Detection in a Live Tissue by Two-Photon Microscopy ...Br B-NH 2 (a) (b) 1 2 O O N O CH 2 OH C C (c)...

S1

Supporting Information

Two-Photon Probes for the Endoplasmic Reticulum: Its

Detection in a Live Tissue by Two-Photon Microscopy

Ji-Woo Choia,‡, Seung-Hye Choia,‡, Seung Taek Honga, Mun Seok Kimb, Seong Shick Ryua, Yeo Uk Yoonb, Kyu Cheol Paikb, Man So Hanb, Taebo Sima,c, and Bong Rae Choa,b,d,*

a KU-KIST Graduate School of Converging Science and Technology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Koreab Department of Chemistry, Daejin University, 1007 Hoguk-ro, Pocheon-si, Gyeonggi-do 11159, Republic of Koreac Severance Biomedical Science Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Koread Department of Chemistry, Korea University, 145 Anam-ro, Sungbuk-gu, Seoul 02841, Republic of Korea* Corresponding author E-mail: [email protected]

TABLE OF CONTENTS Page

Synthesis………………………..…………………………………………………... S5

Spectroscopic Properties……..……………………………………………………. S13

Solubility………………………..………………………………………………….. S13

Two-Photon Microscopy…..………………………………………………………. S13

Measurements of TP Action Cross-Section (Фδmax) and Effective TP Action Cross-Section (Фδeff)……………………………………………………………….. S14

Cell Preparation..…………………………………………………………………... S14

Tissue Preparation…………………..……………………………………………... S14

Detection Windows…………………..…………………………………………….. S15

Cell Imaging by High-resolution S16

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2020

S2

OPM……………………………………………

Cell Imaging by OPM and TPM………………………………………………….. S16

Tissue Imaging by TPM…………………………………………………………… S16

Cytotoxicity………………………………………………………………………… S16

Photostability………………………………………………………………………. S16

A Western Blot Assay……………………………………………………………… S17

Statistical Analysis…………………………………………………………………. S17

Figure S1. Synthesis of BER-blue and FER-green. (a) i) Benzoxazole, Pd(OAc)2, CuI, PPh3, Cs2CO3, dimethylformamide (DMF), 145C. (b) Benzothiazole, Pd(OAc)2, CuI, PPh3, Cs2CO3, DMF, 145C. (c) (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl [TEMPO], NaOCl, CH2Cl2. (d) I, NaBH4, DMF, room temperature (rt). (e) Toluene, 140C. (f) Ac-CFFKDEL, P(CH2CH2CO2H)3, Et3N, dimethyl sulfoxide (DMSO), rt…………………………………………………………………………... S5

Figure S2. (a,b) Partial 1H NMR spectra of B-Mal (a) and BER-blue (b) in DMSO in the region 8.66.3. The protons in the B-Mal and Ac-CFFKDEL moieties are designated as ak, *, F2, and F3, respectively. (c) 2D 1H-1H TOCSY spectrum of BER-blue in the NH-Ha fingerprint region. Each line indicates amide-to-side chain connectivity. (d) Overlay of TOCSY (blue) and NOESY (red) spectra. The lines trace the sequential backbone assignment via intramolecular NH–Hα NOE connections. (e) An overlay of 1H-1H NOESY (black) and TOCSY (red) spectra showing NOE peaks between protons on succinimide and Cys moieties………………...……….... S10

Table S1. Chemical shifts of each proton in the fingerprint region of the TOCSY spectrum of BER-blue in DMSO-d6……………………………………………..…... S10

Figure S3. (a,b) Partial 1H NMR spectra of F-Mal (a) and FER-green (b) in DMSO in the region 8.46.5. The protons in the F-Mal and Ac-CFFKDEL moieties are designated as ak, *, F2, and F3, respectively. (c) 2D 1H-1H TOCSY spectrum of FER-green in the NH-Ha fingerprint region. Each line indicates amide-to-side chain connectivity. (d) Overlay of TOCSY (red) and NOESY (green) spectra. The lines trace the sequential backbone assignment via intramolecular NH–Hα NOE connections………………………………………………………………………….. S12

Table S2. Chemical shifts of each proton in the fingerprint region of the TOCSY spectrum of FER-green in DMSO-d6……………………………………………...... S12

Table S3. Spectroscopic properties…………………………………………………. S13

S3

Table S4. Detection windows for the OPM and TPM imaging…………………….. S15

Figure S4. Normalized absorption (a) and emission (b) spectra of BER-blue and FER-green in phosphate-buffered saline (PBS) and EtOH…………………………... S18

Figure S5. (a,c) Fluorescence spectra of BER-blue (a) and FER-green (c), and plots (b,d) of the fluorescence intensity against probe concentration for BER-blue (b) and FER-green (d) in PBS. The excitation wavelengths for BER-blue and FER-green were 354 and 373 nm, respectively………………………………………………….. S18

Figure S6. (a) Normalized TPEF spectra of BER-blue (blue), FER-green (green), PLT-yellow (orange), and ABI-Nu (purple) and a normalized fluorescence spectrum of ETR (red) in HeLa cells. The wavelengths for the one- and two-photon excitation were 405 and 750 nm, respectively. (b) TP excitation spectra of BER-blue and FER-green in EtOH and PBS, respectively………………………………………………... S19

Figure S7. Viability of HeLa cells in the presence of BER-blue (a,b) or FER-green (c,d), as measured by a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were incubated with the probes (010 M) for 024 h……………………………………………………………………………………... S19

Figure S8. (a,c) Two-photon microscopy images of HeLa cells labeled with BER-blue (a) or FER-green (c). (b,d) Relative TPEF intensity as a function of time. The digitized intensity was recorded with 2 s intervals during 1 h in xyt mode. The TPEF intensities at positions AF were recorded at 380–660 nm upon excitation at 750 nm. Scale bar: 30 µm………………………………………………………………… S20

Figure S9. Effects of pH on the one-photon fluorescence intensity of BER-blue and FER-green (2 μM each in universal buffer: 0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, and 0.1 M KCl)……………………………………………….. S20

Figure S10. Sectional TPM images of a rat hippocampal slice colabeled with BER-blue (10 μM) and FER-green (6 μM). The images were captured in channel 3 (Ch3: BER-blue, 385–450 nm) and Ch6 (FER-green, 535–655 nm) at a depth of 90–165 μm and 10× magnification after excitation at 750 nm…………………….................. S21

Figure S11. 1H NMR spectrum (300 MHz) of compound I in DMSO-d6…………. S22

Figure S12. 13C NMR spectrum (75 MHz) of compound I in DMSO-d6.................. S22

Figure S13. 1H NMR spectrum (300 MHz) of compound B-NH2 in CDCl3…...….. S23

Figure S14. 13C NMR spectrum (75 MHz) of compound B-NH2 in CDCl3…...…... S23

S4

Figure S15. 1H NMR spectrum (300 MHz) of compound 3 in CDCl3.…………….. S24

Figure S16. 13C NMR spectrum (75 MHz) of compound 3 in CDCl3..…………….. S24

Figure S17. 1H NMR spectrum (800 MHz) of B-Mal in DMSO-d6……...………... S25

Figure S18. 13C NMR spectrum (75 MHz) of B-Mal in CDCl3/CD3OD (10/1)…… S25

Figure S19. 1H NMR spectrum (300 MHz) of compound F-NH2 in CDCl3..……… S26

Figure S20. 13C NMR spectrum (75 MHz) of compound F-NH2 in CDCl3……….. S26

Figure S21. 1H NMR spectrum (300 MHz) of compound 4 in CDCl3.…………….. S27

Figure S22. 13C NMR spectrum (75 MHz) of compound 4 in CDCl3..…………….. S27

Figure S23. 1H NMR spectrum (800 MHz) of F-Mal in DMSO-d6….……………. S28

Figure S24. 13C NMR spectrum (75 MHz) of F-Mal in CDCl3……………………. S28

Figure S25. 1H NMR spectrum (800 MHz) of BER-blue in DMSO-d6......……….. S29

Figure S26. 1H NMR spectrum (800 MHz) of FER-green in DMSO-d6…...……... S29

Figure S27. HRMS of BER-blue. TOF MS: time-of-flight mass spectrometry….... S30

Figure S28. HRMS of FER-green. TOF MS: time-of-flight mass spectrometry...... S30

References………………………………………………………………………….. S31

S5

Synthesis

Compounds 1 and 2 were available from earlier works,1,2 and compound C was prepared by

a method from the literature.3 The synthesis of BER-blue and FER-green is shown below.

Figure S1. Synthesis of BER-blue and FER-green. (a) i) Benzoxazole, Pd(OAc)2, CuI, PPh3, Cs2CO3, dimethylformamide (DMF), 145C. (b) Benzothiazole, Pd(OAc)2, CuI, PPh3, Cs2CO3, DMF, 145C. (c) (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl [TEMPO], NaOCl, CH2Cl2. (d) I, NaBH4, DMF, room temperature (rt). (e) Toluene, 140C. (f) Ac-CFFKDEL, P(CH2CH2CO2H)3, Et3N, dimethyl sulfoxide (DMSO), rt.

Compound I

N

O

O

O

O

A solution of NaOCl (aq) (4%, 20 mL) was added dropwise to a mixture of compound C (1.12 g, 5 mmol), NaHCO3 (0.84 g, 10 mmol), KBr (0.71 g, 6 mmol), and TEMPO (0.02 g, 0.13 mmol) in CH2Cl2 (10 mL), and the resultant solution was stirred for 2 h at 0°C. The product was extracted with CH2Cl2 (3 × 20 mL), washed with 10% Na2S2O3 (aq) (3 × 10 mL) and brine (10 mL) and dried over MgSO4, and the solvent was evaporated. The crude product was purified by silica gel chromatography with a 010% gradient of MeOH/CH2Cl2 as the eluent. Yield: 1.01 g (96%); 1H NMR (300 MHz, DMSO-d6): δ 9.57 (1 H, t, J = 1.5 Hz), 6.53 (2 H, s),

Ar O

O

N

NH

Ar NH2Ar = B (B-NH2)

F (F-NH2)

Ar O

O

N

NHS

Ac-CFFKDELAr = B (B-Mal)

F (F-Mal)Ar = B (BER-blue)

F (FER-green)

(f)

Ar O

O

N

NH

Ar = B (3)F (4)

(e)O(d)

N

O

B FN

S

I NH2F-NH2

NH2S

N

N

O

NH2

NH2

Br B-NH2

(a)

(b)1

2

O

NO

OCH2OH

C

C(c)

O

NO

OCHO

I

S6

5.10 (2 H, s), 3.61 (2 H, t, J = 6.9 Hz), 2.90 (2 H, s), 2.61 (1 H, td, J = 7.0, 1.5 Hz) ppm; 13C NMR (75 MHz, DMSO-d6): δ 201.8, 177.0, 137.2(2C), 81.0(2C), 47.9(2C), 41.6, 32.6 ppm.

Compound B-NH2

NH2O

N

A flame-dried round-bottom flask with a magnetic stirring bar was loaded with CuI (0.09 g, 0.47 mmol), Pd(OAc)2 (0.03 g, 0.11 mmol), PPh3 (0.31 g, 0.12 mmol), Cs2CO3 (1.7 g, 4.7 mmol), benzoxazole (0.56 g, 4.7 mmol), and 1 (0.52 g, 2.3 mmol). Anhydrous DMF (1.0 mL) was then added to the mixture using a syringe in an atmosphere of Ar. The reaction mixture was heated to 145°C for 20 min, cooled to rt, and filtered through a small pad of Celite. The solid residue was washed with CH2Cl2 (10 mL), and the combined organic layers were evaporated. The product was purified by silica gel column chromatography with 50% EtOAc in n-hexane. Yield: 0.45 g (74%); 1H NMR (300 MHz, CDCl3): δ 8.62 (1 H, d, J = 1.7 Hz), 8.19 (1 H, dd, J = 8.5, 1.7 Hz), 7.80−7.77 (2 H, m), 7.69 (1 H, d, J = 8.5 Hz), 7.61−7.58 (1 H, m), 7.38–7.32 (2 H, m), 6.98−7.00 (2 H, m), 4.06 (2 H, br, s) ppm; 13C NMR (75 MHz, CDCl3): δ 163.7, 150.7, 146.1, 142.3, 136.6, 130.5, 128.2, 127.1, 126.4, 124.7, 124.5, 124.4, 120.7, 119.6, 118.9, 110.4, 108.1 ppm.

Compound 3

O

O

O

N

N

O

NH

A solution containing B-NH2 (0.72 g, 2.8 mmol) and I (0.61 g, 2.8 mmol) in anhydrous DMF was purged with argon several times with stirring. NaBH4 (0.58 g, 4.2 mmol) was added to this solution, and the reaction mixture was stirred vigorously for 2 h under argon at rt. The organic layer was separated, washed with saturated brine, dried over MgSO4, and filtered. The solvent was evaporated, and the crude product was purified by silica gel column chromatography with 30–50% ethyl acetate in n-hexane. Yield: 0.98 g (76%); 1H NMR (300 MHz, CDCl3): δ 8.58 (1 H, d, J = 1.7 Hz), 8.17 (1 H, dd, J = 8.5, 1.7 Hz), 7.787.68 (3 H, m), 7.60−7.57 (1 H, m), 7.35−7.32 (2 H, m), 6.95 (1 H, dd, J = 8.8, 2.2 Hz), 6.79 (1 H, d, J = 2.2 Hz), 6.52 (2 H, s), 5.29 (2 H, s), 4.52 (1 H, brs), 3.64 (2 H, t, J = 6.5 Hz), 3.25 (2 H, t, J = 6.3 Hz), 2.86 (2 H, s), 1.95 (2 H, quin, J = 6.3 Hz) ppm; 13C NMR (75 MHz, CDCl3): δ 176.6(2C), 163.9, 150.7,

S7

147.2, 142.4, 137.0, 136.5(2C), 130.2, 128.1, 126.5, 126.4, 124.5(2C), 124.4, 120.0, 119.6, 118.8, 110.3, 103.7, 81.0(2C), 47.4(2C), 40.0, 36.3, 26.4 ppm.

Compound B-Mal

NHO

N

N

O

O

A solution of 3 (0.33 g, 0.71 mmol) in toluene (50 mL) was refluxed overnight. The solvent was evaporated, and the crude product was purified by silica gel column chromatography with 30–50% ethyl acetate in n-hexane. Yield: 0.25 g (88%); 1H NMR (800 MHz, DMSO-d6): δ 8.54 (1 H, d, J = 1.4 Hz), 8.05 (1 H, dd, J = 8.4, 1.4 Hz), 7.84 (1 H, d, J = 8.8 Hz), 7.797.77 (2 H, m), 7.73 (1 H, d, J = 8.4 Hz), 7.417.40 (2 H, m), 7.07 (1 H, dd, J = 8.8, 1.9 Hz), 7.04 (2 H, s), 6.76 (1 H, d, J = 1.9 Hz), 6.39 (1 H, t, J = 5.6 Hz), 3.57 (2 H, t, J = 6.9 Hz), 3.16 (2 H, td, J = 6.9, 5.6 Hz), 1.88 (2 H, quin, J = 6.9 Hz); 13C NMR (75 MHz, CDCl3): δ 171.1(2C), 164.0, 150.4, 147.3, 141.8, 137.0, 134.1(2C), 130.1, 128.1, 126.4(2C), 124.6, 124.4, 124.3, 119.6, 119.2, 118.7, 110.3, 103.5, 40.1, 35.2, 27.4 ppm.

Compound F-NH2

NH2N

S

A flame-dried round-bottom flask with a magnetic stirring bar was loaded with CuI (0.05 g, 0.02 mmol), Pd(OAc)2 (0.01 g, 0.006 mmol), PPh3 (0.16 g, 0.06 mmol), Cs2CO3 (0.5 g, 1.4 mmol), benzothiazole (0.13 g, 1.2 mmol), and 2 (0.4 g, 1.2 mmol). Next, anhydrous DMF (1.0 mL) was added to the mixture via a syringe under Ar. The reaction mixture was heated to 145°C for 30 min, cooled to rt, and filtered through a small pad of Celite. The solid residue was washed with CH2Cl2 (10 mL), and the combined organic layers were evaporated. The product was purified by silica gel column chromatography with 10−25% EtOAc in n-hexane. Yield: 0.18 g (44%); 1H NMR (300 MHz, CDCl3): δ 8.26 (1 H, d, J = 1.7 Hz), 8.18 (1 H, d, J = 8.0 Hz), 7.99 (1 H, dd, J = 8.0, 1.7 Hz), 7.88 (1 H, dd, J = 8.0, 1.1 Hz), 7.62 (1 H, d, J = 8.0 Hz), 7.52 (1 H, d, J = 8.2 Hz), 7.52 (1 H, td, J = 7.5, 1.1 Hz), 7.37 (1 H, td, J = 7.5, 1.1 Hz), 6.73 (1 H, d, J = 2.1 Hz), 6.65 (1 H, dd, J = 8.2, 2.1 Hz), 3.97 (2 H, br. s.), 1.53 (6 H, s) ppm ; 13C NMR (75 MHz, CDCl3): δ 168.7, 156.2, 153.9, 153.2, 147.1, 142.7, 134.6, 130.3, 128.5, 127.1, 126.0, 124.6, 122.5, 121.4, 121.3, 120.9, 118.7, 113.9, 108.9, 46.5, 26.9(2C) ppm.

S8

Compound 4

S

NO

O

O

NNH

Compound 4 was prepared from F-NH2 (0.38 g, 1.11 mmol) and I (0.25 g, 1.11 mmol) as described for 3. Yield: 0.31 g (51%); 1H NMR (300 MHz, CDCl3): δ 8.14 (1 H, d, J = 1.7 Hz), 8.07 (1 H, d, J = 8.0 Hz), 7.94 (1 H, dd, J = 8.0, 1.7 Hz), 7.86 (1 H, dd, J = 8.0 Hz), 7.59 (1 H, d, J = 8.0 Hz), 7.53 (1 H, d, J = 8.2 Hz), 7.47 (1 H, td, J = 7.6, 1.1 Hz), 7.34 (1 H, td, J = 7.6, 1.1 Hz), 6.68 (1 H, d, J = 2.1 Hz), 6.59 (1 H, dd, J = 8.2, 2.1 Hz), 6.45 (2 H, s), 5.25 (2 H, s), 3.62 (2 H, t, J = 6.6 Hz), 3.18 (2 H, t, J = 6.5 Hz), 2.80 (2 H, s), 1.89 (2 H, quin, J = 6.4 Hz), 1.52 (6 H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 176.4(2C), 168.8, 156.3, 154.0, 153.2, 148.3, 143.0, 136.3(2C), 134.7, 130.1, 127.7, 127.1, 126.0, 124.6, 122.6, 121.6, 121.4, 121.0, 118.6, 111.8, 106.7, 80.8(2C), 47.3(2C), 46.6, 40.4, 36.2, 27.1(2C), 26.5 ppm.

Compound F-Mal

S

N

O

O

NNH

F-Mal was synthesized from compound 4 (0.31 g, 0.57 mmol) as described for B-Mal. Yield: 0.22 g (81%); 1H NMR (800 MHz, CDCl3): δ 8.31 (1 H, s), 8.13 (1 H, d, J = 1.4 Hz), 8.10 (1 H, d, J = 7.8 Hz), 8.04 (1 H, d, J = 7.8 Hz), 7.94 (1 H, dd, J = 7.8, 1.4 Hz), 7.70 (1 H, d, J = 7.8 Hz), 7.59 (1 H, d, J = 8.3 Hz), 7.52 (1 H, t, J = 7.8 Hz), 7.43 (1 H, t, J = 7.8 Hz), 7.02 (2 H, s), 6.72 (1 H, d, J = 1. Hz), 6.58 (1 H, dd, J = 8.3, 1.2 Hz), 3.54 (2 H, t, J = 7.0 Hz), 3.10 (2 H, t, J = 7.0 Hz), 1.82 (2 H, quin, J = 7.0 Hz), 1.43 (6 H, s) ppm; 13C NMR (75 MHz, CDCl3): δ 170.6(2C), 168.7, 156.2, 153.9, 153.1, 148.3, 142.8, 134.5, 133.7(2C), 130.0, 127.6, 127.1, 125.9, 124.5, 122.5, 121.5, 121.3, 120.9, 118.5, 111.7, 106.5, 46.5, 40.5, 35.0, 27.5, 27.0(2C) ppm.

S9

BER-blue

A mixture of B-Mal (0.05 g, 0.13 mmol), Et3N (0.02 g, 0.17 mmol), (HO2CCH2CH2)P (0.0003 g, 0.001 mmol), and an endoplasmic-reticulum–targeting peptide (Ac-CFFKDEL, InterPharm, Gyeonggi-do, Korea) (0.12 g, 0.13 mmol) in DMSO was stirred at rt for 2 h. The crude product was purified by semipreparative reverse-phase high-performance liquid chromatography (RP-HPLC, SunFire Preparative Column C18 OBD, 5 µm, 19 50 mm) on a Waters HPLC system (Waters Corporation, Milford, MA) using step gradient elution with 30–90% MeOH/H2O at flow rate 25 mL/min during 10 min. Yield: 0.09 g (51%); 1H NMR (800 MHz, DMSO-d6): δ 8.54 (1 H, s), 8.26−8.18 (4 H, m), 8.05 (1 H, d, J = 8.8 Hz), 8.00 (2 H, br. s.), 7.86−7.83 (2 H, m), 7.78−7.77 (2 H, m), 7.73 (1 H, d, J = 8.3 Hz), 7.41−7.40 (2 H, m), 7.25 (4 H, s.), 7.20−7.16 (6 H, m), 7.08 (1 H, d, J = 8.8 Hz), 6.77 (1 H, s), 6.39 (1 H, s), 4.57−4.55 (2 H, m), 4.49−4.45 (2 H, m), 4.27 (2 H, m), 4.16−4.15 (2 H, m), 4.03−4.02 (1 H, m), 3.96−3.95 (1 H, m), 3.60−3.54 (4 H, m), 7.86−7.83 (2 H, m), 3.40 (1 H, m), 3.19−3.13 (5 H, m), 3.07−3.04 (3 H, m), 2.99−2.97 (2 H, m), 2.94−2.87 (2 H, m), 2.84 (2 H, br. s.), 2.75−2.67 (6 H, m), 2.55−2.47 (4 H, m), 2.27−2.21 (2 H, m), 1.95 (1 H, m), 1.86−1.82 (4 H, m), 1.76 (1 H, m), 1.69 (1 H, m), 1.64−1.63 (1 H, m), 1.54−1.50 (5 H, m), 1.36−1.33 (2 H, m), 0.88 (3 H, d, J = 6.4 Hz), 0.83 (3 H, d, J = 6.4 Hz) ppm; high-resolution mass spectrometry (HRMS) (electrospray ionization): m/z calcd. for [C68H81N11O16S+H+]: 1339.5583, found: 1340.5662.

The partial 1H NMR spectrum of BER-blue in DMSO in the region 8.66.3 revealed all the proton peaks of B-Mal [except for the vinylic protons at 7.03 (h)] and new peaks due to the aromatic protons of the phenylalanine moiety at 7.37.1 (10 H) and N-H protons (*) of the CFFKDEL moiety at 8.288.13 (4 H), 8.037.97 (2 H), and 7.887.87 (1 H; Figure S2a,b). In addition, the 2D 1H-1H TOCSY spectrum and an overlay of TOCSY and NOESY spectra are consistent with the amide-to-side chain and amino acid residue connectivity in BER-blue (Figure S2c,d and Table S1). The chemical shifts of each proton in the fingerprint region of the TOCSY spectrum are summarized in Table S1. Furthermore, high-resolution mass spectrometry (HRMS) result on BER-blue molecular-weight ion peak at m/e = 1340.5662 (calcd. 1339.5583) (Figures S27). These results confirm that the fluorophores are linked to HS-Ac-CFFKDEL via a sulfide bond and that BER-blue represents a 1:1 mixture of two diastereomers produced after the addition of a thiol group to the maleimide moiety of B-Mal.

S10

Figure S2. (a,b) Partial 1H NMR spectra of B-Mal (a) and BER-blue (b) in DMSO in the region 8.66.3. The protons in the B-Mal and Ac-CFFKDEL moieties are designated as ak, *, F2, and F3, respectively. (c) 2D 1H-1H TOCSY spectrum of BER-blue in the NH-Ha fingerprint region. Each line indicates amide-to-side chain connectivity. (d) Overlay of TOCSY (blue) and NOESY (red) spectra. The lines trace the sequential backbone assignment via intramolecular NH–Hα NOE connections. (e) An overlay of 1H-1H NOESY (black) and TOCSY (red) spectra showing NOE peaks between protons on succinimide and Cys moieties.

Table S1. Chemical shifts of each proton in the fingerprint region of the TOCSY spectrum of BER-blue in DMSO-d6.

BER-blueResidue NH Hα Hβ, Hβ’ Hγ, Hγ’ Hδ, Hδ’ Hε, Hε’ Hζ

Cys1 8.18 4.47 3.07, 2.84Cys1a 8.18 4.45 3.05, 2.90Phe2 7.99 4.47 2.98, 2.76 7.25 7.16 7.17

Phe2a 8.01 4.48 2.98, 2.76Phe3 8.20 4.55 2.90, 2.70 7.25 7.15 7.17

Phe3a 8.15 4.56 2.90, 2.70Lys4 8.22 4.27 1.68, 1.54 1.33 1.53 2.75Asp5 8.25 4.55 2.66,2.54 6.30Glu6 7.85 4.27 1.94, 1.76 2.24 2.82Leu7 7.99 4.15 1.51 1.62 0.87, 0.82

[2] (BER-1D).esp

8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1Chemical Shift (ppm)

ab

f, gj

e, hc,*i

d *(4H)*(2H)

(b) BER-blue F2,F3 (10H)

k

[1] (BMAL-1D).esp


a

h

bf, gje, hcid

(a) B-Mal

k

[1] (BMAL-1D).esp

8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3 6.2 6.1Chemical Shift (ppm)[2] (BER-1D).esp


//

//

HNO

N N

O

O

O

NH ONH

OHN

ONH

NH2

OHN

O

OH ONH

O OH

OHN

OH

O

S

N

O

O

NH(CH2)3B-Mal

BER-blue

B

*

**

**

**

a

bcde

h ij k

Ac-C1F2

F3

K4

D5

E6

L7

13

h

4,4’

5β αf

g

(5a)

4’ 4’a

C1Hβ:5

C1aHβ:5a

4 4a

C1Hα:5

C1aHα:5a

55a

δ 1H (ppm)

δ1 H

(ppm

)

4.0

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F3

D5

F2

C1Hα:F2NHK4

E6

L7

δ1 H

(ppm

)

δ 1H (ppm)

F2Hα:F3NHF3Hα:K4NHK4Hα:D5NHD5Hα:E6NHE6Hα:L7NH

C1

L7

C1 F2

K4

D5

E6

F3

[2] (BER-TOCSY).esp

8.3 8.2 8.1 8.0 7.9 7.8 7.7F2 Chemical Shift (ppm)

4.1

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hem

ical S

hift

(ppm

)

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)

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S11

FER-green

Compound FER-green was synthesized from F-Mal and the endoplasmic-reticulum peptide as described above. Yield: 0.07 g (64%); 1H NMR (800 MHz, DMSO-d6): δ 8.35−8.33 (2 H, m), 8.27−8.11 (6 H, m), 8.05 (1 H, d, J = 7.8 Hz ), 7.99−7.96 (2 H, m), 7.81 (1 H, d, J = 7.8 Hz), 7.73 (1 H, d, J = 7.8 Hz), 7.61 (1 H, d, J = 8.8 Hz), 7.54 (1 H, t, J = 7.8 Hz), 7.45 (1 H, t, J = 7.8 Hz), 7.26 (4 H, m), 7.20−7.17 (6 H, m), 6.73 (1 H, d, J = 1.0 Hz), 6.58 (1 H, dd, J = 8.3, 1.2 Hz), 4.57−4.55 (2 H, m), 4.49−4.45 (2 H, m), 4.27 (2 H, m), 4.16−4.15 (1 H, m), 4.03 (1 H, m), 3.95 (1 H, m), 3.53−3.51 (3 H, m), 3.20−3.04 (7 H, m), 2.99−2.97 (1 H, m), 2.92−2.90 (1 H, m), 2.83 (1 H, m), 2.75−2.70 (3 H, m), 2.56−2.46 (6 H, m), 2.56−2.46 (2 H, m), 2.26−2.25 (3 H, m), 1.98−1.92 (2 H, m), 1.83−1.80 (8 H, m), 1.65−1.62 (3 H, m), 1.56−1.54 (3 H, m), 1.52−1.43 (9 H, m), 1.27 (1 H, m), 0.89 (3 H, d, J = 6.8 Hz), 0.83 (3 H, d, J = 6.8 Hz) ppm; HRMS (electrospray ionization): m/z calcd. for [C73H87N11O16S2+H+]: 1421.5825, found: 1422.5903.

A partial 1H NMR spectrum of FER-blue in DMSO in the region 8.46.5 manifested all the proton peaks of F-Mal [except for the vinylic protons at 7.03 (h)] and new peaks due to the aromatic protons of the phenylalanine moiety at 7.287.13 (10 H) and N-H protons (*) of the CFFKDEL moiety at 8.308.07 (4 H), 8.037.93 (2 H), and 7.847.78 (1 H; Figure S3a,b). In addition, a 2D 1H-1H TOCSY spectrum and an overlay of TOCSY and NOESY spectra are consistent with the amide-to-side chain and amino acid residue connectivity in FER-green (Figure S3c,d and Table S2). The chemical shifts of each proton in the fingerprint region of the TOCSY spectrum are summarized in Table S2. Furthermore, high-resolution mass spectrometry (HRMS) result on FER-green revealed molecular-weight ion peak at m/e = 1422.5903 (calcd. 1421.5825) (Figure S28). These results confirm that the fluorophore is linked to HS-Ac-CFFKDEL via a sulfide bond and that FER-green represent a 1:1 mixture of two diastereomers produced after the addition of a thiol group to the maleimide moiety of F-Mal.

S12

Figure S3. (a,b) Partial 1H NMR spectra of F-Mal (a) and FER-green (b) in DMSO in the region 8.46.5. The protons in the F-Mal and Ac-CFFKDEL moieties are designated as ak, *, F2, and F3, respectively. (c) 2D 1H-1H TOCSY spectrum of FER-green in the NH-Ha fingerprint region. Each line indicates amide-to-side chain connectivity. (d) Overlay of TOCSY (red) and NOESY (green) spectra. The lines trace the sequential backbone assignment via intramolecular NH–Hα NOE connections.

Table S2. Chemical shifts of each proton in the fingerprint region of the TOCSY spectrum of FER-green in DMSO-d6.

FER-greenResidue NH Hα Hβ, Hβ’ Hγ, Hγ’ Hδ, Hδ’ Hε, Hε’ Hζ

Cys1 8.20 4.44 3.06, 2.68Cys1a 8.18 4.44 3.06, 2.90Phe2 7.98 4.47 2.96, 2.74 7.25 7.16 7.17Phe2a 8.01 4.48 2.98, 2.76Phe3 8.18 4.58 3.08, 2.83 7.25 7.15 7.17Phe3a 8.15 4.58 3.05, 2.83Lys4 8.26 4.26 1.76, 1.68 1.53, 1.46 1.61, 1.60Asp5 8.36 4.55 2.72, 2.55Glu6 7.81 4.28 1.76, 1.93 2.26Leu7 8.10 4.17 1.63, 1.55 1.50 0.89, 0.83

CJW_080819.002.esp

8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5Chemical Shift (ppm)

ke

a f dg h b c

hj i

CJW_072519.002.esp


f g h b c

j

i

(a) F-Mal

(b) FER-green e, a,* (4H)

d,**

K,*

F2, F3 (10H)

(c)

NH

S

N

N

O

O

O

HNS

ONH

OHN

ONH

NH2

OHN

O

OH ONH

O OH

OHN

OH

O

FER-green

N

O

O

TFHN(H2C)3F-Mal

*

**

**

**

Ac-C1F2

F3

K4

D5

E6

L7

β α

dc

ba

f g h i

ej

6

k

31

2 4,4’

5

h

(d)[4] (FER-TOCSY).esp

8.25 8.00 7.75F2 Chemical Shift (ppm)

4.1

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hem

ical

Shi

ft (p

pm)

[4] (FER-TOCSY).esp


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hem

ical

Shi

ft (p

pm)

[4] (FER-TOCSY).esp


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hem

ical

Shi

ft (p

pm)

[4] (FER-TOCSY).esp


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F1 C

hem

ical

Shi

ft (p

pm)L7

C1F2

K4

D5

E6

F3

[4] (FER-TOCSY).esp

4.6 4.5 4.4 4.3 4.2 4.1F2 Chemical Shift (ppm)

7.7

7.8

7.9

8.0

8.1

8.2

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8.5

F1 C

hem

ical

Shi

ft (p

pm)

F3

D5

F2

K4

E6

L7

δ1 H

(ppm

)

δ 1H (ppm)

C1F2Hα:F3NHF3Hα:K4NHK4Hα:D5NHD5Hα:E6NHE6Hα:L7NH

C1Hα:F2NHδ

1 H (p

pm)

δ 1H (ppm)

S13

Spectroscopic Properties

The spectral data were obtained as reported elsewhere, and the fluorescence quantum yield (Ф) was determined by a method from the literature.4,5 The spectral data obtained under various conditions are summarized in Table S3.

Table S3. Spectroscopic properties.

Cpd Solvent (ETN)a λmax/λflb c δd

BER-blueEtOH (0.654)

PBS (1.00)HeLa cell

358/445 354e/465

/460f (460)g

1.01.0

7161

1580h

FER-greenEtOH (0.654)

PBS (1.00)HeLa cell

385/513 373i/553

/480f (497)g

0.930.24

20270

3030h

aThe numbers in parentheses are normalized empirical parameters of solvent polarity.6 bOne-photon absorption and emission maxima unless stated otherwise. cFluorescence quantum yield. dTP action cross-section in GM 15%. eMolar extinction coefficients () = 25,560 cm1M1. fλfl of emission spectra. gλfl of TPEF spectra. hEffective TP action cross-section (eff) values measured in HeLa cells. i = 32,440 cm1M1

Solubility

The solubility of BER-blue and FER-green in phosphate-buffered saline (PBS) was determined by a fluorescence method, as reported before.4

Two-Photon Microscopy

TPM images of probe-labeled cells and tissues were obtained by spectral confocal and multiphoton microscopes (Leica TCS SP2; Leica Camera, Solms, Germany), using the 100× oil objective with a numerical aperture (NA) of 1.30, and a 10× dry objective with a numerical aperture (NA) of 0.30. The excitation was provided using a mode-locked titanium-sapphire laser (Chameleon, 90 MHz, 200 fs; Coherent Inc., Santa Clara, CA, USA) set at 750 nm and output power of 1305 mW, which corresponded to a power of 4 × 109 mW/cm2 (100×) at the focal plane. Images were obtained by collecting the emissions at the indicated channels (see Table S4) with PMTs in an 8-bit unsigned 512 512 pixel format at a scan speed of 400 Hz.

S14

Measurements of TP Action Cross-Section (Фδmax) and Effective TP Action Cross-Section (Фδeff)

Фδmax and Фδeff of BER-blue and FER-green were determined via a comparison of TP excited fluorescence (TPEF) intensities of the probes with that of Rhodamine 6G in methanol in a cuvette or through a comparison of TPEF intensities from the regions of interest in probe-labeled cells with TPEF intensity of 5.0 µM Rhodamine 6G in methanol in the TPM setup, respectively.4

Cell Preparation

Human cervical carcinoma HeLa cells were acquired from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured as described before and seeded 24 h prior to imaging at a density of 5.0 104 cells/mL on glass coverslips coated with 0.1 mg/mL poly-L-lysine for high-resolution OPM with Airyscan (LSM 800, Carl Zeiss) or in glass-bottomed dishes (SPL Life Sciences, Gyeonggi-do, Korea) for OPM and TPM live imaging.4

Tissue Preparation

All animal procedures were approved by the Korea Institute of Science and Technology Laboratory Animal Facilities and Use Committee and carried out at Association for Assessment and Accreditation of Laboratory Animal Care–accredited facilities. Ex vivo brain slices were obtained from the hippocampus of Sprague–Dawley rats aged 2 weeks (Orient Bio Inc., Gyeonggi-do, Korea). Hippocampal coronal slices were cut into 400-µm-thick slices on a vibrating-blade microtome in artificial cerebrospinal fluid and were used for TPM imaging as described before.7

S15

Detection Windows

The detection windows of the probes were determined ensuring good separation of the emission bands and similar emission intensities from the two probes under comparison. For the co-localization experiments with the probe-labeled cells, the detection windows were selected via a comparison of TPEF spectra of BER-blue, FER-green, ABI-Nu, and PLT-yellow (a TP probe for lysosomes), and a one-photon fluorescence spectrum of ETR in HeLa cells (Figure S6a and Table S4).4,8 The detection windows determined for the colocalization experiments in the HeLa cells colabeled with a pair of probes are summarized in Table S4. In each channel, the fluorescence of one probe contributed to that of another by 0–35% (Table S4).

Table S4. Detection windows for the OPM and TPM imaging.

OPM TPM

BERa

/ETRcFERb

/ETRcBERa

/FERbBERa

/FERbBERa

/PLTdBERa

/ABIe

Ch1 (0)f(400–550)g

Ch1 (0)f

(400-550)gCh2 (30)f (410‒460)g

Ch3 (20)f (385‒450)g

Ch4 (1)f (385‒500)g

Ch5 (35)f

(385‒435)g

Ch8 (1)f

(580–700)gCh8 (15)f

(580–700)gCh6 (20)f

(535‒655)gCh6 (17)f (535‒655)g

Ch7 (0)f (600‒660)g

Ch6 (35)f

(535‒655)g

aBER-blue. bFER-green. cER-tracker Red. dPLT-yellow. eABI-Nu. fThe degree (%) to which the emission intensity of one probe contributes to that of the other probe in a given channel. gWavelength ranges for each channel.

S16

Cell Imaging by High-resolution OPM

High-resolution OPM images of HeLa cells colabeled with BER-blue (5 μM) and ER-tracker Red (ETR; 1 μM) as well as with FER-green (3 μM) and ETR (1 μM) were captured by detection of the emission at 400550 nm (channel 1 [Ch1], BER-blue and FER-green) and 580–700 nm (Ch7, ETR) upon excitation at 405 nm for BER-blue and FER-green and at 561 nm for ETR during the OPM in Airyscan mode (LSM 800, Carl Zeiss) with an alpha Plan-Apochromat 63×/ 1.40 oil immersion objective lens. The obtained raw images were processed using the Zen software (Carl Zeiss). Pearson’s coefficient was calculated in the ImageJ software (NIH).

Cell Imaging by OPM and TPM

OPM images of HeLa cells colabeled with BER-blue (3 μM) and FER-green (1.5 μM) were captured by the emission acquired at 410–460 nm (Ch2, BER-blue) and 535–655 (Ch6, FER-green) after excitation at 405 nm under a confocal fluorescence microscope (LSM 700, Carl Zeiss) with a 63× oil objective lens. TPM images of HeLa cells colabeled with BER-blue (3 μM) and FER-green (1.5 μM) were obtained via recording of the emission at 385–450 nm (Ch3, BER-blue) and 535–655 nm (Ch6, FER-green) upon excitation at 750 nm under a multiphoton microscopy with a 100× oil objective lens as described elsewhere.4

Tissue Imaging by TPM

TPM images of ex vivo brain slices colabeled with BER-blue (10 μM) and ABI-Nu (a TP probe for nucleus, 3 μM) and those colabeled with BER-blue (10 μM) and FER-green (6 μM) were obtained across wavelength ranges 380–435 nm (Ch5, BER-blue) and 535–655 nm (Ch6, ABI-Nu) as well as 385–450 nm (Ch3, BER-blue) and 535–655 nm (Ch6, FER-green), respectively, after excitation at 750 nm. In all cases, 50–100 sectional TPM images were captured as reported before.9 Enlarged cellular TPM images of the brain slices were captured by means of a 100× oil objective lens.

Cytotoxicity

This property of BER-blue and FER-green was determined by the CellTiter-Glo Luminescent Cell Viability Assay (G7572, Promega, USA).

Photostability

This characteristic was determined as reported before.4

S17

A Western Blot Assay

HeLa cells were treated with 5 g/mL tunicamycin for 0−16 h and washed twice with ice-cold PBS. The cells were lysed in NP40 buffer (50 mM Tris-HCl pH 7.5, 1% of NP40, 1 mM EDTA, 150 mM NaCl, 5 mM Na3VO4, and 2.5 mM NaF) containing a 1× protease inhibitor cocktail (Roche). The mixture was centrifuged for 20 min at 15,871 g and 4°C, and the supernatants were collected. The protein concentrations were determined with the Bradford Protein Assay Kit (Bio-Rad, Inc., USA).Samples of the cell lysates containing equal amounts of total protein (25 μg) were separated

by SDS-PAGE. The proteins were transferred to a nitrocellulose membrane (0.45 m pore size) in a Wet/Tank Blotting System. The membranes were blocked for 1 h in Tris-buffered saline containing 0.1% of Tween 20 (TBST) and 5% skim milk at room temperature. The membrane was hybridized at 4°C for 18 h with one of the following primary antibodies:

anti-BIP (catalog No. 3183S, Cell Signaling Technology; dilution, 1:1000), anti-CHOP (catalog No. 5554S, Cell Signaling Technology; dilution, 1:1000), anti-cleaved caspase-3 (catalog No. 9661L; dilution, 1:1000), anti-PARP 1/2 (catalog No. 5625L; dilution, 1:3000), and anti-β-actin (catalog No. sc-47798, Santa Cruz Biotechnology; dilution, 1:10000). The membranes were washed three times with TBST (15 min each time) and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000, an anti-mouse immunoglobulin G [IgG] antibody and anti-rabbit IgG antibody, GenDEPOT) in TBST with 1% skim milk at room temperature for 2 h. The protein bands were detected using the Enhanced Chemiluminescence Kit (PierceTM ECL Western Blotting Substrate and Super-SignalTM West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific, Inc.).

Statistical Analysis

Numerical data are presented as mean ± standard deviation. The significance of data after a comparison was evaluated by one-way ANOVA in GraphPad Prism 6.0 (GraphPad Software, Inc., San Diego, CA, USA). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

S18

Figure S4. Normalized absorption (a) and emission (b) spectra of BER-blue and FER-green in phosphate-buffered saline (PBS) and EtOH.

Figure S5. (a,c) Fluorescence spectra of BER-blue (a) and FER-green (c), and plots (b, d) of the fluorescence intensity against probe concentration for BER-blue (b) and FER-green (d) in PBS. The excitation wavelengths for BER-blue and FER-green were 354 and 373 nm,

0 2 4 6 80

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Fluo

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ence

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nsity

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00.10.20.30.40.50.60.812357BER-blue

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00.10.20.30.40.50.60.812357FER-green

(a) (b)

(c) (d)

400 500 600 7000.0

0.5

1.0

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Nor

mal

ized

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ores

cenc

e

BER in PBSBER in EtOH

FER in PBSFER in EtOH

(a) (b)

300 350 400 4500.0

0.5

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wavelength, nm

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ized

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orba

nce



400 500 600 7000.0

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mal

ized

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ores

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e



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300 350 400 4500.0

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wavelength, nm

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ized

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orba

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S19

respectively.

Figure S6. (a) Normalized TPEF spectra of BER-blue (blue), FER-green (green), PLT-yellow (orange), and ABI-Nu (purple) and a normalized fluorescence spectrum of ETR (red) in HeLa cells. The wavelengths for the one- and two-photon excitation were 405 and 750 nm, respectively. (b) TP excitation spectra of BER-blue and FER-green in EtOH and PBS, respectively.

Figure S7. Viability of HeLa cells in the presence of BER-blue (a,b) or FER-green (c,d), as

0.1 1 100

20

40

60

80

100

120

Concentration, M

Rel

ativ

e Si

gnal

Inte

nsity

(% o

f con

trol

)

(a) (b)

0.1 1 100

20

40

60

80

100

120

Concentration, M

Rel

ativ

e Si

gnal

Inte

nsity

(% o

f con

trol

)

(c) (d)

0.5 1 2 4 8 24 0

20

40

60

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120

BER-blue

Time, h

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ativ

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0.5 1 2 4 8 24 0

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Time, h

Rel

ativ

e Si

gnal

Inte

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(% o

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ized

Flu

ores

cenc

e

BER

ABI-Nu PLTFER

ETR

720 750 780 810 840 8700

50

100

150

200

250

Wavelength, nm

(GM

)

BER-blue in EtOH

FER-green in EtOHBER-blue in PBS

FER-green in PBS

(a) (b)

S20

measured by a 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were incubated with the probes (010 M) for 024 h.

S21

Figure S8. (a,c) Two-photon microscopy images of HeLa cells labeled with BER-blue (a) or FER-green (c). (b,d) Relative TPEF intensity as a function of time. The digitized intensity was recorded with 2 s intervals during 1 h in xyt mode. The TPEF intensities at positions AF were recorded at 380–660 nm upon excitation at 750 nm. Scale bar: 30 µm.

Figure S9. Effects of pH on the one-photon fluorescence intensity of BER-blue and FER-green (2 μM each in universal buffer: 0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, and 0.1 M KCl).

3 4 5 6 7 8 9 100.0

0.5

1.0

1.5

FER-green

pH

Nor

mal

ized

Flu

ores

cenc

e

3 4 5 6 7 8 9 100.0

0.5

1.0

1.5

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pH

Nor

mal

ized

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0 500 1000 1500 2000 2500 3000 35000

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resc

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0 400 800 1200 1600 2000 2400 2800 3200 36000

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S22

Figure S10. Sectional TPM images of a rat hippocampal slice colabeled with BER-blue (10 μM) and FER-green (6 μM). The images were captured in channel 3 (Ch3: BER-blue, 385–450 nm) and Ch6 (FER-green, 535–655 nm) at a depth of 90–165 μm and 10× magnification after excitation at 750 nm.

(a) (b) (c)

S23

Mal-Furna-CHO-DMSOd6-H1.esp

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)


3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3Chemical Shift (ppm)










11.1 11.0 10.9 10.8 10.7 10.6 10.5 10.4 10.3 10.2 10.1 10.0 9.9 9.8 9.7 9.6 9.5 9.4 9.3 9.2 9.1 9.0 8.9 8.8 8.7 8.6 8.5Chemical Shift (ppm)

//////////

N O

O

O

O

Figure S11. 1H NMR spectrum (300 MHz) of compound I in DMSO-d6.

Figure S12. 13C NMR spectrum (75 MHz) of compound I in DMSO-d6.

ALDEHYDE-MAL-FURAN-DMSOD6-C13-1.ESP

216 208 200 192 184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0Chemical Shift (ppm)

N

O

O

OO

S24

H1)H2N-NAPH-BZ-H-1-CDCL3-H1.ESP

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)

H1)H2N-NAPH-BZ-H-1-CDCL3-H1.ESP

9.0 8.9 8.8 8.7 8.6 8.5 8.4 8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.4 6.3Chemical Shift (ppm)

NH2

O

N

Figure S13. 1H NMR spectrum (300 MHz) of compound B-NH2 in CDCl3.

C1)B-CDCL3-C13.ESP

168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0Chemical Shift (ppm)

C1)B-CDCL3-C13.ESP

131.0 130.5 130.0 129.5 129.0 128.5 128.0 127.5 127.0 126.5 126.0 125.5 125.0 124.5 124.0 123.5 123.0 122.5Chemical Shift (ppm)

NH2

O

N

Figure S14. 13C NMR spectrum (75 MHz) of compound B-NH2 in CDCl3.

S25

H2)B1-CDCL3-H1.ESP

8.65 8.60 8.55 8.50 8.45 8.40 8.35 8.30 8.25 8.20 8.15 8.10 8.05 8.00 7.95 7.90 7.85 7.80 7.75 7.70 7.65 7.60 7.55 7.50Chemical Shift (ppm)

H2)B1-CDCL3-H1.ESP


H2)B1-CDCL3-H1.ESP


H2)B1-CDCL3-H1.ESP


H2)B1-CDCL3-H1.ESP


// // // //

N

O

NH

N

O

O

O

H2-1)B1-CDCL3-H1.ESP



3.7 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7Chemical Shift (ppm)





////

Figure S15. 1H NMR spectrum (300 MHz) of compound 3 in CDCl3.

N

O

NH

N

O

O

O

C2)BZ-MAL-FURAN-CDCL3-C13.ESP


C2)BZ-MAL-FURAN-CDCL3-C13.ESP


//C2)B1-C13.ESP

176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0Chemical Shift (ppm)

C2)B1-C13.ESP

50 49 48 47 46 45 44 43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24Chemical Shift (ppm)

C2)B1-C13.ESP


//

C2)B1-C13.ESP


C2)B1-C13.ESP


////

Figure S16. 13C NMR spectrum (75 MHz) of compound 3 in CDCl3.

S26

N

O

NH

N

O

O

NMR2D_1072519.027-BMal.esp

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0Chemical Shift (ppm)

Water















// // // // // //



WaterNMR2D_1072519.027-BMal.esp


WaterNMR2D_1072519.027-BMal.esp


// //



DMSO-d6

Water

8.53

47

8.04

838.

0361

7.83

567.

8246

7.77

82 7.77

217.

7672

7.72

447.

7134 7.39

947.

3945

7.38

967.

0731

7.02

91

6.75

16

6.38

386.

3765

3.57

423.

5657

3.55

71 3.34

69 3.3

029

3.16

363.

1550

3.14

773.

1392

2.50

00

1.88

28 1.87

431.

8657



DMSO-d6

Water

8.53

47

8.04

838.

0361

7.83

567.

8246

7.77

82 7.77

217.

7672

7.72

447.

7134 7.39

947.

3945

7.38

967.

0731

7.02

91

6.75

16

6.38

386.

3765

3.57

423.

5657

3.55

71 3.34

69 3.3

029

3.16

363.

1550

3.14

773.

1392

2.50

00

1.88

28 1.87

431.

8657

Figure S17. 1H NMR spectrum (800 MHz) of B-Mal in DMSO-d6.

N

O

NH

N

O

O

C3-1)B-MAL-CDCL3_10-CD3OD_1-C13.ESP

184 176 168 160 152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8 0Chemical Shift (ppm)


43 42 41 40 39 38 37 36 35 34 33 32 31 30 29 28 27 26 25 24Chemical Shift (ppm)C3-1)B-MAL-CDCL3_10-CD3OD_1-C13.ESP

174 172 170 168 166 164 162 160 158 156 154 152 150 148 146 144 142 140 138 136Chemical Shift (ppm)





// //


130 128 126 124 122 120 118 116 114 112 110 108 106 104 102Chemical Shift (ppm)



//



//

Figure S18. 13C NMR spectrum (75 MHz) of B-Mal in CDCl3/CD3OD (10/1).

S27

NH2S

N

H6)F-CDCL3-H1.ESP


H6)F-CDCL3-H1.ESP


H6)F-CDCL3-H1.ESP


//

Figure S19. 1H NMR spectrum (300 MHz) of F-NH2 in CDCl3.

NH2S

N

C6)F-CDCL3-C13.ESP


C6-1)F-CDCL3-C13.ESP


Figure S20. 13C NMR spectrum (75 MHz) of F-NH2 in CDCl3.

S28

NH

N

O

O

S

N

O

H7)F1-CDCL3-H1.ESP


H7)F1-CDCL3-H1.ESP


//

H7-2)F1-CDCL3-H1.ESP


Figure S21. 1H NMR spectrum (300 MHz) of compound 4 in CDCl3.

NH

N

O

O

S

N

OC7)F1-CDCL3-C13.ESP

138 137 136 135 134 133 132 131 130 129 128 127 126 125 124 123 122 121 120 119 118Chemical Shift (ppm)

C7)F1-CDCL3-C13.ESP

138 137 136 135 134 133 132 131 130 129 128 127 126 125 124 123 122 121 120 119 118Chemical Shift (ppm)

//C7-1)F1-CDCL3-C13.ESP


C7-1)F1-CDCL3-C13.ESP






// //

Figure S22. 13C NMR spectrum (75 MHz) of compound 4 in CDCl3.

S29

CJW_080819.003.esp


CJW_080819.003.esp


CJW_080819.003.esp


CJW_080819.003.esp


// //

CJW_080819.003.esp


CJW_080819.003.esp


CJW_080819.003.esp


CJW_080819.003.esp


CJW_080819.003.esp


CJW_080819.003.esp


// ////////

NH

N

O

O

S

N

CJW_080819.003.esp

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5 -1.0 -1.5 -2.0 -2.5Chemical Shift (ppm)

DMSO-d6

Water

3.55

22 3.54

373.

5339

3.11

103.

1013

3.09

27

1.84

251.

8340

1.82

421.

8156

1.80

59

1.45

76

Figure S23. 1H NMR spectrum (800 MHz) of F-Mal in DMSO-d6.

NH

N

O

O

S

NC8)F-MAL-CDCL3-C13.ESP


C8)F-MAL-CDCL3-C13.ESP


C8-1)F-MAL-CDCL3-C13.ESP


C8-1)F-MAL-CDCL3-C13.ESP


//

Figure S24. 13C NMR spectrum (75 MHz) of F-Mal in CDCl3.

S30

NMR2D_1072519.201-BER1D1.ESP


NHO

N

N

O

O

O

NHS

ONH

OHN

ONH

NH2

OHN

O

OH ONH

O OH

OHN

OH

OBER-blue

Figure S25. 1H NMR spectrum (800 MHz) of BER-blue in DMSO-d6.NMR2D_1072519.002.esp


NH

N

O

O

S

NO

NHS OHN

O

NH

OHN

H2N

O

NH

O

OH OHN

OHO

O

NH

OH

O

FER-green

Figure S26. 1H NMR spectrum (800 MHz) of FER-green in DMSO-d6.

S31

Figure S27. HRMS of BER-blue. TOF MS: time-of-flight mass spectrometry.

Figure S28. HRMS of FER-green. TOF MS: time-of-flight mass spectrometry.

m/z result

Molecular formula: C68H81N11O16SExact mass: 1339.5583335.8974 (4+)447.5273 (3+)670.7870 (2+)1340.5662 (1+)

[M+H]+Isotope modeling

[M+H]+

[M+2H]2+

m/z result

Molecular formula: C73H87N11O15S2Exact mass: 1421.5825356.4034 (4+)474.8686 (3+)711.7991 (2+)1422.5903 (1+)

[M+H]+Isotope modeling

[M+H]+

[M+2H]2+

S32

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2. A. Ganguly, J. Zhu and T.L. Kelly, J. Phys. Chem. C., 2017, 121, 9110.

3. B.J. Neubert and B.B. Snider, Org. Lett. 2003, 5, 765.

4. C.S. Lim, S.T. Hong, S.S. Ryu, D.E. Kang and B.R. Cho, Chem. Asian J., 2015, 10, 2240.

5. K. Rurack and M. Spieles, Anal. Chem., 2011, 83, 1232.

6. C. Reichardt, Chem Rev, 1994, 94, 2319.

7. P.S. Mohan, C.S. Lim, Y.S. Tian, W.Y. Roh, J.H. Lee and B.R. Cho, Chem. Commun., 2009 5365.

8. C.S. Lim, E.S. Kim, J.Y. Kim, S.T. Hong, H.J. Chun, D.E. Kang and B.R. Cho, Sci Rep., 2015, 5, 18521.

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Detection in a Live Tissue by Two-Photon Microscopy ...Br B-NH 2 (a) (b) 1 2 O O N O CH 2 OH C C (c)...

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Transcript of Detection in a Live Tissue by Two-Photon Microscopy ...Br B-NH 2 (a) (b) 1 2 O O N O CH 2 OH C C (c)...