Supporting Information. Mitochondrial thioredoxin-responding off-on fluorescent probe

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1 Supporting Information Mitochondrial thioredoxin-responding off-on fluorescent probe 5 Min Hee Lee, a, Ji Hye Han, b, Jae-Hong Lee, a Hyo Gil Choi, c Chulhun Kang, b, * and Jong Seung Kim a, * a Department of Chemistry, Korea University, Seoul, , Korea, jongskim@korea.ac.kr b The School of East-West Medical Science, Kyung Hee University, Yongin, , Korea, kangch@khu.ac.kr c Protected Horticulture Research Station, ational Institute of Horticultural and Herbal Science, Rural 1 Development Administration, Busan, 618-8, Korea These authors contributed equally to the work *Corresponding authors: kangch@khu.ac.kr (C. Kang); jongskim@korea.ac.kr (J. S. Kim) 15 Contents 1. Additional Scheme page S2 2. Supplementary Data page S H-MR, 13 C-MR and ESI-MS Spectra page S1 4. Additional Cell Imaging Data page S18 25 S1

2 1. Additional Scheme Scheme S1. Synthetic route to Scheme S2. Synthetic route to 7. S2

3 4 2. Supplementary Data 45 Figure S1. Absorption (a) and fluorescence (b) spectra of Mito-aph (5. µm) toward Trx (5. µm) and other biologically relevant analytes, including amino acids (5. mm of Val, Tyr, Thr, Tau, Ser, Pro, Phe, Met, Lys, Leu, Ile, His, Gly, Gluc, Glu, Gln, Asp, Asn, Arg, Ala, Trp, respectively), H 2 2 (5. mm), and metal ions (1. mm of K(I), a(i); 1 µm of Ca(II), Cu(II), Fe(II), Fe(III), Mg(II), Zn(II), respectively). All spectra were acquired min after analytes addition at 37 C in PBS buffer (ph 7.4), with an excitation λ = 428 nm. a) b) Fluorescence Intensity (a.u.) at 54 nm Mito-aph + Trx Fluorescence Intensity (a.u.) at 54 nm Time (sec) c) d) Mito-aph + GSH Time (sec) Fluorescence Intensity (a.u.) at 54 nm Time (sec) Mito-aph + Cys Fluorescence Intensity (a.u.) at 54 nm Mito-aph + Hcy Time (sec) 5 Figure S2. Time course of the fluorescence response of Mito-aph (1. µm) in the presence of 1. µm of Trx (a), 1. mm of GSH (b), 1. µm of Cys (c) and Hcy (d). The fluorescence intensity was recorded at 54 nm with an excitation at 428 nm. All fluorescence changes were measured at 37 C in 55 PBS buffer (ph 7.4). S3

4 .15 Mito-aph + GSH linear fitting.15 Mito-aph + DTT linear fitting ln2/t1(/sec).1.5. Y = A + B * X Parameter Value Error A E E-5 B E E R SD P E ln2/t1 (/sec).1.5. Y = A + B * X Parameter Value Error A E E-5 B E R SD P E-5 6 < GSH (mm) DTT (mm).15 Mito-aph + Cys linear fitting.15 Mito-aph + Hcy linear fitting ln2/t1 (/sec).1.5. Y = A + B * X Parameter Value Error A E E-5 B E R SD P E-5 7 < ln2/t1 (/sec).1.5. Y = A + B * X Parameter Value Error A E E-5 B E E R SD P E-5 6 < Cys (mm) Hcy (mm) 6 Figure S3. Plot of ln2/t1 vs concentration of thiol including GSH, DTT, Cys, and Hcy. In ln2/t1, t1 is the life time of Mito-aph (1. µm) in various concentrations of each thiol based on pseudo-first order model. The boxes indicate linear fitting results. All kinetic experiments were performed under physiological conditions (at 37 C in PBS buffer, ph 7.4) S4

5 6 Trx.56 - first trial Trx.56 - second trial Trx.56 - third trial 6 Trx.7 - first trial Trx.7 - second trial Trx.7 - third trial ln([b][a]/[a][b]) 4 2 [B][A] ln = k([b] [A][B] -[A] )t ln([b][a]/[a][b]) 4 2 [B][A] ln = k([b] [A][B] -[A] )t Time (sec) Time (sec) 8 Repeat experiments R a [B] b [A] c k([b] -[A] ) X X X X X X 1 3 k d Average of k = (4.4 ±.26) X 1 3 a The linear correlation in the plots of ln([b][a] /[A][B] ) vs time. b The initial concentration of Mito- aph with unit of M. c The initial concentration of Trx with unit of M. d The slope of the plot, which is the second order rate constant with unit of /(Ms). 85 Figure S4. Plot of ln([b][a] /[A][B] ) vs time of Mito-aph (1. µm) in various concentrations of Trx (.56, and.7 µm, respectively). In ln([b][a] /[A][B] ), [A] and [B] are [A] - (F/F max )[A], and [B] - (F/F max )[A], respectively, at given time. The second order rate constant (k) for reaction of Mito- aph with Trx was obtained from repeat experiments (n = 6). All kinetic experiments were performed under physiological conditions (at 37 C in PBS buffer, ph 7.4). 9 S5

6 GSH linear fitting DTT linear fiitting ln2/t1(/sec).12.9 Y = A + B * X Parameter Value Error A E E-5 B 4.965E E R SD P E ln2/t1(/sec).15.1 Y = A + B * X Parameter Value Error A E E-5 B E R SD P E E GSH (mm) DTT (mm) Cys linear fitting Hcy linear fitting ln2/t1(/sec) Cys (mm) Y = A + B * X Parameter Value Error A E E-5 B E R SD P E E ln2/t1(/sec) Y = A + B * X Parameter Value Error A 3.95E E-5 B E E R SD P E E Hcy (mm) 95 Figure S5. Plot of ln2/t1 vs concentration of thiol including GSH, DTT, Cys, and Hcy. In ln2/t1, t1 is the life time of 7 (1. µm) in various concentrations of each thiol based on pseudo-first order model. The boxes indicate linear fitting results. All kinetic experiments were performed under physiological conditions (at 37 C in PBS buffer, ph 7.4) S6

7 6 Trx.56 - first trial Trx.56 - second trial Trx.56 - third trial 6 Trx.7 - first trial Trx.7 - second trial Trx.7 - third trial ln([b][a]/[a][b]) 4 2 [B][A] ln = k([b] [A][B] -[A] )t ln([b][a]/[a][b]) 4 2 [B][A] ln = k([b] [A][B] -[A] )t 6 12 Time (sec) Time (sec) Repeat experiments R a [B] b [A] c k([b] -[A] ) k d X X X X X X 1 3 Average of k = (4.1 ±.11) X 1 3 a The linear correlation in plot of ln([b][a] /[A][B] ) vs time. b The initial concentration of 7 with unit of M. c The initial concentration of Trx with unit of M. d The slope of the plot, which is the second order rate constant with unit of /(Ms). 12 Figure S6. Plot of ln([b][a] /[A][B] ) vs time of 7 (1. µm) in various concentrations of Trx (.56, and.7 µm, respectively). In ln([b][a] /[A][B] ), [A] and [B] are [A] - (F/F max )[A], and [B] - (F/F max )[A], respectively, at given time. The second order rate constant (k) for reaction of 7 with Trx was obtained from repeat experiments (n = 6). All kinetic experiments were performed under physiological conditions (at 37 C in PBS buffer, ph 7.4). 125 S7

8 Table S1. The second order rate constants for reaction of 7 with GSH, Trx, Cys, Hcy, and DTT. 1 Thiols GSH Trx Cys Hcy DTT k a.49 ±.4 (1.) b ± (8375.6) b 1.15 ±.4 (2.34) b.48 ±.2 (.97) b 3.42 ±.23 (6.97) b a The second order rate constants with unit of /(Ms). b The parentheses represent the ratio of the reaction rate of 7 with a thiol to that with GSH E % Intensity Mass (m/z) Figure S7. MALDI-TF MS spectrum of Trx (from Escherichia coli.). S8

9 % Intensity Mass (m/z) E % Intensity Mass (m/z) Figure S8. MALDI-TF MS spectrum of Mito-aph recorded upon the addition of Trx S9

10 3. 1 H-MR, 13 C-MR and ESI-MS Spectra Ph 3 P H H S H S Exact Mass: Figure S9. 13 C-MR spectrum of Mito-aph in CDCl 3. Ph 3 P H H S H S Exact Mass: Figure S1. 13 C-MR spectrum of Mito-aph in CDCl 3. S1

11 Ph 3 P H H S H S Exact Mass: Figure S11. ESI-MS spectrum of Mito-aph f1 (ppm) Figure S12. 1 H-MR spectrum of 4 in CD 3 D S11

12 f1 (ppm) 17 Figure S C-MR spectrum of 4 in CD 3 D Figure S14. ESI-MS spectrum of 4. S12

13 H S S H Exact Mass: Figure S15. 1 H MR spectrum of 9 recorded in CDCl 3. H S S H Exact Mass: Figure S C MR spectrum of 9 recorded in CDCl 3. S13

14 H S S H Exact Mass: Figure S17. ESI-MS spectrum of f1 (ppm) Figure S18. 1 H MR spectrum of 6 recorded in CDCl S14

15 f1 (ppm) Figure S C MR spectrum of 6 recorded in CDCl Figure S2. ESI-MS spectrum of 6. S15

16 Ph 3 P H H 3 S H S Exact Mass: Figure S21. 1 H MR spectrum of 7 recorded in CDCl 3 with 1% CD 3 D. Ph 3 P H H 3 S H S Exact Mass: Figure S C MR spectrum of 7 recorded in CDCl 3 with 1% CD 3 D. S16

17 Ph 3 P H H 3 S H S Exact Mass: Figure S23. ESI-MS spectrum of S17

18 4. Additional Cell Imaging Data Figure S24. Confocal microscopic analysis of HepG2 cells treated with Mito-aph (5. µm) (A). Images of the cells were obtained using excitation at 458 nm and a band-path (5-6 nm) emission filter. For the PX-12-treated samples, before the media were finally replaced with PBS containing Mito-aph, the cells were preincubated with media containing PX-12 at 2. µm (B) and 1. µm (C) for 24 h at 37 C. Bottom panels show an overlay of the image with a non-confocal contrast image S18

19 Figure S25. Confocal microscopic analysis of HepG2 cells treated with 9 (1. µm) (A). Images of the cells were obtained using excitation at 458 nm and a band-path (5-6 nm) emission filter. For the 25 PX-12-treated samples, before the media were finally replaced with PBS containing 9, the cells were preincubated with media containing PX-12 at 2. µm (B) and 1. µm (C) for 24 h at 37 C. Bottom panels show an overlay of the image with a non-confocal contrast image. 255 Figure S26. Colocalization experiments using Mito-aph and (a-c) ER and (d-f) Lyso Tracker Red in HeLa cells. The cells were incubated with 5. µm of Mito-aph for 2 min at 37 C and the medium was replaced with fresh medium containing.5 µm of ER or Lyso Tracker Red and incubated for 1 min. Images were then obtained using excitation at 458 nm and band-path at (a) 47 5 nm and (b) nm, (d) 47 6 nm, (e) 65 7 nm. Panels (c,f) are merged images of (a) and (b), (d) and (e). S19