Supplementary Material. Norbornene probes for the study of cysteine oxidation

Transcription

1 upplementary Material Norbornene probes for the study of cysteine oxidation Lisa J. Alcock, a Kyle D. Farrell, a Mawey T. Akol, a Gregory H. Jones, b Matthew M. Tierney, c Holger B. Kramer, d Tara L. Pukala, e Gonçalo J. L. Bernardes, f,g Michael V. Perkins a and Justin M. Chalker a, a Flinders University, College of cience and Engineering, turt Road, Bedford Park, outh Australia 542, Australia b California Institute of Technology, Division of Chemistry and Chemical Engineering, 12 East California Boulevard, Pasadena, CA 91125, UA c The University of North Carolina at Chapel Hill, Department of Chemistry, Chapel Hill, NC 27599, UA d Imperial College London, MRC London Institute of Medical ciences, Hammersmith Hospital Campus, Du Cane Road, London, W12 NN, UK e The University of Adelaide, chool of Physical ciences, Adelaide, outh Australia 55, Australia f University of Cambridge, Department of Chemistry, Lensfield Road, Cambridge CB2 1EW, UK g Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, , Lisboa, Portugal Table of Contents General considerations xidation of N-acetylcysteine (9) to its disulfide using hydrogen peroxide (unbuffered, NMR) cis-5-norbornene-endo-2,3-dicarboxylic acid (8) as probe for N-acetylcysteine oxidation (NMR) cis-5-norbornene-endo-2,3-dicarboxylic acid (8) as probe for N-acetylcysteine oxidation (LC-M) exo-5-norbornenecarboxylic acid (12) as a probe for N-acetylcysteine oxidation (NMR) exo-5-norbornenecarboxylic acid (12) as a probe for N-acetylcysteine oxidation (LC-M) Dimedone as a probe for N-acetylcysteine oxidation (unbuffered, NMR) Dimedone as a probe for N-acetylcysteine oxidation (unbuffered, LC-M) Cyclooctyne BCN (6) as a probe for N-acetylcysteine oxidation (unbuffered, NMR) Cyclooctyne BCN (6) as a probe for N-acetylcysteine oxidation (unbuffered, LC-M) Cyclooctyne DBC (7) as a probe for N-acetylcysteine oxidation (unbuffered, NMR) Cyclooctyne DBC (7) as a probe for N-acetylcysteine oxidation (unbuffered, LC-M) Norbornene derivative 8 as a probe for N-acetylcysteine oxidation (pd 5. NaAc buffer, NMR) Norbornene derivative 8 as a probe for N-acetylcysteine oxidation (ph 5. NaAc buffer, LC-M) Norbornene derivative 8 does not react with sulfinates or sulfonates at pd 5. Dimedone as probe for N-acetylcysteine oxidation (pd 5. NaAc buffer, NMR) Dimedone reacting with H 2 2 over 24 hrs (ph 5. NaAc buffer, NMR) Dimedone as probe for N-acetylcysteine oxidation (ph 5. NaAc buffer, LC-M) Norbornene derivative 8 as a probe for N-acetylcysteine oxidation (pd 7.4 phosphate buffer, NMR) xidation of N-acetylcysteine to its disulfide using hydrogen peroxide (pd 5. NaAc buffer, NMR) Dimedone as a probe for N-acetylcysteine oxidation (pd 7.4 phosphate buffer, NMR) Norbornene derivative 8 does not react with sulfinates or sulfonates at pd 7.4 ynthesis of alkyne-tagged probe 19 Hydrolysis study of alkyne-tagged probe 19 ynthesis of norbornene NH derivative 21 ynthesis of 22 (norbornene probe containing an alkyne reporter group) ynthesis of norbornene amine derivative 24 ynthesis of biotin NH-ester 26 ynthesis of 27 (norbornene probe 27 containing a biotin reporter group) References Corresponding author. Tel.: ; justin.chalker@flinders.edu.au; web: 1

2 General considerations All reagents were used directly from commercial suppliers without further purification. All reactions without water as a solvent were carried out under an inert atmosphere of nitrogen in flame-dried glassware. CH 2 Cl 2 was distilled over CaH 2 and THF was distilled over sodium and benzophenone prior to use. All other solvents were used directly from commercial suppliers without further purification. Analytical thin layer chromatography was performed on aluminium sheets coated with silica gel containing a fluorescent indicator (.15-.2mm thickness, 8 µm granularity), with visualisation carried out using an ultraviolet lamp (254 nm) and/or development with potassium permanganate. Column chromatography was performed using silica gel (23 4 mesh, 6Å pore diameter). High resolution mass spectra (HRM) were recorded on a Waters ynapt HDM Q-ToF by electrospray ionization (EI) or a Perkin Elmer, AxIN, DA-ToF by atmospheric-pressure chemical ionization (APCI) and are reported as the observed molecular ion. Infrared spectra were recorded on an FTIR spectrometer with the absorptions reported in wavenumbers (cm 1 ). 1 H and 13 C NMR spectra were recorded on a Bruker 6 MHz pectrometer. NMR were assigned using CY, HMQC and HMBC where required. Where D 2 was used as the solvent and internal lock, spectra were referenced to residual solvent for HD (δ H 4.79 ppm) for 1 H NMR. For MeD-d 4, spectra were referenced to residual solvent (δ H 3.33 ppm) for 1 H NMR and (δ C 49. ppm) for 13 C NMR. For DM-d 6, spectra were referenced to residual solvent for DM-d 6 (δ H 2.5 ppm) for 1 H NMR and (δ C 39.5 ppm) for 13 C NMR. Chemical shift values are reported in parts per million, 1 H- 1 H coupling constants are reported in hertz and H multiplicity is abbreviated as; s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, br = broad signal. Liquid chromatography mass spectrometry (LC-M) analyses were carried out on a Waters Acquity UPLC coupled to a Micromass Quattro Micro triple quadrupole mass spectrometer using electrospray ionization in both positive and negative mode, as specified. A kinetex C18 column with particle size 2.6 µm, and dimensions mm length was used for all experiments. UHPLC grade solvents and milli Q water were used in these experiments. Before injection, samples were diluted to approximately 5 µg/ml in milli Q water, mixed, and then filtered through.2 µm nylon syringe filters. For all experiments, the mobile phases were run with water.1 formic acid (solvent A) and acetonitrile (solvent B). All samples had an injection volume of 2 µl and ran with a flow rate of.2 ml min -1. The gradient was programmed as followed: 95 A 5 B ( minutes) and maintained for 5 minutes (isocratic). 5 A 95 B over 7 minutes and maintained at 5 A for a further 3 minutes. 95 A 5 B over.5 minutes and held for a further 4.5 minutes to wash/equilibrate the column. The total run time was 2 minutes. The electrospray source was operated with a capiliary voltage of 3 kv and cone voltage of 3 V. The source temperature was operated at 8 C and the desolvation temperature of 35 C. All data was analysed using Masslynx software. 2

3 xidation of N-acetylcysteine (9) to its disulfide (1) using hydrogen peroxide Reference 1 H-NMR spectra for 9 under the reaction conditions: N-acetylcysteine (9, 36 mg,.22 mmol) was added to a vial and suspended in D 2 (.8 ml). odium carbonate (47 mg,.22 mmol) was added and the capped mixture was stirred for 3 minutes and then analysed directly by 1 H-NMR (6 MHz, D 2 ): δ = 4.32 (1H, t, J = 5.4, CHα) 2.85 (m, CH 2 ), 2. (3H, s, NHAc). H Na2 C 3 (1 equiv) C 2 H D 2, 3 min 9 H C 2 Na 1 H NMR for N-acetylcysteine disulfide (1) (6 MHz, D 2 ) N-acetylcysteine (9, 36 mg,.22 mmol) was added to a vial and suspended in D 2 (.8 ml). odium carbonate (47 mg,.22 mmol) was added and the mixture was stirred to provide a homogeneous solution. Hydrogen peroxide (5 µl of a 3 wt aq. solution,.44 mmol) was then added and the reaction was stirred for an additional 3 minutes. The reaction was analysed directly by NMR, with full conversion to disulfide 1 observed. 1 H-NMR (6 MHz, D 2 ): δ = 4.41 (2H, dd, J = 8.9, 4.1, CHα) 3.18 (2H, ABX, J = 14., 4.1, CHH), 2.9 (2H, ABX, J = 14., 8.9, CHH), 1.98 (6H, s, NHAc). H H Na2 C 3 (1 equiv) H 2 2 C 2 H D 2 C 2 Na 3 min C 2 Na 9 1 C 2 Na 3

4 NMR studies using cis-5-norbornene-endo-2,3-dicarboxylic acid (8) as a probe for cysteine oxidation [NMR data and control experiments associated with Figure 4A]. General experimental procedure for NMR experiments A-D: For reactions A-D (pages 4-5), stock solutions were prepared for both cis-5-norbornene-endo-2,3- dicarboxylic acid (8) (solution 1) and N-acetylcysteine (9) (solution 2): olution 1: In a vial, a mixture of cis-5-norbornene-endo-2,3-dicarboxylic acid 8 (45 mg,.25 mmol), D 2 (2 ml) and Na 2 C 3 (25 mg,.24 mmol) was stirred or shaken until fully dissolved. olution 2: In a vial, a mixture of N-acetylcysteine (125 mg,.77 mmol), D 2 (2 ml) and Na 2 C 3 (25 mg,.24 mmol) was stirred or shaken until fully dissolved. A: cis-5-norbornene-endo-2,3-carboxycylic acid disodium salt Na 2 C 3 (1 equiv) C 2 H C D 2 2 Na 8 C 2 H C 2 Na To a vial, D 2 (.4 ml) was added to solution 1 (.4 ml,.5 mmol 8) and stirred for 2 minutes before analysing directly by NMR. B: cis-5-norbornene-endo-2,3-dicarboxylic acid disodium salt does not react with H 2 2 Na 2 C 3 (1 equiv) H 2 2 (3 equiv) C 2 H C D 2, 2 min 2 Na 8 C 2 H C 2 Na To a vial, D 2 (.4 ml) was added to solution 1 (.4 ml,.5 mmol 8) and stirred for a few seconds before the addition of H 2 2 (2 µl, 3 wt aq. solution). The solution was stirred for 2 minutes before analysing directly by NMR. C: No reaction between 8 and N-acetylcysteine (9) was detected by NMR H C 2 Na C 2 Na C 2 Na 3 equiv 1 equiv D 2, 2 min C 2 Na To a vial, solution 1 (.4 ml,.5 mmol 8) was added followed by solution 2 (.4 ml,.15 mmol 9) and stirred for 2 minutes before analysing directly by NMR. D: Norbornene derivative 8 reacts with the sulfenic acid of N-acetylcysteine H C 2 Na C 2 Na C 2 Na 3 equiv 1 equiv H 2 2 (3 equiv) To a vial, solution 1 (.4 ml,.5 mmol 8) was added followed by H 2 2 (2 µl, 3 wt in H 2 ) and stirred for a few seconds before adding solution 2 (.4 ml,.15 mmol, 9) dropwise over 1 minute. The mixture was stirred for 2 minutes before analysing directly by NMR. The ph of this reaction mixture was 4.3. NMR data for experiments A-D are shown on the following page. H D 2, 2 min C 2 Na 1 C 2 Na C 2 Na C 2 Na 11 C 2 Na C 2 Na C 2 Na (mixture of four diastereomers) 4

5 Conclusion: at ph 4.3, 8 only reacts with N-acetylcysteine in the presence of hydrogen peroxide (6 MHz, D 2 ). The olefin is consumed by reaction with the sulfenic acid (reaction D). D C B A 5

6 LC-M studies using cis-5-norbornene-endo-2,3-dicarboxylic acid (8) as a probe for cysteine oxidation [LC-M data and control experiments associated with Figure 4A]. General experimental procedure: For each reaction, stock solutions were prepared for both cis-5-norbornene-endo-2,3-dicarboxylic acid (8) (solution 1) and N-acetylcysteine (solution 2) and used for all experiments on pages 6-8. olution 1: In a vial, a mixture cis-5-norbornene-endo-2,3-dicarboxylic acid 8 (18 mg,.1 mmol), H 2 (.8 ml), and Na 2 C 3 (13 mg,.12 mmol) was stirred or shaken until fully dissolved. olution 2: In a vial, a mixture of N-acetylcysteine (36 mg,.22 mmol), H 2 (.6 ml) and Na 2 C 3 (9.8 mg,.9 mmol) was stirred or shaken until fully dissolved. LC-M data for control experiment in which 8 and N-acetylcysteine were reacted (in the absence of hydrogen peroxide): H C 2 Na C 2 Na C 2 Na 3 equiv 1 equiv H 2, 3 min H C 2 Na C 2 Na C 2 Na C 2 Na C 2 Na C 2 Na To a vial, solution 1 (.2 ml,.25 mmol, 8) was added followed by solution 2 (.2 ml,.73 mmol, 9) and stirred for 3 minutes before analysing directly by LC-M The LC-M data are shown on the following page. The thiol-ene product was detected as a minor product at 7.3 minutes. 6

7 Lisa A 11 can E TIC 9.54e6 H 6.92 C 2 H C 2 H C 2 H.54 C 2 H C 2 H C 2 H Time N Ncys 2 old Lisa A (7.318) can E e6 C 2 H C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 344 Found: m/z = Lisa A (6.948) can E e6 181 C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 181 Found: m/z = Lisa A (1.312) can E e6 162 H C 2 H Nominal mass [M-H] Calculated: m/z = 162 Found: m/z = m/z

8 LC-M study indicating cis-5-norbornene-endo-2,3-dicarboxylic (8) can trap the sulfenic acid of N- acetylcysteine at ph 4.3 [LC-M data associated with Figure 4A]. H C 2 Na C 2 Na C 2 Na 3 equiv 1 equiv H 2 2 (3 equiv) H 2, 3 min C 2 Na To a vial, solution 1 (.2 ml,.25 mmol 8) was added followed by H 2 2 (1 µl, 3 wt in H 2 ) and stirred for a few seconds before adding solution 2 (.2 ml,.73 mmol 9) dropwise over 1 minute. The mixture was stirred for 3 minutes before analysing by LC-M. The ph of this reaction was 4.3. The LC-M data are shown below. Disulfide 1 and sulfoxide adduct 11 were the major products. No product of the thiol-ene reaction was detected. 1 C 2 Na 11 C 2 Na C 2 Na C 2 Na (mixture of four diastereomers) Lisa A 13 can E-.82 TIC e6 C 2 H C 2 H C 2 H C 2 H C 2 H Time N H22 Ncys 3 old Lisa A (4.222) can E- 91 Nominal mass [M-H] C 2.72e6 2 H Calculated: m/z = 36 C 2 H Found: m/z = C 36 2 H Lisa A (3.511) can E- 91 Nominal mass [M-H] C 2.91e6 2 H Calculated: m/z = 36 C H Found: m/z = C 2 H Lisa A (3.38) can E- 91 Nominal mass [M-H] C 2.57e6 2 H Calculated: m/z = 36 C 2 H Found: m/z = C 36 2 H Lisa A (3.77) can E- 91 Nominal mass [M-H] C 2.78e6 2 H Calculated: m/z = 36 C 2 H Found: m/z = C 2 H Lisa A (2.569) Nominal mass [M-H] can E- 91 Calculated: m/z = e6 C 2 H C 2 H Found: m/z = m/z

9 NMR studies using exo-5-norbornenecarboxylic acid (12) as a probe for cysteine oxidation [NMR data and control experiments associated with Figure 4B]. 1 H-NMR spectrum for exo-5-norbornenecarboxylic acid sodium salt (6 MHz, D 2 ) exo-5-norbornenecarboxylic acid (12, 3 mg,.22 mmol) was added to a vial and suspended in D 2 (.8 ml). odium carbonate (47 mg,.44 mmol) was then added and the mixture was stirred for 3 minutes before analysing directly by NMR. A representative 1 H-NMR spectrum is shown below: C 2 Na exo-5-norbornenecarboxylic acid sodium salt does not react with hydrogen peroxide (6 MHz, D 2 ) Na 2 C 3 (2 equiv) C 2 H H 2 2 (2 equiv) C 2 Na 12 D 2, 3 min No reaction with H 2 2 exo-5-norbornenecarboxylic acid (12, 3 mg,.22 mmol) was added to a vial and suspended in D 2 (.8 ml). odium carbonate (47 mg,.44 mmol) was then added, followed by hydrogen peroxide (5 µl of a 3 wt solution in H 2,.44 mmol). The mixture was stirred for 3 minutes before analysing directly by NMR. No reaction was observed: 9

10 exo-5-norbornenecarboxylic acid sodium salt does not react with N-acetylcysteine (6 MHz, D 2 ) [NMR data and control experiment associated with Figure 4B]. N-acetylcysteine (9, 18 mg,.66 mmol) was added to a vial along with exo-5-norbornenecarboxylic acid (12, 3 mg,.22 mmol) and suspended in D 2 (.8 ml). odium carbonate (47 mg,.44 mmol) was then added and the resulting solution was stirred for 3 minutes before analysing directly by NMR. No thiol-ene reaction was observed: 1 equiv C 2 Na H C2 Na 3 equiv D 2 3 min C 2 Na no reaction H C2 Na exo-5-norbornenecarboxylic acid (12) does not react with N-acetylcysteine disulfide (1) (6 MHz, D 2 ). [NMR data and control experiment associated with Figure 4B]. H C 2 Na 1 equiv H equiv D 2 C 2 Na 1 12 C 2 Na C 2 H 1 equiv 3 min C 2 Na no reaction with disulfide C 2 Na 1 C 2 Na N-acetylcysteine (9, 36 mg,.22 mmol) was added to a vial and suspended in D 2 (.8 ml). odium carbonate (47 mg,.44 mmol) was added and the mixture was stirred to provide a homogeneous solution. Next, hydrogen peroxide (5 µl of a 3 wt solution in H 2,.44 mmol) was added. The reaction was stirred for 1 minutes to convert 9 into disulfide 1. After 1 minutes, exo-5-norbornenecarboxylic acid (12, 3 mg,.22 mmol) was added. The reaction was stirred for an additional 3 minutes and then analysed by NMR. nly disulfide 1 and the sodium carboxylate of 12 were observed: 1

11 exo-5-norbornenecarboxylic acid (12) can trap the sulfenic acid of N-acetylcysteine (6 MHz, D 2 ). [NMR data associated with Figure 4B]. H C 2 Na C 2 Na H 2 3 equiv 2 1 equiv 2 equiv D 2, 3 min C 2 Na 1 C 2 Na C 2 Na C 2 Na 13 C 2 Na 14 (mixture of eight diastereomers) C 2 Na exo-5-norbornenecarboxylic acid (12, 3 mg,.22 mmol) was added to a vial and suspended in D 2 (.4 ml). odium carbonate (23 mg,.22 mmol) was then added and the mixture was stirred to provide a homogeneous solution. Next, hydrogen peroxide (5 µl of a 3 wt solution in H 2,.44 mmol) was added to the solution of 12. In a separate vial, N-acetylcysteine (9, 18 mg,.66 mmol) and sodium carbonate (23 mg,.22 mmol) were dissolved in D 2 (.4 ml). The solution of 9 was then added dropwise by pipette over 1 minute at room temperature to the solution of 12. The reaction was stirred for 3 minutes and then analysed by NMR. All alkene from 12 was consumed in the reaction: 11

12 LC-M studies using exo-5-norbornenecarboxylic acid (12) as a probe for cysteine oxidation [LC-M data associated with Figure 4B]. C 2 Na H C 2 Na H 2 3 equiv 2 1 equiv 2 equiv H 2, 3 min C 2 Na 1 exo-5-norbornenecarboxylic acid (12, 3 mg,.22 mmol) was added to a vial and suspended in H 2 (.4 ml). odium carbonate (23 mg,.22 mmol) was then added and the mixture was stirred to provide a homogeneous solution. Next, hydrogen peroxide (5 µl of a 3 wt solution in H 2,.44 mmol) was added. In a separate vial, N-acetylcysteine (9, 18 mg,.66 mmol) and sodium carbonate (23 mg,.22 mmol) were dissolved in H 2 (.4 ml). The solution of 9 was then added dropwise by pipette over 1 minute at room temperature to the solution of 12. The reaction was stirred for 3 minutes and then analysed by LC- M. LC-M data are shown below and on page 13. At the ph of this reaction (ph = 4.6), exo-5- norbornenecarboxylic acid (12) can trap the sulfenic acid of N-acetylcysteine. No product of the thiolene reaction was detected. EX-5-norbN-AcetylH _ C 2 Na 13 C 2 Na 14 C 2 Na (mixture of eight diastereomers) C 2 Na C 2 Na can E- TIC 4.12e6 C 2 H C 2 H 2.52 C 2 H C 2 H C 2 H C 2 H Time TIC for selected ions: m/z = 316 (top) and m/z = 323 (bottom) EX-5-norbN-AcetylH _ can E e5 2.1 C 2 H C 2 H C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 316 Found: m/z = _ can E e6 C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 323 Found: m/z = 323 Time

13 Representative mass spectra (EI - ) C 2 H C 2 H C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 316 Found: m/z = _ (3.779) can E- 1.75e _ (3.659) can E- 2.63e _ (3.539) 316 can E- 5.34e _ (3.4) can E- 3.49e5 m/z EX-5-norbN-AcetylH _ (2.421) can E e6 C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 323 Found: m/z = m/z

14 1 H NMR for dimedone (6 MHz, CDCl 3 ) 1 H NMR for dimedone enolate (6 MHz, D 2 ) Na 2 C 3 (1 equiv.) D 2 14

15 NMR studies using dimedone as a probe for cysteine oxidation [NMR data and control experiments associated with Figure 5A]. For each reaction, stock solutions were prepared for both dimedone (solution 1) and N-acetylcysteine (solution 2) and used for all experiments on pages olution 1: In a vial, a mixture of dimedone (35 mg,.25 mmol), D 2 (2 ml) and Na 2 C 3 (36 mg,.34 mmol) was stirred or shaken until fully dissolved. olution 2: In a vial, a mixture of N-acetylcysteine (211 mg, 1.3 mmol), D 2 (3.2 ml), and Na 2 C 3 (54 mg,.5 mmol) was stirred or shaken until fully dissolved. A: Dimedone enolate Na Na 2 C 3 (1 equiv) D 2, 2 min To a vial, D 2 (.4 ml) was added to solution 1 (.4 ml,.5 mmol dimedone). The solution was stirred for 2 minutes before analysing directly by NMR. B: The dimedone enolate reacts slowly with hydrogen peroxide Na 2 C 3 (1 equiv) H 2 2 (3 equiv) D 2, 2 min major Na To a vial, D 2 (.4 ml) was added to solution 1 (.4 ml,.5 mmol dimedone) with stirring followed by addition of H 2 2 (2 µl, 3 wt in H 2 ). The solution was stirred for 2 minutes before analysing directly by NMR. C: The dimedone enolate does not react with N-acetylcysteine H C 2 Na 3 equiv 1 equiv To a vial, solution 1 (.4 ml,.5 mmol dimedone) was added followed by solution 2 (.4 ml,.16 mmol 9). The solution was stirred for 2 minutes before analysing directly by NMR. D: The dimedone enolate does not trap the N-acetylcysteine sulfenic acid To a vial, solution 1 (.4 ml,.5 mmol dimedone) was added followed by H 2 2 (2 µl, 3 wt in H 2 ). After a few seconds of stirring, solution 2 (.4 ml,.16 mmol 9) was added dropwise over 1 minute. The solution was stirred for 2 minutes before analysing directly by NMR. E: The dimedone enolate does not react with N-acetylcysteine disulfide H To a vial, solution 2 (.4 ml,.16 mmol 9) was added followed by H 2 2 (2 µl, 3 wt in H 2 ) and stirred for 2 minutes before adding solution 1 (.4 ml,.5 mmol dimedone) dropwise over 1 minute. The mixture was left to stir for 2 minutes. minor (tentative assignment) Na H Na 2 C 3 (1 equiv) D 2, 2 min C 2 Na H Na Na 2 C 3 (1 equiv) H 2 2 (3 equiv) C 2 Na C 2 Na C 2 Na D 2, 2 min 3 equiv 1 equiv H 3 equiv H 2 2 (3 equiv) C 2 Na D 2, 2 min C 2 Na C 2 Na 1 equiv Na 2 C 3 (1 equiv) D 2, 2 min C 2 Na C 2 Na Na 15

16 NMR data for experiments A-E are shown below: Conclusions: Dimedone reacts slowly with hydrogen peroxide (see new minor peaks in B) Dimedone did not react with N-acetylcysteine (C) Under these conditions there was no evidence that dimedone trapped the sufenic acid of 9 (i.e. 15 was not detected in D). Dimedone did not react with the disulfide of N-acetylcysteine, 1 (E) E D C B A 16

17 Lisa 6 58 (.536) can E e6 LC-M studies using dimedone as a probe for cysteine oxidation [LC-M data and control experiment associated with Figure 5A]. For each reaction, stock solutions were prepared for both dimedone (solution 1) and N-acetylcysteine (solution 2) and used for all experiments on pages olution 1: In a vial, a mixture of dimedone (16 mg,.11 mmol), H 2 (.8 ml) and Na 2 C 3 (12 mg,.11 mmol) was stirred until fully dissolved. olution 2: In a vial, a mixture of N-acetylcysteine (38 mg,.23 mmol), H 2 (.6 ml) and Na 2 C 3 (9.1 mg,.9 mmol) was stirred until fully dissolved. Dimedone reacts slowly with hydrogen peroxide (LC-M). H 2 2 Na 2 C 3 H 2, 2 min major To a vial, H 2 (.2 ml) was added to solution 1 (.2 ml,.28 mmol dimedone) with stirring followed by addition of H 2 2 (1 µl, 3 wt in H 2 ). The solution was stirred for 2 minutes before analysing by LC- M. LC-M data are shown below. The minor product at 8.34 minutes is consistent with an oxidative dimerisation. A possible structure is proposed below. H minor (tentative assignment) Lisa A 8 can E TIC 1.59e7 H Time D H Lisa 6 96 (8.373) Nominal mass [M-H] can E- 9.6 H Calculated: m/z = e6 Found: m/z = Lisa (7.347) can E- 9.6 Nominal mass [M-H] 2.78e Calculated: m/z = 139 Found: m/z = Lisa 6 68 (6.284) can E e

18 Dimedone does not react with N-acetylcysteine (LC-M) [LC-M data and control experiment associated with Figure 5A]. H C 2 H Na 2 C 3 H 2, 2 min Na H C 2 Na C 2 Na C 2 Na To a vial, solution 1 (.2 ml,.28 mmol dimedone) was added followed by solution 2 (.2 ml,.77 mmol 9). The solution was stirred for 2 minutes before analysing by LC-M. LC-M data are shown below Lisa 7 can E TIC 1.31e7 H C 2 H 1.29 C 2 H C 2 H Time D cys Lisa (1.285) can E e6 H Nominal mass [M-H] Calculated: m/z = 162 C 2 H Found: m/z = Lisa (2.643) can E e6 Nominal mass [M-H] Calculated: m/z = 323 C 2 H C 2 H Found: m/z = Lisa (7.338) can E e6 9.6 Nominal mass [M-H] Calculated: m/z = 139 Found: m/z = m/z

19 Dimedone does not trap the N-acetylcysteine sulfenic acid (LC-M) [LC-M data associated with Figure 5A]. H C 2 H H 2 2 Na 2 C 3 H 2, 2 min Na H C 2 Na C 2 Na C 2 Na H To a vial, solution 1 (.2 ml,.28 mmol dimedone) was added followed by H 2 2 (1 µl, 3 wt in H 2 ). After stirring for a few seconds, solution 2 (.2 ml,.77 mmol 9) was added dropwise over 1 minute. The reaction mixture was stirred for 2 minutes before analysing by LC-M. LC-M data are shown below. C 2 Na Not detected Lisa 8 can E TIC 7.82e6 C 2 H C 2 H H Unknown Time D H22 cys Lisa (7.347) can E e6 Nominal mass [M-H] 9.6 Calculated: m/z = 139 Found: m/z = Lisa (6.238) can E e unknown product (sometimes detected when dimedone is treated with carbonate in water) [M-H] = 32; [M-2H]/2 = Lisa 8 28 (2.588) can E e6 Nominal mass [M-H] Calculated: m/z = 323 C 2 H C 2 H Found: m/z = m/z

20 NMR studies using cyclooctyne BCN as a probe for cysteine oxidation [NMR data and control experiments associated with Figure 5B]. For each reaction, stock solutions were prepared for both BCN (solution 1) and N-acetylcysteine (solution 2) and used for all experiments on pages olution 1: In a vial, BCN (2 mg,.13 mmol) was dissolved in DM-d6 (.4 ml) and then D 2 (1.6 ml) and Na 2 C 3 (15 mg,.14 mmol) were added and the mixture was stirred or shaken until fully dissolved. Note: BCN has very low solubility in aqueous media. olution 2: N-acetylcysteine (6 mg,.37 mmol), D 2 (2 ml) and Na 2 C 3 (15 mg,.14 mmol) were added to a vial and stirred or shaken until fully dissolved. A: BCN is stable but has poor solubility in aqueous solutions H Na 2 C 3 (1 equiv) H DM-d 6, D 2, H 2 min 6 6 H H To a vial, D 2 (.4 ml) was added to solution 1 (.4 ml,.26 mmol 6). The solution was stirred for 2 minutes before analysing directly by NMR. B: BCN reacts slowly with hydrogen peroxide Na 2 C 3 (1 equiv) H 2 2 (3 equiv) H H DM-d 6, D 2, 2 min H 6 H 6 H H H unknown products To a vial, D 2 (.4 ml) was added to solution 1 (.4 ml,.26 mmol 6) with stirring followed by addition of H 2 2 (2 µl, 3 wt in H 2 ). The solution was stirred for 2 minutes before analysing directly by NMR. C: BCN does not appear to react with N-acetylcysteine in an amount observable by NMR H H 6 H 1 equiv H C2 H 9 3 equiv Na 2 C 3 (1 equiv) DM-d 6, D 2, 2 min H 6 H H To a vial, solution 1 (.4 ml,.26 mmol 6) was added followed by solution 2 (.4 ml,.74 mmol 9). The solution was stirred for 2 minutes before analysing directly by NMR. D: BCN does not appear to trap the N-acetylcysteine sulfenic acid in an amount observable by NMR H C 2 Na H Na 2 C 3 (1 equiv) H 2 2 (3 equiv) H H C 2 H DM-d 6, D 2, 2 min C 2 Na 6 H 9 1 To a vial, solution 1 (.4 ml,.26 mmol 6) was added followed by H 2 2 (2 µl, 3 wt in H 2 ). After stirring for a few seconds, solution 2 (.4 ml,.74 mmol 9) was added dropwise over 1 minute. The solution was stirred for 2 minutes before analysing directly by NMR. C 2 Na C 2 Na 1 H 6 H H C 2 Na C 2 Na Not detected 16 C 2 Na H H H H Not detected H H 2

21 E: BCN does not react with N-acetylcysteine disulfide H 3 equiv H 2 2 (3 equiv) C 2 Na D 2, 2 min C 2 Na To a vial, solution 2 (.4 ml,.74 mmol 9) was added followed by H 2 2 (2 µl, 3 wt in H 2 ) and stirred for 2 minutes before adding solution 1 (.4 ml,.26 mmol 6) dropwise over 1 minute. The solution was stirred for 2 minutes before analysing directly by NMR. NMR data for experiments A-E are shown below. 1 C 2 Na Na 2 C 3 (1 equiv) H H DM-d C 2 Na C 2 Na 6, D 2, 2 min H H 6 H equiv H Conclusions: Under these conditions B: BCN reacts slowly hydrogen peroxide (unknown decomposition products) C: BCN does not appear to react with N-acetylcysteine in an amount observable by NMR D: BCN does not appear to trap the N-acetylcysteine sulfenic acid in an amount observable by NMR E: BCN does not react with N-acetylcysteine disulfide E D C B A 21

22 LC-M studies using cyclooctyne BCN (6) as a probe for cysteine oxidation [LC-M data and control experiments associated with Figure 5B]. For each reaction, stock solutions were prepared for both BCN (solution 1) and N-acetylcysteine (solution 2) and used for all experiments on pages olution 1: In a vial, BCN (13 mg,.9 mmol) was dissolved in DM (.2 ml), followed by the addition of H 2 (.6 ml) and Na 2 C 3 (12 mg,.11 mmol). The mixture was stirred or shaken until fully dissolved. Note: BCN has very low solubility in aqueous media. olution 2: N-acetylcysteine (38 mg,.23 mmol), H 2 (.6 ml), and Na 2 C 3 (1 mg,.9 mmol) were added to a vial and stirred or shaken until fully dissolved. The direct reaction of cyclooctyne BCN (6) and N-acetylcysteine (9) is observable by LC-M H H H H Na 2 C 3 (1 equiv) H H H C 2 H C 2 H C 2 Na DM:H 2, C 2 H min 1 H To a vial, solution 1 (.2 ml,.23 mmol 6) was added followed by solution 2 (.2 ml,.77 mmol 9). The solution was stirred for 2 minutes before analysing by LC-M. Note: BCN itself was not detectable by EI under the conditions of this experiment. LC-M BCN Ncys data are shown below Lisa 4 can E- H 8.1 H TIC 1.44e6 H H C 2 Na 1.73 C 2 H C 2 H.53 C 2 H C 2 H BCN Ncys Time H H Lisa (8.23) can E e5 9.6 C 2 H H Nominal mass [M-H] Calculated: m/z = 312 Found: m/z = Lisa (4.28) can E e4 Nominal mass [M-H] Calculated: m/z = 323 C 2 H C 2 H Found: m/z = Lisa (1.637) can E H Nominal mass [M-H] e5 Calculated: m/z = 162 C 2 H Found: m/z = m/z

23 The cyclooctyne BCN does not trap the N-acetylcysteine sulfenic acid (LC-M) [LC-M data and control experiments associated with Figure 5B]. H Na 2 C 3 (1 equiv) H 2 2 (3 equiv) C 2 H H H DM, H C 2 Na C 2 Na C 2 Na min 1 16 Not detected H To a vial, solution 1 (.2 ml,.23 mmol 6) was added followed by H 2 2 (1 µl, 3 wt in H 2 ). The mixture was stirred for a few seconds before adding solution 2 (.2 ml,.77 mmol 9) dropwise over 1 minute. The solution was stirred for 2 minutes before analysing by LC-M. Note: BCN itself was not detectable by EI under the conditions of this experiment. LC-M data are shown below. H H H Lisa 5 can E TIC 1.93e6 C 2 H C 2 H Time BCN H22 Ncys Lisa (4.224) can E e6 C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 323 Found: m/z = m/z

24 NMR studies using cyclooctyne DBC as a probe for cysteine oxidation For each reaction, stock solutions were prepared for both DBC (solution 1) and N-acetylcysteine (solution 2) and used for all experiments on pages olution 1: In a vial, DBC (35 mg,.13 mmol) was dissolved in DM-d6 (.4 ml) and then D 2 (1.6 ml) and Na 2 C 3 (13 mg,.12 mmol) were added. The mixture was stirred or shaken until fully dissolved. Note: DBC has very low solubility. olution 2: N-acetylcysteine (63 mg,.39 mmol), D 2 (2 ml) and Na 2 C 3 (13 mg,.12 mmol) were added to a vial and stirred or shaken until fully dissolved. A: DBC is stable but with poor solubility in aqueous solutions Na 2 C 3 (1 equiv) N DM-d 6, D 2 2 min 7 7 NH 2 N NH 2 To a vial, D 2 (.4 ml) was added to solution 1 (.4 ml,.26 mmol 7). The mixture was stirred for 2 minutes before analysing directly by NMR. B: DBC appeared to react with hydrogen peroxide under the experimental conditions, leading to either decomposition and/or precipitation. This reaction could occur at the alkyne or the amine. 7 N NH 2 Na 2 C 3 (1 equiv) H 2 2 (3 equiv) DM-d 6, D 2 2 min N NH 2 not observed To a vial, D 2 (.4 ml) was added to solution 1 (.4 ml,.26 mmol 7) with stirring followed by addition of H 2 2 (2 µl, 3 wt in H 2 ). The reaction mixture was stirred for 2 minutes before analysing directly by NMR. unknown decomposition products and precipitation C: DBC may react with N-acetylcysteine, but the subtle changes in the NMR spectra did not allow a firm conclusion to be drawn. H N C 2 H 3 equiv 7 NH equiv Na 2 C 3 (1 equiv) DM-d 6 :D 2, 2 min To a vial, solution 1 (.4 ml,.26 mmol 7) was added followed by solution 2 (.4 ml,.78 mmol 9). The reaction mixture was stirred for 2 minutes before analysing directly by NMR. C 2 H N? ( regioisomer) NH 2 H C 2 H 24

25 D: DBC reacted with hydrogen peroxide and thus its reaction with N-acetylcysteine sulfenic acid could not be properly assessed. H Na 2 C 3 (1 equiv) H 2 2 (3 equiv) N C 2 H DM-d 6, D 2 C 2 Na 7 NH min 1 C 2 Na N unknown decomposition products NH 2 ( regioisomer) Not detected To a vial, solution 1 (.4 ml,.26 mmol 7) was added followed by H 2 2 (2 µl, 3 wt in H 2 ) and stirred for a few seconds before adding solution 2 (.4 ml,.78 mmol 9) dropwise over 1 minute. The reaction mixture was stirred for 2 minutes before analysing directly by NMR. C 2 H E: DBC reacted with excess hydrogen peroxide in this control experiment, so its reaction with N- acetylcysteine disulfide could not be properly assessed. H H 2 2 (3 equiv) C 2 Na D 2, 2 min C 2 Na C 2 Na 3 equiv To a vial, solution 2 (.4 ml,.26 mmol 9) was added followed by H 2 2 (2 µl, 3 wt in H 2 ). The reaction was then stirred for 2 minutes before adding solution 1 (.4 ml,.78 mmol 7) dropwise over 1 minute. The reaction mixture was stirred for 2 minutes before analysing directly by NMR. NMR data for experiments A-E are shown on the following page. N 1 equiv NH 2 Na 2 C 3 (1 equiv) DM-d 6, D 2, 2 min 1 C 2 Na unknown decomposition products C 2 Na 25

26 E D C B A 26

27 LC-M studies using cyclooctyne DBC as a probe for cysteine oxidation For each reaction, stock solutions were prepared for both the DBC 7 (solution 1) and N-acetylcysteine 9 (solution 2) and used for all experiments on pages olution 1: In a vial, DBC 7 (28.6 mg,.1 mmol) was dissolved in DM (.2 ml) and then H 2 (.6 ml) and Na 2 C 3 (11 mg,.1 mmol) were added. The resulting mixture was stirred or shaken until fully dissolved. Note: solubility of DBC was very low. olution 2: N-acetylcysteine 9 (38 mg,.24 mmol), H 2 (.6 ml) and Na 2 C 3 (9.5 mg,.9 mmol) were added to a vial and the mixture was stirred or shaken until fully dissolved. The cyclooctyne DBC reacts with hydrogen peroxide (LC-M) N Na 2 C 3 (1 equiv) H 2 2 (3 equiv) NH 2 DM, H 2 2 min N NH 2 not observed unknown decomposition products In a vial, H 2 (.2 ml) was added to solution 1 (.2 ml,.25 mmol 7) and stirred followed by addition of H 2 2 (1 µl, 3 wt in H 2 ). The reaction mixture was stirred for 2 minutes before analysing directly by LC-M. No mass corresponding to DBC was found, indicating possible decomposition or precipitation. Two unknown masses of 295. and 327. m/z were observed (EI). The peaks at 8.11 and 8.49 min were likely impurities in the DBC. (Unmodified DBC elutes at 7.8 minutes see 28) LC-M data are shown below. DBC H Lisa 7 can E.64 TIC 3.1e Time DBC H Lisa (5.832) can E e m/z

28 The cyclooctyne DBC reacts directly with cysteine (LC-M) H N C 2 H 3 equiv 7 NH 2 1 equiv 9 Na 2 C 3 (1 equiv) DM, D 2, 2 min C 2 H N NH 2 ( regioisomer) H C 2 H N NH 2 To a vial, solution 1 (.2 ml,.25 mmol 7) was added followed by solution 2 (.2 ml,.77 mmol 9). The reaction mixture was stirred for 2 minutes before analysing directly by LC-M. The peaks at 8.1 and 8.51 min were impurities in the DBC. LC-M data are shown below Lisa 8 can E.64 TIC 3.42e7 N H NH C 2 H N NH C 2 H ( regioisomer) DBC cys Lisa (8.115) can E Time 2.93e Lisa (7.8) can E 277. Nominal mass [MH] 1.84e6 N Calculated: m/z = 277 Found: m/z = NH Lisa (7.55) can E N NH e6 Nominal mass [MH] Calculated: m/z = C 2 H ( regioisomer) Found: m/z = Lisa (1.691) can E H Nominal mass [MH] 3.48e6 Calculated: m/z = 164 C 2 H Found: m/z = m/z

29 NMR studies using cis-5-norbornene-endo-2,3-dicarboxylic acid (8) as a probe for cysteine oxidation at pd 5. (NaAc buffer, 2 mm) [NMR data and control experiments associated with Figure 6A]. Preparation of buffer: In a beaker, sodium acetate trihydrate (1.35 g,.1 mol) was dissolved in approx. 2 ml of D 2. The ph was adjusted by adding acetic acid until a reading of ph = 4.6 was measured using a ph meter, which corresponds to a pd = 5.. The solution was then diluted to 25 ml with D 2 using a volumetric flask so that the concentration of the buffer was 4 mm. For each reaction, stock solutions were prepared for both cis-5-norbornene-endo-2,3-dicarboxylic acid 8 (solution 1), N-acetylcysteine 9 (solution 2), and N-acetylcysteine disulfide 1 (solution 3). These solutions were used for all experiments on pages 29-3 olution 1: cis-5-norbornene-endo-2,3-dicarboxylic acid (17 mg,.9 mmol 8), D 2 (1. ml), and NaH (3.8 mg,.9 mmol) were added to a vial and stirred or shaken until dissolved. Finally, the pd 5. acetate buffer in D 2 (4 mm, 1. ml) was added and the solution was stirred. olution 2: N-acetylcysteine (42 mg,.26 mmol 9), D 2 (1. ml), and NaH (3.3 mg,.8 mmol) were added to a vial and shaken or stirred until fully dissolved. Finally, the pd 5. acetate buffer in D 2 (4 mm, 1. ml) was added and the solution was stirred. olution 3: N-acetylcysteine (9.8 mg,.6 mmol 9), D 2 (.25 ml), and NaH (2.4 mg,.6 mmol) were added to a vial and stirred or shaken until fully dissolved. A solution of H 2 2 (15 µl, 3 wt in H 2 ) was then added and the reaction was stirred for 1 hour or until N-acetylcysteine was fully converted to the disulfide (as monitored by 1 H-NMR). Finally, the pd 5. acetate buffer in D 2 (4 mm,.25 ml) was added and the solution was stirred. A: cis-5-norbornene-endo-2,3-dicarboxycylic acid (8) 8 C 2 H C 2 H NaH (1 equiv) NaAc buffer pd 5. C 2 Na C 2 Na To a vial, solution 1 (.5 ml,.2 mmol 8) was added followed by pd 5. acetate buffer (.5 ml, 2 mm). The solution was stirred for 2 minutes before analysing directly by NMR. B: cis-5-norbornene-endo-2,3-dicarboxylic acid (8) does not react with H C 2 H C 2 H NaH(1 equiv) H 2 2 (3 equiv) NaAc buffer pd 5., 2 min C 2 Na C 2 Na To a vial, solution 1 (.5 ml,.2 mmol 8) was added followed by pd 5. acetate buffer (.5 ml, 2 mm) and H 2 2 (7 µl, 3 wt in H 2 ). The solution was stirred for 2 minutes before analysing directly by NMR. C: ome reaction between cis-5-norbornene-endo-2,3-dicarboxylic acid (8) and N-Acetylcysteine 9 was detected by NMR (LC-M indicated that the thiol-ene reaction generated a minor by-product). H C 2 Na C 2 Na C 2 Na 9 3 equiv 1 equiv NaAc buffer pd 5., 2 min H C 2 Na C 2 Na C 2 Na To a vial, solution 1 (.5 ml,.2 mmol 8) was added followed by solution 2 (.5 ml,.6 mmol 9). The solution was stirred for 2 minutes before analysing directly by NMR. C 2 Na C 2 Na C 2 Na minor (also see LC-M) 29

30 D: cis-5-norbornene-endo-2,3-dicarboxylic acid (8) reacts with the sulfenic acid of N-acetylcysteine (9). The alkene signal in the 1 H-NMR from 8 is completely consumed in the reaction. Not all cysteine was consumed in the reaction. C 2 H 8 C 2 H (1 eq.) 1. NaH (1 eq.), H 2 2 (3 eq.) 2. H (3 eq.) NaH C 2 H (1 eq.) 9 ph 5. NaAc buffer, 2 min, rt To a vial, solution 1 (.5 ml,.2 mmol 8) was added followed by H 2 2 (7 µl, 3 wt in H 2 ). The resulting solution was stirred for a few seconds before the dropwise addition of solution 2 (.5 ml,.6 mmol 9) over 1 minute. The solution was stirred for 2 minutes before analysing directly by NMR. E: No reaction between cis-5-norbornene-endo-2,3-dicarboxylic acid (8) and N-acetylcysteine disulfide was detected by NMR H 3 equiv H 2 2 (3 equiv) C 2 Na D 2, 2 min C 2 Na C 2 Na To a vial, solution 3 (.5 ml,.6 mmol 1) was added followed by dropwise addition of solution 1 (.5 ml,.2 mmol 8). The solution was stirred for 2 minutes before analysing directly by NMR. NMR data for experiments A-E are shown below. 1 H C 2 Na C 2 Na 1 8 C 2 Na C 2 Na NaAc buffer pd 5., 2 min 1 equiv 11 C 2 Na C 2 Na C 2 Na C 2 Na (mixture of four diastereomers) C 2 Na Full consumption of alkene C 2 Na 1 no reaction C 2 Na C 2 Na E D C B A 3

31 LC-M studies using cis-5-norbornene-endo-2,3-dicarboxylic acid (8) as a probe for cysteine oxidation at ph 5. (NaAc buffer, 2 mm) [LC-M data and control experiments associated with Figure 6A]. For each reaction, stock solutions were prepared for both cis-5-norbornene-endo-2,3-dicarboxylic acid (8) (solution 1) and N-acetylcysteine 9 (solution 2) and used for all experiments on pages olution 1: cis-5-norbornene-endo-2,3-dicarboxylic acid (14.8 mg,.8 mmol 8), H 2 (1. ml) and NaH (3.8 mg,.9 mmol) were added to a vial and stirred or shaken until fully dissolved. Finally, ph 5. sodium acetate buffer in H 2 (4 mm, 1. ml) was added and the solution was stirred. olution 2: N-acetylcysteine (38.1 mg,.23 mmol 9), H 2 (1. ml), and NaH (3.1 mg,.8 mmol) were added to a vial and stirred to dissolve. Finally, ph 5. sodium acetate buffer in H 2 (4 mm, 1. ml) was added and the solution was stirred. LC-M data for a control experiment in which cis-5-norbornene-endo-2,3-dicarboxylic acid (8) and N- acetylcysteine (9) were reacted at ph 5.. The thiol-ene product was detected as a major product at 7.3 minutes. The conversion of this reaction may depend on the amount of oxygen available to initiate the thiolene radical chain reaction. (Note the 1 H-NMR spectrum on page 3 where 8 was largely unreacted under what were intended to be the same conditions). In any event, it is clear that the thiol-ene reaction must be considered when using norbornene probes for the analysis of cysteine oxidation. H C 2 Na C 2 Na C 2 Na 9 3 equiv 8 1 equiv NaAc buffer ph 5., 2 min To a vial, solution 1 (.5 ml,.2 mmol 9) was added followed by solution 2 (.5 ml,.6 mmol 8). The reaction mixture was stirred for 2 minutes before analysing directly by LC-M. The LC-M data are shown on the following page. H C 2 Na C 2 Na C 2 Na C 2 Na 31

32 N NCys Lisa A 3 can E TIC H 5.6e6 C 2 H 7.35 C 2 H C 2 H C 2 H 3 Time N NCys Lisa A (1.321) can E e6 H C 2 H Nominal mass [M-H] Calculated: m/z = 162 Found: m/z = m/z N NCys Lisa A (7.338) can E e6 91 C 2 H C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 344 Found: m/z = m/z

33 LC-M study indicating cis-5-norbornene-endo-2,3-dicarboxylic acid (8) can trap the sulfenic acid of N- acetylcysteine at ph 5.. The product for the thiol-ene reaction was also detected. C 2 H 8 C 2 H (1 eq.) 1. NaH (1 eq.), H 2 2 (3 eq.) 2. H (3 eq.) NaH C 2 H (1 eq.) 9 ph 5. NaAc buffer, 2 min, rt H C 2 Na C 2 Na C 2 Na 1 Full consumption of alkene (NMR, LC-M) 11 C 2 Na C 2 Na C 2 Na (mixture of four diastereomers) Product of trapping sulfenic acid C 2 Na C 2 Na C 2 Na Product of thiol-ene reaction To a vial, solution 1 (.5 ml,.2 mmol 9) was added followed by H 2 2 (7 µl, 3 wt in H 2 ). The solution was then stirred for a few seconds before dropwise addition of solution 2 (.5 ml,.6 mmol 9) over 1 minute. The reaction mixture was stirred for 2 minutes before analysing directly by LC-M. The LC-M data are shown on the following pages (34-35). 33

34 N H22 NCys 3 C 2 H C 2 H Lisa A 4 can E- 2.5 TIC 3.77e6 C 2 H C 2 H C 2 H C 2 H C 2 H C 2 H Time N H22 NCys Lisa A 4 27 (2.495) can E e6 C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 323 Found: m/z = m/z N H22 NCys Lisa A (2.976) can E e5 36 C 2 H C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 36 Found: m/z = m/z

35 N H22 NCys Lisa A (4.39) can E e C 2 H C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 36 Found: m/z = 36 m/z N H22 NCys Lisa A (7.356) can E e6 C 2 H C 2 H C 2 H Nominal mass [M-H] Calculated: m/z = 344 Found: m/z = m/z

36 NMR studies showing cis-5-norbornene-endo-2,3-dicarboxylic acid (8) does not react with sulfinates or sulfonates at pd 5. (NaAc buffer, 2 mm) Preparation of buffer: In a beaker, sodium acetate trihydrate (.53 g,.4 mol) was dissolved in approximately 8 ml of D2. The ph was adjusted by adding acetic acid until a reading of ph = 4.6 was measured using a ph meter, which corresponds to a pd = 5.. The solution was then diluted to 1 ml with D2 using a volumetric flask so that the concentration of the buffer was 4 mm. For each reaction, stock solutions were prepared for both cis-5-norbornene-endo-2,3-dicarboxylic acid 8 (solution 1), sodium p-toluenesulfinate (solution 2), and sodium p-toluenesulfonate (solution 3). olution 1: cis-5-norbornene-endo-2,3-dicarboxylic acid (17.4 mg,.1 mmol 8), D2 (1. ml), and NaH (3.3 mg,.8 mmol) were added to a vial and stirred or shaken until dissolved. The resulting solution was then diluted with the pd 5. acetate buffer in D2 (4 mm, 1. ml). olution 2: sodium p-toluenesulfinate (43.2 mg,.24 mmol) and D2 (1. ml) were added to a vial and shaken or stirred until fully dissolved. The resulting solution was then diluted with the pd 5. acetate buffer in D2 (4 mm, 1. ml). olution 3: p-toluenesulfonic acid (48.9 mg,.23 mmol), D2 (1. ml), and NaH (3.2 mg,.8 mmol) were added to a vial and stirred or shaken until dissolved. The resulting solution was then diluted with the pd 5. acetate buffer in D2 (4 mm, 1. ml). A: No reaction between 8 and sodium p-toluenesulfinate was detected by NMR Na C 2 Na C 2 Na 3 equiv 1 equiv NaAc buffer pd 5., 2 min Na C 2 Na C 2 Na To a vial, solution 1 (.5 ml,.2 mmol 8) was added followed by solution 2 (.5 ml,.6 mmol sodium p- toluenesulfinate). The solution was stirred for 2 minutes before analysing directly by NMR. B: No reaction between 8 and sodium p-toluenesulfonate was detected by NMR Na C 2 Na C 2 Na 3 equiv 1 equiv NaAc buffer pd 5., 2 min To a vial, solution 1 (.5 ml,.2 mmol 8) was added followed by solution 3 (.5 ml,.6 mmol sodium p- toluenesulfonate). The solution was stirred for 2 minutes before analysing directly by NMR. NMR data for experiments A-B are shown on the following page. No reactions were observed. no reaction Na C 2 Na C 2 Na 36

37 A B 37

38 NMR studies using dimedone (2) as a probe for cysteine oxidation at pd 5. (NaAc buffer, 2 mm) [NMR data and control experiments associated with Figure 6B]. Preparation of buffer: ee page 29 for the preparation of the pd 5. acetate buffer in D 2 (4 mm). olution 1: Dimedone (13 mg,.9 mmol), D 2 (1. ml) and NaH (3.4 mg,.8 mmol) were added to a vial and stirred or shaken until fully dissolved. Finally, the pd 5. acetate buffer in D 2 (4 mm, 1. ml) was added and the solution was stirred. olution 2: N-acetylcysteine (42 mg,.26 mmol), D 2 (1. ml) and NaH (3.3 mg,.8 mmol) were added to a vial and stirred or shaken until fully dissolved. Finally, the pd 5. acetate buffer in D 2 (4 mm, 1. ml) was added and the solution was stirred. olution 3: N-acetylcysteine (9.8 mg,.6 mmol), D 2 (.25 ml) and NaH (2.4 mg,.6 mmol) were added to a vial and stirred or shaken until fully dissolved. A solution of H 2 2 (15 µl, 3 wt in H 2 ) was then added and stirred for 1 hr or until N-acetylcysteine was fully converted to the disulfide (as monitored by 1 H-NMR). Finally, the pd 5. acetate buffer in D 2 (4 mm,.25 ml) was added and the solution was stirred. A: Dimedone NaH (1 equiv) Na NaAc buffer pd 5., 2 min 2 To a vial, solution 1 (.5 ml,.2 mmol dimedone) was added followed by pd 5. acetate buffer in D 2 (.5 ml, 2 mm). The solution was stirred for 2 minutes before analysing directly by NMR. B: Dimedone reacts slowly with hydrogen peroxide 2 NaH (1 equiv) H 2 2 (3 equiv) NaAc buffer pd 5., 2 min Na H To a vial, solution 1 (.5 ml,.2 mmol dimedone) was added followed by pd 5. acetate buffer in D 2 (.5 ml, 2 mm) and H 2 2 (7 µl, 3 wt in H 2 ). The solution was stirred for 2 minutes before analysing directly by NMR. minor (tentative assignment) C: Dimedone does not react with N-acetylcysteine 1 equiv Na 9 H 3 equiv C 2 Na NaAc buffer pd 5., 2 min To a vial, solution 1 (.5 ml,.2 mmol dimedone) was added followed by solution 2 (.5 ml,.6 mmol 9). The solution was stirred for 2 minutes before analysing directly by NMR. Na No reaction H C 2 Na 38

39 D: Dimedone does not trap the N-acetylcysteine sulfenic acid 1 equiv 2 9 H 3 equiv C 2 Na H 2 2 (3 equiv) NaAc buffer pd 5., 2 min To a vial, solution 1 (.5 ml,.2 mmol) was added followed by H 2 2 (7 µl, 3 wt in H 2 ) and stirred for a few minutes before dropwise addition of solution 2 (.5 ml,.6 mmol). The solution was stirred for 2 minutes before analysing directly by NMR. Na H C 2 H 1 C 2 Na C 2 Na E: The dimedone enolate does not react with N-acetylcysteine disulfide 9 H 3 equiv H 2 2 (6 equiv) C 2 Na D 2, 2 min C 2 Na 1 C 2 Na 1 equiv NaAc buffer pd 5., 2 min To a vial, solution 3 (.5 ml,.6 mmol) was added followed by dropwise addition of solution 1 (.5 ml,.2 mmol). The solution was stirred for 2 minutes before analysing directly by NMR. 2 C 2 Na C 2 Na Na NMR data for experiments A-E are shown below. Under these conditions: Dimedone reacts slowly with hydrogen peroxide (B) [Also see page 4] Dimedone does not react with N-acetylcysteine (C) Dimedone does not trap the sulfenic acid of N-acetylcysteine (D) Dimedone does not react with N-acetylcysteine disulfide (E) E D C B A 39