δ 8.33 (d, J = 8.4 Hz), 8.17

3

(

d, J = 8.5 Hz, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.81 (t, J = 7.9 Hz, 1H), 7.67 (t, J = 7.5Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H),

1

3

6

1

3

.00 (s, 2H), 2.57 (s, 6H), 1.22 (s, 6H). C NMR (75 MHz, CDCl ) δ 156.5, 154.0, 178.1, 172.6, 138.8, 137.3, 131.3,

3

+

30.5, 129.7, 127.8, 127.7, 127.4, 121.7, 121.3, 14.7, 13.9. HRMS (ESI ): m/z calcd for C H BF N [M+H] :

22

20

2

3

76.1797; found 376.1791.

2

.2.10.

5,5-difluoro-1,3,7,9-tetramethyl-10-(naphthalen-2-yl)-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-

f][1,3,2]diazaborinine (13).

Following the general procedure, 2,4-dimethyl-1H-pyrrole (0.73 mL, 6.9 mmol), 2-naphthaldehyde (500 mg, 3.0

mmol), TFA (23

µL, 0.3 mmol) and dry DCM (70 mL) were employed. Then p-chloranil (737 mg, 3.0 mmol) was

dissolved in DCM (80 mL) and added to the mixture. Afterwards, Et N (5 mL) and BF ·OEt (5 mL) were added.

3

2

The product was purified by flash column chromatography (petroleum ether/EtOAc = 90 : 10) affording 13

785.6 mg, 70% yield) as an orange solid (m.p. = 310–312 °C). The spectroscopic data correspond with the

(

4

0 1

previously reported ones. H NMR (400 MHz, CDCl ) δ 8.00 (d, J = 8.3 Hz, 1H), 7.94–7.92 (m, 1H), 7.87–7.85 (m,

3

1

3

1

H), 7.79 (s, 1H), 7.57 (m, 2H), 7.38 (dd, J = 1.4, 8.3 Hz, 1H), 5.98 (s, 2H), 2.57 (s, 6H), 1.30 (s, 6H). C NMR (75

MHz MHz, CDCl ) δ 155.5, 143.2, 141.6, 133.3, 133.1, 132.3, 131.6, 129.0, 128.1, 127.9, 127.3, 126.9, 126.8,

3

+

1

25.6, 121.2, 14.6. HRMS (ESI ): m/z calcd for C23

2 2

H21BF N [M+H] : 375.1884; found 375.1890.

2

.2.11. ((2-(5,5-Difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-

yl)quinolin-8-yl)oxy)methyl acetate (14).

Following the general procedure, 2,4-dimethyl-1H-pyrrole (0.29 mL, 2.8 mmol), 9 (300 mg, 1.2 mmol), TFA (10

µ

L, 0.12 mmol) and dry DCM (60 mL) were employed. Then p-chloranil (295 mg, 1.2 mmol) was dissolved in DCM

30 mL) and added to the mixture. Afterwards, Et N (2.8 mL) and BF ·OEt (2.8 mL) were added. The product was

purified by flash column chromatography (gradient petroleum ether/EtOAc = 90 : 10 – 80 : 20) affording 14

(

3

2

1

(

186.1 mg, 35% yield) as a brown solid (m.p. = 250–252 °C). H NMR (400 MHz, CDCl

3

) δ 8.31 (d, J = 8.5 Hz, 1H),

7

.64 (d, J = 8.15 Hz, 1H), 7.59 – 7.55 (m, 2H), 7.40 (d, J = 7.8 Hz, 1H), 6.07 (s, 1H), 6.00 (s, 1H), 2.57 (s, 6H), 2.09

13

(

s, 3H), 1.23 (s, 6H). C NMR (100 MHz, CDCl

22.5, 122.3, 121.2, 115.1, 87.0, 20.8, 14.5, 13.9. HRMS (ESI ): m/z calcd for C23

found 375.1890.

3

) δ 169.7, 156.3, 153.2, 152.9, 142.6, 140.3, 137.3, 128.9, 127.5,

+

1

2 2

H21BF N [M+H] : 375.1884;

2

8

.2.12. 2-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)quinolin-

-yl methanesulfonate (15).

Compound 3 (100 mg, 0.25 mmol) was dissolved in dry DCM (3 mL) in a sealed reaction tube under N at 0 °C.

2

Then Et N (0.01 mL, 0.64 mmol) and mesyl chloride (0.07 mL, 0.85 mmol) were added and the reaction was

3

allowed to reach room temperature. After stirring for 4 h. the mixture was diluted with water and extracted

with DCM 3 times. The organic layer was dried with MgSO , filtered and the solvent was removed under reduced

4

pressure. Finally, the product was purified by flash column chromatography (DCM) affording 15 (77 mg, 64%

1

yield) as an orange solid (m.p. = 176–178 °C). H NMR (400 MHz, CDCl ) δ 8.38 (d, J = 8.4 Hz), 7.90 (d, J = 8.2 Hz,

3

1

H), 7.77 (d, J = 7.55 Hz, 1H), 7.69–7.65 (m, 1H), 7.59 (d, J = 7.5 Hz, 1H), 6.0 (s, 2H), 3.47 (s, 3H), 2.58 (s, 6H), 1.22

13

(s, 6H). C NMR (75 MHz, CDCl ) δ 155.8, 153.6, 144.9, 141.6, 140.4, 137.1, 136.4, 130.1, 127.9, 126.6, 126.0,

3

+

1

4

23.7, 121.7, 120.5, 38.9, 13.7, 12.8. HRMS (ESI ): m/z calcd for C H BF N O S [M+H] : 470.1443; found

23 22 2 3 3

70.1519.

2

8

.2.13. 2-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)quinolin-

-yl 4-methylbenzenesulfonate (16).

Compound 3 (100 mg, 0.25 mmol) was dissolved in dry DCM (3 mL) in a sealed reaction tube under N at 0 °C.

2

Then Et N (0.09 mL, 0.64 mmol) and tosyl chloride (162 mg, 0.85 mmol) were added and the reaction was

3

allowed to reach room temperature. After stirring for 4 h. the mixture was diluted with water and extracted

with DCM 3 times. The organic layer was dried with MgSO , filtered and the solvent was removed under reduced

4

pressure. Finally, the product was purified by flash column chromatography (DCM) affording 16 (110 mg, 79%

1

yield) as an orange solid (m.p. = 244–245 °C). H NMR (400 MHz, CDCl ) δ 8.30 (d, J = 8.4 Hz, 1H), 7.88–7.79 (m,

3

4

2

1

H), 7.64 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 8.3Hz, 1H), 7.04 (d, J = 8.0 Hz, 2H), 6.0 (s, 2H), 5.2 (s, 2H), 2.60 (s, 6H),

13

.27 (s, 3H), 1.09 (s, 6H). C NMR (75 MHz, CDCl ) δ 156.3, 154.1, 146.1, 145.0, 142.7, 141.3, 138.4, 136.9, 133.7,

3

+

31.2, 129.1, 128.8, 128.5, 127.4, 126.8, 124.1, 122.5, 121.3, 53.4, 21.5, 17.7, 13.9. HRMS (ESI ): m/z calcd for

+

C H BF N O S [M+H] : 546.1834; found 546.1830.

29

26

2

3

2

8

.2.14. 2-(5,5-Difluoro-1,3,7,9-tetramethyl-5H-4l4,5l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-10-yl)quinolin-

-yl pentanoate (17).

Compound 3 (100 mg, 0.25 mmol) and pyridine (40 μL, 0.5 mmol) were dissolved in dry DCM (10 mL) under N₂.

Then valeroyl chloride (0.1 mL, 0.75 mmol) was added at 0 °C and the mixture was stirred for another 10 min at

0

°C and then for 3 h at room temperature. Afterwards, the mixture was diluted with water and extracted with

DCM 3 times. Further, the DCM extract was washed 5 times with a diluted solution of NaHCO (0.1 M) and finally

3

washed again with water. The organic layer was dried with MgSO

4

, filtered and the solvent was removed under

reduced pressure. The product was purified by flash column chromatography (gradient petroleum ether/EtOAc

1

=

3

90 : 10) affording 17 (59 mg, 80% yield) as a brown solid (m.p. = 179–180 °C). H NMR (400 MHz, CDCl ) δ 8.32

(

d, J = 8.3 Hz, 1H), 7.80 (d, J = 8.3 Hz, 1H), 7.63 (t, J = 8.3 Hz, 1H), 7.50 (dd, J = 8.3, 13.5 Hz, 2H), 5.97 (s, 2H), 2.66

t, J = 7.7 Hz, 2H), 5.57 (s, 6H), 1.69 (quint, J = 7.7 Hz, 2H), 1.39 (sext, J = 7.9 Hz, 2H), 1.20 (s, 6H), 0.78 (t, J = 7.0

1

3

Hz, 3H). C NMR (75 MHz, CDCl ) δ 172.9, 156.2, 153.8, 148.2, 142.9, 141.0, 139.0, 137.1, 131.3, 128.6, 127.4,

1

4

+

25.6, 122.2, 122.1, 33.9, 27.3, 22.1, 14.7, 13.9, 13.5. HRMS (ESI ): m/z calcd for C27

2 3 2

H28BF N O [M+H] :

76.2321; found 476.2315.

2

.2.15.

1-(10-(8-(benzyloxy)quinolin-2-yl)-5,5-difluoro-1,3,7,9-tetramethyl-5H-5l4,6l4-dipyrrolo[1,2-c:2',1'-

f][1,3,2]diazaborinin-2-yl)pentan-1-one (18).

Compound 10 (100 mg, 0.21 mmol) was dissolved in dry DCM (10 mL) under N . Then the solution was cooled at

2

0

°C and immediately, BF ·OEt (0.08 mL, 0.62 mmol) was added and stirred for 10 min. Afterwards, valeroyl

3 2

chloride (0.06 mL, 0.46 mmol) was added at 0 °C. The mixture was stirred for another 10 min at 0 °C and then for

2 h at room temperature. Afterwards, the mixture was diluted with water and extracted with DCM 3 times.

7

Further, the DCM extract was washed 5 times with a diluted solution of NaHCO (0.1 M) and finally washed

3

again with water. The organic layer was dried with MgSO , filtrated and the solvent was removed under reduced

4

pressure. The product was purified by flash column chromatography (petroleum ether/EtOAc = 90 : 10)

1

affording 18 (83 mg, 70% yield) as a brow solid (m.p. = 300–302 °C). H NMR (400 MHz, CDCl ) δ 8.30 (d, J = 8.4

3

H, 1H), 7.53 (d, J = 8.23 Hz, 1H), 7.46–7.41 (m, 4H), 7.31 (t, J = 7.6 Hz, 2H), 7.27–7.24 (m, 1H), 7.10 (t, J = 7.37 Hz,

1

H), 6.10 (s, 1H), 5.51 (s, 2H), 2.75 (s, 3H), 2.66 (t, J = 7.6 Hz, 2H), 2.61 (s, 3H), 1.62 (quintet, J = 7.6 Hz, 2H), 1.42

1

3

(s, 3H), 1.34–1.26 (m + s, 5H), 0.89 (t, J = 7.40 Hz, 3H). C NMR (75 MHz, CDCl ) δ 200.1, 160.6, 154.5, 152.3,

3

1

40.7, 140.4, 137.4, 137.0, 128.9, 128.6, 127.9, 126.6, 123.2, 122.1, 119.9, 111.8, 70.7, 43.5, 26.7, 22.5, 15.0,

+

4.8, 14.5, 13.9, 12.3. HRMS (ESI ): m/z calcd for C H BF N O [M+H] : 566.2790; found 566.2786.

34

2

3

2

.2.16.

Ethyl

5-(10-(8-(benzyloxy)quinolin-2-yl)-5,5-difluoro-1,3,7,9-tetramethyl-5H-5l4,6l4-dipyrrolo[1,2-

c:2',1'-f][1,3,2]diazaborinin-2-yl)-5-oxopentanoate (19).

Compound 10 (100 mg, 0.21 mmol) was dissolved in dry DCM (10 mL) under N . Then it was cooled at 0 °C and

2

immediately, BF ·OEt (0.18 mL, 0.62 mmol) was added and stirred for 10 min. Afterwards, ethyl glutaryl chloride

3

2

(0.13 mL, 0.81 mmol) was added at 0 °C. The mixture was stirred for another 10 min at 0 °C and then for 72h at

room temperature Afterwards, the mixture was diluted with water and extracted with DCM 3 times. Further, the

DCM extract was washed 5 times with a diluted solution of NaHCO (0.1 M) and finally washed again with water.

3

The organic layer was dried with MgSO , filtrated and the solvent was removed under reduced pressure. The

4

product was purified by flash column chromatography (gradient petroleum ether/EtOAc = 80 : 20 – DCM)

1

affording 19 (59 mg, 45% yield) as a brow solid (m.p. = 320–321 °C). H NMR (400 MHz, CDCl ) δ 8.30 (d, J = 8.3

3

Hz), 7.52 (d, J = 8.2 Hz, 1H), 7.46–7.43 (m, 4H), 7.32 (t, J = 7.7 Hz, 2H), 7.28–7.24 (m, 1H), 7.11 (t, J = 4.24 Hz, 1H),

6

2

1

7

6

.10 (s, 1H), 5.51 (s, 2H), 4.09 (q, J = 7.2 Hz, 2H), 2.76–2.72 (s + t, J = 7.2 Hz, 5H), 2.61 (s, 3H), 2.34 (t, J = 7.2 Hz,

13

H), 1.97 (quintet, J = 7.2, 2H), 1.43 (s, 3H), 1.27–1.20 (m, 6H). C NMR (75 MHz, CDCl ) δ 198.5, 173.2, 161.0,

3

54.5, 152.2, 146.0, 140.9, 140.4, 137.5, 136.9, 128.9, 128.6, 128.0, 127.7, 126.6, 123.4, 122.1, 119.9, 111.8,

+

0.7, 60.3, 42.5, 33.4, 29.7, 19.6, 15.1, 14.5, 14.2, 13.4. HRMS (ESI ): m/z calcd for C H BF N O [M+H] :

3

6

36

2

3

4

24.2845; found 624.2866.

2

.3. Spectroscopic measurements.

Solutions were prepared using commercially available spectroscopic grade solvents without further purification.

Absorption was measured using a Lambda 40 spectrophotometer with blank correction. The fluorescence

spectra and quantum yields were obtained using a Horiba Jobin-Yvon fluorimeter, with corrections for the

wavelength dependence of the detector, fluctuations of the lamp intensity and the optical path. Absolute

5

2,53

quantum yields were determined using an integrating sphere.

density filters of 0.3 % were used for all solutions except for the Triton X-100 in Milli-Q water solutions, for

which a neutral density filter of 1 % was used. Solutions of the compound 3, 10 - 19 (1 M) were prepared in

toluene (tol), acetonitrile (ACN) and methanol (MeOH). The compounds were first dissolved in DMSO (10 mM)

When recording the Rayleigh scatter, neutral

µ

and then dissolved in a solution water Milli-Q + 0.5% triton (5

supporting information.

µM). The acquired spectra are shown in

2

.4. Cell culture.

Human cervical cancer cells (HeLa) were cultured in high glucose containing Dulbecco’s modified Eagle’s

medium (DMEM), supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, and

1

% penicillin/streptomycin (Thermofisher, Belgium). Cells were maintained in a humidified atmosphere at 37°C

and 5% CO and split upon reaching 80% confluency.

2

.5. High-content imaging.

For high-content imaging and colocalization studies, HeLa cells were seeded at 3000 cells/well in 96 well plate

and placed in the incubator overnight. Before imaging, cells were incubated for 1 hour at 37˚C using the

TM

appropriate combination of BODIPY-derivatives (10 µM), commercial organelle markers (1 µM of ER-tracker

TM

Red for Endoplasmatic reticulum, 200 nM of Mitotracker red CMXRos for mitochondria and 1 µM of

TM

Lysotracker

red DND-99 for lysosomes, Thermofisher, Belgium) and 0.2 µg/mL of Hoescht 33342

(Thermosfisher, Belgium) in 100 µL of complete DMEM medium. After staining, the cells were washed twice

with DMEM (without serum) and fixed with 2% paraformaldehyde for 4 min at 37 °C. The fixative was removed,

the cells were washed once with DMEM and 100 µL of PBS was added to each well. The 96-well plates were

imaged using the InCell 2000 analyser (GE Healthcare Life Sciences, Belgium) where fluorescence-based images

for the blue (λex=350, λem=455 nm), green (λex=490, λem=525 nm) and red ((λex=579, λem=624 nm) channel

were collected at minimum 1000 cells/well. To calculate the colocalization coefficient, the images were

processed in Fiji. Briefly, the background-subtracted images were filtered using an unsharp mask (radius sigma =

1

.1 pixels and mask weight 0.8). The Pearson’s colocalization coefficient of the fluorescence image of the

BODIPY-derivates and the commercial organelle markers was calculated using a function present in Fiji.

2

.6. Cytotoxicity.

HeLa cells were plated at a density 10.000 cells/well onto flat bottom 96-well microtiter plates (Greiner Bio-One,

Belgium) and left to attach overnight. Next day cells were treated with compounds at the concentration of 10

µM dissolved in the cell specific medium during 5 to 240 minutes at 37 °C in a humidity-controlled incubator

with 5% CO . After the treatment, media was gently removed and cells were incubated with 5 µM PI

2

+

2+

(

Invitrogen/Life Technologies, USA) in PBS containing Ca and Mg at 37 °C during 30 min.

As positive control the cells were treated with 0.2 % Triton X-100 for 15 minutes.

Wells containing no cells and PI solution were used to determine the level of background signal. Fluorescence

was recorded at 530 nm/630 nm from the top of the plate using a FlexStation 3 Benchtop Multi-Mode

Microplate Reader and the SoftMax Pro 7 Microplate Data Acquisition & Analysis Software (Molecular Devices,

USA). Data obtained from at least 4 samples was background subtracted and normalized to values of control

cells not treated with compounds. Analysis was done using OriginPro9 (OriginLab Corporation, Northampton,

MA, USA) software.

2

.7. Confocal fluorescence imaging.

The day before imaging, HeLa cells were seeded on a 29 mm glass bottom dish (Cellvis). After 24 hours the cells

were washed 3 times with Hank’s Balanced Salt Solution (HBSS, Thermo Fisher) and stained using a solution

TM

containing 1 µM of the BODIPY derivative, 1 µM of ER-tracker Red for Endoplasmatic reticulum (Thermofisher,

Belgium) and 0.2 mg/ml of Hoechst (Sigma-Aldrich). After 1 hour of incubation at 37°C the cells were washed 3

times with HBSS and fixed with 4% formaldehyde for 10 minutes at room temperature. Finally, the cells were

washed twice with HBSS and kept in HBSS during imaging. Fluorescence images were acquired using a Leica TCS

SP8 X microscope equipped with a water objective (HC PL APO 63x/1,40, motCORR, Leica). As excitation sources

we used a supercontinuum White Light Laser (470 – 670 nm, pulsed, 80 MHz, NKT Photonics) at 587 nm for the

ER tracker, a 405 nm diode laser (pulsed, 40 MHz) for the Hoechst dye and MaiTai DeepSee (690-1040 nm,

pulsed, 80 MHz) at 900 nm for BODIPY derivative. The fluorescence of the Hoechst and ER tracker were detected

using an internal hybrid photomultiplier tube (HYD-SMD, Leica). For BODIPY dye an external HYD RLD detector

was used. The sample was scanned using a field-of-view scanner (200 Hz, bidirectional). All measurements were

executed in a temperature-controlled environment. The images showed in Figure 5 were deconvolved using

Huygens Professional software.

3

. Results and discussion

.1. Synthesis of BODIPY-derivatives library

The compound 3 was synthesized starting from the aldehyde 4, following the previously procedure reported by

3

37

Moon et al. (Scheme 1). Starting from this compound, a small library of ten BODIPY derivatives was

synthesized. To evaluate the influence the OH group in the 8-hydroxyquinoline moiety in compound 3,

molecules presenting different groups at the OH position were synthesized (10 – 17, Scheme 1). These included

other O- or N- groups (10 – 11, 14 – 17, Scheme 1) or just a H atom (12 – 13, Scheme 1). The influence of the N

in the quinoline was evaluated by synthesizing a naphthalene derivative (compound 13). In addition, the

introduction of carbonyl groups in the 6-position of the BODIPY core was also explored (compounds 18 – 19).

In short, the target compounds 10 – 14 (Scheme 1) were synthesized starting from the corresponding aldehydes

5

– 9 using the classical “one pot” protocol for the synthesis of BODIPYs in 34, 45, 34, 70 and 35 % yield

3

8

respectively. Then, using 3 as reagent the mesyl (15), tosyl (16) and ester (17) derivatives were obtained in 64,

7

9 and 80 % yield respectively (Scheme 1). Finally, compounds 18 and 19 were synthesized by electrophilic

39

aromatic substitution starting from 10 in 70 and 45 % yield respectively.

MsO

BnO

N

ii)

15 (Y = 64%)

R²

O

v)

N

B

HO

TsO

BnO

3

iii)

F

N

vi)

18 (Y = 70%)

Y

R²

Y

3

iv)

1

6 (Y = 79%)

10

BnO

N

O

3

O

N

B

3

_R2

OEt

F

1

7 (Y = 80%)

Scheme 1. Synthetic route for the target compounds 3, 10 – 19. i) 2,4-dimethyl-1H-pyrrole, TFA, p-chloranil,

Et N, BF .OEt, dry DCM; ii) MsCl, dry pyridine (Py), DCM; iii) TsCl, dry Py, DCM; iv) valeroyl chloride, dry Py, DCM;

19 (Y = 45%)

3

v) valeroyl chloride, BFt .OEt, dry DCM; vi) glutaric acid monoethyl ester chloride; BF .OEt, dry DCM.

3

.2. Spectroscopic measurements

To study the spectroscopic properties of 3 and 10 - 19, absorption and emission spectra were recorded in

toluene, acetonitrile and methanol. The spectral properties were also determined in Milli-Q water with 0.5%

Triton X-100. Triton X-100 was used to increase the solubility of the compounds in aqueous solutions. The

3

8

spectra are shown in supporting information (Figures S31 – S32), and a summary of the data can be found in

Table 1.

In toluene, methanol and acetonitrile, the S → S transition of all measured compounds features a narrow

0

1

absorption band with a vibrational shoulder at shorter wavelengths, which is characteristic for BODIPY dyes. The

absorption is shifted hypsochromically, going from toluene to methanol and acetonitrile, reflecting the

40-42

decreasing polarizability of the solvent and indicating an absent or small dipole moment in the ground state.

Also the emission spectra showed the typical characteristics of BODIPY dyes consisting of a blue shift in less

-1

polarizable solvents and a small Stokes shift of 550 to 800 cm . For most compounds, the FWHM of the emission

band is however nearly twice the value typically obtained for BODIPY dyes. For 8-phenyltetramethyl-BODIPY for

-

1

-1

example, a FWHMem of ≈ 800 cm is reported in methanol and a FWHMem of ≈ 900 cm in acetonitrile, similar to

4

3,44

those published for the unsubstituted BODIPY.

The broadening of the fluorescence band suggests a more

extensive conjugation with the quinoline moiety compared to the corresponding 8-phenyl BODIPY.

The absorption of compound 13 is slightly blue shifted with respect to literature values reported for

unsubstituted tetramethyl-BODIPY in methanol and acetonitrile and almost identical to values reported for the

corresponding 8-phenyl BODIPY (497 nm and 498 nm in acetonitrile and methanol respectively, Figure S31,

43,44

Table 1).

Bai et al. reported the same absorption maximum for 13 in methanol and acetonitrile, but found a

40

slightly different emission maximum and quantum yield of fluorescence. The quantum yield decreases slightly

from 53 % in toluene to 40 % in acetonitrile and 34 % in methanol, but the emission band does not broaden

significantly, and the Stokes shift remains more or less the same. The quenching of the fluorescence with

respect to unsubstituted BODIPY originates from non-radiative decay induced by rotation of the naphthalene

group with respect to the BODIPY core, which also explains the decreased quantum yield with respect to the

analogue coupled at the 1-position of the naphthalene (89 % in acetonitrile and 96 % in methanol), where this

45,46,25

rotation is more sterically hindered.

For 8-phenyltetramethyl-BODIPY, quantum yields of 60 % in

43

acetonitrile and 65 % in methanol are reported, due to the same quenching mechanism. Compared to the 8-

quinoline substituted BODIPYs, a small blue shift is accompanied by a reduction of the FWHM_e_mby 30 %.

Furthermore, 13 also has the smallest Stokes shift of this series of 8-substituted BODIPYs. These data suggest

that the conjugation with the electron accepting quinoline moiety is more outspoken than with naphthalene.

Table 1. Spectroscopic data and quantum yields of the compounds 3 – 19.

-1

Solvent

Compound

ꢀ_ꢁ_ꢂ_ꢃ_ꢄ/nm

ꢀ_ꢅ_ꢆ/nm

( ꢇꢈ_ꢁ_ꢂ_ꢃ− ꢇꢈ_ꢅ_ꢆ) cm

FWHMem/cm

ꢉ (%)

3

509

508

504

508

507

510

509

502

501

498

502

503

502

503

502

503

504

502

527

526

523

524

518

525

524

528

523

530

528

518

515

516

512

517

520

516

523

519

518

521

523

670 ± 50

570 ± 50

600 ± 50

540 ± 50

640 ± 50

600 ± 50

750 ± 50

600 ± 50

740 ± 50

710 ± 50

620 ± 50

660 ± 50

540 ± 50

580 ± 50

550 ± 50

580 ± 50

650 ± 50

540 ± 50

760 ± 50

650 ± 50

580 ± 50

650 ± 50

800 ± 50

1480 ± 50

1620 ± 50

1360 ± 50

1420 ± 50

990 ± 50

5 ± 1

1 ± 0.5

3 ± 0.5

2 ± 0.5

53 ± 5

3 ± 0.5

6 ± 1

1

0

1

2

3

4

5

6

7

8

9

1620 ± 50

1870 ± 50

1390 ± 50

1600 ± 50

1540 ± 50

1350 ± 50

1190 ± 50

1290 ± 50

940 ± 50

12 ± 1

11 ± 1

2 ± 0.5

3 ± 0.5

8 ± 1

3

1

0

1

2

3

4

5

6

7

8

9

3 ± 0.5

1 ± 0.5

3 ± 0.5

40 ± 4

4 ± 0.5

7 ± 1

1320 ± 50

1450 ± 50

1840 ± 50

1280 ± 50

1960 ± 50

1450 ± 50

1350 ± 50

1510 ± 50

1160 ± 50

8 ± 1

9 ± 1

4 ± 0.5

5 ± 1

3

9 ± 1

1

0

1

9 ± 1

1 ± 0.5

1

2

3

4

5

6

7

8

9

504

498

503

504

503

507

506

509

503

507

506

519

513

519

518

524

518

524

520

525

524

521

515

526

523

526

524

528

527

570 ± 50

590 ± 50

610 ± 50

580 ± 50

760 ± 50

580 ± 50

800 ± 50

650 ± 50

680 ± 50

450 ± 50

460 ± 50

710 ± 50

600 ± 50

710 ± 50

680 ± 50

820 ± 50

790 ± 50

1310 ± 50

1010 ± 50

1480 ± 50

1890 ± 50

1390 ± 50

1560 ± 50

1500 ± 50

1280 ± 50

1420 ± 50

1280 ± 50

1370 ± 50

930 ± 50

10 ± 1

34 ± 3

10 ± 1

8 ± 1

2 ± 0.5

13 ± 1

12 ± 1

33 ± 3

41 ± 4

7 ± 1

3

1

0

1

2

3

4

5

6

7

8

9

23 ± 2

79 ± 8

40 ± 4

51 ± 5

44 ± 4

55 ± 6

33 ± 3

30 ± 3

1410 ± 50

1420 ± 50

1830 ± 50

1320 ± 50

1540 ± 50

1510 ± 50

Substituting the naphthalene moiety for quinoline in compound 12 shifts the absorption bathochromically by a

few nanometers and decreases the quantum yield to 2 % and 3 % in toluene and acetonitrile, respectively. In

methanol, however, the quantum yield increases to 10 %. This trend, which is also observed for most other

quinoline substituted compounds, can perhaps be attributed to damping of the rotation around the meso bond

through hydrogen bonding of the nitrogen with solvent molecules. For 3 and its derivatives 10 and 14 similar low

2+

37

quantum yields were obtained. As was mentioned before, 3 was previously reported as a Hg -selective sensor

4

7

and as the product of a reaction of a BODIPY probe with biothiols. However, in the first case no quantum yields

were reported and in the second case a quantum yield of 16 % was obtained in a 1:1 DMSO/PBS mixture in the

presence of cysteine, which is difficult to compare with the present study.

When an acyl substituent is introduced on the 2-position of the tetramethyl-BODIPY core, a very modest red

shift and increase in quantum yield is observed, e.g., from 9 % to 12 % for 18 and 19 with respect to 10 in

methanol, but the other features remain the same. Comparing 15 and 16, the FWHM_e_mof 16 is consistently

larger, and their quantum yields do not follow the same trend, indicating they behave differently, despite their

structural similarities. For 11, a broad red shifted emission band with a maximum around 650 nm was observed

in toluene, which can be either due to the presence of aggregates or a charge transfer (CT) state. The excitation

spectrum resembles the absorption spectrum, so no aggregates are directly excited. The excited state of the

aggregates would then need to be populated through energy transfer, but this seems unlikely when measuring

in dilute conditions. However, when there is a CT state in equilibrium with the local excited state (the excited

state populated when exciting the BODIPY core) in toluene, we would expect significant stabilization of this CT

state in acetonitrile and methanol and hence further quenching of the LE emission in favor of the CT

33,47

emission.

It is however not appropriate to draw any conclusions based solely on the obtained quantum

yields of 3 % in toluene and 1 % in acetonitrile and methanol. Since the obtained results in organic solvents are

not necessarily representative for the performance of the synthesized dyes in microscopy on biological samples,

also the steady-state spectroscopy in Milli-Q water containing 0.5 % Triton X-100 was evaluated. Neutral dyes

48

are expected to be bound by hydrophobic interactions in the Stern layer of the micelles. This case represents a

much more viscous environment than the organic solvents reported here. For all dyes, the quantum yield was

enhanced markedly, indicating a hindered rotation around the meso bond, leading to slower non-radiative

decay. This result is not surprising, considering that meso-substituted BODIPY’s are often used as ‘turn-on’

3

6

viscosity probes. For two dyes, unpredictable behavior was noted. For compound 12, the absorption spectrum

is substantially broadened, and a shoulder at longer wavelengths can be discerned, and compound 16 even

38

features a distinct peak at lower energy than the 0-0 transition. In both cases, no separate emission could be

discerned when exciting this shoulder, and the FWHM_e_mis not changed with respect to the other reported

solvents, leading us to tentatively assume that these bands originate from non-fluorescent J-type aggregates or

dimers that form in the Stern layer of the micelles. For 11, again a red tail was observed in the emission. Since

1

1, 12, and 16 show a more complex and not completely understood behavior compared to the other

compounds, they might be less suitable for microscopy purposes. Based on the spectroscopic properties,

compounds 10, 13, 14, 15 and 17 are the most suitable for in vivo labelling.

3

.3. Cytotoxicity

The cytotoxicity of compounds 3, 10 - 19 was evaluated using propidium iodide (PI) staining. Briefly, HeLa cells

were treated with 10 µM of each compound for different time intervals (5-240 minutes), washed with PBS

38

buffer and stained with PI dye. A higher fluorescence signal corresponds to a lower cell viability. As a control

38

for cell death, cells were treated with Triton X-100 (concentration 0.2 %, 15 min).

Compound 17 present almost no cytotoxicity, even for incubation times of 240 min (Figure 3 and Figure S29).

Compound 3 had a small effect on the viability of the cells, reaching 25% of the viability measured when using

Triton X-100. Addition of compounds 12, 13, 14, 18 and 19 (Figure 3) for 1h had higher cytotoxicity,

(approximately 30-35%, when compared with Triton X-100, Figure 3). Compounds 10 and 15 lead to a reduced

cell viability (lower than treatment with Triton X-100). For both compounds, severe cell death was already

measurable after 5 min of treatment (Figure S29 and S34). Cytotoxicity of compound 11 increased with

treatment time showing similar toxicity to Triton X-100 after 180 minutes of incubation (Figure S30). The

cytotoxicity of compound 16 was approximately 60% of that of Triton X-100 (Figure 3). Summarizing, while

compounds 10, 11, 15 and 16 induced relatively high levels of cytotoxicity. Compounds 3 and 17 have negligible

effects in cell viability.

Figure 3. Viability of HeLa cells in the presence of compound 3, 10 – 19 compared with cell death inducer Triton

X-100. The cells were incubated with 10 µM of the compounds for 60 min. Data is presented as mean ± s.e.m.

from 4 repeats.

3

.4. Intracellular localization

To investigate the intracellular localization of the derivatives 3, 10 - 19, we examined their colocalization with

3

8

known organelles probes. Organelle probes (Mito-Tracker, ER-Tracker and Lyso-Tracker) and the synthesized

derivatives 3, 10 - 19 (10 μM) were incubated 1 hour in HeLa cells and then fixed. Afterwards, fluorescence

38

images were acquired using high content imaging (details in the methods section). The results showed that

practically all the tested compounds, except 12, could easily be loaded in the cells (Figure S17 – S26). Compound

1

2 could not be detected intracellularly in the imaging conditions employed. This is a consequence of its low

solubility in the incubation medium (DMEM) and subsequent low cellular loading. The Pearson’s colocalization

coefficient value (Rr) of the fluorescence images of the ER-tracker and the other BODIPY-derivatives were in the

38

range of 0.72 – 0.91. For mitochondria and lysosomes fluorescence images, the Rr were in the range of 0.27 –

.58 and 0.01 – 0.34, respectively (Figure 4). Using high content imaging, it was observed that all the derivatives

0

38

were mainly localized in the ER and partially in mitochondria. Compound 19 showed the highest value (Rr =

.91). Furthermore, the compound 16 was the one which showed the highest ER localization relation (Rr = 0.88)

0

compared with mitochondria (Rr = 0.31), and lysosomes (Rr = 0.15) (Figure 4).

Figure 4. Pearson’s coefficient values for the compounds 3, 10 – 19, except 12. Mito – tracker red (Blue); ER –

Tracker (Red); Lyso – Tracker (Light blue).

Based on the results obtained, we selected 17 as the best candidate for ER staining, due to its high quantum

yield (Φ = 55 %) in water, in combination with its low toxicity under the tested conditions. Therefore, to confirm

the ability of this dye as ER fluorescence tracer, HeLa cells were incubated 1 hour with 17 (10 mM) and ER –

Tracker red (2) (1 µM) and imaged using confocal fluorescence microscopy (Figure 5). Obtained results

confirmed that compound 17 is mainly staining the ER and highly colocalizes with the ER – Tracker (2) (Figure

5

G). We can also see how 17 has a similar intensity pattern compared with 2 when the fluorescence intensity

was determined in a cross-section of the image (Figure 5F).

Figure 5. Confocal fluorescence images of HeLa cells. A) Fluorescence image of compound 17 (10 µM, λex = 405

nm, collected at 420 - 480). B) Fluorescence image of ER – Tracker red 2 (500 nm, λex = 559 nm, collected at 590

–

640 nm). C) Merged image. D-F) Zoom in of intracellular structures marked with white square, displaying the

fluorescence signal of compound 17 (D) , ER-tracker (E) and both (F). G) Cross-section of the image in panels D

and E along the cyan dashed line.

4

. Conclusions

In conclusion, a small library of 11 compounds was designed and studied as potential molecules for fluorescence

staining of the ER. The compounds were easily obtained with a yield between 34 – 80%. Only the compounds 10

and 15 showed to be more cytotoxic than Triton X-100 (for an incubation time of 60 min). Compounds 3 and 17

showed negligible cytotoxic effects. According to the high content fluorescence images, all the compounds

labeled mainly the ER (Pearson colocalization coefficient between 0.72 – 0.91). Due to its high quantum yield in

water (Φ = 55 %) and low cytotoxicity, compound 17 was selected as a candidate for further colocalization

studies using confocal fluorescence microscopy. The results confirmed that the compound 17 highly colocalizes

with the commercial ER – Tracker Red, making it a potential fluorescent ER tracker. In conclusion, this class of

molecules may serve as a tool for ER imaging.

Acknowledgement

The authors thank Rik Nuyts and Carla Rios-Luci for the assistance with the confocal microscope and the high

content images, respectively. F. de Jong and J. Vandaele thank the Research Foundation – Flanders (FWO) for

PhD fellowship 1SC4719N and 1186220N. G. S-D also thanks Dr. Manuel Morales Fund for the postdoctoral

fellowship. Mass spectrometry was made possible by the support of the Hercules Foundation of the Flemish

Government (grant: 20100225-7).

References

1

2

3

4

5

. Vance, J. E. Phospholipid synthesis and transport in mammalian cells. Traffic 2015, 16 (1), 1–18.

https://doi: 10.1111/tra.12230.

. Anelli, T.; Sitia, R. Protein quality control in the early secretory pathway. EMBO J. 2008, 27 (2), 315–327.

https://doi: 10.1038/sj.emboj.7601974.

. Pizzo, P.; Pozzan, T. Mitochondria-endoplasmic reticulum choreography: structure and signaling. Trends

Cell Biol. 2007, 17 (10), 511–517.

. Ma, Y.; Hendershot, L. M. ER chaperone functions during normal and stress conditions. J. Chem.

Neuroanat. 2004, 28 (1-2), 51–65.

. Kim, I.; Xu, W.; Reed, J. C. Cell death and endoplasmic reticulum stress: diseases relevance and

therapeuthic opportunities. Nat. Rev. Drug Discovery, 2008, 7 (12), 1013–1030. https://doi:

1

0.1038/nrd2755.

6

. Shoulders, M. D.; Ryno, L. M.; Genereux, J. C.; Moresco, J. J.; Tu, P. G.; Wu, C.; Yates, J. R.; Su, Al.; Kelly, J.

W.; Wiseman, R. L. Stress-independent activation of XBP1s and/or ATF6 reveals three functionally

diverse ER proteostasis environments. Cell Rep., 2013,

0.1016/j.celrep.2013.03.024.

3

(4), 1279–1292. https://doi:

1

7

. Hetz, C. The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat.

Rev. Mol. Cell Biol., 2012, 13 (2), 89–102. https://doi: 10.1038/nrm3270.

8

. Raina, K.; Noblin, D. J.; Serebrenik, Y. V.; Adams,. A.; Zhao, C.; Crews, C. M. Targeted protein

destabilization reveals an estrogen-mediated ER stress response. Nat. Chem. Biol., 2014, 10 (11), 957–

9

62. https://doi: 10.1038/nchembio.1638.

. Maly, D.; Papa, F. R. Druggable sensors of the unfolded protein response. Nat. Chem. Biol., 2014, 10 (11),

92–901. https://doi: 10.1038/nchembio.1664.

0. Wang, M.; Kaufman, R. J. Protein misfolding in the endoplasmic reticulum as a conduit to human

disease. Nature, 2016, 529 (7586), 326–335. https://doi: 10.1038/nature17041.

1. Smith, M. H.; Ploegh, H. L.; Weissman, J. S. Road to ruin: targeting proteins for degradation in the

endoplasmic reticulum. Science, 2011, 334 (6059), 1086–1090. https://doi:10.1126/science.1209235.

2. Hotamisligil, G. S. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease Cell

9

1

8

2

010, 140 (6), 900-917. https://doi: 10.1016/j.cell.2010.02.034.

3. Lin, J.H. et al. Endoplasmic reticulum stress in disease pathogenesis. Annu. Rev. Pathol. Mech. Dis. 2008,

, 399-425.

3

4. Tse, Y. C.; Weissman, A. M. The Unfolded Protein Response, Degradation from Endoplasmic Reticulum

and Cancer. Genes Cancer 2010, 1, 764-778.

5. Xu, C.; Bailly-Maitre, B.; Reed, J. C. Endoplasmic reticulum stress: cell life and death decisions. J. Clin.

Invest. 2005, 115 (10), 2656-2664.

1

6. Lin, W.; Popko, B. Endoplasmic reticulum stress in disorders of myelinating cells. Nat. Neurosci. 2009, 12

4), 379-385. https://doi: 10.1038/nn.2273.

7. van Vliet AR, et.al. The ER Stress Sensor PERK Coordinates ER-Plasma Membrane Contact Site Formation

through Interaction with Filamin-A and F-Actin Remodeling. Mol Cell. 2017, 65 (5), 885-899.

https://doi.org/10.1016/j.molcel.2017.01.020.

(

1

8. Coffey, C. M.; Sheh, A.; Kim, I. S.; Chandran, K.; Nibert, M. L.; Parker, J. S. Reovirus outer capsid protein

micro1 induces apoptosis and associates with lipid droplets, endoplasmic reticulum, and mitochondria.

J. Virol. 2006, 80 (17), 8422-8438.

9. Nezu, A.; Parvin, M. N.; Turner, R. J. A conserved hydrophobic tetrad near the C terminus of the

+

-

secretory Na -K -2Cl cotransporter (NKCC1) is required for its correct intracellular processing. J. Biol.

Chem. 2009, 284 (11), 6869-6876. https://doi: 10.1074/jbc.M804302200.

2

0. Rizzuto, R.; Brini, M.; Pizzo, P.; Murgia, M.; Pozzan, T. Chimeric green fluorescent protein as a tool for

visualizing subcellular organelles in living cells. Curr. Biol. 1995, 5 (6), 635-642.

1. Chalfie, M.; Tu Y.; Euskirchen, G.; Ward, W. W.; Prasher, D. C. Green fluorescent protein as a marker for

gene expression. Science 1994, 263 (5148), 802-805.

2. Dolman, N. J.; Kilgore, J. A.; Davidson Focus, M. W. A Review of Reagents for Fluorescence Microscopy of

Cellular Compartments and Structures, Part I: BacMam Labeling and Reagents for Vesicular Structures.

Curr Protoc Cytom. 2013 12, 1-12.

2

3. Liu, Y.; Lok, C. N.; Ko, B. C.; Shum, T. Y.; Wong, M. K.; Che, C. M. Subcellular localization of a fluorescent

artemisinin derivative to endoplasmic reticulum. Org Lett. 2010, 12 (7), 1420-1423. https://doi:

1

0.1021/ol902890j.

4. Lee, H. W.; Cho, M. K.; Kim, H. R.; Lim, C. S.; Kang, C.; Kim, H. M. Visualization of vesicular transport from

the endoplasmic reticulum to lysosome using an amidine derived two-photon probe. Chem. Commun.

2

017, 53 (45), 6097-6100. https://doi: 10.1039/c7cc01518f.

5. Sunahara, H.; Urano, Y.; Kojima, H.; Nagano, T. Design and synthesis of a library of BODIPY-based

environmental polarity sensors utilizing photoinduced electron-transfer-controlled fluorescence ON/OFF

switching. J. Am. Chem. Soc. 2007, 129 (17), 5597-5604.

6. Colston, J.; Horobin, R. W.; Rashid-Doubell, F.; Pediani, J.; Johal, K. K. Why fluorescent probes for

endoplasmic reticulum are selective: an experimental and QSAR-modelling study. Biotech. Histochem.

2

003, 78 (6), 323-332.

7. Cole, L.; Davies, D. G.; Hyde, J.; Ashford, A. E. ER-Tracker dye and BODIPY-brefeldin A differentiate the

endoplasmic reticulum and golgi bodies from the tubular-vacuole system in living hyphae of Pisolithus

tinctorius. J. Microsc. 2000, 197 (Pt 3), 239 - 249.

2

8. Liu, H. S.; Jan, M. S.; Chou, C. K.; Chen, P. H.; Ke, N. J. Is green fluorescent protein toxic to the living cells?

Biochem. Biophys. Res. Commun. 1999, 260 (3), 712-717.

9. Hoogenkamp, M. A.; Crielaard, W.; Krom, B. P. Uses and limitations of green fluorescent protein as a

viability marker in Enterococcus faecalis: An observational investigation. J. Microbiol. Methods, 2015,

1

15, 57-63. https://doi: 10.1016/j.mimet.2015.05.020.

3

0. Kalyanaraman, B.; Zielonka, J. Green fluorescent proteins induce oxidative stress in cells: A worrisome

new wrinkle in the application of the GFP reporter system to biological systems? Redox. Biol., 2017, 12,

7

55 - 757. https://doi: 10.1016/j.redox.2017.03.019.

3

1. Information found in Termo-fisher https://www.thermofisher.com/be/en/home.html.

2. Zünkler, B. J.; Wos-Maganga, M.; Panten, U. Fluorescence microscopy studies with a fluorescent

+

glibenclamide derivative, a high-affinity blocker of pancreatic beta-cell ATP-sensitive K currents.

Biochem. Pharmacol. 2004, 67 (8), 1437−1444.

3

3. Qin, W.; Dou, W.; Leen, V.; Dehaen, W.; Van der Auweraer, M.; Boens N. A ratiometric, fluorescent

BODIPY-based probe for transition and heavy metal ions. RSC Advances. 2016, 6, 7806-7816

.

https://doi.org/10.1039/C5RA23751C

3

4. Chong, H.; Fron, E.; Liu. Z.; Boodts, S.; Thomas, J.; Harvey, J. N.; Hofkens, J.; Dehaen, W.; Van der

3

Auweraer, M.; Smet, M. Acid-Sensitive BODIPY Dyes: Synthesis through Pd-Catalyzed Direct C(sp )-H

Arylation and Photophysics. Chem. Eur. J. 2017, 23 (19), 4687-4699. https://doi:

1

0.1002/chem.201700015.

5. Yin, S.; Leen, V.; Van Snick, S.; Boens, N.; Dehaen, W. A highly sensitive, selective, colorimetric and near-

2+

infrared fluorescent turn-on chemosensor for Cu based on BODIPY. Chem. Commun. 2010, 46 (34),

329-31. https://doi.org/10.1039/C0CC01772H.

6. Boens, N, Leen, V.; Dehaen, W. Fluorescent indicators based on BODIPY. Chem. Soc. Rev. 2012, 41 (3),

130-1172. https://doi: 10.1039/c1cs15132k.

7. Moon, S. Y.; Cha, N. R.; Kim, Y. H.; Chang, S. K. New Hg -selective chromo- and fluoroionophore based

upon 8-hydroxyquinoline. J. Org. Chem. 2004, 69 (1), 181-183.

6

3

1

2

+

3

8. See supporting information

9. Mirri, G.; Schoenmakers, D. C.; Kouwer, P. H.; Veranič, P.; Muševič, I.; Štefane, B. Synthesis of Functional

Fluorescent BODIPY-based Dyes through Electrophilic Aromatic Substitution: Straightforward Approach

towards Customized Fluorescent Probes. Chemistry Open 2016, 5 (5), 450-454.

4

0. Bai, Y.; Shi, X.; Chen, Y.; Zhu, C.; Jiao, Y.; Han, Z.; He, W.; Guo, Z. Coumarin/BODIPY Hybridisation for

Ratiometric Sensing of Intracellular Polarity Oscillation. Chem. Eur. J. 2018, 24 (29), 7513-7524.

https://doi:10.1002/chem.201800915.

1. Qin, W.; Baruah, M.; Van der Auweraer, M.; De Schryver, F. C.; Boens, N. Photophysical Properties of

Borondipyrromethene Analogues in Solution. J. Phys. Chem. A 2005, 109 (33), 7371–7384.

https://doi.org/10.1021/jp052626n.

2. Baruah, M.; Qin, W.; Flors, C.; Hofkens, J.; Vallée, R. A. L.; Beljonne, D.; Van Der Auweraer, M.; De

Borggraeve, W. M.; Boens, N. Solvent and PH Dependent Fluorescent Properties of a

Dimethylaminostyryl Borondipyrromethene Dye in Solution. J. Phys. Chem. A 2006, 110 (18), 5998–6009.

https://doi.org/10.1021/jp054878u.

4

3. Bricks, J. L.; Kovalchuk, A.; Trieflinger, C.; Nofz, M.; Büschel, M.; Tolmachev, A. I.; Daub, J.; Rurack, K. On

the Development of Sensor Molecules That Display Fe III -Amplified Fluorescence. J. Am. Chem. Soc.

2

005, 127 (39), 13522–13529. https://doi.org/10.1021/ja050652t.

4. Cunha Dias De Rezende, L.; Menezes Vaidergorn, M.; Biazzotto Moraes, J. C.; Da Silva Emery, F.

Synthesis, Photophysical Properties and Solvatochromism of Meso-Substituted Tetramethyl BODIPY

Dyes. J. Fluoresc. 2014, 24 (1), 257–266. https://doi.org/10.1007/s10895-013-1293-8.

4

5. Prlj, A.; Fabrizio, A.; Corminboeuf, C. Rationalizing Fluorescence Quenching in Meso-BODIPY Dyes. Phys.

Chem. Chem. Phys. 2016, 18 (48), 32668–32672. https://doi.org/10.1039/C6CP06799A.

6. Li, F.; Yang, S. I.; Ciringh, Y.; Seth, J.; Martin, C. H.; Singh, D. L.; Kim, D.; Birge, R. R.; Bocian, D. F.; Holten,

D.; et al. Design, Synthesis, and Photodynamics of Light-Harvesting Arrays Comprised of a Porphyrin and

One, Two, or Eight Boron-Dipyrrin Accessory Pigments. J. Am. Chem. Soc. 1998, 120 (39), 10001–10017.

https://doi.org/10.1021/ja9812047.

4

7. Wang, L.; Qian, Y. A Novel Quinoline-BODIPY Fluorescent Probe for Fast Sensing Biothiols via Hydrogen

Bonds Assisted-Deprotonation Mechanism and Its Application in Cells and Zebrafish Imaging. J.

Photochem.

Photobiol.

A

Chem.

2019,

372,

122–130.

https://doi.org/10.1016/j.jphotochem.2018.12.013.

4

8. de Jong, F.; Feldt, M.; Feldt, J.; Harvey, J. N. Modelling Absorption and Emission of a: Meso -Aniline-

BODIPY Based Dye with Molecular Mechanics. Phys. Chem. Chem. Phys. 2018, 20 (21), 14537–14544.

https://doi.org/10.1039/c8cp01877d.

9. Van der Auweraer, M.; Dederen, C.; Palmans-Windels, C.; De Schryver, F. C. Fluorescence Quenching by

Neutral Molecules in Sodium Dodecyl Sulfate Micelles. J. Am. Chem. Soc. 1982, 104 (7), 1800–1804.

5

0. Shavaleev, N. M.; Scopelliti, R.; Gumy, F.; Bünzli, J. C. Modulating the near-infrared luminescence of

neodymium and ytterbium complexes with tridentate ligands based on benzoxazole-substituted 8-

hydroxyquinolines. Inorg. Chem., 2009, 48 (7), 2908-2918. https://doi: 10.1021/ic8020468.

1. Baig, M. F.; Shaik, S. P.; Nayak, V. L.; Alarifi, A.; Kamal, A. Iodine-catalyzed Csp3-H functionalization of

methylhetarenes: One-pot synthesis and cytotoxic evaluation of heteroarenyl-benzimidazoles and

benzothiazole. Bioorg. Med. Chem. Lett. 2017, 27 (17), 4039

0.1016/j.bmcl.2017.07.051.

2. De Mello, J. C.; Wittmann, H. F.; Friend, R. H. An Improved Experimental Determination of External

Photoluminescence Quantum Efficiency. Adv. Mater. 1997, (3), 230–232.

https://doi.org/10.1002/adma.19970090308.

–

4043. https://doi:

1

5

9

3. Porres, L.; Holland, A.; Palsson, L. O.; Monkman, A. P.; Kemp, C.; Beeby, A. Absolute Measurements of

Photoluminescence Quantum Yields of Solutions Using an Integrating Sphere. J. Fluoresc. 2006, 16 (2),

2

67–272. https://doi.org/10.1007/s10895-005-0054-8.

Highlights

•

Visualization of the structure of the Endoplasmic Reticulum by fluorescence microscopy

Fluorescent dyes for Endoplasmic Reticulum visualization

Cytotoxicity of Fluorescent dyes in HeLa cells

Photophysical characterization and quantum yields determination of novel fluorescence dyes

Declaration of interests

☒

The authors declare that they have no known competing financial interests or personal relationships

that could have appeared to influence the work reported in this paper.

☐

The authors declare the following financial interests/personal relationships which may be considered

as potential competing interests:

Article Doi

Development and characterization of BODIPY-derived tracers for fluorescent labeling of the endoplasmic reticulum

DOI: 10.1016/j.dyepig.2020.108200

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.dyepig.2020.108200

Full text of DOI:10.1016/j.dyepig.2020.108200

Products guided by the article

R&D Labs maybe for 51219-89-7

Relevant to this article

Hot Product