Supplementary Information

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1 Supplementary Information Transthiocarbamoylation of proteins by thiolated isothiocyanates Takahiro Shibata 1,Yuuki Kimura 1, Akihiro Mukai 1, Hitoshi Mori 1, Sohei Ito 2, Yukio Asaka 3, Sho Oe 3, Hiroshi Tanaka 3, Takashi Takahashi 3, and Koji Uchida 1,4 1 Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya , Japan, 2 School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka , Japan, 3 Graduate School of Science and Engineering, Tokyo Institute of Technology, Tokyo , Japan S1

2 Supplementary Text Synthesis of the isothiocyanate derivative and dithiocarbamate derivative Synthesis of the isothiocyanate derivative 4 and dithiocarbamate derivative 5 is shown in Scheme X. Chemoselecitve protection of the amino group of 6-aminohexanol with Boc group, followed by O-tosylation provided the tosylate 1 in 78% yield. Conversion of the tosylate 1 to thioester 2 was archived by the treatment with potassium thioacetate to afford 2 in 91% yield. The thioacetate was converted to 3-butynylsulfide 3. Treatment with thiophosgen after removal of the Boc group of 3 provided isothiocyanate, followed by oxidation of the sulfide with IBXresin to provide sulfoxide 4 in 56%. Addition of 2-mercaptethanol to isothiocyanate 4 afforded dithiocarbamate 5 in 72%. Synthesis of 6-(tert-butoxycarbonylamino)hexyl p-toluenesulfonate (1). To a solution of 6-amino-1-hexanol (2.34 g, 20.7 mmol) and K 2 CO 3 (7.14g, 51.8 mmol) in tetrahydrofuran (20 ml) and water (20 ml) was added di-tert-butyldicarbonate (5.42 g, 24.8 mmol) at room temperature. After being stirred at the same temperature for 12 h, the reaction mixture was poured into ethyl acetate (100 ml) and 1 M HCl aq. (100 ml) at 0 o C. The aqueous layer was extracted with ethyl acetate (100 ml x2). The combined organic layer was washed with 1 M HCL aq. (100 ml) and brine (100 ml), dried over MgSO 4 and filtered through a filter paper to remove MgSO 4. The filtrate was concentrated in vacuo. The residue was used for the next reaction without further purification. To a solution of a mixture of the residue and triethylamine (8.66 ml, 62.2 mmmol) and trimethylamine hydrochloride (198 mg, 2.07 ml) in dichloromethane (40.0 ml) was added p- toluenesulfonyl chloride (4.74 ml, 24.9 mmmol) at 0 o C. After being stirred at the same temperature at 1.5 h, the reaction the reaction mixture was poured into ethyl acetate (150 ml) and water (150 ml) at 0 o C. The aqueous layer was extracted with ethyl acetate (100 ml x2). The combined organic layer was washed with saturated NH 4 Cl aq. (100 ml) and brine (100 ml), dried over MgSO 4 and filtered through a filter paper to remove MgSO 4. The filtrate was concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluent with 80:20 hexane:ethyl acetate) to afford 6-(tert-butoxycarbonylamino)hexyl p-toluenesulfonate 1 (6.80 g, 18.3 mmol, 78% in 2 steps); IR(neat) 3413, 2977, 2934, 2864, 1712, 1520, 1365, 1177, 960 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 7.79 (d, 2H, J = 8.2 Hz), 7.35 (d, 2H, J = 8.2 Hz), 4.49 (brs, 1H), 4.01 (t, 2H, J = 6.8 Hz) (m, 2H), 2.45 (s, 3H), (m, 2H), (m, 6H), 1.44 (s, 9H); 13 C NMR (100 MHz, CDCl 3 ) δ 156.0, 144.8, 133.2, 129.9, 127.9, 79.1, 70.5, 40.4, 29.9, 28.8, 28.5, 26.2, 25.2, Synthesis of S-6-(tert-butoxycarbonylamino)hexyl thioacetate (2) To a solution of 6-(tert-butoxycarbonylamino)hexyl p-toluenesulfonate 1 (5.5 g, 14.8 mmol) in N,N-dimethylformamide (15.0 ml) was added potassium thioacetate (2.5 g, mmol) at room temperature. After being stirred at 60 o C for 1 h, the reaction the reaction mixture was poured into ethyl acetate (100 ml) and water (100 ml) at 0 o C. The aqueous layer was extracted with ethyl acetate (50 ml x2). The combined organic layer was washed with brine (50 ml), dried over MgSO 4 and filtered through a filter paper to remove MgSO 4. The filtrate was concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluent with 80:20 hexane:ethyl acetate) to afford S-6-(tert-butoxycarbonylamino)hexyl thioacetate 2 (3.70 g, 13.4 mmol, 91%) IR (neat) 3371, 2977, 2933, 2860, 12694, 1520, 1366, 1172, 1136, 954 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 4.49 (brs, 1H), (m, 2H), 2.86 (t, 2H, J = 7.3 Hz), 2.32 (s, S2

3 3H), (m, 8H), 1.44 (s, 9H); 13 C NMR (100 MHz, CDCl 3 ) δ196.0, 156.0, 79.1, 40.5, 30.7, 30.0, 29.5, 29.0, 28.48, 28.45, Synthesis of 6-(3-butynylsulfinyl)hexyl isothiocyanate (4). To a solution of a mixture of S-6-(tert-butoxycarbonylamino)hexyl thioacetate 2 (25 mg, 90.8 µmol) and 1. M NaOMe in MeOH (0.25 ml) in 1,4-dioxane (0.25 ml) was added 3-butynyl p- toluenesulfonate (22 µl, 99.9 µmol) at room temperature. The reaction mixture was stirred at the same temperature for 30 min. 8 M HCl in 1,4-dioxane (0.5 ml) was added to the reaction mixture at room temperature to remove the N-Boc group. After additional 30 min, the reaction mixture was diluted with dichloromethane (2 ml) and neutralized with 4 M NaOH aq. (1 ml) at 0 o C. Subsequently, thiophosgen (8.3 µl, 109 µmol) was added to the reaction mixture at 0 o C. After being stirred at the same temperature for 30 min, the reaction mixture was poured into ethyl acetate (10 ml) and 10 wt% Na 2 S 2 O 3 aq. (10 ml). The aqueous layer was extracted with ethyl acetate (10 ml x2). The combined organic layer was washed with brine (10 ml), dried over MgSO 4 and filtered through a filter paper to remove MgSO 4. The filtrate was concentrated in vacuo. The residue was used for the next reaction without further purification. To a solution of the residue in dichloromethane (0.5 ml) was added IBX-resin (60 mg, 1.0 mmol) at room temperature. After being stirred at the same temperature at 17 h, the reaction mixture was filtered through a filter paper to remove the IBX-resin. The filtrate was concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluent: toluene:acetonitrile = 85:15) to afford 6-(3-butynylsulfinyl)hexyl isothiocyanate 4 (12.3 mg, 50.5 µmol, 56% in 4 steps); IR (neat) 3442, 3291, 3222, 2934, 2860,2183, 2106, 1679, 1455, 1348, 1036, 643 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 3.53 (t, 2H, J = 6.8 Hz), (m, 2H), 2.11 (t, 2H, J = 2.4 Hz) (m, 2H), (m, 2H), 1.43 (m, 6H). 13 C NMR (100 MHz, CDCl 3 ) δ 130.3, 80.9, 70.6, 52.2, 50.8, 45.0, 29.7, 28.0, 26.2, 225., 126; HRMS (ESI-TOF) m/z calcd for C 11 H 18 NOS 2 : [M+H] + ; found: Synthesis of S-2-hydroxyethyl N-6-(3-butynylsulfinyl)hexyldithiocarbamate (5) To a solution of 6-(3-butynylsulfinyl)hexyl isothiocyanate 4 (11.0 mg, 45.2 mmol) in methanol (0.500 ml) was added 2-mercaptoethanl (6.34 µl, 90.4 µmol) and a trace amount of triethylamine at room temperature. After being stirred at the same temperature 30 o C for 1 h, the reaction mixture was concentrated in vacuo. The residue was purified by column chromatography on silica gel (eluent: chloroform:methanol = 98:2) to afford S-2-hydroxyethyl N- 6-(3-butynylsulfanyl)hexyldithiocarbamate (10.5 mg, 32.7 µmol, 72%); IR (neat) 3245, 2934, 2861, 1531, 1369, 1336, 1011, 647 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.70 (brs, 1H), 3.92 (brs, 2H), (brs, 2H), 3.31 (brs, 2H), (m, 2H), 2.10 (t, 1H, J = 2.4 Hz), 1.85 (tt, 2H, J = 7.2, 7.2 Hz), 1.43 (m, 6H) ; 13 C NMR (100 MHz, CDCl 3 ) δ 197.2, 80.7, 70.8, 62.7, 60.5, 51.9, 50.8, 47.0, 27.7, 127.6, 26.12, 22.4, 12.7; HRMS (ESI-TOF) m/z calcd for C 13 H 24 NO 2 S 3 : [M+H] + ; found: Synthesis of N-2-hydroxyethyl-N -6-(3-butynylsulfanyl)hexylthiourea (6) To a solution of isothiocyanate X (11.0 mg, mmol) in methanol (0.500 ml) was added 2- aminoethanol (5.46 µl, mmol). After stirring at the same temperature for 3 h, the reaction mixture was evaporated in vacuo. The residue was purified by column chromatography on silica gel (eluent with 5:95 methaol:chloroform) to afford thiourea X (10.2 mg, mmol, 74%) as a colorless oil; IR (neat): 3292, 3091, 2933, 2860, 1558, 1014, 677 cm-1; 1H NMR (400 MHz, CD3Cl) δ 6.80 (brs, 1H), 6.62 (brs, 1H), 3.78 (brs, 2H), 3.62 (br, 2H), 3.49, (br, 2H), 2.88 (t, 2H, S3

4 J = 7.2 Hz), 2.71 (dt, 2H, J = 2.4, 6.8 Hz), (m, 2H), 2.11 (t, 1H, J = 2.4 Hz), 1.83 (tt, 2H, J= 7.2, 7.2 Hz), 1.43 (m, 6H): 13C NMR δ 209.9, 80.7, 70.9, 62.4, 51.9, 50.8, 47.02, 47.00, 28.2, 27.8, 26.1, 22.4, 12.7; HRMS (ESI-TOF) m/z calcd for C 14 H 24 N 2 O 2 S 2 : [M+H] + ; found: S4

5 Supplementary Tables Table S1. Identification of cellular proteins labeled with Al-ITC in human intestinal Caco-2 cells S5

6 Table S2. Cysteine-containing peptides identified by MALDI-TOF MS/MS from the recombinant Hsp90β treated with 6-HITC. Mr (expt): experimental molecular weight of each peptide. Mr (calc): molecular weight calculated for the indicated peptide sequence. S6

7 Supplementary Figures Fig. S1. Rearrangement reaction between the 6-HITC-bound β-mercaptoethanol and NAC. (A) Reaction of the 6-HITC-β-mercaptoethanol conjugate (6-HITC-ME) with NAC. The reaction mixture, containing 1 mm NAC, was incubated with 1 mm 6-HITC-ME in 50 mm sodium phosphate buffer (ph 7.4). After incubation for 2 h at 37 C, the reaction mixtures were analyzed by reverse-phase HPLC on a Develosil ODS-HG-5 column (4.6 mm id. 250 mm, Nomura Chemicals) eluted with a linear gradient of water containing 0.1% TFA (solvent A)-acetonitrile (solvent B) (time=0 min, 10% B; 40min, 80% B) at the flow rate of 0.8 ml/min. The elution profiles were monitored by absorbance at nm. (B) LC-MS analysis of 6-HITC-ME incubated with NAC. Upper, Selected ion-current chromatograms obtained from the LC-MS analysis monitored with m/z 369. Lower, HPLC profile. (C) A proposed mechanism for the rearrangement reaction between the 6-HITC-ME and NAC. S7

8 Fig. S2. Schematic of the proposed reactions of alkynylated isothiocyanate reagents with proteins, and the click chemistry labeling with a fluorescent tag to visualize the covalently bound isothiocyanates on the protein. S8

9 Fig. S3. Synthesis of alkynylated 6-HITC analogues. (A) Chemical structures of 6-HITC and its alkynylated analogue, Al-ITC, and a fluorescent tag, TAMRA-N 3, used in this study to visualize the protein-bound isothiocyanates. (B) Synthesis of Al-ITC and its β-mercaptoethanol (Al-ITC-ME) and aminoethanol (Al-ITC-AE) derivatives. S9

10 Fig. S4. Validation of Hsp70, Keap1, and GSTP1 as the selected targets of the alkynylated 6-HITC analogues in Caco-2 cells. Caco-2 cells were pretreated with the alkynylated 6-HITC analogues or vehicle for 2 h. The cell lysates were subjected to the click reaction with biotin-n 3 on beads and the labeled proteins were precipitated with NeutrAvidin beads followed by Western blot. S10

11 Fig. S5. Effect of down-regulation of Hsp90β on the expression of Hsp70. (A) Effect of specific Hsp90β sirna on the expression of Hsp70. Changes in the protein levels of Hsp90β and Hsp70 were assessed by an immunoblot analysis. (B) Effect of 17-AAG, an inhibitor of Hsp90 on the expression of Hsp70. Caco-2 cells were treated with 2 µm 17- AAG for the indicated time intervals. Changes in the Hsp70 protein levels were assessed by an immunoblot analysis. S11

12 Fig. S6. Nuclear translocation of HSF-1 in Caco-2 cells treated with 6-HITC. The cells were treated with 6-HITC for the indicated time intervals. HSF-1 in the nucleus and cytosol was analyzed by immunoblot analysis using rabbit anti-hsf-1 polyclonal antibody. S12