A Selective Fluorescent Probe for Live Cell-Monitoring of Sulfide

Transcription

1 Supplementary Information A Selective Fluorescent Probe for Live Cell-Monitoring of Sulfide Yong Qian 1, 2, Jason Karpus 1, Omer Kabil 3, Hai-Liang Zhu 2, Ruma Banerjee 3, Jing Zhao 2 & Chuan He 1 1 Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA. 2 State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing , China. 3 Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI , USA. Correspondence should be addressed to C. H. (chuanhe@uchicago.edu) Supplementary figures and text: Supplementary Figure 1. Crystal structure diagrams of SFP-1 (12). Supplementary Figure 2-3. High resolution mass spectrometry identification of 12, 12a. Supplementary Figure 4. Fluorescence spectra of SFP-1 with Na 2 S in PBS buffer after different incubation times. Supplementary Figure 5. Fluorescence spectra of SFP-1 in PBS buffer incubated with different concentrations of Na 2 S. Supplementary Figure 6. Fluorescence spectra of SFP-1 in the presence of H 2 S. Supplementary Figure 7-8. High resolution mass spectrometry identification of 20 and 20a. Supplementary Figure 9. Fluorescence spectra of SFP-2 with Na 2 S in PBS buffer after different incubation times. Supplementary Figure Fluorescence spectra of SFP-2 in PBS buffer incubated with different concentrations of Na 2 S. Supplementary Figure 13. Cytotoxicity assay of SFP-1. Supplementary Figure 14. Additional imaging experiments with various sulfur sources. Supplementary Figure NMR spectra of synthesized compounds. Supplementary Tables 1-5: Crystal and structure refinement parameters for compound 12. Supplementary Methods S1

2 Supplementary References Supplementary Figure 1. Crystal structure diagrams of compound 12. Molecular structure diagram with displacement ellipsoids being at the 50% probability level. S2

3 Supplementary Figure S2. HRMS identification of SFP-1 probe 12 (calculated for C 26 H 19 F 2 N 2 O 3 (M+H) ; found ) prior to the addition of sulfide. S3

4 Supplementary Figure S3. HRMS identification for the sulfide addition product of SFP-1 probe 12a (calculated for C 26 H 21 F 2 N 2 O 3 S (M+H) ; found ). S4

5 Supplementary Figure S4. Fluorescence spectra of 10 μm SFP-1 probe in 10 mm PBS buffer (ph 7.4, 10% CH 3 CN) with 50 μm Na 2 S. Probe was allowed to incubate with Na 2 S for various amounts of time (5, 15, 30, 45, 60, 90, 120 min) at 37 o C prior to measurement. Fluorescence intensity at 388 nm was monitored at each time point. Excitation: 300 nm, emission: nm. The data represent the mean ± SD of at least three independent experiments. Supplementary Figure S5. Fluorescence spectra of 10 μm SFP-1 probe in 10 mm PBS buffer (PH 7.4, 10% CH 3 CN) with increasing amounts of Na 2 S. Probe was incubated with concentrations of 10, 20, 30, 40, 50, 60, and 80 μm Na 2 S at 37 o C for 60 min. Fluorescence intensity at 388 nm was monitored as a function of analyte concentration. Excitation: 300 nm, emission: nm. The data represent the mean ± SD of at least three independent experiments. S5

6 Supplementary Figure S6. Fluorescence spectra of 10 μm SFP-1 measured in the presence of PBS buffers that H 2 S gas had been bubbled through for varying lengths of time. SFP-1 probe was allowed to incubate in the buffers for 60 min at 37 o C. Measurements performed in 10 mm PBS buffer (ph 7.4, 10% CH 3 CN). Excitation: 300 nm, emission: nm. All H 2 S buffers were prepared by adding 10 ml DI H 2 O to a 25 ml Schlenk tube and subsequently bubbling nitrogen through for 30 min. H 2 S was then bubbled through for various lengths of time using a 0.8 mm needle. Ventilation rate was maintained at 1 bubble per second. The data represents the average of three independent experiments. S6

7 Supplementary Figure S7. HRMS identification of SFP-2 probe 20 (calculated for C 24 H 24 BF 2 N 2 O 3 (M+H) ; found ) prior to the addition of sulfide. S7

8 Supplementary Figure S8. HRMS identification of the sulfide addition product of SFP-2 probe 20a (calculated for C 24 H 26 BF 2 N 2 O 3 S (M+H) ; found ). S8

9 Supplementary Figure S9. Fluorescence spectra of 5 μm SFP-2 probe in 20 mm PBS buffer (ph 7.0, 1% DMSO) with 50 μm Na 2 S. Probe was allowed to incubate with Na 2 S for various amounts of time (5, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210 and 240 min) at 37 o C prior to measurement. Fluorescence intensity at 510 nm was monitored at each time point. Excitation: 465 nm, emission: nm. The data represent the mean ± SD of at least three independent experiments. Supplementary Figure 10. Fluorescence spectra of 5 μm SFP-2 probe in 20 mm PBS buffer (PH 7.0, 1% DMSO) with increasing amounts of Na 2 S. Probe was incubated with concentrations of 5, 10, 20, 40, 60, 80, and 100 μm Na 2 S at 37 o C for 120 min. Fluorescence intensity at 510 nm was monitored as a function of analyte concentration. Excitation: 465 nm, emission: nm. The data represent the mean ± SD of at least three independent experiments. S9

10 Supplementary Figure 11a Supplementary Figure S11b Supplementary Figure S11a b. (a) Fluorescence spectra of 5 μm SFP-2 probe in 20 mm PBS buffer (PH 7.0, 1% DMSO) with increasing amounts of Na 2 S. Probe was incubated with concentrations of 5, 10, 20, 40, 60, 80, and 100 μm Na 2 S at 37 o C for overnight (14 h). (b) Fluorescence intensity at 510 nm was monitored as a function of analyte concentration. Excitation: 465 nm, emission: nm. The data represent the mean ± SD of at least three independent experiments. S10

11 SI Fig. S12a SI Fig. S12b Supplementary Figure S12a b. (a) Fluorescence spectra of 5 μm SFP-2 probe measured in the presence of 1 μl of PBS buffers that H 2 S gas had been bubbled through for varying lengths of time. (b) Fluorescence spectra of 5 μm SFP-2 probe measured in the presence of 10 μl of PBS buffers that H 2 S gas had been bubbled through for varying lengths of time. Probe was allowed to incubate in the buffers for 20 min at 25 o C. Measurements performed in 20 mm PBS buffer (ph 7.0, 1% DMSO). Excitation: 465 nm, emission: nm. All H 2 S buffers were prepared by adding 10 ml DI H 2 O to a 25 ml Schlenk tube and subsequently bubbling nitrogen through for 30 min. H 2 S was then bubbled through for various lengths of time using a 0.8 mm needle. Ventilation rate was maintained at 1 bubble per second. The data represents the average of three independent experiments. S11

12 Supplementary Figure S13. Evaluation of the potential cytotoxicity of SFP-1 probe to live cells. Hela cells were allowed to incubate with increasing concentrations of the probe overnight. Viability measured using a CellTiter 96 Non-Radioactive Cell Proliferation Assay from Promega using the vendor s protocol. Absorbance of cells read at 570 nm in a 96-well plate reader. The data represent the mean ± SD for three separate experiments. Supplementary Figure S14a e. Additional imaging of sulfur compounds in Hela cells after 30 min incubation using SFP-2 (20) or thiol tracker (Invitrogen). Images were obtained by using confocal fluorescence capture with 2 µm probe, with either: (a) 0 μm sulfide source with 2 μm thiol tracker, (b) 0 μm sulfide source with 2 μm SFP-2, (c) 100 μm biotin (a form of thioether control) with 2 μm SFP-2, (d) 100 μm methyl disulfide with 2 μm SFP-2, (e) 100 μm cysteine with 2 μm SFP-2. S12

13 (1) Supplementary Figure S15. 1 H NMR spectrum of compound 1. S13

14 Supplementary Figure S C NMR spectrum of compound 1. S14

15 (2) Supplementary Figure S17. 1 H NMR spectrum of compound 2. S15

16 Supplementary Figure S C NMR spectrum of compound 2. S16

17 (3) Supplementary Figure S19. 1 H NMR spectrum of compound 3. S17

18 Supplementary Figure S C NMR spectrum of compound 3. S18

19 (4) Supplementary Figure S21. 1 H NMR spectrum of compound 4. S19

20 Supplementary Figure S C NMR spectrum of compound 4. S20

21 (5) Supplementary Figure S23. 1 H NMR spectrum of compound 5. S21

22 Supplementary Figure S C NMR spectrum of compound 5. S22

23 (6) Supplementary Figure S25. 1 H NMR spectrum of compound 6. S23

24 Supplementary Figure S C NMR spectrum of compound 6. S24

25 (7) Supplementary Figure S27. 1 H NMR spectrum of compound 7. S25

26 Supplementary Figure S C NMR spectrum of compound 7. S26

27 (8) Supplementary Figure S29. 1 H NMR spectrum of compound 8. S27

28 Supplementary Figure S C NMR spectrum of compound 8. S28

29 (9) Supplementary Figure S31. 1 H NMR spectrum of compound 9. S29

30 Supplementary Figure S C NMR spectrum of compound 9. S30

31 (10) Supplementary Figure S33. 1 H NMR spectrum of compound 10. S31

32 Supplementary Figure S C NMR spectrum of compound 10. S32

33 (11) Supplementary Figure S35. 1 H NMR spectrum of compound 11. S33

34 Supplementary Figure S C NMR spectrum of compound 11. S34

35 (12) Supplementary Figure S37. 1 H NMR spectrum of compound 12. S35

36 Supplementary Figure S C NMR spectrum of compound 12. S36

37 (13) Supplementary Figure S39. 1 H NMR spectrum of compound 13. Supplementary Figure S C NMR spectrum of compound 13. S37

38 (14) Supplementary Figure S41. 1 H NMR spectrum of compound 14. Supplementary Figure S C NMR spectrum of compound 14. S38

39 (15) Supplementary Figure S43. 1 H NMR spectrum of compound 15. Supplementary Figure S C NMR spectrum of compound 15. S39

40 (16) Supplementary Figure S45. 1 H NMR spectrum of compound 16. Supplementary Figure S C NMR spectrum of compound 16. S40

41 (17) Supplementary Figure S47. 1 H NMR spectrum of compound 17. S41

42 Supplementary Figure S C NMR spectrum of compound 17. S42

43 (18) Supplementary Figure S49. 1 H NMR spectrum of compound 18. S43

44 Supplementary Figure S C NMR spectrum of compound 18. S44

45 (19) Supplementary Figure S51. 1 H NMR spectrum of compound 19. S45

46 Supplementary Figure S C NMR spectrum of compound 19. S46

47 (20) Supplementary Figure S53. 1 H NMR spectrum of compound 20. S47

48 Supplementary Figure S C NMR spectrum of compound 20. S48

49 Supplementary Table 1: Crystal and structure refinement for compound 12. Identification Code compound 12 Empirical formula C 26 H 18 F 2 N 2 O 3 Formula weight Temperature 100 K Wavelength Å Crystal system Triclinic Space Group P1(bar) Unit cell dimensions a = 8.781(2) Å α = (4) o b = (3) Å β = (4) o c = (3) Å γ = (4) o Volume (4) Å 3 Z 2 Density (calculated) Mg/m 3 Absorption coefficient mm -1 F(000) 460 Crystal size, color, habit 0.40 x 0.20 x 0.20mm, clear, fragment Theta range for data collection o Index ranges -11 h 11, -15 k 15, -15 l 15 Reflections collected 12,683 Independent reflections 4,989 (R int = ) Reflections with I > 4σ(F o ) 4,423 Absorption correction SADABS based on redundant diffractions Max. and min. transmission 1.0, Refinement method Full-matrix least squares on F 2 Weighting scheme w = q [σ 2 (F 2 o ) + (ap) 2 + bp] -1 where: P = (F 2 o +2 F 2 c )/3, a = , b = 0.128, q =1 Data / restraints / parameters 4989 / 0 / 299 Goodness-of-fit on F Final R indices [I > 2 sigma(i)] R1 = , wr2 = R indices (all data) R1 = , wr2 = Largest diff. peak and hole 0.332, eå -3 S49

50 Supplementary Table 2. Atomic coordinates [ x 10 4 ] and equivalent isotropic displacement parameters [Å 2 x 10 3 ] for compound 12. U(eq) is defined as one third of the trace of the orthogonalized U ij tensor. x y z U(eq) SOF C(1) 4349(1) 3651(1) 4096(1) 22(1) C(2) 4494(2) 2395(1) 4176(1) 26(1) C(3) 5341(2) 2061(1) 3329(1) 31(1) C(4) 6023(2) 2968(1) 2409(1) 32(1) C(5) 5847(2) 4226(1) 2320(1) 29(1) C(6) 5016(1) 4569(1) 3168(1) 25(1) C(7) 3299(1) 3256(1) 6188(1) 22(1) C(8) 2168(1) 4069(1) 6512(1) 23(1) C(9) 1733(1) 5340(1) 5420(1) 22(1) C(10) 687(1) 6614(1) 5287(1) 22(1) C(11) 186(2) 6687(1) 6367(1) 27(1) C(12) -736(2) 7899(1) 6239(1) 30(1) C(13) -1172(2) 9069(1) 5097(1) 29(1) C(14) -633(1) 8959(1) 4054(1) 26(1) C(15) 260(1) 7774(1) 4102(1) 24(1) C(16) 4332(1) 1893(1) 7023(1) 22(1) C(17) 5976(1) 1582(1) 6990(1) 24(1) C(18) 6915(1) 309(1) 7829(1) 24(1) C(19) 6266(1) -682(1) 8701(1) 22(1) C(20) 4608(1) -380(1) 8748(1) 21(1) C(21) 3680(1) 922(1) 7906(1) 22(1) C(22) 7422(2) -1991(1) 9538(1) 26(1) C(23) 3839(1) -1406(1) 9582(1) 23(1) C(24) 2385(2) -1127(1) 9907(1) 24(1) C(25) 1585(1) -2168(1) 10641(1) 25(1) C(26) 1544(2) -4417(1) 11379(1) 35(1) F(1) -1225(1) 7947(1) 7299(1) 46(1) F(2) -1007(1) 10097(1) 2913(1) 33(1) N(1) 3465(1) 4032(1) 4938(1) 22(1) N(2) 2502(1) 5318(1) 4455(1) 23(1) O(1) 396(1) -1967(1) 11128(1) 32(1) O(2) 2282(1) -3356(1) 10681(1) 31(1) O(3) 7154(1) -2992(1) 10372(1) 36(1) S50

51 Supplementary Table 3. Bond lengths [Å] and angles [ o ] for compound 12. C(1)-C(6) (17) C(1)-C(2) (17) C(1)-N(1) (15) C(2)-C(3) (18) C(3)-C(4) 1.382(2) C(4)-C(5) (19) C(5)-C(6) (17) C(7)-C(8) (16) C(7)-N(1) (15) C(7)-C(16) (16) C(8)-C(9) (16) C(9)-N(2) (15) C(9)-C(10) (16) C(10)-C(11) (17) C(10)-C(15) (17) C(11)-C(12) (17) C(12)-F(1) (15) C(12)-C(13) (19) C(6)-C(1)-C(2) (11) C(6)-C(1)-N(1) (10) C(2)-C(1)-N(1) (11) C(3)-C(2)-C(1) (12) C(4)-C(3)-C(2) (12) C(3)-C(4)-C(5) (12) C(4)-C(5)-C(6) (12) C(1)-C(6)-C(5) (11) C(8)-C(7)-N(1) (10) C(8)-C(7)-C(16) (11) N(1)-C(7)-C(16) (10) C(7)-C(8)-C(9) (11) N(2)-C(9)-C(8) (10) N(2)-C(9)-C(10) (10) C(8)-C(9)-C(10) (11) C(11)-C(10)-C(15) (11) C(11)-C(10)-C(9) (11) C(15)-C(10)-C(9) (11) C(12)-C(11)-C(10) (12) F(1)-C(12)-C(11) (12) F(1)-C(12)-C(13) (11) C(11)-C(12)-C(13) (12) C(12)-C(13)-C(14) (11) F(2)-C(14)-C(15) (11) F(2)-C(14)-C(13) (11) C(15)-C(14)-C(13) (12) C(14)-C(15)-C(10) (11) C(21)-C(16)-C(17) (11) C(21)-C(16)-C(7) (11) C(17)-C(16)-C(7) (10) C(18)-C(17)-C(16) (11) C(17)-C(18)-C(19) (11) C(18)-C(19)-C(20) (11) C(18)-C(19)-C(22) (11) C(20)-C(19)-C(22) (11) C(21)-C(20)-C(19) (11) C(21)-C(20)-C(23) (11) C(19)-C(20)-C(23) (10) C(16)-C(21)-C(20) (11) C(13)-C(14) (18) C(14)-F(2) (14) C(14)-C(15) (17) C(16)-C(21) (16) C(16)-C(17) (17) C(17)-C(18) (16) C(18)-C(19) (16) C(19)-C(20) (17) C(19)-C(22) (16) C(20)-C(21) (16) C(20)-C(23) (16) C(22)-O(3) (15) C(23)-C(24) (17) C(24)-C(25) (17) C(25)-O(1) (15) C(25)-O(2) (15) C(26)-O(2) (15) N(1)-N(2) (13) O(3)-C(22)-C(19) (12) C(24)-C(23)-C(20) (11) C(23)-C(24)-C(25) (11) O(1)-C(25)-O(2) (12) O(1)-C(25)-C(24) (11) O(2)-C(25)-C(24) (10) N(2)-N(1)-C(7) (9) N(2)-N(1)-C(1) (9) C(7)-N(1)-C(1) (10) C(9)-N(2)-N(1) (9) C(25)-O(2)-C(26) (10) S51

52 Supplementary Table 4. Anisotropic displacement parameters [Å 2 x 10 3 ] for compoud 12. The anisotropic displacement factor exponent takes the form: -2π 2 [h 2 a *2 U hka * b * U 12 ] U 11 U 22 U 33 U 23 U 13 U 12 C(1) 20(1) 22(1) 22(1) -11(1) 0(1) -2(1) C(2) 28(1) 21(1) 25(1) -10(1) -1(1) -5(1) C(3) 36(1) 25(1) 32(1) -17(1) -4(1) 0(1) C(4) 31(1) 36(1) 30(1) -20(1) 3(1) -2(1) C(5) 26(1) 33(1) 28(1) -14(1) 5(1) -7(1) C(6) 24(1) 23(1) 27(1) -12(1) 2(1) -6(1) C(7) 22(1) 19(1) 23(1) -8(1) 2(1) -7(1) C(8) 23(1) 20(1) 24(1) -9(1) 3(1) -6(1) C(9) 20(1) 20(1) 23(1) -9(1) 3(1) -6(1) C(10) 19(1) 20(1) 27(1) -11(1) 3(1) -6(1) C(11) 26(1) 23(1) 28(1) -10(1) 6(1) -6(1) C(12) 30(1) 31(1) 33(1) -19(1) 11(1) -8(1) C(13) 24(1) 23(1) 40(1) -17(1) 4(1) -4(1) C(14) 22(1) 20(1) 31(1) -8(1) -1(1) -5(1) C(15) 21(1) 22(1) 26(1) -11(1) 3(1) -5(1) C(16) 24(1) 18(1) 21(1) -9(1) 1(1) -4(1) C(17) 25(1) 22(1) 22(1) -8(1) 3(1) -7(1) C(18) 22(1) 24(1) 23(1) -11(1) 2(1) -4(1) C(19) 24(1) 21(1) 19(1) -10(1) 1(1) -4(1) C(20) 25(1) 19(1) 20(1) -10(1) 3(1) -6(1) C(21) 22(1) 21(1) 24(1) -11(1) 3(1) -4(1) C(22) 26(1) 24(1) 22(1) -9(1) 1(1) -3(1) C(23) 26(1) 18(1) 21(1) -8(1) 0(1) -5(1) C(24) 28(1) 21(1) 22(1) -9(1) 3(1) -6(1) C(25) 24(1) 23(1) 21(1) -7(1) 0(1) -4(1) C(26) 30(1) 24(1) 41(1) -6(1) 0(1) -11(1) F(1) 57(1) 40(1) 40(1) -24(1) 19(1) -6(1) F(2) 36(1) 19(1) 34(1) -7(1) -2(1) -1(1) N(1) 22(1) 17(1) 23(1) -8(1) 2(1) -4(1) N(2) 22(1) 17(1) 25(1) -8(1) 1(1) -2(1) O(1) 28(1) 31(1) 30(1) -11(1) 8(1) -7(1) O(2) 30(1) 22(1) 37(1) -10(1) 6(1) -8(1) O(3) 32(1) 26(1) 32(1) -2(1) 1(1) -4(1) S52

53 Supplementary Table 5. Hydrogen coordinates [ x 10 4 ] and isotropic displacement parameters [Å 2 x 10 3 ] for compound 12. x y z U(eq) H(2) H(3) H(4) H(5) H(6) H(8) H(11) H(13) H(15) H(17) H(18) H(21) H(22) H(23) H(24) H(26A) H(26B) H(26C) S53

54 Supplementary Methods Synthetic protocols of compounds 1-12 Methyl 3-bromo-4-methylbenzoate (1) A suspension of 3-bromo-4-methylbenzoic acid (5 g, 23.2 mmol) was prepared in MeOH (30 ml). H 2 SO 4 (760 μl) was added and the mixture was heated to reflux and allowed to stir for 12 h. The solvent was removed by rotary evaporation and the residue was quenched with saturated aqueous NaHCO 3. Extraction was performed with ethyl acetate (3 x 100 ml) and the combined organic layers were dried with sodium sulfate and concentrated in vacuo. The crude product was purified by column chromatography on SiO 2 giving a colorless oil. Yield 87 %. TLC (silica, hexane: EtOAc, 4:1 v/v): R f = 0.7; 1 H NMR (400 MHz, CDCl 3 ): δ 8.20 (s, 1 H), 7.86 (d, J = 8.0 Hz, 1 H), 7.30 (d, J = 8.0 Hz, 1 H), 3.91 (s, 3 H), 2.45 (s, 3 H); 13 C NMR (125 MHz, CDCl 3 ): δ 165.8, 143.3, 133.5, 130.7, 129.5, 128.4, 124.8, 52.2, 23.3; HRMS (m/z, ESI + ): (M+H) + calcd. for C 9 H 10 BrO 2, ; found, (2-Bromo-4-(methoxycarbonyl)phenyl)methylene diacetate (2) 3-bromo-4-methylbenzoate (1) (4.56 g, 20 mmol) was dissolved in a mixture of AcOH (33 ml, 30 eq) and Ac 2 O (34 ml, 18 eq) containing H 2 SO 4 (5 ml, 4.5 eq). The solution was cooled to 0 o C in an ice-bath and CrO 3 (6 g, 3 eq) was added in portions over 30 min. The mixture was allowed to stir in the ice bath for one hour. The reaction mixture was poured onto H 2 O (320 ml) and stirred vigorously for 20 min. After, the product was filtered and the resulting solid was washed with 20 ml water three times. The crude product was purified by column chromatography on SiO 2 giving a white solid as the purified product. Yield 57%. TLC (silica, hexane: EtOAc, 8:1 v/v): R f = 0.2; 1 H NMR (500 MHz, CDCl 3 ): δ 8.26 (s, 1 H), 8.02 (dd, J = 8.5, 6.5 Hz, 1 H), 7.91 (s, 1 H), 7.63 (d, J = 8.0 Hz, 1 H), 3.94 (s, 3 H), 2.16 (s, 6 H); 13 C NMR (125 MHz, CDCl 3 ): δ 168.3, 165.3, 139.3, 134.4, 132.8, 128.7, 128.0, 122.6, 88.6, 52.7, 20.7; HRMS (ESI + ): (M+Na) + calcd. for C 13 H 13 BrNaO 6, ; found, S54

55 Methyl 3-bromo-4-formylbenzoate (3) A solution of (2-bromo-4-(methoxycarbonyl)phenyl)methylene diacetate (2) (3.8 g, 11 mmol) was prepared in MeOH H 2 O (1:1, 30 ml). H 2 SO 4 (440 μl) was added and the mixture was heated under reflux at 100 C for 30 min. The mixture was then diluted with H 2 O and extracted with EtOAc (3 x 100 ml). The solution was dried over Na 2 SO 4, and the solvent was removed by rotary evaporation to yield a white solid. The solid was dissolved in THF (25 ml) and 1 N HCl (7 ml) and the mixture was heated under reflux at 80 C for 2 h. The THF was removed by rotary evaporation and the residue was extracted with ethyl acetate (3 x 100 ml) and then dried over Na 2 SO 4. The solvent was removed by rotary evaporation to yield a white solid. Yield 78 %. TLC (silica, hexane: EtOAc, 8:1 v/v): R f = 0.6; 1 H NMR (500 MHz, CDCl 3 ): δ (s, 1 H), 8.26 (s, 1 H), 8.02 (d, J = 8.0 Hz, 1 H), 7.91 (d, J = 8.0 Hz, 1 H), 3.93 (s, 3 H); 13 C NMR (125 MHz, CDCl 3 ): δ 191.2, 164.8, 136.2, 136.0, 135.0, 129.8, 128.8, 126.6, 52.9; HRMS (ESI + ): (M+H) + calcd. for C 9 H 8 BrO 3, ; found, Methyl 3-bromo-4-(1,3-dioxan-2-yl)benzoate (4) Propane-1,3-diol (1.04 ml, 14.4 mmol), para toluenesulfonic acid monohydrate (17 mg, 0.88 mmol), and anhydrous Na 2 SO 4 (200 mg) were added to a solution of methyl 3-bromo-4-formylbenzoate (3) (581 mg, 2.4 mmol) in toluene (10 ml). Subsequently, the reaction solution was heated at 80 C for 24 h. The reaction mixture was quenched with approximately 200 ml of water. After three separate extractions with 100 ml EtOAc, the combined organic layers were dried over Na 2 SO 4 and concentrated. The crude product was purified by column chromatography on SiO 2 to give the purified product, which was a white solid. Yield 80 %. TLC (silica, hexane: EtOAc, 8:1 v/v): R f = 0.25; 1 H NMR (500 MHz, CDCl 3 ): δ 8.21 (s, 1 H), 7.99 (dd, J = 8.0, 1.5 Hz, 1 H), 7.77 (d, J = 8.0 Hz, 1 H), 5.77 (s, 3 H), (m, 2 H), (m, 2 H), 3.92 (s, 2 H), (m, 1 H), (m, 1 H); 13 C NMR (125 MHz, CDCl 3 ): δ 165.7, S55

56 141.9, 133.9, 132.1, 128.7, 128.3, 122.4, 100.5, 52.6, 25.8; HRMS (ESI + ): (M+H) + calcd. for C 12 H 14 BrO 4, ; found, (3-Bromo-4-(1,3-dioxan-2-yl)phenyl)methanol (5) NaBH 4 (600 mg, 16.7 mmol) was slowly added to a solution of methyl 3-bromo-4-(1,3-dioxan-2-yl)benzoate (4) (500 mg, 1.67 mmol) in 1,4 dioxane/h 2 O (3:2, 10 ml) at 0 o C. The reaction mixture was stirred at 65 o C for 12 h, and then the mixture was quenched with 6 M HCl in an ice bath. An extraction was performed with EtOAc (3 x 100 ml). The combined organic layers were dried over Na 2 SO 4 and concentrated to obtain the crude product, which was then purified by column chromatography on SiO 2 to give a colorless oil. Yield 70 %. TLC (silica, hexane: EtOAc, 1:1 v/v): R f = 0.5; 1 H NMR (500 MHz, CDCl 3 ): δ 7.65 (d, J = 8.0 Hz, 1 H), 7.53 (s, 1 H), 7.27 (d, J = 8.0 Hz, 3 H), 5.76 (s, 1 H), 4.62 (s, 2 H), (m, 2 H), (m, 2 H), (m, 1 H), (m, 1 H); 13 C NMR (125 MHz, CDCl 3 ): δ 143.7, 136.6, 130.7, 128.2, 125.8, 122.5, 100.9, 67.7, 64.1, 25.8; HRMS (ESI + ): (M+H) + calcd. for C 11 H 14 BrO 3, ; found, Bromo-4-(1,3-dioxan-2-yl)benzaldehyde (6) A mixture of (3-bromo-4-(1,3-dioxan-2-yl)phenyl)methanol (5) (218 mg, 0.8 mmol), PCC (244 mg, 1.2 mmol) and Celite (350 mg) in CH 2 Cl 2 (5 ml) was stirred at room temperature for 2 h. The reaction mixture was filtered through Celite and a silica gel pad and then evaporated to obtain the crude product. This was purified by column chromatography on SiO 2, resulting with our pure product, a white solid. Yield 90 %. TLC (silica, hexane: EtOAc, 4:1 v/v): R f = 0.5; 1 H NMR (500 MHz, CDCl 3,): δ 9.95 (s, 1 H), 8.03 (s, 1 H), 7.88 (d, J = 8.0 Hz, 1 H), 7.83 (d, J = 8.0 Hz, 1 H), 5.76 (s, 1 H), 4.27 (dd, J = 11.0, 4.5 Hz, 2 H), 4.03 (dd, J = 12.0, 10.5 Hz, 2 H), (m, 1 H), 1.47 (d, J = 6.8 Hz, 1 H); 13 C NMR (125 MHz, CDCl 3,): δ 190.7, 143.3, 137.8, 133.7, 129.0, 128.7, 123.3, 100.4, 67.7, 25.7; HRMS (ESI + ): (M+H) + calcd. for C 11 H 12 BrO 3, ; found, S56

57 (E)-3-(3-bromo-4-(1,3-dioxan-2-yl)phenyl)-1-(3,5-difluorophenyl)prop-2-en-1-one (7) 5 N NaOH (2.6 ml) was added dropwise into a stirred solution of aldehyde 6 (711 mg, 2.61 mmol) and 3,5-difluoroacetophenone (448 mg, 2.87 mmol) in 10 ml EtOH. The reaction mixture was continuously stirred at room temperature for 2 h. After, the mixture was filtered to collect the solid, which was then washed with water to obtain the crude product. The crude product was then purified by column chromatography on SiO 2 to give the purified product as a white solid. Yield 91 %. TLC (silica, hexane:etoac, 4:1 v/v): R f = 0.45; 1 H NMR (500 MHz, CDCl 3 ): δ 7.82 (d, J = 1.5 Hz, 1 H), (m, 2 H), 7.60 (dd, J = 8.0, 1.5 Hz, 1 H), 7.52 (dd, J = 7.5, 2.0 Hz, 2 H), 7.38 (d, J = 8.0 Hz, 1 H), (m, 1 H), 4.29 (dd, J = 6.0, 5.0 Hz, 2 H), 4.04 (dd, J = 10.5, 10.0 Hz, 2 H), (m, 1 H), 1.47 (d, J = 1.5 Hz, 1 H); 13 C NMR (125 MHz, CDCl 3 ): δ 187.5, 164.2, 162.2, 144.2, 141.0, 139.9, 136.7, 132.4, 128.8, 127.8, 123.1, 122.4, 111.7, 111.6, 111.5, 108.4, 100.6, 67.8, 25.8; HRMS (ESI + ): (M+H) + calcd. for C 19 H 16 BrF 2 O 3, ; found, (E)-2-bromo-4-(3-(3,5-difluorophenyl)-3-oxoprop-1-enyl)benzaldehyde (8) 10 N HCl (9 ml) was added to a solution of compound 7 (735 mg, 1.8 mmol) in THF (11 ml) and stirred at room temperature for 5 h. The reaction was quenched with water (ca. 200 ml) and extracted with EtOAc (3 x 100 ml). The combined organic layers were washed with water, a saturated NaHCO 3 solution, and brine, and then dried with Na 2 SO 4 to obtain the product, a white solid. Yield 99 %. TLC (silica, hexane:etoac, 4:1 v/v): R f = 0.6; 1 H NMR (500 MHz, DMSO d 6 ): δ (s, 1 H), 8.45 (s, 1 H), 8.13 (d, J = 15.5 Hz, 1 H), 8.06 (d, J = 8.0 Hz, 1 H), 7.90 (dd, J = 15.5, 6.0 Hz, 3 H), 7.79 (d, J = 11.5 Hz, 1 H), 7.63 (t, J = 9.0, 5.0 Hz, 1 H); 13 C NMR (125 MHz, DMSO d 6 ): δ 191.2, 186.8, 164.1, 161.7, 142.1, 141.5, 140.1, 133.9, 133.6, 130.3, 128.9, 126.1, 125.2, 112.0, 111.8, 109.0; HRMS (ESI + ): (M+H) + calcd. for C 16 H 10 BrF 2 O 2, ; found, S57

58 (E)-3-(3-bromo-4-(hydroxymethyl)phenyl)-1-(3,5-difluorophenyl)prop-2-en-1-one (9) HCOOH (206 μl, 5.46 mmol) was added dropwise by a syringe to a stirred suspension of Et 3 N (462 μl, 3.32 mmol) in THF (2 ml) and cooled to room temperature under nitrogen. RuCl 2 (PPh 3 ) 3 (13 mg, mmol) was subsequently added and stirred for 3 min. A solution of compound 8 in THF (6 ml) was added into the mixture. The reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with 1 N HCl and extracted with EtOAc (3 x 100 ml). The combined organic layers were washed with water and brine and then dried with Na 2 SO 4. The solvent was evaporated and the crude product was purified by column chromatography on SiO 2 to give the purified product, a white solid. Yield 83 %. TLC (silica, hexane:etoac, 2:1 v/v): R f = 0.2; 1 H NMR (500 MHz, DMSO d 6 ): δ 8.25 (s, 1 H), 8.00 (d, J = 15.5 Hz, 1 H), (m, 3 H), 7.76 (d, J = 15.5 Hz, 1 H), 5.56 (s, 1 H), 4.55 (s, 2 H); 13 C NMR (125 MHz, DMSO d 6 ): δ 186.8, 163.7, 161.7, 161.6, 143.8, 140.6, 135.0, 131.8, 128.8, 128.1, 121.9, 121.5, 111.9, 108.6, 62.6; HRMS (ESI + ): (M+H) + calcd. for C 16 H 12 BrF 2 O 2, ; found, (2-Bromo-4-(3-(3,5-difluorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)phenyl)methanol (10) Phenylhydrazine (206.7 μl, 2.1 mmol) and HCl ( 63.8 μl, 2.1 mmol) were added to a solution of compound 9 (569 mg, 1.62 mmol) and K 2 CO 3 (56 mg, 0.4 mmol) in EtOH (13 ml). The reaction mixture was stirred at 90 o C for 12 h. The reaction was quenched with water (100 ml) and extracted with EtOAc (3 x 100mL). The combined organic layers were washed with water and brine and then dried with Na 2 SO 4. They were then concentrated under reduced pressure. The crude product was purified by column chromatography on SiO 2 to give the purified product, a yellow solid. Yield 65 %. TLC (silica, hexane:etoac, 2:1 v/v): R f = 0.5; 1 H NMR (500 MHz, CDCl 3 ): δ S58

59 7.49 (s, 1 H), 7.44 (d, J = 16.0 Hz, 1 H), 7.37 (s, 1 H), (m, 4 H), 7.06 (d, J = 16.0 Hz, 1 H), 6.85 (t, J = 7.0 Hz, 1 H), 5.26 (dd, J = 12.5, 7.5 Hz, 1 H), 4.70 (s, 2 H), 3.76 (dd, J = 17.0, 12.5 Hz, 1 H), 3.03 (dd, J = 17.0, 7.0 Hz, 1 H); 13 C NMR (125 MHz, CDCl 3 ): δ 164.3, 164.2, 162.3, 162.2, 144.6, 144.0, 143.4, 139.5, 135.8, 123.0, 129.8, 129.2, 125.2, 123.4, 120.2, 113.7, 108.6, 108.4, 103.9, 64.8, 64.0, 43.2; HRMS (ESI ): (M-H) calcd. for C 22 H 16 BrF 2 N 2 O, ; found, Bromo-4-(3-(3,5-difluorophenyl)-1-phenyl-1H-pyrazol-5-yl)benzaldehyde (11) A mixture of compound 10 (453 mg, 1.03 mmol), PCC (1.11 g, 5.17 mmol) and Celite (1 g) in CH 2 Cl 2 (20 ml) was stirred at room temperature for 2 h. The reaction mixture was filtered through Celite and a silica gel pad. The solvent was evaporated to obtain the crude product, which was then purified by column chromatography on SiO 2 to give the purified product, a white solid. Yield 86%. TLC (silica, hexane: EtOAc, 2:1 v/v): R f = 0.85; 1 H NMR (500 MHz, CDCl 3 ): δ (s, 1 H), 7.82 (d, J = 7.0 Hz, 1 H), 7.62 (s, 1 H), (m, 5 H), (m, 2 H), 7.24 (d, J = 8.0 Hz, 1 H), 6.91 (s, 1 H), 6.79 (t, J = 6.5 Hz, 1 H); 13 C NMR (125 MHz, CDCl 3 ): δ 191.1, 164.6, 164.5, 162.6, 162.5, 150.4, 142.0, 139.4, 136.9, 135.9, 133.6, 133.0, 130.0, 129.5, 128.7, 128.0, 127.1, 125.5, 108.8, 108.6, 106.4, 103.6; HRMS (ESI + ): (M+H) + calcd. for C 22 H 14 BrF 2 N 2 O, ; found, (E)-methyl 3-(5-(3-(3,5-difluorophenyl)-1-phenyl-1H-pyrazol-5-yl)-2-formylphenyl)acrylate (12) Tetrabutylammonium acetate (207 mg, 0.68 mmol), K 2 CO 3 (47.3 mg, 0.34 mmol), and Pd(OAc) 2 (5 mg, 0.02 mmol) were added into 25 ml schlenk tube under nitrogen. A solution of methyl acrylate (24.7 μl, 0.27 mmol) in DMF (500 μl) and compound 11 (100 mg, 0.23 mmol) in DMF (2 ml) was also added, and stirred at room temperature for 5 min. The reaction mixture was then stirred at 90 o C for 2 h. The resulting mixture was diluted with EtOAc and filtered through a short pad of Celite. The filtrate was then diluted with water and extracted with EtOAc (3 x 100mL). The combined organic layers were washed with water and brine and then dried with Na 2 SO 4, and concentrated under reduced pressure. The crude product was purified by column chromatography on SiO 2 to give the final product as a yellow solid. Yield 56 %. TLC (silica, hexane:etoac, 2:1 v/v): R f = 0.5; 1 H NMR (500 MHz, CDCl 3 ): δ (s, 1 H), 8.44 (d, J = 16.0 Hz, 1 H), 7.83 (d, J S59

60 = 7.5 Hz, 1 H), (m, 7 H), (m, 2 H), 6.93 (s, 1 H), 6.80 (t, J = 2.0 Hz, 1 H), 6.03 (d, J = 16.0 Hz, 1 H), 6.82 (s, 3 H); 13 C NMR (125 MHz, CDCl 3 ): δ 191.0, 166.4, 164.4, 162.6, 162.5, 150.4, 142.8, 140.6, 139.5, 137.0, 135.9, 135.3, 133.2, 132.6, 129.6, 129.5, 128.7, 128.0, 125.7, 123.6, 108.8, 108.7, 108.6, 106.1, 103.5, 77.4, 77.1, 76.9, 52.1; HRMS (ESI + ): (M+H) + calcd. for C 26 H 19 F 2 N 2 O 3, ; found, Synthetic protocols of compounds Methyl 3-bromo-4-(hydroxymethyl)benzoate (13) HCOOH (650 μl, mmol) was added dropwise by a syringe to a stirred suspension of Et 3 N (1.47 ml, mmol) in THF (4 ml) and cooled to room temperature under nitrogen. RuCl 2 (PPh 3 ) 3 (42 mg, mmol) was subsequently added and stirred for 3 min. A solution of compound 3 in THF (23 ml) was added into the mixture. The reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with 1 N HCl and extracted with EtOAc (3 x 100 ml). The combined organic layers were washed with water and brine and then dried with Na 2 SO 4. The solvent was evaporated and the crude product was purified by column chromatography on SiO 2 to give the purified product, a white solid. Yield 92 %. TLC (silica, hexane:etoac, 4:1 v/v): R f = 0.3; 1 H NMR (500 MHz, CDCl 3 ): δ 8.22 (s, 1 H), 8.21 (d, J = 1.5 Hz, 1 H), 8.01 (dd, J = 1.5, 8 Hz, 1 H), 4.81 (s, 2 H), 3.94 (s, 3 H); 13 C NMR (125 MHz, CDCl 3 ): δ 165.8, 144.9, 133.6, 130.9, 128.8, 128.2, 121.9, 64.8, 52.5; HRMS (ESI + ): (M+H) + calcd. for C 9 H 10 BrO 3, ; found, Methyl 3-bromo-4-((tert-butyldimethylsilyloxy)methyl)benzoate (14) A solution of TBSCl (958 mg, 6.39 mmol) in dry DMF (5 ml) was added by using a syringe to a solution of compound 13 (1.3 g, 5.33 mmol) and imidazole (727 mg, 76.4 mmol) in dry DMF (5 ml) in a 25 ml Schlenk tube. The reaction mixture was stirred at room temperature for 12 h. The S60

61 reaction was quenched with deionized water (100 ml) and extracted with EtOAc (3 x 100 ml). The combined organic layers were washed with water and brine and then dried with Na 2 SO 4. The solvent was evaporated and the crude product was purified by column chromatography on SiO 2 to give the purified product, colorless oil. Yield 99 %. TLC (silica, hexane:etoac, 4:1 v/v): R f = 0.8; 1 H NMR (500 MHz, CDCl 3 ): δ 8.17 (s, 1 H), 8.00 (d, J = 1.5, 8 Hz, 1 H), 7.65 (d, J = 8 Hz, 1 H), 4.76 (s, 2 H), 3.92 (s, 3 H), 0.97 (s, 9 H), 0.15 (s, 6 H); 13 C NMR (125 MHz, CDCl 3 ): δ 166.0, 145.7, 133.2, 130.3, 128.6, 127.4, 120.7, 64.7, 52.4, 26.0, 18.5, -5.2; HRMS (ESI + ): (M+H) + calcd. for C 15 H 24 BrO 3 Si, ; found, (3-Bromo-4-((tert-butyldimethylsilyloxy)methyl)phenyl)methanol (15) NaBH 4 (2.64 g, 69.8 mmol) was slowly added to a solution of compound 14 (2.50 g, 6.98 mmol) in 1,4 dioxane/h 2 O (3:2, 34 ml) at 0 o C. The reaction mixture was stirred at 65 o C for 12 h, and then the mixture was quenched with 6 M HCl in an ice bath. An extraction was performed with EtOAc (3 x 100 ml). The combined organic layers were dried over Na 2 SO 4 and concentrated to obtain the crude product, which was then purified by column chromatography on SiO 2 to give a colorless oil. Yield 72 %. TLC (silica, hexane:etoac, 4:1 v/v): R f = 0.4; 1 H NMR (500 MHz, CDCl 3 ): δ 7.54 (d, J = 8.0 Hz, 1 H), 7.52 (s, 1 H), 7.30 (d, J = 7.5 Hz, 1 H), 4.74 (s, 2 H), 4.63 (s, 2 H), 0.99 (s, 9 H), 0.15 (s, 6 H); 13 C NMR (125 MHz, CDCl 3 ): δ 141.3, 139.6, 130.6, 127.8, 125.9, 121.2, 64.6, 64.4, 26.0, 18.4, -5.3; HRMS (ESI + ): (M+H) + calcd. for C 14 H 24 BrO 2 Si, ; found, Bromo-4-((tert-butyldimethylsilyloxy)methyl)benzaldehyde (16) A mixture of compound 15 (1.37 g, 4.15 mmol), PCC (1.34 g, 6.23 mmol) and Celite (1 g) in CH 2 Cl 2 (30 ml) was stirred at room temperature for 1 h. The reaction mixture was filtered through Celite and a silica gel pad and then evaporated to obtain the crude product. This was purified by column chromatography on SiO 2 to give an yellow oil. Yield 64 %. TLC (silica, hexane:etoac, 4:1 v/v): R f = 0.8; 1 H NMR (500 MHz, CDCl 3 ): δ 9.92 (s, 1 H), 7.97 (s, 1 H), 7.81 (d, J = 8.0 Hz, 1 H), 7.73 (d, J = 8.0 Hz, 1 H), 4.75 (s, 2 H), 0.96 (s, 9 H), 0.13 (s, 6 H); 13 C NMR (125 MHz, CDCl 3 ): δ 190.6, 147.4, 136.5, 132.8, 128.9, 128.0, 121.6, 64.7, 26.0, 18.4, -5.3; HRMS (ESI + ): (M+H) + calcd. for C 14 H 22 BrO 2 Si, ; found, S61

62 4, 4-Difluoro-8-(3-Bromo-4-((tert-butyldimethylsilyloxy)methyl)phenyl)-1,3,5,7-tetramethyl-4- bora-3a,4a-diaza-s-indacene (17) Compound 16 (783mg, mmol) and 2,4-dimethylpyrrole (492μL, 4.77 mmol) were dissolved in 100 ml of dry CH 2 Cl 2 under N 2. One drop of TFA was added, after the solution was stirred at room temperature for 12 h, DDQ (543 mg, mmol) was added, and stirring was continued for 1 h. The reaction mixture was washed with water three times and brine once, dried over Na 2 SO 4. The compound was purified by short column chromatography on SiO 2 (eluent: EtOAc with 1% Et 3 N). The Red powder thus obtained and 4 ml of DIPEA were dissolved in 20 ml of CH 2 Cl 2 under N 2. Then 8 ml of BF 3 Et 2 O was added, and the solution was stirred at room temperature for 2 h. The reaction mixture was washed with water three times and brine once, dried over Na 2 SO 4, filtered, and evaporated. the crude product was purified by column chromatography on SiO 2 to give the purified product, a red foam. Yield 58 %. TLC (silica, hexane:etoac, 2:1 v/v): R f = 0.85; 1 H NMR (500 MHz, CDCl 3 ): δ 7.70 (d, J = 7.5 Hz, 1 H), 7.47 (s, 1 H), 7.27 (d, J = 7.5 Hz, 1 H), 6.00 (s, 2 H), 4.83 (s, 2 H), 2.56 (s, 6 H), 1.44 (s, 6 H), 1.00 (s, 9 H), 0.18 (s, 6 H); 13 C NMR (125 MHz, CDCl 3 ): δ 155.9, 143.1, 141.6, 139.8, 135.1, 131.5, 131.4, 128.2, 127.3, 121.5, 121.4, 64.6, 26.0, 18.5, 14.8, 14.7, -5.2; HRMS (ESI + ): (M+H) + calcd. for C 26 H 35 BBrF 2 N 2 OSi, ; found , 4-Difluoro-8-(3-bromo-4-(hydroxymethyl)phenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-i ndacene (18) A solution of 17 (187 mg, mmol) and TBAF (1.0M in THF, 326μL, mmol) in THF (3 ml) was stirred at RT for 30 min, then it was quenched with aq. sat. NaHCO 3 (2 ml) and S62

63 extracted with EtOAc (3 x 100 ml), dried over Na 2 SO 4, filtered, and evaporated. The crude product was purified by column chromatography on SiO 2 to give the purified product, a red powder. Yield 83 %. TLC (silica, hexane:etoac, 2:1 v/v): R f = 0.3; 1 H NMR (500 MHz, CDCl 3 ): δ 7.58 (d, J = 7.5 Hz, 1 H), 7.43 (s, 1 H), 7.20 (d, J = 8.0 Hz, 1 H), 5.92 (s, 2 H), 4.75 (s, 2 H), 2.47 (s, 6 H), 1.35 (s, 6 H); 13 C NMR (125 MHz, CDCl 3 ): δ 156.0, 142.9, 140.9, 139.3, 135.7, 131.9, 131.2, 128.9, 127.4, 122.5, 121.5, 64.5, 14.8; HRMS (ESI + ): (M+H) + calcd. for C 20 H 21 BBrF 2 N 2 O, ; found, , 4-Difluoro-8-(3-bromo-4-formylphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (19) A mixture of compound 18 (499 mg, mmol), PCC (495 mg, 2.31 mmol) and MgSO 4 (480 mg) in CH 2 Cl 2 (200 ml) was stirred at room temperature for 30 min under N 2. The reaction mixture was filtered through Celite and then evaporated to obtain the crude product. The crude product was purified by column chromatography on SiO 2 to give the purified product, a red powder. Yield 65 %. TLC (silica, hexane:etoac, 2:1 v/v): R f = 0.8; 1 H NMR (500 MHz, CDCl 3 ): δ (s, 1 H), 8.05 (d, J = 9.5 Hz, 1 H), 7.67 (s, 1 H), 7.42 (d, J = 10.0 Hz, 1 H), 6.02 (s, 2 H), 2.56 (s, 6 H), 1.44 (s, 6 H); 13 C NMR (125 MHz, CDCl 3 ): δ 191.1, 156.8, 142.7, 142.6, 137.8, 133.9, 133.8, 130.7, 130.6, 128.3, 127.5, 120.0, 15.0, 14.8; HRMS (ESI + ): (M+H) + calcd. for C 20 H 19 BBrF 2 N 2 O, ; found, SFP-2 probe (20) Triphenylphosphine (9.1 mg, mmol), Et 3 N (24μL, mmol), Pd(OAc) 2 (3 mg, 0.1 equiv.) and CH 3 CN (500μL) were added into 25 ml schlenk tube under N 2. A solution of methyl acrylate (42 μl, mmol) in CH 3 CN (2.50 ml) was added. The reaction mixture was then stirred at 90 o C for 12 h. The resulting mixture was diluted with EtOAc and filtered through a short pad of Celite. The filtrate was then diluted with water and extracted with EtOAc (3 x 20mL). S63

64 The combined organic layers were washed with water and brine and then dried with Na 2 SO 4, and concentrated under reduced pressure. The crude product was purified by column chromatography on SiO 2 to give the final product as a red solid. Yield 38 %. TLC (silica, hexane:etoac, 2:1 v/v): R f = 0.8; 1 H NMR (500 MHz, CDCl 3 ): δ 10.4 (s, 1 H), 8.55 (d, J = 16.0 Hz, 1 H), 8.04 (dd, J = 2.0, 8.0 Hz, 1 H), 7.63 (s, 1 H), 7.54 (dd, J = 1.5, 8.0 Hz, 1 H), 6.39 (dd, J = 4.0, 16.0 Hz, 1 H), 6.01 (s, 2 H), 3.83 (s, 3 H), 2.56 (s, 6 H), 1.38 (s, 6 H); 13 C NMR (125 MHz, CDCl 3 ): δ 191.1, 166.4, 156.7, 142.6, 141.1, 140.1, 138.8, 137.6, 134.1, 133.0, 132.2, 130.0, 128.1, 123.9, 121.9, 52.2, 14.9, 14.8; HRMS (ESI + ): (M+H) + calcd. for C 24 H 24 BF 2 N 2 O 3, ; found, Crystallographic data collection. An irregular broken fragment (0.40 x 0.20 x 0.20 mm) from crystals of 12 grown by solvent evaporation was selected under a stereo-microscope while immersed in Fluorolube oil to avoid possible drying in air. The crystal was removed from the oil using a tapered glass fiber that also served to hold the crystal for data collection. The crystal was mounted and centered on a Bruker SMART APEX system at 100 K. Rotation and still images showed the diffractions to be sharp. Frames separated in reciprocal space were obtained and provided an orientation matrix and initial cell parameters. Final cell parameters were obtained from the full data set. A full sphere data set was obtained which samples approximately all of reciprocal space to a resolution of 0.75 Å using 0.3 o steps in ω using 10 second integration times for each frame. Data collection was made at 100 K. Integration of intensities and refinement of cell parameters were done using SAINT 1. Absorption corrections were applied using SADABS 30 based on redundant diffractions. Structure solution and refinement. The space group was determined as P1(bar) based on systematic absences and intensity statistics. Direct methods were used to locate most C atoms from the E-map. Repeated difference Fourier maps allowed recognition of all expected C, N, O and F atoms. Following anisotropic refinement of all non-h atoms, ideal H-atom positions were calculated. Final refinement was anisotropic for all non-h atoms, and isotropic-riding for H atoms. No anomalous bond lengths or thermal S64

65 parameters were noted. All ORTEP diagrams have been drawn with 50% probability ellipsoids. CCDC contains the supplementary crystallographic data for this paper. The data can be obtained free of charge from the Cambridge Crystallographic Data Centre Via Equations of interest: R int = Σ F 2 o - <F 2 o > / Σ F 2 o R1 = Σ F o - F c / Σ F o wr2 = [Σ [w (F 2 o F 2 c ) 2 ] / Σ [w (F 2 o ) 2 ]] 1/2 2 GooF = S = [Σ [w (F o F 2 c ) 2 ] / (n-p) 1/2 where: w = q /σ 2 (F o 2 ) + (ap) 2 + bp; n = number of independent reflections; q, a, b, P as defined in [1]; p = number of parameters refined. Fluorometric analysis. All fluorescence measurements were carried out at room temperature on a Varian Cary Eclipse Fluorescence Spectrophotometer. The samples were excited at 300 nm with the excitation and emission slit widths set at 5 nm and 10 nm. The emission spectrum was scanned from 310 nm to 550 nm at 120 nm/min. The photomultiplier voltage was set at 1000 V. The probe was dissolved in CH 3 CN or DMSO to make a 10 mm stock solution, which was diluted to the required concentration for measurement. Na 2 S stock solution (20 mm) preparation mg EDTA was dissolved in 10 ml DI H 2 O in a 25 ml Schlenk tube. The solution was purged vigorously with nitrogen for 15 min. Then 48 mg sodium sulfide (Na 2 S 9H2O) was dissolved in the solution under nitrogen. The resulting solution was 20 mm Na 2 S, which was then diluted to 1 mm stock solution for general use. Cystathionine β-synthase assay Recombinant human cystathioinine β synthatse (CBS) was purified as described previously 32. It was mixed with either 10 mm homocysteine or 10 mm cysteine and probe (5 or 10 µm) in 100 mm phosphate buffer (ph 7.4). Solutions were prepared in quartz fluorescence cuvettes. Reactions were started by addition of CBS and fluorescence emission was monitored in a Varian Cary S65

66 Eclipse fluorescence spectrophotometer. The excitation wavelength was set at 300 nm with both the excitation and emission slit widths set at 10 nm. Changes in fluorescence were monitored over 30 min at one min intervals. All reactions were monitored at room temperature (25 C). Reaction mixtures lacking CBS and/or substrates were recorded as controls. CBS catalyzes the generation of H 2 S through one of three methods: β-replacement of cysteine by water (equation 1), via reaction of two cysteines (equation 2), or via reaction of cysteine and homocysteine (equation 3) 8. Cysteine + H 2 O Serine + H 2 S [1] Cysteine + Cysteine Lanthionine + H 2 S [2] Cysteine + Homocysteine Cystathionine + H 2 S [3] Cytotoxicity assay Hela cells were grown in DMEM media with 10% FBS and penicillin/streptomycin (Invitrogen). Cells were allowed to grow to 80% confluency before being harvested using trypsin-edta. The cell number was determined and solution was diluted to a final concentration of 2.22 x 10 5 cells/ml in the aforementioned media. A final number of 2 x 10 4 cells (90 μl) was transferred to each well in a 96 well plate (BD Falcon). Cells were incubated overnight at 37 o C in a 5% CO 2 atmosphere. A serial dilution of probe 12 was performed in DMEM media, with 10 μl added to each well to give final concentrations of 0.4, 0.8, 1.6, 3.1, 12.5, 25, 50, and 100 μm probe. Cells were allowed to incubate for 20 h. Wells containing only cells and only probe were also set up to serve as positive and negative controls. Dye solution and stop/solubilization mix were obtained from a CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega). Cytotoxicity assay was performed as per manufacturer s instructions. Absorbance at 570 was monitored using a Synergy Plate Reader (BioTek). Data was collected for three separate serial dilutions and averaged. Cellular Imaging Experiments Hela cells were grown as previously described. Cells were allowed to grow to 80% confluency before being harvested and transferred to a 6-well plate (BD Falcon). These cells were allowed to S66

67 grow overnight at 37 o C in a 5% CO 2 atmosphere. Cells were maintained at these conditions until immediately prior to imaging experiments. At this time, a final concentration of 10 μm probe 12 was added to the cells and they were allowed to incubate at the previous conditions for 15 min. Media was then removed and fresh media was added to remove any probe left in solution and optimize the background signal. Sulfur source was then added (Na 2 S, cysteine, or GSH) to the desired concentration and cells were incubated for min at room temperature prior to imaging. All imaging experiments were performed on a fixed cell DSU spinning confocal microscope (Olympus). Wide-field fluorescence capture was used to visualize probe 12 under all conditions. Excitation and emission monitored using the DAPI filters provided with the scope, set at 387 nm and 440 nm, respectively. Imaging performed using either the 20x or 40x dry objectives that are provided with the scope. Images were captured using Slidebook software References 30. All software and sources of scattering factors are contained in the SHELXTL (version 5.1) program library (G. Sheldrick, Bruker Analytical X-ray Systems, Madison, WI). 31. Zhao, Y., Wang, H., & Xian, M. J. Am. Chem. Soc., 133, (2011). 32. Taoka, S., Ohja, S., Shan, X., Kruger, W. D., and Banerjee, R. J Biol Chem 273, (1998). S67

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