S1

Supplementary Information

A Selective Fluorescent Probe for Live Cell-Monitoring of Sulfide

Yong Qian1, 2, Jason Karpus1, Omer Kabil3, Hai-Liang Zhu2, Ruma Banerjee3, Jing Zhao2 & Chuan He1

1Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA. 2State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093, China. 3Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109-0600, 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 Na2S in PBS buffer after different

incubation times.

Supplementary Figure 5. Fluorescence spectra of SFP-1 in PBS buffer incubated with different

concentrations of Na2S.

Supplementary Figure 6. Fluorescence spectra of SFP-1 in the presence of H2S.

Supplementary Figure 7-8. High resolution mass spectrometry identification of 20 and 20a.

Supplementary Figure 9. Fluorescence spectra of SFP-2 with Na2S in PBS buffer after different

incubation times.

Supplementary Figure 10–12. Fluorescence spectra of SFP-2 in PBS buffer incubated with different

concentrations of Na2S.

Supplementary Figure 13. Cytotoxicity assay of SFP-1.

Supplementary Figure 14. Additional imaging experiments with various sulfur sources.

Supplementary Figure 15-54. NMR spectra of synthesized compounds.

Supplementary Tables 1-5: Crystal and structure refinement parameters for compound 12.

Supplementary Methods

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Supplementary References

Supplementary Figure 1. Crystal structure diagrams of compound 12. Molecular structure diagram

with displacement ellipsoids being at the 50% probability level.

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Supplementary Figure S2. HRMS identification of SFP-1 probe 12 (calculated for C26H19F2N2O3

(M+H)+ 445.1363; found 445.1360) prior to the addition of sulfide.

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Supplementary Figure S3. HRMS identification for the sulfide addition product of SFP-1 probe 12a

(calculated for C26H21F2N2O3S (M+H)+ 479.1241; found 479.1243).

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Supplementary Figure S4. Fluorescence spectra of 10 μM SFP-1 probe in 10 mM PBS buffer (pH 7.4, 10% CH3CN) with 50 μM Na2S. Probe was allowed to incubate with Na2S for various amounts of time (5, 15, 30, 45, 60, 90, 120 min) at 37 oC prior to measurement. Fluorescence intensity at 388 nm was monitored at each time point. Excitation: 300 nm, emission: 310–550 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% CH3CN) with increasing amounts of Na2S. Probe was incubated with concentrations of 10, 20, 30, 40, 50, 60, and 80 μM Na2S at 37oC for 60 min. Fluorescence intensity at 388 nm was monitored as a function of analyte concentration. Excitation: 300 nm, emission: 310–550 nm. The data represent the mean ± SD of at least three independent experiments.

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Supplementary Figure S6. Fluorescence spectra of 10 μM SFP-1 measured in the presence of PBS buffers that H2S gas had been bubbled through for varying lengths of time. SFP-1 probe was allowed to incubate in the buffers for 60 min at 37oC. Measurements performed in 10 mM PBS buffer (pH 7.4, 10% CH3CN). Excitation: 300 nm, emission: 310–550 nm. All H2S buffers were prepared by adding 10 ml DI H2O to a 25 ml Schlenk tube and subsequently bubbling nitrogen through for 30 min. H2S 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.

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Supplementary Figure S7. HRMS identification of SFP-2 probe 20 (calculated for C24H24BF2N2O3

(M+H)+ 437.1848; found 437.1874.) prior to the addition of sulfide.

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Supplementary Figure S8. HRMS identification of the sulfide addition product of SFP-2 probe 20a

(calculated for C24H26BF2N2O3S (M+H)+ 471.1724; found 471.1732).

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Supplementary Figure S9. Fluorescence spectra of 5 μM SFP-2 probe in 20 mM PBS buffer (pH 7.0, 1% DMSO) with 50 μM Na2S. Probe was allowed to incubate with Na2S for various amounts of time (5, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210 and 240 min) at 37 oC prior to measurement. Fluorescence intensity at 510 nm was monitored at each time point. Excitation: 465 nm, emission: 480–580 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 Na2S. Probe was incubated with concentrations of 5, 10, 20, 40, 60, 80, and 100 μM Na2S at 37 oC for 120 min. Fluorescence intensity at 510 nm was monitored as a function of analyte concentration. Excitation: 465 nm, emission: 480–580 nm. The data represent the mean ± SD of at least three independent experiments.

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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 Na2S. Probe was incubated with concentrations of 5, 10, 20, 40, 60, 80, and 100 μM Na2S at 37 oC for overnight (14 h). (b) Fluorescence intensity at 510 nm was monitored as a function of analyte concentration. Excitation: 465 nm, emission: 480–580 nm. The data represent the mean ± SD of at least three independent experiments.

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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 H2S 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 H2S gas had been bubbled through for varying lengths of time. Probe was allowed to incubate in the buffers for 20 min at 25oC. Measurements performed in 20 mM PBS buffer (pH 7.0, 1% DMSO). Excitation: 465 nm, emission: 480–580 nm. All H2S buffers were prepared by adding 10 ml DI H2O to a 25 ml Schlenk tube and subsequently bubbling nitrogen through for 30 min. H2S 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.

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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.

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Supplementary Figure S15. 1H NMR spectrum of compound 1.

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Supplementary Figure S16. 13C NMR spectrum of compound 1.

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Supplementary Figure S17. 1H NMR spectrum of compound 2.

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Supplementary Figure S18. 13C NMR spectrum of compound 2.

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Supplementary Figure S19. 1H NMR spectrum of compound 3.

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Supplementary Figure S20. 13C NMR spectrum of compound 3.

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Supplementary Figure S21. 1H NMR spectrum of compound 4.

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Supplementary Figure S22. 13C NMR spectrum of compound 4.

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Supplementary Figure S23. 1H NMR spectrum of compound 5.

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Supplementary Figure S24. 13C NMR spectrum of compound 5.

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Supplementary Figure S25. 1H NMR spectrum of compound 6.

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Supplementary Figure S26. 13C NMR spectrum of compound 6.

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Supplementary Figure S27. 1H NMR spectrum of compound 7.

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Supplementary Figure S28. 13C NMR spectrum of compound 7.

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Supplementary Figure S29. 1H NMR spectrum of compound 8.

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Supplementary Figure S30. 13C NMR spectrum of compound 8.

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Supplementary Figure S31. 1H NMR spectrum of compound 9.

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Supplementary Figure S32. 13C NMR spectrum of compound 9.

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Supplementary Figure S33. 1H NMR spectrum of compound 10.

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Supplementary Figure S34. 13C NMR spectrum of compound 10.

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Supplementary Figure S35. 1H NMR spectrum of compound 11.

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Supplementary Figure S36. 13C NMR spectrum of compound 11.

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Supplementary Figure S37. 1H NMR spectrum of compound 12.

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Supplementary Figure S38. 13C NMR spectrum of compound 12.

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Supplementary Figure S39. 1H NMR spectrum of compound 13.

Supplementary Figure S40. 13C NMR spectrum of compound 13.

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Supplementary Figure S41. 1H NMR spectrum of compound 14.

Supplementary Figure S42. 13C NMR spectrum of compound 14.

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Supplementary Figure S43. 1H NMR spectrum of compound 15.

Supplementary Figure S44. 13C NMR spectrum of compound 15.

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Supplementary Figure S45. 1H NMR spectrum of compound 16.

Supplementary Figure S46. 13C NMR spectrum of compound 16.

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Supplementary Figure S47. 1H NMR spectrum of compound 17.

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Supplementary Figure S48. 13C NMR spectrum of compound 17.

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Supplementary Figure S49. 1H NMR spectrum of compound 18.

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Supplementary Figure S50. 13C NMR spectrum of compound 18.

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Supplementary Figure S51. 1H NMR spectrum of compound 19.

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Supplementary Figure S52. 13C NMR spectrum of compound 19.

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Supplementary Figure S53. 1H NMR spectrum of compound 20.

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Supplementary Figure S54. 13C NMR spectrum of compound 20.

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Supplementary Table 1: Crystal and structure refinement for compound 12.

Identification Code compound 12

Empirical formula C26H18F2N2O3

Formula weight 444.42

Temperature 100 K

Wavelength 0.71073 Å

Crystal system Triclinic

Space Group P1(bar)

Unit cell dimensions a = 8.781(2) Å α = 62.420(4) o

b = 11.624(3) Å β = 89.149(4) o

c = 11.964(3) Å γ = 75.148(4) o

Volume 1038.6(4) Å3

Z 2

Density (calculated) 1.421 Mg/m3

Absorption coefficient 0.106 mm-1

F(000) 460

Crystal size, color, habit 0.40 x 0.20 x 0.20mm, clear, fragment

Theta range for data collection 1.93 – 28.31 o

Index ranges -11 ≤ h ≤ 11, -15 ≤ k ≤ 15, -15 ≤ l ≤ 15

Reflections collected 12,683

Independent reflections 4,989 (Rint = 0.0189)

Reflections with I > 4σ(Fo) 4,423

Absorption correction SADABS based on redundant diffractions

Max. and min. transmission 1.0, 0.824

Refinement method Full-matrix least squares on F2

Weighting scheme w = q [σ2 (Fo2) + (aP)2 + bP]-1 where:

P = (Fo2

+2 Fc2)/3, a = 0.0703, b = 0.128, q =1

Data / restraints / parameters 4989 / 0 / 299

Goodness-of-fit on F2 1.063

Final R indices [I > 2 sigma(I)] R1 = 0.0423, wR2 = 0.1162

R indices (all data) R1 = 0.0479, wR2 = 0.1198

Largest diff. peak and hole 0.332, -0.254 eÅ -3

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Supplementary Table 2. Atomic coordinates [ x 104] and equivalent isotropic displacement parameters [Å2 x 103] for compound 12. U(eq) is defined as one third of the trace of the orthogonalized Uij 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) _____________________________________________________________________

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Supplementary Table 3. Bond lengths [Å] and angles [o] for compound 12. _______________________________________________________________________ C(1)-C(6) 1.3846(17) C(1)-C(2) 1.3899(17) C(1)-N(1) 1.4250(15) C(2)-C(3) 1.3869(18) C(3)-C(4) 1.382(2) C(4)-C(5) 1.3843(19) C(5)-C(6) 1.3875(17) C(7)-C(8) 1.3708(16) C(7)-N(1) 1.3715(15) C(7)-C(16) 1.4678(16) C(8)-C(9) 1.4051(16) C(9)-N(2) 1.3354(15) C(9)-C(10) 1.4668(16) C(10)-C(11) 1.3915(17) C(10)-C(15) 1.3978(17) C(11)-C(12) 1.3725(17) C(12)-F(1) 1.3533(15) C(12)-C(13) 1.3748(19)

C(13)-C(14) 1.3771(18) C(14)-F(2) 1.3562(14) C(14)-C(15) 1.3746(17) C(16)-C(21) 1.3888(16) C(16)-C(17) 1.3999(17) C(17)-C(18) 1.3789(16) C(18)-C(19) 1.3950(16) C(19)-C(20) 1.4143(17) C(19)-C(22) 1.4750(16) C(20)-C(21) 1.3955(16) C(20)-C(23) 1.4710(16) C(22)-O(3) 1.2096(15) C(23)-C(24) 1.3331(17) C(24)-C(25) 1.4699(17) C(25)-O(1) 1.2117(15) C(25)-O(2) 1.3388(15) C(26)-O(2) 1.4415(15) N(1)-N(2) 1.3602(13)

C(6)-C(1)-C(2) 120.58(11) C(6)-C(1)-N(1) 118.91(10) C(2)-C(1)-N(1) 120.48(11) C(3)-C(2)-C(1) 119.14(12) C(4)-C(3)-C(2) 120.61(12) C(3)-C(4)-C(5) 119.86(12) C(4)-C(5)-C(6) 120.17(12) C(1)-C(6)-C(5) 119.62(11) C(8)-C(7)-N(1) 106.53(10) C(8)-C(7)-C(16) 128.18(11) N(1)-C(7)-C(16) 124.67(10) C(7)-C(8)-C(9) 105.38(11) N(2)-C(9)-C(8) 111.58(10) N(2)-C(9)-C(10) 120.28(10) C(8)-C(9)-C(10) 127.96(11) C(11)-C(10)-C(15) 119.57(11) C(11)-C(10)-C(9) 119.33(11) C(15)-C(10)-C(9) 121.00(11) C(12)-C(11)-C(10) 118.90(12) F(1)-C(12)-C(11) 118.05(12) F(1)-C(12)-C(13) 118.34(11) C(11)-C(12)-C(13) 123.61(12) C(12)-C(13)-C(14) 115.69(11) F(2)-C(14)-C(15) 118.41(11) F(2)-C(14)-C(13) 117.54(11) C(15)-C(14)-C(13) 124.05(12) C(14)-C(15)-C(10) 118.16(11) C(21)-C(16)-C(17) 119.53(11) C(21)-C(16)-C(7) 119.50(11) C(17)-C(16)-C(7) 120.88(10) C(18)-C(17)-C(16) 119.21(11) C(17)-C(18)-C(19) 121.62(11) C(18)-C(19)-C(20) 119.75(11) C(18)-C(19)-C(22) 115.30(11) C(20)-C(19)-C(22) 124.94(11) C(21)-C(20)-C(19) 117.79(11) C(21)-C(20)-C(23) 119.74(11) C(19)-C(20)-C(23) 122.33(10) C(16)-C(21)-C(20) 122.08(11)

O(3)-C(22)-C(19) 127.64(12) C(24)-C(23)-C(20) 124.47(11) C(23)-C(24)-C(25) 123.37(11) O(1)-C(25)-O(2) 123.29(12) O(1)-C(25)-C(24) 123.91(11) O(2)-C(25)-C(24) 112.76(10) N(2)-N(1)-C(7) 111.77(9) N(2)-N(1)-C(1) 118.20(9) C(7)-N(1)-C(1) 129.72(10) C(9)-N(2)-N(1) 104.71(9) C(25)-O(2)-C(26) 115.40(10)

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Supplementary Table 4. Anisotropic displacement parameters [Å2 x 103]

for compoud 12. The anisotropic displacement factor exponent takes the

form:

-2π2[h2a*2U11+ ... + 2hka*b*U12] _____________________________________________________________________

U11 U22 U33 U23 U13 U12

_____________________________________________________________________

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)

_____________________________________________________________________

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Supplementary Table 5. Hydrogen coordinates [ x 104] and isotropic

displacement parameters [Å2 x 103] for compound 12.

________________________________________________________________

x y z U(eq)

________________________________________________________________ H(2) 4020 1773 4803 31

H(3) 5453 1202 3382 37

H(4) 6611 2727 1838 38

H(5) 6296 4857 1677 35

H(6) 4905 5429 3114 30

H(8) 1765 3826 7307 28

H(11) 477 5910 7180 32

H(13) -1804 9899 5032 35

H(15) 579 7744 3352 28

H(17) 6439 2241 6397 29

H(18) 8031 101 7813 28

H(21) 2567 1150 7939 27

H(22) 8503 -2054 9408 31

H(23) 4420 -2330 9912 27

H(24) 1837 -209 9650 29

H(26A) 1468 -4539 12244 53

H(26B) 2184 -5262 11417 53

H(26C) 478 -4170 10953 53

________________________________________________________________

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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).

H2SO4 (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

NaHCO3. 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 SiO2 giving a colorless oil. Yield 87 %. TLC (silica, hexane:

EtOAc, 4:1 v/v): Rf = 0.7; 1H NMR (400 MHz, CDCl3): 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); 13C NMR (125 MHz, CDCl3): 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 C9H10BrO2,

228.9864; found, 228.9869.

(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 Ac2O (34 mL, 18 eq) containing H2SO4 (5 mL, 4.5 eq). The solution was cooled to 0 oC in

an ice-bath and CrO3 (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 H2O (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 SiO2 giving

a white solid as the purified product. Yield 57%. TLC (silica, hexane: EtOAc, 8:1 v/v): Rf = 0.2;

1H NMR (500 MHz, CDCl3): 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); 13C NMR (125 MHz, CDCl3): 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

C13H13BrNaO6, 366.9794; found, 366.9797.

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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–H2O (1:1, 30 mL). H2SO4 (440 μL) was added and the mixture was heated

under reflux at 100 °C for 30 min. The mixture was then diluted with H2O and extracted with

EtOAc (3 x 100 mL). The solution was dried over Na2SO4, 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

Na2SO4. The solvent was removed by rotary evaporation to yield a white solid. Yield 78 %. TLC

(silica, hexane: EtOAc, 8:1 v/v): Rf = 0.6; 1H NMR (500 MHz, CDCl3): δ 10.36 (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); 13C NMR (125 MHz, CDCl3):

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

C9H8BrO3, 242.9657; found, 242.9675.

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 Na2SO4 (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 Na2SO4 and concentrated. The crude product was purified by column

chromatography on SiO2 to give the purified product, which was a white solid. Yield 80 %. TLC

(silica, hexane: EtOAc, 8:1 v/v): Rf = 0.25; 1H NMR (500 MHz, CDCl3): 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), 4.26–4.30 (m, 2 H), 4.01–4.06 (m, 2

H), 3.92 (s, 2 H), 2.24–2.27 (m, 1 H), 1.46–1.49 (m, 1 H); 13C NMR (125 MHz, CDCl3): δ 165.7,

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141.9, 133.9, 132.1, 128.7, 128.3, 122.4, 100.5, 52.6, 25.8; HRMS (ESI+): (M+H)+ calcd. for

C12H14BrO4, 301.0075; found, 301.0065.

(3-Bromo-4-(1,3-dioxan-2-yl)phenyl)methanol (5)

NaBH4 (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/H2O (3:2, 10 mL)

at 0 oC. The reaction mixture was stirred at 65 oC 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 Na2SO4 and concentrated to obtain the crude product,

which was then purified by column chromatography on SiO2 to give a colorless oil. Yield 70 %.

TLC (silica, hexane: EtOAc, 1:1 v/v): Rf = 0.5; 1H NMR (500 MHz, CDCl3): 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), 4.03–4.29 (m, 2 H),

4.01–4.02 (m, 2 H), 2.24–2.30 (m, 1 H), 1.45–1.49 (m, 1 H); 13C NMR (125 MHz, CDCl3): 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 C11H14BrO3, 273.0121; found, 273.0129.

3-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 CH2Cl2 (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 SiO2, resulting with our pure

product, a white solid. Yield 90 %. TLC (silica, hexane: EtOAc, 4:1 v/v): Rf = 0.5; 1H NMR (500

MHz, CDCl3,): 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), 2.21–2.29 (m, 1

H), 1.47 (d, J = 6.8 Hz, 1 H); 13C NMR (125 MHz, CDCl3,): 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 C11H12BrO3, 270.9970; found,

270.9966.

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(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 SiO2 to give the purified product as a white solid. Yield

91 %. TLC (silica, hexane:EtOAc, 4:1 v/v): Rf = 0.45; 1H NMR (500 MHz, CDCl3): δ 7.82 (d, J =

1.5 Hz, 1 H), 7.73–7.76 (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), 7.03–7.07 (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), 2.25–2.28 (m, 1 H), 1.47 (d, J = 1.5 Hz, 1 H); 13C NMR (125 MHz, CDCl3): 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 C19H16BrF2O3, 409.0251; found,

409.0240.

(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 NaHCO3 solution, and brine, and then dried with Na2SO4 to obtain the product, a white

solid. Yield 99 %. TLC (silica, hexane:EtOAc, 4:1 v/v): Rf = 0.6; 1H NMR (500 MHz, DMSO–d6):

δ 10.22 (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); 13C NMR (125 MHz,

DMSO–d6): 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 C16H10BrF2O2, 350.9832; found,

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350.9827.

(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 Et3N

(462 μL, 3.32 mmol) in THF (2 mL) and cooled to room temperature under nitrogen.

RuCl2(PPh3)3 (13 mg, 0.136 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 Na2SO4.

The solvent was evaporated and the crude product was purified by column chromatography on

SiO2 to give the purified product, a white solid. Yield 83 %. TLC (silica, hexane:EtOAc, 2:1 v/v):

Rf = 0.2; 1H NMR (500 MHz, DMSO–d6): δ 8.25 (s, 1 H), 8.00 (d, J = 15.5 Hz, 1 H), 7.89–7.91

(m, 3 H), 7.76 (d, J = 15.5 Hz, 1 H), 5.56 (s, 1 H), 4.55 (s, 2 H); 13C NMR (125 MHz, DMSO–d6):

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 C16H12BrF2O2, 352.9988; found, 352.9998.

(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 K2CO3 (56 mg, 0.4 mmol) in EtOH (13 mL). The reaction

mixture was stirred at 90 oC 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 Na2SO4. They were then concentrated under reduced pressure. The crude

product was purified by column chromatography on SiO2 to give the purified product, a yellow

solid. Yield 65 %. TLC (silica, hexane:EtOAc, 2:1 v/v): Rf = 0.5; 1H NMR (500 MHz, CDCl3): δ

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7.49 (s, 1 H), 7.44 (d, J = 16.0 Hz, 1 H), 7.37 (s, 1 H), 7.19–7.26 (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); 13C NMR (125 MHz, CDCl3): 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 C22H16BrF2N2O,

441.0414; found, 441.0400.

2-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

CH2Cl2 (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 SiO2 to give the purified product, a white solid. Yield

86%. TLC (silica, hexane: EtOAc, 2:1 v/v): Rf = 0.85; 1H NMR (500 MHz, CDCl3): δ 10.33 (s, 1

H), 7.82 (d, J = 7.0 Hz, 1 H), 7.62 (s, 1 H), 7.41–7.45 (m, 5 H), 7.34–7.36 (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); 13C NMR (125 MHz, CDCl3): 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 C22H14BrF2N2O,

439.0257; found, 439.0235.

(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), K2CO3 (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 oC 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 Na2SO4, and

concentrated under reduced pressure. The crude product was purified by column chromatography

on SiO2 to give the final product as a yellow solid. Yield 56 %. TLC (silica, hexane:EtOAc, 2:1

v/v): Rf = 0.5; 1H NMR (500 MHz, CDCl3): δ 10.26 (s, 1 H), 8.44 (d, J = 16.0 Hz, 1 H), 7.83 (d, J

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= 7.5 Hz, 1 H), 7.39–7.46 (m, 7 H), 7.35–7.37 (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); 13C NMR (125 MHz, CDCl3): 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 C26H19F2N2O3, 445.1363; found, 445.1360.

Synthetic protocols of compounds 13-20

Methyl 3-bromo-4-(hydroxymethyl)benzoate (13)

HCOOH (650 μL, 17.23 mmol) was added dropwise by a syringe to a stirred suspension of Et3N

(1.47 mL, 10.56 mmol) in THF (4 mL) and cooled to room temperature under nitrogen.

RuCl2(PPh3)3 (42 mg, 0.439 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

Na2SO4. The solvent was evaporated and the crude product was purified by column

chromatography on SiO2 to give the purified product, a white solid. Yield 92 %. TLC (silica,

hexane:EtOAc, 4:1 v/v): Rf = 0.3; 1H NMR (500 MHz, CDCl3): 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); 13C NMR (125 MHz, CDCl3): 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

C9H10BrO3, 244.9813; found, 244.9827.

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

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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 Na2SO4. The

solvent was evaporated and the crude product was purified by column chromatography on SiO2 to

give the purified product, colorless oil. Yield 99 %. TLC (silica, hexane:EtOAc, 4:1 v/v): Rf = 0.8;

1H NMR (500 MHz, CDCl3): 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); 13C NMR (125 MHz, CDCl3): 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 C15H24BrO3Si, 359.0678; found, 359.0658.

(3-Bromo-4-((tert-butyldimethylsilyloxy)methyl)phenyl)methanol (15)

NaBH4 (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/H2O (3:2, 34 mL) at 0 oC. The reaction mixture was stirred at 65 oC 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 Na2SO4 and concentrated to

obtain the crude product, which was then purified by column chromatography on SiO2 to give a

colorless oil. Yield 72 %. TLC (silica, hexane:EtOAc, 4:1 v/v): Rf = 0.4; 1H NMR (500 MHz,

CDCl3): 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); 13C NMR (125 MHz, CDCl3): 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 C14H24BrO2Si,

331.0729; found, 331.3404.

3-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

CH2Cl2 (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 SiO2 to give an yellow oil. Yield 64 %. TLC (silica, hexane:EtOAc,

4:1 v/v): Rf = 0.8; 1H NMR (500 MHz, CDCl3): 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); 13C NMR (125 MHz,

CDCl3): 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 C14H22BrO2Si, 329.0572; found, 329.0567

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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, 2.387 mmol) and 2,4-dimethylpyrrole (492μL, 4.77 mmol) were dissolved

in 100 mL of dry CH2Cl2 under N2. One drop of TFA was added, after the solution was stirred at

room temperature for 12 h, DDQ (543 mg, 2.387 mmol) was added, and stirring was continued for

1 h. The reaction mixture was washed with water three times and brine once, dried over Na2SO4.

The compound was purified by short column chromatography on SiO2 (eluent: EtOAc with 1%

Et3N). The Red powder thus obtained and 4 mL of DIPEA were dissolved in 20 mL of CH2Cl2

under N2. Then 8 mL of BF3⋅Et2O 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 Na2SO4,

filtered, and evaporated. the crude product was purified by column chromatography on SiO2 to

give the purified product, a red foam. Yield 58 %. TLC (silica, hexane:EtOAc, 2:1 v/v): Rf = 0.85;

1H NMR (500 MHz, CDCl3): 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); 13C NMR (125

MHz, CDCl3): 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 C26H35BBrF2N2OSi, 547.1763;

found. 547.1777.

4,

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, 0.3425 mmol) and TBAF (1.0M in THF, 326μL, 0.3425 mmol) in THF

(3 mL) was stirred at RT for 30 min, then it was quenched with aq. sat. NaHCO3 (2 mL) and

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extracted with EtOAc (3 x 100 mL), dried over Na2SO4, filtered, and evaporated. The crude

product was purified by column chromatography on SiO2 to give the purified product, a red

powder. Yield 83 %. TLC (silica, hexane:EtOAc, 2:1 v/v): Rf = 0.3; 1H NMR (500 MHz, CDCl3):

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); 13C NMR (125 MHz, CDCl3): 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

C20H21BBrF2N2O, 433.0898; found, 433.0899.

4, 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, 1.155 mmol), PCC (495 mg, 2.31 mmol) and MgSO4 (480

mg) in CH2Cl2 (200 mL) was stirred at room temperature for 30 min under N2. The reaction

mixture was filtered through Celite and then evaporated to obtain the crude product. The crude

product was purified by column chromatography on SiO2 to give the purified product, a red

powder. Yield 65 %. TLC (silica, hexane:EtOAc, 2:1 v/v): Rf = 0.8; 1H NMR (500 MHz, CDCl3):

δ 10.43 (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); 13C NMR (125 MHz, CDCl3): 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

C20H19BBrF2N2O, 431.0742; found, 431.0755.

SFP-2 probe (20)

Triphenylphosphine (9.1 mg, 0.0347mmol), Et3N (24μL, 0.1736 mmol), Pd(OAc)2 (3 mg, 0.1

equiv.) and CH3CN (500μL) were added into 25 mL schlenk tube under N2. A solution of methyl

acrylate (42 μL, 0.4628 mmol) in CH3CN (2.50 mL) was added. The reaction mixture was then

stirred at 90 oC 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).

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The combined organic layers were washed with water and brine and then dried with Na2SO4, and

concentrated under reduced pressure. The crude product was purified by column chromatography

on SiO2 to give the final product as a red solid. Yield 38 %. TLC (silica, hexane:EtOAc, 2:1 v/v):

Rf = 0.8; 1H NMR (500 MHz, CDCl3): 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); 13C NMR (125 MHz, CDCl3): 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 C24H24BF2N2O3, 437.1848; found, 437.1874.

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 0.3o 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 SAINT1. Absorption corrections were applied using SADABS30 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

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parameters were noted. All ORTEP diagrams have been drawn with 50% probability ellipsoids.

CCDC 843032 contains the supplementary crystallographic data for this paper. The data can be

obtained free of charge from the Cambridge Crystallographic Data Centre Via

www.ccdc.cam.ac.uk/data_request/cif.

Equations of interest:

Rint = Σ | Fo2 - <Fo

2> | / Σ | Fo2| R1 = Σ | | Fo| - | Fc|| / Σ| Fo|

wR2 = [Σ [w (Fo2

– Fc2)2] / Σ [w (Fo

2) 2]]1/2 GooF = S = [Σ [w (Fo2 – Fc

2) 2] / (n-p)1/2

where: w = q /σ2 (Fo2) + (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 CH3CN or DMSO to make a 10 mM stock solution, which was diluted to the required

concentration for measurement.

Na2S stock solution (20 mM) preparation31.

5 mg EDTA was dissolved in 10 mL DI H2O in a 25 mL Schlenk tube. The solution was purged

vigorously with nitrogen for 15 min. Then 48 mg sodium sulfide (Na2S·9H2O) was dissolved in

the solution under nitrogen. The resulting solution was 20 mM Na2S, 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 previously32. 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

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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 H2S 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 + H2O→ Serine + H2S [1]

Cysteine + Cysteine → Lanthionine + H2S [2]

Cysteine + Homocysteine → Cystathionine + H2S [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 105

cells/ml in the aforementioned media. A final number of 2 x 104 cells (90 μL) was transferred to

each well in a 96 well plate (BD Falcon). Cells were incubated overnight at 37oC in a 5% CO2

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

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grow overnight at 37oC in a 5% CO2 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 (Na2S, cysteine, or GSH) to the

desired concentration and cells were incubated for 15-30 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, 15–17 (2011).

32. Taoka, S., Ohja, S., Shan, X., Kruger, W. D., and Banerjee, R. J Biol Chem 273, 25179-25184 (1998).

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