Real-time Monitoring of Cell Apoptosis and Drug Screening Using. Fluorescent Light-up Probe with Aggregation-Induced Emission

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1 Supporting Informtion Rel-time Monitoring of Cell Apoptosis nd Drug Screening Using Fluorescent Light-up Probe with Aggregtion-Induced Emission Chrcteristics Hibin Shi, Ryn T. K. Kwok, # Jinzho Liu, # Bengng Xing, Ben Zhong Tng, #,, nd Bin Liu,, Deprtment of Chemicl nd Biomoleculr Engineering, 4 Engineering Drive 4, Ntionl University of Singpore, Singpore, # Deprtment of Chemistry, Institute for Advnced Study, Division of Biomedicl Engineering, Stte Key Lbortory of Moleculr Neuroscience & Institute of Moleculr Functionl Mterils, The Hong Kong University of Science nd Technology, Cler Wter By, Kowloon, Hong Kong, Chin Institute of Mterils Reserch Engineering, 3 Reserch Link, 11762, Singpore Division of Chemistry nd Biologicl Chemistry, School of Physicl nd Mthemticl Sciences, Nnyng Technologicl University, , Singpore Tble of Contents Experimentl Section Scheme S1. Synthetic route to compound 5. Figure S1. (A) 1 H nd (B) 13 C NMR spectr of compound 1 in CDCl 3. Figure S2. High resolution mss spectrum (MALDI-TOF) of compound 1. Figure S3. (A) 1 H nd (B) 13 C NMR spectr of compound 2 in CDCl 3. Figure S4. High resolution mss spectrum (MALDI-TOF) of compound 2. Figure S5. (A) 1 H nd (B) 13 C NMR spectr of compound 3 in CDCl 3. (S4) (S5) (S6) (S6) (S7) (S7) (S8) S1

2 Figure S6. High resolution mss spectrum (MALDI-TOF) of compound 3. (S8) Figure S7. (A nd B) HPLC spectr nd (C) high resolution mss spectrum (MALDI-TOF) of probe Ac-DEVDK-TPE. Figure S8. (A) 1 H nd (B) 13 C NMR spectr of Ac-DEVDK-TPE in DMSO-d 6. (S9) (S1-S11) Figure S9. (A) UV-vis bsorption spectr of TPE-N 3 nd Ac-DEVDK-TPE in DMSO/wter (v/v = 1:199). [TPE-N 3 ] = [Ac-DEVDK-TPE] = 1 µm. (B) Hydrodynmic dimeters of TPE-N 3 prticles in DMSO/wter (v/v = 1/199) obtined from LLS. (S12) Figure S1. PL spectr of TPE-N 3 nd Ac-DEVDK-TPE in DMSO/wter (v/v = 1/199) with vried concentrtions of NCl (, 3, 6, 12, 24, 48 nd 96 mm) nd in cell culture medium (DMEM). [TPE-N 3 ] = [Ac-DEVDK-TPE] = 1 µm; λ ex = 312 nm. (S12) Figure S11. The cspse-ctlyzed hydrolysis of Ac-DEVDK-TPE (t = 1 h) monitored by LC-MS. (S13) Figure S12. (A) Hydrodynmic dimeters of the residue of Ac-DEVDK-TPE fter cspse-3 clevge in DMSO/wter (v/v = 1/199) obtined from LLS. (B) Plot of (I-I )/I versus cspse-3 concentrtion in PIPEs buffer. I nd I re the PL intensities of the ssy in the presence nd bsence of cspse-3. [Ac- DEVDK-TPE] = 1 µm. λ ex = 312 nm. (S13) Tble S1. Photophysicl properties of quinoline sulfte, TPE-N 3, K-TPE nd Ac-DEVDK-TPE in 1 PBS buffer. (S14) Figure S13. Michelis-Menton plot for the hydrolysis of three substrtes (Ac-DEVDK-TPE, Ac- DEVD-AFC nd Z-DEVD-AFC) in PIPES buffer by cspse-3 (1 pm). The concentrtion of substrtes vried from to 2 µm. The fluorescence intensities were monitored t 47 nm for Ac- DEVDK-TPE nd 55 nm for Ac-DEVD-AFC nd Z-DEVD-AFC, nd the excittion wvelengths were 312 nm for Ac-DEVDK-TPE nd 4 nm for Ac-DEVD-AFC nd Z-DEVD-AFC, respectively. (S15) Tble S2. Kinetic dt of Ac-DEVDK-TPE, Ac-DEVD-AFC nd Z-DEVD-AFC with cspse-3. (S15) Figure S14. Inhibition ssy of 5-[(S)-(+)-2-(methoxymethyl)pyrrolidino]sulfonylistin to cspse-3 with Ac-DEVDK-TPE s the substrte. (S16) S2

3 Figure S15. Metbolic vibility of MCF-7 cncer cells fter incubtion with TPE-N 3, Ac-DEVDK-TPE nd K-TPE t concentrtions of 5, 1 nd 25 µm for 48 h. (S16) Figure S16. Fluorescence microscope imges. (A & D) norml MCF-7 cells treted with Ac-DEVDK- TPE; (B & E) poptotic MCF-7 cells treted with Ac-DEVDK-TPE; (C & F) poptotic MCF-7 cells pretreted with 1 µm inhibitor 5-[(S)-(+)-2-(methoxymethyl)pyrrolidino]sulfonylistin. Nuclei were stined with propidium iodide (Invitrogen). The imges were cquired using fluorescence microscope (Nikon) equipped with DAPI nd Texs Red filter. All imges shre the sme scle br (1 µm). [Ac- DEVDK-TPE] = 5 µμ, [STS] = 1 µm. (S17) Figure S17. Fluorescence microscope imges. (A) Apoptotic MCF-7 cells treted with Ac-DEVDK- TPE (5 µm, 1% DMSO); (B) Apoptotic MCF-7 cells treted with Ac-DEVDK-TPE (5 µm, 1% DMSO) nd Annexin V-Alex Fluor. (C) Overly imges. 1 µm STS ws used to induce cell poptosis. Blue = probe fluorescence; green = Annexin V-Alex Fluor. The imges were cquired using fluorescence microscope (Nikon) equipped with DAPI nd FITC filter. All imges shre the sme scle br of 1 µm. (S18) Movie. Rel-time dynmic imging of cell poptotic process of MCF-7 cells with Ac-DEVDK-TPE t room temperture (cell poptosis.vi) S3

4 Experimentl Section 1. Synthesis nd Chrcteriztion Synthesis of 1-(4-Methylphenyl)-1,2,2-triphenylethene (1, Scheme 1): In 25 ml two necked round bottom flsk, 5.5 g (3 mmol) of diphenylmethne ws dissolved in 1 ml of distilled THF under N 2. After the mixture ws cooled to C, 15 ml (2.5 M in hexne, 37.5 mmol) of n-butyllithium ws slowly dded by syringe. The mixture ws stirred t C for 1 h g (25 mmol) of 4- methylbenzophenone ws then dded into the rection mixture. The mixture ws wrmed to room temperture nd stirred overnight. The rection mixture ws quenched with sturted NH 4 Cl solution nd then extrcted with DCM. The orgnic lyers were collected nd concentrted. The crude product nd.2 g of p-toluenesulfonic cid were dissolved into 1 ml of toluene. The mixture ws heted to reflux for 4 h. After cooled down to room temperture, the rection mixture ws extrcted with DCM. The orgnic lyer ws collected nd concentrted. The crude product ws purified by silic-gel chromtogrphy using hexne s eluent to give 1 s white solid (3.34, 78% yield). 1 H NMR (CDCl 3, 4 MHz), δ (TMS, ppm): (m, 15H), 6.9 (s, 4H), 2.24 (s, 3H). 13 C NMR (CDCl 3, 1 MHz), δ (TMS, ppm): 144.6, , 141.4, , , 132.2, , , , 129.5, , , 127., , HR-MS (MALDI-TOF): m/z [(M) +, clcd ]. Synthesis of 1-[(4-Bromomethyl)phenyl]-1,2,2-triphenylethene (2): In 25 ml round bottom flsk, solution of 5.2 g (15. mmol) of 1, 2.94 g (16. mmol) of N-bromosuccinimide,.36 g of benzoyl peroxide in 8 ml of CCl 4 ws refluxed for 12 h. After the rection ws completed, the mixture ws extrcted with dichloromethne nd wter. The orgnic lyers were combined, dried over mgnesium sulfte, nd removed under reduced pressure. The crude product ws purified by silic-gel chromtogrphy using hexne s eluent to give 2 s white solid (3.83 g, 6% yield). 1 H NMR (CDCl 3, 4 MHz), δ (TMS, ppm): (m, 11H), (m, 8H), 4.42(s, 2H). 13 C NMR (CDCl 3, 1 MHz), δ (TMS, ppm): , , 144.9, 142.2, 14.88, , , 132.1, , 129.9, S4

5 128.42, , , , HR-MS (MALDI-TOF): m/z [(M + 2) +, clcd ]. Scheme S1. Synthetic route to compound 5 Synthesis of Fmoc-Lys(lkyne)-COOH (5). Fmoc-Lys(Boc)-COOH (.47 g, 1. mmol) ws vigorously stirred in 2% TFA/DCM solution for 3 h. The rection solution ws then concentrted nd dried in vcuum to fford compound 4. The crude compound 4 ws directly used without further purifiction. To solution of compound 4 in DMF, hex-5-ynoic cid-nhs (.25 g, 1.2 mmol) nd DIEA (.4 ml, 2.5 mmol) were dded. The rection mixture ws stirred t room temperture overnight. The resulting solution ws then concentrted in vcuum nd purified by flsh chromtogrphy using ethyl cette nd hexne (v/v = 1:2) to fford 5 s white solid (.31 g, 67%). 1 H NMR (3 MHz, CDCl 3 ), δ (TMS, ppm): 7.64 (d, J = 6. Hz, 2H), 7.49 (t, J = 6. Hz, 2H), 7.28 (t, J = 9. Hz, 2H), 7.19 (t, J = 9. Hz, 2H), 6.2 (s, 1H), 5.82 (s, 1H), 4.25 (d, J = 6. Hz, 4H), (m, 2H), 3.1 (s, 2H), 2.19 (t, J = 6. Hz, 2H), (m, 2H), (m, 2H), (m, 2H), (m, 2H). 13 C NMR (75 MHz, CDCl 3 ), δ (TMS, ppm): 175.6, 173.5, 156.4, 143.8, 14.5, 127.7, 127.1, 125.1, 12.6, 83.4, 69.4, 67.1, 65.2, 6.4, 53.6, 5.2, 47., 45.2, 39.1, 36., 34.9, 33.9, 31.6, 28.7, 27.3, 23.8, 21.5, 17.7, MS (IT-TOF): m/z [(M+H) +, clcd ]. S5

6 A B Ar-H (19H) (CH 3, 3H) c,d,i,n m o n j i h m l m' l' n' o' k f g h e d c b j,o e,g,l b f,k Chemicl shift (ppm) Chemicl shift (ppm) Figure S1. (A) 1 H nd (B) 13 C NMR spectr of compound 1 in CDCl 3. ry16, MW=346 tn11419_4 81 (1.35) Cm (81-1:5) TOF MS CI+ 1.47e % m/z Figure S2. High resolution mss spectrum (MALDI-TOF) of compound 1. S6

7 A B o j n i Ar-H (19H) (CH 2 Br, 2H) c,d,i,n m l m' l' k n' o' f g h e d c b Br h m Br g b f,k,l e j,o Figure S3. (A) 1 H nd (B) 13 C NMR spectr of compound 2 in CDCl 3. ry17, MW=424 tn1142_5 78 (1.3) Cm (78-1:63) TOF MS CI+ 7.7e3 % m/z Figure S4. High resolution mss spectrum (MALDI-TOF) of compound 2. S7

8 A B i,n o j Ar-H (19H) N3 h,m n m l m' l' k n' o' f g h e d c b N 3 i (CH 2 N 3, 2H) c,d geb f,k,l j,o Figure S5. (A) 1 H nd (B) 13 C NMR spectr of compound 3 in CDCl 3. ry67, MW=387; t-bpmp, LP2; tn11921_2 18 (.6) Cn (Cen,2, 9., Ht); Sb (99,4. ); Sm (SG, 2x3.); Cm (17:21) TOF LD % m/z Figure S6. High resolution mss spectrum (MALDI-TOF) of compound 3. S8

9 mau 254nm,4nm (1.) 75 5 A min mau 36nm,4nm (1.) B min C Figure S7. (A nd B) HPLC spectr nd (C) high resolution mss spectrum (MALDI-TOF) of Ac- DEVDK-TPE. S9

10 A i h b,e c,d,f # g j,j ppm Chemicl shift (ppm) h B d c f e b ppm ppm i g j,j ppm Chemicl shift (ppm) Figure S8. (A) 1 H nd (B) 13 C NMR spectr of Ac-DEVDK-TPE in DMSO-d 6 (# overlp with wter pek). 2. Opticl Property Mesurements. S1

11 Absorbnce (.u.) A Ac-DEVDK-TPE TPE-N 3 Intensity (.u.) B TPE-N Wvelength (nm) Dimeter (nm) Figure S9. (A) UV-vis bsorption spectr of TPE-N 3 nd Ac-DEVDK-TPE in DMSO/wter (v/v = 1:199). [TPE-N 3 ] = [Ac-DEVDK-TPE] = 1 µm. (B) Hydrodynmic dimeters of TPE-N 3 prticles in DMSO/wter (v/v = 1/199) obtined from LLS. 3. Enzymtic Assy with Ac-DEVDK-TPE. The stock solution of Ac-DEVDK-TPE in DMSO ws diluted with cspse-3/7 ssy buffer (5 mm PIPES, 1 mm NCl, 1 mm EDTA,.1% w/v CHAPS, 25% w/v sucrose, ph = 7.2) to mke 5 µm working solution. 5 µl of the recombinnt cspse-3 nd -7 (.1 µg/µl stock solution in ssy buffer) ws dded into the working solution. The rection mixture ws incubted t room temperture for 6 min nd ws then diluted to totl of 3 µl with deionized wter for photoluminescence (PL) mesurement. The solution ws excited t 312 nm, nd the emission ws collected from 32 to 6 nm. S11

12 PL Intensity (.u.) TPE-N 3 Ac-DEVDK-TPE [NCl] ( 96 mm) DMEM Wvelength (nm) Figure S1. PL spectr of TPE-N 3 nd Ac-DEVDK-TPE in DMSO/wter (v/v = 1/199) with vried concentrtions of NCl (, 3, 6, 12, 24, 48 nd 96 mm) nd in cell culture medium (DMEM). [TPE-N 3 ] = [Ac-DEVDK-TPE] = 1 µm; λ ex = 312 nm. 1. mau(x1,) 254nm,4nm (1.).5. K-TPE DEVDK-TPE min Inten.(x1,) Inten.(x1,,) m/z m/z S12

13 Figure S11. The cspse-ctlyzed hydrolysis of Ac-DEVDK-TPE (t = 1 h) monitored by LC-MS. 1 A 35 B R 2 =.957 Intensity (.u.) K-TPE (I-I )/I Dimeter (nm) [Cspse-3] (pm) Figure S12. (A) Hydrodynmic dimeters of the residue of Ac-DEVDK-TPE fter cspse-3 clevge in DMSO/wter (v/v = 1/199) obtined from LLS. (B) Plot of (I-I )/I versus cspse-3 concentrtion in PIPEs buffer. I nd I re the PL intensities of the ssy in the presence nd bsence of cspse-3. [Ac- DEVDK-TPE] = 1 µm. λ ex = 312 nm. Tble S1. Photophysicl properties of quinoline sulfte, TPE-N 3, K-TPE nd Ac-DEVDK-TPE in 1 PBS. Smples λ ex [] λ em [b] Φ [c] Quinoline sulfte TPE-N K-TPE Ac-DEVDK-TPE [] λ ex is the excittion mximum; [b] λ em is the emission mximum; [c] Φ is quntum yield which is determined using quinoline sulfte in 1 PBS s the stndrd. 4. Kinetic Assy of Ac-DEVDK-TPE. The ssy conditions were s previously described. 1 Briefly, pproprite dilutions of Ac-DEVDK- TPE, Ac-DEVD-AFC or Z-DEVD-AFC were dded to rection mixtures contining 1 pm enzyme S13

14 nd buffer in totl volume of 5 µl. Libertion of AFC or TPE ws monitored continuously t room temperture using BioTek Synergy 4 plte reder. Kinetic constnts were computed by direct fitting the dt to the Michelis-Menton Eqution using non-liner regression vi GrphPd Prism softwre. The vlue ws tken in men ± S.D. 1.4 V (µm min -1 ) Ac-DEVD-AFC Ac-DEVD-TPE Z-DEVD-AFC [Substrte] (µm) Figure S13. Michelis-Menton plots for the hydrolysis of three substrtes (Ac-DEVDK-TPE, Ac- DEVD-AFC nd Z-DEVD-AFC) in PIPES buffer by cspse-3 (1 pm). The concentrtion of substrtes vried from to 2 µm. The fluorescence intensities were monitored t 47 nm for Ac- DEVDK-TPE nd 55 nm for Ac-DEVD-AFC nd Z-DEVD-AFC, nd the excittion wvelengths were 312 nm for Ac-DEVDK-TPE nd 4 nm for Ac-DEVD-AFC nd Z-DEVD-AFC, respectively. Tble S2. Kinetic dt of Ac-DEVDK-TPE, Ac-DEVD-AFC nd Z-DEVD-AFC with cspse-3. Probes Cspse-3 K m (µm) K ct (s -1 ) K ct /K m (µm -1 s -1 ) Ac-DEVDK-TPE 5.38 ± ± ±.2 Ac-DEVD-AFC 12.7 ± ±.1.21 ±.1 Z-DEVD-AFC ± ±.3.16 ±.3 S14

15 1 IC 5 = 63.4 nm % Activity Cspse-3 O N S O OCH 3 N H Log[Inhibitor]/nM O O Figure S14. Inhibition ssy of 5-[(S)-(+)-2-(methoxymethyl)pyrrolidino]sulfonylistin to cspse-3 with Ac-DEVDK-TPE s the substrte. Cell Vibility (%) µm 1 µm 25 µm 2 TPE-N 3 DEVDK-TPE K-TPE Figure S15. Metbolic vibility of MCF-7 cncer cells fter incubtion with TPE-N 3, Ac-DEVDK-TPE nd K-TPE t concentrtions of 5, 1 nd 25 µm for 48 h. S15

16 A B C D E F Figure S16. Fluorescence microscope imges. (A & D) norml MCF-7 cells treted with Ac-DEVDK- TPE; (B & E) poptotic MCF-7 cells treted with Ac-DEVDK-TPE; (C & F) poptotic MCF-7 cells pretreted with 1 µm inhibitor 5-[(S)-(+)-2-(methoxymethyl)pyrrolidino]sulfonylistin. Nuclei were stined with propidium iodide (Invitrogen). The imges were cquired using fluorescence microscope (Nikon) equipped with DAPI nd Texs Red filter. All imges shre the sme scle br of 1 µm. [Ac- DEVDK-TPE] = 5 µμ, [STS] = 1 µm. S16

17 Ac-DEVDK-TPE Anti Annexin V Overly A B C Figure S17. Fluorescence microscope imges. (A) Apoptotic MCF-7 cells treted with Ac-DEVDK- TPE (5 µm, 1% DMSO); (B) Apoptotic MCF-7 cells treted with Ac-DEVDK-TPE (5 µm, 1% DMSO) nd Annexin V-Alex Fluor. (C) Overly imges. 1 µm STS ws used to induce cell poptosis. Blue = probe fluorescence; green = Annexin V-Alex Fluor. The imges were cquired using fluorescence microscope (Nikon) equipped with DAPI nd FITC filter. All imges shre the sme scle br of 1 µm. Reference (1) Hu, M.; Li, L.; Wu, H.; Su, Y.; Yng, P.-Y.; Uttmchndni, M.; Xu, Q.-H.; Yo, S. Q. J. Am. Chem. Soc. 211, 133, S17