suementary inrmatin10.1038/nchem.2601... · meeee) at room temperature - lc-ms i. reaction of 1...

NATURE CHEMISTRY | www.nature.com/naturechemistry 1

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NCHEM.2601

Supplementary Information

Click and Chemically Triggered Declick Reactions Through Reversible Amine and Thiol Coupling via a Conjugate Acceptor

Katharine L. Diehla, Igor V. Kolesnichenkob, Scott A. Robothamb, J. Logan Bachmanb, Ye

Zhongc, Jennifer S. Brodbeltb, and Eric V. Anslynb aDepartment of Chemistry, Princeton University, Princeton NJ, 08544, USA

bDepartment of Chemistry, The University of Texas at Austin, Austin TX, 78712, USA

cUnilever, Shanghai, China

© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.



Table of Contents

1. General Information 2. Shorthand Notation 3. Synthesis of 1 4. General Reactivity Studies of 1

a. Reaction of 1 with amine (4-(methylamino)benzoic acid, AMBA) - 1H-NMR b. Reaction of AMBA-1 with thiol (N-acetylcysteine, NAC) - 1H-NMR c. Reaction of 1 with amine (4-(methylamino)benzoic acid, AMBA) at room

temperature - LC-MS d. Reaction of 1 with amine (α-boc-lysine, Lys) at room temperature - LC-MS e. Reaction of 1 with amine (α-boc-lysine, Lys) at 45°C - LC-MS f. Reaction of Lys-1 with thiol (N-acetylcysteine, NAC) at room temperature - LC-

MS g. Reaction of Lys-1 with thiol (N-acetylcysteine, NAC) at 45°C - LC-MS h. Reaction of Lys-1 with thiol (2-{2-[2-(2-mercaptoethoxy)ethoxy]ethoxy}ethanol,

MEEEE) at room temperature - LC-MS i. Reaction of 1 with an excess of amine at room temperature - LC-MS j. Reaction of 1 with thiol first (NAC) and then amine (Lys) - LC-MS

5. Reactivity studies of 1 with amine and thiol simultaneously a. Reaction of 1, NAC, and AMBA – 1H-NMR b. Reaction of 1, NAC, and benzylamine – LC/MS studies c. Reaction of 1, OctSH, and benzylamine – LC/MS studies d. Reaction of 1, OctSH, and propargylamine – LC/MS studies e. Reaction of 1, OctSH, and 1-amino-3-butyne – LC/MS studies f. Reaction of 1, PhSH, and propargylamine – LC/MS studies

6. Measuring pKa of NH in 2 7. Testing Decoupling Agents

a. Screening by 1H-NMR b. Reaction of DTT with Lys-1-NAC at room temperature - LC-MS c. Reaction of DTT with Lys-1-NAC at 45 °C - LC-MS d. Reaction of DTT with Lys-1-Lys at 45 °C - LC-MS

8. Triamine and Resin a. Reacting triamine with 1 (6) b. Adding thiols to 6 (7, 8, 9, and 10) c. Adding DTT to functionalized triamine mixture d. Synthesis of 11 e. Synthesis of 12 f. Removal of 1 from resin by DTT g. Thiol library on the resin

9. Peptide Labeling a. Peptide synthesis b. Reaction of acIAHRFK(1)DLGE with VVKLKC c. Reaction of acIAHRFK(1)DLGE with MEEEE d. Mass spectrometry analysis of modified peptides e. Decoupling of acIAHRFKDLGE and VVKLKC by DTT f. Crosslinking of myogloblin and MEEEE with 1




g. Mass spectrometry analysis of modified myoglobin h. Decoupling of myoglobin and MEEEE by DTT

10. Oligomers a. Synthesis of 13 b. Synthesis of oligomers c. Reaction of oligomers with DTT

1. General Information

Chemicals and reagents were obtained from Sigma Aldrich and used without further purification. The resins, amino acids, and coupling reagents were obtained from NovaBioChem. Methyl mercaptan, which is a gas released in reactions with 1, is commonly added in small amounts to natural gas in order to make detection of leaks easier, since humans are very sensitive to the smell. However, at higher concentrations, this gas is toxic and has an extremely putrid smell. In the experiments described in this paper, the unpleasantness of the smell released from the reactions was equivalent to or slightly worse than the odor of natural gas. However, as a precaution against even these levels of exposure, the reactions were carried out in a fume hood.

HPLC-grade solvents were prepared with 0.1% TFA (v/v) and filtered through a 0.2 micron filter. HPLC was performed on a Shimadzu instrument with a preparative C-18 column using a water and acetonitrile mobile phase. LC-MS analysis was performed on an Agilent 1200 Series HPLC with an Agilent 6130 single quadrupole mass spectrometer (ESI and APCI ionization). 1H-NMR spectra were obtained on a 400 MHz Varian.

When LC chromatograms were used to approximate the extent of reaction, it should be noted that the extinction coefficients of the starting material and product may differ. Furthermore, the baseline of the spectra may also affect the quantitative accuracy of the reaction extents. Thus, in these cases, the extents of reaction were obtained semi-quantitatively as percentages calculated based on the integration of the starting material and product peaks. This approach is essentially equivalent to using thin-layer chromatography (TLC) for reaction monitoring.

A general mechanism for amine and thiol exchange on 1:

O O

O O

SS

R NH2

O O

O O

SSNH

R

O O

OO

SHN

R+ S CH3

O O

O O

SSN HRH

B

+ H B

OO

OO

NH

SR

SR'

OO

OO+

SHN

R R'

SH3C

SR'




2. Shorthand Notation

3. Synthesis of 1

Reagent 1 was synthesized in one step, according to the literature procedure: Ben Cheikh, A. et al. Synthesis of .alpha.-cyano carbonyl compounds by flash vacuum thermolysis of (alkylamino)methylene derivatives of Meldrum’s acid. Evidence for facile 1,3-shifts of alkylamino and alkylthio groups in imidoylketene intermediates. J. Org. Chem. 56, 970–975 (1991).

4. General Reactivity Studies of 1

a. Reaction of 1 with amine (4-(methylamino)benzoic acid, AMBA)- 1H-NMR

The 4-(methylamino)benzoic acid (AMBA, 45.6 mg, 301 µmol) was dissolved in 5 mL D2O. The pH was adjusted to 7 with NaOD. The 1 (74.8 mg, 301 µmol) was dissolved in 2 mL CD3CN. The solutions were combined (43 mM), and the pH was adjusted to 7 with NaOD. NaOD was added periodically to maintain a pH of 7, since the solution was not buffered. The reaction was allowed to proceed for 18 h. The product was confirmed by 1H-NMR (400 MHz, D2O):




Supplementary Figure 1. AMBA-1 spectrum. Peak at 4.11 ppm is from unreacted amine CH2

b. Reaction of AMBA-1 with thiol (N-acetylcysteine, NAC)- 1H-NMR

To the AMBA-1 (301 µmol) was added N-acetylcysteine (NAC, 51 mg, 313 µmol). The pH was adjusted to 7 with NaOD and remained constant throughout the reaction without further adjustment. The reaction was sparged with nitrogen gas in an open vial and was allowed to proceed for 24 h. The product was confirmed by 1H-NMR (400 MHz, D2O):




Supplementary Figure 2. AMBA-1-NAC spectrum. Peak at 4.11 ppm is from unreacted amine CH2. Peaks at 1.88, 2.74, and 4.22 ppm are from unreacted NAC

c. Reaction of 1 with amine (4-(methylamino)benzoic acid, AMBA) at room temperature- LC-MS 1 (39.5 mg, 158 µmol) was dissolved in 1 mL MeCN. AMBA (23.9 mg, 158 µmol) was dissolved in 3 mL of 0.1 M phosphate buffer, 0.15 M NaCl, pH 7.4. The solutions were combined and stirred at room temperature (20°C) for 4 h and analyzed by LC-MS. Based on the extracted chromatogram at 320 nm (below), the reaction was about 90% complete. LRMS (ESI): 350.1 (M-1).




Supplementary Figure 3. LC trace (λ = 320 nm) of AMBA + 1 reaction @ RT after 4 h

d. Reaction of 1 with amine (α-boc-lysine, Lys) at room temperature- LC-MS

1 (20.4 mg, 82 µmol) was dissolved in 0.5 mL MeCN. α-boc-lysine (20.2, 82 µmol) was dissolved in 1.5 mL of 0.1 M phosphate buffer, 0.15 M NaCl, pH 7.4. The solutions were combined (41 mM) and stirred at room temperature (20°C). The reaction was monitored at 4, 22, and 70 h by LC-MS. Based on the extracted chromatogram at 320 nm (below), the reaction was about 50% complete after 4 h, about 70% complete after 22 h, and about 90% complete after 70 h. LRMS (ESI): 445.1 (M-1).




Supplementary Figure 4. LC trace (λ = 320 nm) of α-boc-lysine + 1 reaction @ RT after indicated times

e. Reaction of 1 with amine (α-boc-lysine, Lys) at 45°C - LC-MS

1 (19.8 mg, 80 µmol) was dissolved in 0.5 mL MeCN. α-boc-lysine (19.8, 80 µmol) was dissolved in 1.5 mL of 0.1 M phosphate buffer, 0.15 M NaCl, pH 7.4. The solutions were combined (41 mM) and stirred at 45°C for 4 h and then analyzed by LC-MS. Based on the extracted chromatogram at 320 nm (below), the reaction was about 90% complete. LRMS (ESI): 445.1 (M-1).




Supplementary Figure 5. LC trace (λ = 320 nm) of α-boc-lysine + 1 reaction @ 45 ºC after 4 h

f. Reaction of Lys-1 with thiol (N-acetylcysteine, NAC) at room temperature- LC-MS

To Lys-1 (82 µmol, 41 mM) was added NAC (13.4 mg, 82 µmol). The pH was adjusted to 7, and the solution was sparged with N2 at room temperature (20°C). The reaction was monitored at 7, 23, and 46 h. Based on the extracted chromatogram at 320 nm (below), the reaction was about 50% complete at 7 h, 85% complete at 23 h, and more than 90% complete at 46 h. LRMS (ESI): 560.0 (M-1).

Supplementary Figure 6. LC trace (λ = 320 nm) of Lys-1 + NAC reaction @ RT after indicated times




g. Reaction of Lys-1 with thiol (N-acetylcysteine, NAC) at 45°C - LC-MS

To Lys-1 (80 µmol, 41 mM) was added NAC (13.1 mg, 80 µmol). The pH was adjusted to 7, and the solution was sparged with N2 at 45°C for 8 h at which time the reaction had completed. LRMS (ESI): 560.0 (M-1).

Supplementary Figure 7. LC trace (λ = 320 nm) of Lys-1 + NAC reaction @ 45 ºC after indicated times

h. Reaction of Lys-1 with thiol (2-{2-[2-(2-mercaptoethoxy)ethoxy]ethoxy}ethanol, MEEEE) at room temperature - LC-MS

Lys-1 was prepared as described (38 µmol). To Lys-1 was added 2-{2-[2-(2-mercaptoethoxy)ethoxy]ethoxy}ethanol, MEEEE (22 µL, 115 µmol, 3 eq), and the reaction was stirred in an open container for 18 h. The reaction was analyzed by LC-MS to confirm LysEvaMEEEE. LRMS (ESI): 607.2 (M-1).

Supplementary Figure 8. LC-MS trace (neg. mode) of Lys-1 + MEEEE reaction @ RT after 18 h




i. Reaction of 1 with an excess of amine at room temperature - LC-MS

1 (5.8 mg, 23.5 µmol, 1 eq) was dissolved in 300 µL acetonitrile, and α-Boc-lysine (33.7 mg, 137 µmol, 5.8 eq) was dissolved in 2.5 mL of 50 mM phosphate buffer, H2O, 10 mM EDTA, pH 7.2. The solution of 1 was added to the lysine solution. The reaction was heated to 45 °C for three days. LC-MS analysis indicated that a small amount of Lys-1-Lys product had formed. LRMS (ESI): 643.2 (M+1)

Supplementary Figure 9. LC-MS trace (pos. mode) of Lys-1-Lys reaction @ RT after three days

j. Reaction of 1 with thiol first (NAC) and then amine (Lys) - LC-MS

1 (6.0 mg, 24.2 µmol, 1 eq) was dissolved in 1 mL acetonitrile. NAC (8.3 mg, 50.7 µmol, 2.1 eq) was dissolved in 5 mL of 50 mM phosphate buffer, H2O, 10 mM EDTA, pH 7.5. The two solutions were combined. The reaction was gently sparged with nitrogen and reacted overnight and then analyzed by LC-MS. LRMS (ESI): 477.2 (M-1).

Supplementary Figure 10. LC-MS trace (neg. mode) of NAC-1-NAC reaction @ RT after 18 h

Then α-Boc-lysine (6.0 mg, 24.2 µmol, 1 eq) was added, and the reaction was stirred for 3 days. It was monitored by LC-MS. LRMS (ESI): 560.2 (M-1).




Supplementary Figure 11. LC-MS trace (neg. mode) of NAC-1-NAC + α-boc-lysine reaction @ RT after 3 days

5. Reactivity studies of 1 with amine and thiol simultaneously

a. Reaction of 1, NAC, and AMBA – 1H-NMR

D2O, CD3CN, and NaOD were purged with nitrogen in separate vials for ~2 hours each before being used. 45.60 mg (302 µmol) of 4-(aminomethyl)benzoic acid and 49.34 mg (302 µmol) of N-acetylcysteine were then weighed into a vial, and this was purged with nitrogen. The mixture of solids was then dissolved in 5 mL of D2O and the pD was adjusted to ~8 with NaOD, while keeping the solution under nitrogen the entire time. In a separate purged vial 74.90 mg (302 µmol) of 1 were dissolved in 2 mL of CD3CN. The two solutions were then combined, while being kept under a continuous stream of nitrogen, and the pD was found to be ~7. An aliquot of this was immediately removed, and a 1H-NMR was taken, while the remainder of the solution was allowed to stir. The spectrum showed evidence of partial addition of the amine to the conjugate acceptor and no addition of the thiol. Over the course of the next 18 hours small amounts of nitrogen-purged D2O and CD3CN were occasionally added to the reaction mixture and the pD was adjusted to ~7, as the original solvents evaporated off. A second aliquot was removed after ~5 hours and another 1H-NMR was taken, which showed further addition of the amine and partial addition of the thiol. A final aliquot was removed after ~18 hours and a last 1H-NMR was taken. The spectrum showed that the amine had fully added to the conjugate acceptor, while the thiol addition was still not complete. At this point it was concluded that addition of the amine to the conjugate acceptor is much faster than the thiol addition, beginning almost immediately after combining all of the components.




Supplementary Figure 12. 1H-NMR spectra from the simultaneous reaction of 1, AMBA, and NAC after 0, 5, and 18 h. The AMBA begins to add immediately to 1 (4.88 ppm- CH2). After 5 h, the AMBA has not completely added, but NAC is also beginning to add (3.36 ppm- CH2). After 18 h, the spectrum is consistent with AMBA-1-NAC that was synthesized stepwise. The spectra from Figure S2 is reproduced at the bottom for comparison.

b. Reaction of 1, NAC, and benzylamine – LC/MS studies

NOTE: In each of the following reactions (b-f) 1 equivalent (120.82 µmol) of thiol was dissolved or suspended, depending on solubility, in 2 mL of a 2:1 mixture of aqueous phosphate buffer (100 mM phosphate, 150 mM NaCl, pH 7.4) and acetonitrile. 1 equivalent (120.82 µmol) of amine was then added to the mixture, immediately followed by 30 mg (120.82 µmol, 1 equivalent) of 1. The reaction was then covered with a septum, an exhaust needle was added to allow for the escape of methyl mercaptan, and was stirred vigorously (to the point of frothing, in an attempt to facilitate expulsion of methyl mercaptan) at room temperature. It was then monitored over the course of the next few days by LC/MS.




Supplementary Figure 13. LC/MS trace (neg. mode) of NAC combined simultaneously with benzylamine and 1 after 36 h @ RT.

Supplementary Figure 14. LC/MS analysis (neg. mode) of NAC combined simultaneously with benzylamine and 1 after 36 h @ RT. Scanned over the range of 6.8-11.0 mins, as indicated by the blue line in the LC/MS trace above. 162.1 corresponds to an anionic NAC fragment off of the target compound.

min2 4 6 8 10 12 14

200000

400000

600000

800000

1000000

MSD2 TIC, MS File (M:\NHB_5342_LCMS\01-16\KOLESNICHENKO\280116-Z12MIN_LC-IVK_3_002_PBS_MECN_MS31.D) MM-ES, Neg, Sca

0.10

9 0.

317

0.67

4 0.86

3 0.93

1 1.

188

1.43

9 1.54

3 1.

643

1.73

9 1.

847

1.98

0 2.09

4 2.

244

2.36

3 2.

549

2.78

6 2.

937

3.04

3 3.

270 3.

647

3.79

4 4.

056

4.23

0 4.

342

4.48

7

4.78

4 4.

867

5.00

1 5.

085

5.19

9

5.51

0 5.68

7 5.

885

6.06

7 6.

171

6.34

1 6.

469

6.61

4 6.

731

6.93

7 7.

148

7.42

1 7.

643 7.80

8 8.

015

8.17

7 8.25

5 8.

399

8.61

4 8.

719 8.

935

9.16

2 9.

286

9.41

4 9.

566

9.75

8 9.

829

9.95

4 10

.114

10.18

4 10

.324

10.54

7 10

.650

10.71

5 10

.957

11.06

0 11

.260

11.35

5 11

.477

11.58

3 11

.670

11.77

9 11

.928

12.27

3 12

.361

12.57

1 12

.690

12.83

7 12

.975

13.48

0 13.57

5

13.90

2 14

.122

14.40

6 14

.497

14.61

8 14

.855

m/z0 200 400 600 800 1000 1200 1400

0

20

40

60

80

100

*MSD2 SPC, time=6.647:10.973 of M:\NHB_5342_LCMS\01-16\KOLESNICHENKO\280116-Z12MIN_LC-IVK_3_002_PBS_MECN_MS31.D MM-E

Max: 31323

164

.0 190

.1

422

.1

292

.1

421

.1

162

.1




c. Reaction of 1, OctSH, and benzylamine – LC/MS studies

1, 1-octanethiol (OctSH), and benzylamine were reacted in the above manner and the reaction was monitored by LC/MS:

Supplementary Figure 15. LC/MS trace (neg. mode) of 1-octanethiol combined simultaneously with benzylamine and 1 after 1 h @ RT.

Supplementary Figure 16. LC/MS analysis (neg. mode) of 1-octanethiol combined simultaneously with benzylamine and 1 after 1 h @ RT. The above LC/MS trace was scanned

min2 4 6 8 10 12 14

200000

400000

600000

800000

1000000

1200000

1400000

MSD2 TIC, MS File (M:\NHB_5342_LCMS\12-15\KOLESNICHENKO\141215-LC12MIN_M-IVK_2_183_PBS_MECN_MS11.D) MM-ES+APCI, Neg

0.13

2 0.

297

0.47

8 0.

627

0.69

7 0.

805

1.01

1 1.

135

1.20

6 1.

450

1.62

9 1.

695

1.78

4 1.

836

1.89

7 2.

074

2.15

3 2.

249

2.43

9 2.

526

2.63

2 2.

778

2.90

8 3.

033

3.25

2 3.

333

3.48

6 3.67

1 3.

805

3.88

9 3.

959

4.19

8 4.

360

4.46

8 4.

582

4.68

6 4.

833

5.05

0 5.

311

5.41

9 5.

491

5.58

8 5.

774

5.94

9 6.

099

6.19

4 6.

262

6.41

5 6.

606

6.89

1

7.20

7 7.

425

7.49

6 7.

605

7.69

0 7.88

5 7.

994

8.21

5 8.

327

8.49

7 8.

724

8.98

3 9.

131

9.25

1 9.

396

9.49

7 9.

607 9.83

7 9.

990

10.12

8 10

.221

10.40

9 10

.526

10.58

6 10

.680

10.99

7

11.31

0 11

.538

11.68

4 11

.811

11.93

7

12.27

7 12

.358

12.52

1 12

.631

12.82

2 12

.983

13.23

4 13.36

1 13

.600

13.82

2 13

.955

14.14

5 14

.257

14.51

3 14

.634

14.74

0 14

.881

14.96

1

m/z0 200 400 600 800 1000 1200 1400

0

20

40

60

80

100

*MSD2 SPC, time=6.667:11.276 of M:\NHB_5342_LCMS\12-15\KOLESNICHENKO\141215-LC12MIN_M-IVK_2_183_PBS_MECN_MS11.D MM-E

Max: 3760

66.

1

409

.0

635

.0

365

.1

193

.1

263

.1 157

.1 1

90.1 258

.1

204

.1

831

.2

302

.1

131

.0

306

.1

156

.1

405

.2 4

04.2




over the range of 6.5-11.3 mins. After 1 h the predominant species in the crude reaction mixture is already the target compound shown above. There is also the amine-1 adduct (306.1/635.0, 2M-H-H+Na) and a small amount of the di adduct of amine to 1 (365.1).

d. Reaction of 1, OctSH, and propargylamine – LC/MS studies

1, OctSH, and propargylamine were reacted in the above manner and the reaction was monitored by LC/MS:

Supplementary Figure 17. LC/MS trace (neg. mode) of 1-octanethiol combined simultaneously with propargylamine and 1 after 36 h @ RT.

min2 4 6 8 10 12 14

200000

400000

600000

800000

1000000

1200000

1400000

1600000


0.29

0

0.64

0 0.

753

0.85

1 0.

935

1.06

7 1.

155

1.35

1 1.

466

1.55

9 1.78

9 1.

919

2.00

7 2.

140

2.25

1 2.

347

2.55

8 2.

657

2.77

9 3.

011

3.11

3 3.

211

3.34

7 3.

521

4.16

1 4.

253 4.

403

4.71

6 4.

801

4.90

0 4.

985 5.18

3 5.

331 5.

495 5.65

7 5.

739

6.01

1

6.30

6 6.

452

6.64

5 6.

765

6.85

0 7.

060

7.13

9 7.

211

7.27

9 7.

365

7.46

3 7.

543

7.61

9 7.

869

7.98

0

8.27

4 8.

373

8.44

9 8.

606

8.68

7 8.

805

9.07

2 9.

255

9.34

4 9.

488

9.68

6

10.01

4

10.32

6

10.62

4 10

.761

11.08

9 11

.234

11.36

5 11.56

2 11

.713

11.94

7 12

.212

12.41

0 12

.641

12.98

6 13

.226

13.33

2 13

.414

13.58

6

14.10

7 14

.184

14.29

5 14

.502

14.76

7 14

.874




Supplementary Figure 18. LC/MS analysis (neg. mode) of 1-octanethiol combined simultaneously with propargylamine and 1 after 36 h @ RT. The LC/MS trace shown above was scanned over the range of 3.5-12.2 mins. There still some amine-1 adduct present (254.1), but the target compound is most abundant, along with a fragment of it excluding the anionic 1-octanethiol (206.1).

e. Reaction of 1, OctSH, and 1-amino-3-butyne – LC/MS studies

1, OctSH, and 1-amino-3-butyne were reacted in the above manner and the reaction was monitored by LC/MS:

m/z0 200 400 600 800 1000 1200 1400

0

20

40

60

80

100


Max: 3059

328

.9

486

.9 727

.2

145

.1

190

.0 546

.0

168

.1

66.

2

409

.1 354

.1 3

53.2

254

.1

104

.1 339

.2

206

.1

352

.2




Supplementary Figure 19. LC/MS trace (neg. mode) of 1-octanethiol combined simultaneously with 1-amino-3-butyne and 1 after 1 h @ RT.

Supplementary Figure 20. LC/MS analysis (neg. mode) of 1-octanethiol combined simultaneously with 1-amino-3-butyne and 1 after 1 h @ RT. Scanning the LC/MS trace above over the range of 4.3-10.6 mins showed the predominance of the target compound after only 1 h of reaction time. Also present were the amine-1 adduct (268.1) and unreacted 1-octanethiol (145.2).

min2 4 6 8 10 12 14

200000

400000

600000

800000

1000000

1200000

1400000


0.20

5 0.

301

0.37

9 0.

482

0.53

0 0.

762

0.85

0 0.

996

1.11

7 1.

304 1.54

4 1.

678

1.76

0 1.

897

2.04

0 2.

241

2.41

5 2.

569

2.64

4 2.

761

2.92

2 3.

018 3.16

4 3.

384

3.49

4 3.

582

3.66

3 3.

820

3.96

2 4.

081

4.22

8 4.

302

4.71

6 4.81

3 4.

916

5.12

2 5.

236

5.32

8

5.70

5 5.

867

5.96

3 6.

029

6.13

4 6.

255

6.43

2 6.

638

6.80

5 6.

910

7.10

4 7.

210

7.28

4 7.

369

7.51

6 7.

674

7.88

9 8.

152

8.45

0 8.

530

8.58

9 8.

770

8.87

7 8.

971

9.20

7 9.

276

9.41

7 9.57

5 9.

668

9.77

4

10.16

6

10.60

4 10

.714

10.81

2 10

.962

11.13

1 11

.339

11.47

7 11

.580

11.70

1 11

.795 11.95

5 12

.141

12.28

5 12

.447

12.64

1

12.97

8 13

.200

13.34

0 13.58

6 13

.687

13.84

8 13

.954

14.07

3 14

.177

14.30

7 14

.400

14.58

1 14

.702

14.82

9

m/z0 200 400 600 800 1000 1200 1400

0

20

40

60

80

100


Max: 2412

135

.1

755

.2

409

.0

368

.1

233

.0

269

.0

167

.0

83.

2

264

.1

145

.2

268

.1

66.

1

331

.1

187

.1

367

.1 118

.2

166

.1

366

.2




f. Reaction of 1, PhSH, and propargylamine – LC/MS studies

1, thiophenol (PhSH), and propargylamine were reacted in the above manner and the reaction was monitored by LC/MS:

Supplementary Figure 21. LC/MS trace (neg. mode) of thiophenol combined simultaneously with propargylamine and 1 after 1 h @ RT.

Supplementary Figure 22. LC/MS analysis (neg. mode) of thiophenol combined simultaneously with propargylamine and 1 after 1 h @ RT. Scanning the above LC/MS trace over the range of

min2 4 6 8 10 12 14

200000

400000

600000

800000

1000000

1200000

1400000

1600000


0.26

8 0.

398

0.55

5 0.

627

0.74

4 0.

868

0.98

6 1.

157

1.37

6 1.

501

1.70

6 1.

790

1.91

5 2.

013

2.15

5 2.

258

2.40

4 2.

498

2.56

0 2.

707

2.83

4 2.

927

2.99

2 3.

082

3.36

8 3.

441

3.54

5 3.

721

4.19

8 4.28

8 4.

359 4.42

3

4.75

0 4.

913

5.02

5 5.

147

5.23

3 5.

324

5.48

7 5.

577

5.64

7 5.

721

6.02

2 6.

197

6.24

4 6.

359

6.78

8 7.

034

7.45

1 7.

527 7.71

5 7.

879

8.03

9 8.

315 8.

544

8.65

6 8.

946

9.10

3

9.59

6 9.

833

10.01

3 10

.189

10.44

1 10

.568

10.71

7 10

.837 11

.094

11.25

9 11

.432 11.58

7 11

.835

11.90

7 11

.984

12.08

1 12

.182

12.35

3 12

.477

12.61

0 12

.761

12.98

1

13.38

1 13

.486

13.68

3

14.04

5 14

.243

14.37

2 14

.459

14.58

7 14

.780

14.87

3

m/z0 200 400 600 800 1000 1200 1400

0

20

40

60

80

100

*MSD2 SPC, time=6.564:7.011 of M:\NHB_5342_LCMS\01-16\KOLESNICHENKO\120116-LC12MIN_M-IVK_2_190_PBS_MECN_MS11.D MM-ES

Max: 28074

656.0

66.2

104.1

110.1

168.1

109.1

448.0 316.0

207.1 65

5.0

206.1




6.5-7.1 mins showed the desired product, along with its fragments. The thiophenolate fragments off (109.1), leaving the fragment of the product still containing the propargylamine (206.1).

6. Measuring pKa of NH in 2

A 4 mM solution of 2, with R=Bn (Meldrum’s acid conjugate acceptor – benzylamine adduct), was prepared in 2:1 H2O/MeOH. Separately, a 50 mM sodium hydroxide solution was prepared in 2:1 H2O/MeOH. A pH reading of the 50 mM NaOH solution alone was taken, giving a value of 12.93. 4 mL of the analyte solution were then titrated with the 50 mM NaOH solution, starting at a pH of 6.36 (the pH of the 4 mM solution alone) and going until the endpoint.

Supplementary Figure 23. Determining pKa of Meldrum’s acid conjugate acceptor – benzylamine adduct: 8.95

6

7

8

9

10

11

12

0 100 200 300 400 500 600 700 800

pH

uL of 50 mM NaOH Added

Titra6on of Conjugate Acceptor -‐ Benzylamine Adduct




200 220 240 260 280 300 320 340 360 380 4000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

A

nm

pH 7 .07 pH 7 .83 pH 8 .02 pH 8 .39 pH 8 .56 pH 8 .70 pH 9 .06 pH 9 .42 pH 10.02 pH 10.46 pH 10.96 pH 11.48 pH 11.99

Supplementary Figure 24. pH titration of 1 (absorbance)

7. Testing Decoupling Agents

a. Screening by 1H-NMR

A solution of AMBA-1-NAC (60 mM) was split into 0.6 mL portions, and to each portion was added one of the nucleophiles as described in Table S1. The pH of the solutions was adjusted to pH 7 with NaOH/DCl. The reactions were stirred for 24 h at room temperature. The DTT, cysteine, and EDT reactions had completed. The other reactions were continued for 48 more hours. Precipitate formed in the EDT, PDT, and cysteamine reactions. The solid in the cysteamine and PDT reaction dissolved somewhat upon addition of more CD3CN. The solid in the EDT reaction was taken up in CDCl3 to obtain a 1H-NMR spectrum.

Supplementary Table 1. Reaction set up for decoupling agents

Reagent mg µL µmol molar equivalents

dithiothreitol (DTT)

5.6 --- 36 1

2-mercapoethanol (BME)

--- 2.5 36 1




cysteine HCl

5.7 --- 36 1

ethanedithiol (EDT)

--- 3.0 36 1

propanedithiol (PDT)

--- 3.6 36 1

thioglycolic acid (TGA)

--- 2.5 36 1

cysteamine HCl

4.1 --- 36 1

ethylenediamine (EDA)

--- 2.5 36 1




Supplementary Figure 25. Stacked spectra of decoupling reactions for comparison. All of the reagents released NAC, but only DTT, cysteine, cysteamine, EDT, and PDT released AMBA.




Supplementary Figure 26. 1H-NMR spectrum of DTT reaction. The DTT cleanly released NAC and AMBA.




Supplementary Figure 27. 1H-NMR spectrum of cysteine reaction. The cysteine cleanly released NAC and AMBA.




Supplementary Figure 28. 1H-NMR spectrum of BME reaction. The BME released NAC, but did not fully decouple AMBA. As evidenced by the numerous peaks, many products were formed, and the reaction did not proceed cleanly.




Supplementary Figure 29. 1H-NMR spectrum of TGA reaction. The TGA released NAC, but did not fully decouple AMBA. As evidenced by the numerous peaks, many products were formed, and the reaction did not proceed cleanly.




Supplementary Figure 30. 1H-NMR spectrum of cysteamine reaction. The cysteamine released AMBA and NAC, however the AMBA was not completely decoupled, and several products were formed.




Supplementary Figure 31. 1H-NMR spectrum of EDT reaction. The EDT cleanly released NAC and AMBA.




Supplementary Figure 32. 1H-NMR spectrum of EDT adduct with 1 in CHCl3.




Supplementary Figure 33. 1H-NMR spectrum of PDT reaction. The PDT released NAC, although the AMBA was not fully decoupled, and the PDT-1 adduct was only slightly soluble in D2O.




Supplementary Figure 34. 1H-NMR spectrum of EDA reaction. The EDA reaction did not release an appreciable amount of AMBA or NAC (see Figure S2 for a comparison).

b. Reaction of DTT with Lys-1-NAC at room temperature- LC-MS

To Lys-1-NAC (40 µmol) in 2 mL of 0.1 M phosphate buffer, 0.15 M NaCl, pH 7.4 was added DTT (8.9 mg, 58 µmol). The reaction was stirred at room temperature and monitored at 1, 10, and 46 h by LC-MS.




Supplementary Figure 35. LC-MS analysis (neg. mode) of Lys-1-NAC + DTT reaction at RT after indicated times.

c. Reaction of DTT with Lys-1-NAC at 45 °C - LC-MS

To Lys-1-NAC (40 µmol) in 2 mL of 0.1 M phosphate buffer, 0.15 M NaCl, pH 7.4 was added DTT (8.9 mg, 58 µmol). The reaction was stirred at 45 °C and monitored at 1, 3, and 10 h by LC-MS.




Supplementary Figure 36. LCMS analysis (neg. mode) of Lys-1-NAC + DTT reaction at 45 ºC after indicated times.

d. Reaction of DTT with Lys-1-Lys at 45 °C - LC-MS

DTT (42 mg, 272 µmol, 11.6 eq) was added to the Lys-1-Lys reaction described above, and the heating was continued (45 ºC) for 24 h. LC-MS analysis indicated that the Lys-1 in the solution reacted with DTT, but the Lys-1-Lys did not react with DTT.




Supplementary Figure 37. LC-MS analysis (neg. mode) of Lys-1-Lys + DTT reaction at 45 ºC after indicated times.

8. Triamine and Resin

a. Reacting triamine with 1 (6)

The 1,3,5-triaminomethylbenzene (2.7 mg, 16 µmol) was dissolved in 1.33 mL of 0.1 M phosphate buffer, 0.15 NaCl, pH 7.2. 1 (12.3 mg, 49 µmol) was dissolved in 0.67 mL MeCN. The solutions were combined and stirred at room temperature for 24 h. Some product precipitated, so MeCN was added to aid in dissolution.

b. Adding thiols to 6 (7, 8, 9, and 10)

NAC (16 mg, 100 µmol) was added to 6. The solution was sparged with N2 for 48 h. The reaction had mostly completed: LRMS (ESI) 1109.0 (M-1). Then, 3-mercaptopropionic acid (MPA, 8.7 µL, 100 µmol) was added, and the reaction was monitored by LC-MS for ten days: LRMS (ESI) 938.0 (M-1), 994.8 (M-1), 1051.8 (M-1), 1108.8 (M-1). After 8 days, no further changes were observed in the spectra, indicating that the reaction equilibrated fully in about a week.

Supplementary Figure 38. LC-MS analysis (neg. mode) of 6.




Supplementary Figure 39. MS analysis (neg. mode) of the thiol exchange reaction over several days. The species were not resolvable on the LC, so the extracted ion chromatogram of the single peak was used.

c. Adding DTT to functionalized triamine mixture

The 1,3,5-triaminomethylbenzene (6.72 mg, 41 µmol) was dissolved in 3 mL 0.1 M phosphate buffer, 0.15 NaCl, pH 7.2. 1 (30.1 mg, 121 µmol) was dissolved in 2 mL MeCN. The solutions were combined and stirred for 24 h at room temperature. Then, N-acetylcysteine (40 mg, 245 µmol) was added. The solution was sparged with N2, and the reaction was stirred for 36 h. MeCN was added as the solvent evaporated. The formation of 7 was confirmed by LC-MS. Then, DTT (63 mg, 408 µmol) was added, and the reaction was stirred for 48 h.




Supplementary Figure 40. LCMS analysis (neg. mode) of decoupling reaction of DTT and 7 showing conversion to 5. Note that the NAC elutes with the buffer salts in less than 1 min, and the 1,3,5-triaminomethylbenzene ion did appear in the MS analysis of any of the reactions.

d. Synthesis of 11

Tentagel S Br (0.26 mmol/g, 107 mg, 27.8 µmol, 1 eq) was placed in 5 mL of 1:1 water/MeCN and stirred for 15 min to swell the resin. The 1,3,5-triaminomethylbenzene (14.1 mg, 85.5 µmol, 3.1 eq), proton sponge (18.5 mg, 86.3 µmol, 3.1 eq), and NaI (16 mg, 107 µmol, 3.8 eq) were added to the solution. The reaction was heated to 80 °C for 20 h. The beads were washed with water and methanol and subjected to a Kaiser test. The test was positive for amine. The resin was dried thoroughly under vacuum.

e. Synthesis of 12

Tentagel-triamine (107 mg, 27.8 µmol, 0.52 mmol amine/g) was placed in 3 mL of 0.1 M phosphate buffer, 0.15 M NaCl, H2O, pH 7. 1 (28 mg, 113 µmol, 2 eq) was dissolved in 2 mL MeCN and added to the resin. The reaction was gently sparged with nitrogen for 24 h. Then the resin was washed with water and methanol and subjected to a Kaiser test (negative for amine).

f. Removal of 1 from resin by DTT

12 (11.67 mg, 0.52 mmol EvaCA/g) was placed in 1 mL of 0.1 M phosphate buffer, 0.15 M NaCl, H2O, pH 7.2, and NAC (2 mg, 12.14 µmol) was added. The reaction was gently sparged with nitrogen for 2.5 days. The resin was washed with water and methanol. Then, a solution of DTT (6.15 mg, 40 µmol) was added to the resin. The reaction was stirred for 36 h. Then the solution was collected and analyzed by LC-MS. The resin was washed and subjected to a Kaiser test (positive for amine).




Supplementary Figure 41. LC-MS analysis (neg. mode) of decoupling reaction of DTT and NAC-functionalized 12.

g. Thiol library on the resin

The thiols in the table below were placed in 1 mL MeCN and 1 mL of 0.1 M phosphate buffer, 0.15 M NaCl, H2O, pH 7.4 and then added to 12 (32 mg, 17 µmol) . Another 2 mL MeCN and 2 mL buffer were added to the reaction. The reaction was stirred for 3 days. Then, the resin was washed thoroughly with water and methanol. DTT (27.4 mg, 178 µmol) in 3 mL MeCN and 3 mL buffer was added to the resin. The reaction was stirred for 18 h. This reaction solution was analyzed by LCMS (Figure S31).

Supplementary Table 2. Thiol library setup

Thiol mg µL µmol 1-phenyethylmercaptan (PEM)

---- 7.0 52

MEEEE ---- 10.0 52 1,4-bis(mercaptomethyl)benzene (BMMB)

9.6 ---- 52

4-methoxy-α-toluenethiol (MTT)

---- 7.2 52

3-(dimethoxymethylsilyl)-1-propanethiol (SPT)

---- 9.4 52




Supplementary Figure 42. LC-MS analysis of the thiol library before exposure to 12 (top) and of the DTT reaction with the resin (below). The left side shows the LC trace for λ = 214 nm, and the right side shows the TIC in positive mode. Note that 5 was visible in negative mode (not shown) after DTT treatment of the resin. Note that SPT and MTT elute at precisely the same time. Since MTT, BMMB, and PEM are aromatic, their extinction coefficients at 214 nm were much higher than for SPT and MEEEE.

9. Peptide Labeling

a. Peptide synthesis The peptides IAHRFKDLGE and VVKLKC were synthesized on an automated peptide

synthesizer (Protein Technologies) using Fmoc chemistry and Wang resin. For a 200 µmol-scale reaction, the Wang resin was swelled in DMF and then deprotected with 20% piperidine in DMF twice. In between the resin was washed with DMF and DCM. Each amino acid was dissolved in DMF (100 mM) and introduced to the resin after the deprotection step. PyBOP (300 mM) and DIPEA (1.2 M) in DMF were added along with the amino acid to perform each coupling (twice). The final residue was deprotected at the end. The resin was then removed from the synthesizer.

Before IAHRFKDLGE was cleaved from the resin, the N-terminus was acetylated by placing the resin in 5 mL of 50% acetic anhydride in DMF and 100 µL pyridine. The reaction was allowed to proceed for 3 h with shaking to mix. The resins were washed with DMF, DCM, MeOH, and glacial acetic acid and then dried overnight on the high vacuum line. The peptides were cleaved from the resin with 95% TFA, 2.5% TIPS, and 1% H2O for 4 h. The TFA solution was filtered from the resin, and the TFA was removed. To the residue was added diethyl ether to precipitate the peptides.

The crude acIAHRFKDLGE (100 µmol) was dissolved in 3 mL acetonitrile and 2 mL water. DIPEA (120 µL, 600 µmol, 6 eq) and 1 (50 mg, 200 µL, 2 eq) were added, and the reaction was stirred in an open vial for 24h. The presence of the product was confirmed by MS (FIA, ESI-Neg). The solvent was removed under reduced pressure.




The crude peptides were dissolved in water/DMSO (no more than 50% DMSO) and

purified by HPLC. Fractions containing the desired peptide were verified by LC-MS, combined, and evaporated. The peptides acIAHRFK(1)DLGE and acAHRFK(1)DLGE were not able to be resolved completely, so the mixture was used for labeling reactions.

Supplementary Scheme 1. Structures of the 1-containing peptides.

Supplementary Figure 43. MS analysis of the peptides (neg. mode)

Characterization:




acIAHRFK(1)DLGE and acAHRFK(1)DLGE: 34.8 mg (25% yield); LRMS (ESI): 657.6, 714.1 ((M+1)/2), 1313.8, 1426.8 (M+1); HRMS (ESI): expected 1314.58, found 1314.58, expected 1427.66, found 1427.66 VVKLKC: 59.3 mg (58% yield). LRMS (ESI): 689.4 (M+1); HRMS (ESI): expected 689.44, found 689.44 NOTE:

For the following modified peptide and protein preparations, we used mass spectrometric analysis of digested and intact samples as described. This type of analysis simply does not provide very rigorous quantification of the number of modifications to a protein. In general, exhaustively characterizing a protein that has been chemically modified is quite challenging.

For the peptide crosslinking to VVKLKC and MEEEE, some idea of the extent of modification can be gleaned from the ion abundances of the starting materials and products. For the crosslinking to MEEEE, direct infusion analysis of the reaction mixture showed only the excess MEEEE and the crosslinked product (Figure S45). No starting peptide (acIAHRFK(1)DLGE, acAHRFK(1)DLGE) was observed, which indicates that the reaction proceeded to a significant extent. The same was true for crosslinking of the two peptides (Figure S44). For the decoupling reaction of the crosslinked peptide, only the free thiol-containing peptide (VVKLKC) and the model peptide (acAHRFKDLGE, acIAHRFKDLGE) were observed, again indicating that the reaction was complete to the extent that starting material could not be detected using our characterization method.

As far as the crosslinking to myoglobin, it was difficult to obtain quantitative information from the analysis of the digested peptide because of the complicated nature of the data that requires an algorithm to even identify modification sites qualitatively. However, from the direct infusion MS analysis of the crosslinked myoglobin sample compared before and after DTT treatment, it is clear that the myoglobin was modified at many lysines based on the large mass shift from native myoglobin. Since the crosslinked myoglobin sample is not homogenous, what we observe is a mixture of species centered around 20,500 m/z, which comes out to around 10 crosslinked MEEEE per protein. While it isn’t possible to know precisely how many modifications are actually crosslinked MEEEE or just 1 on each protein in the sample, it is clear that the myoglobin in the sample was on average modified at more than a few sites and that the population of proteins, while heterogeneous, is concentrated in the 20,000 to 21,000 m/z range, which is significantly higher than the 17,000 m/z or unaltered myoglobin and comes out to a difference of about three modifications from the low to the high end of the mass range. For the DTT reaction, this population of myoglobin around 20,500 m/z is completely gone, and we observe a peak for native myoglobin. There are additional peaks from 17,000 to 17,500 m/z, and we would hypothesize that these could be attributed to 1-2 sites that were modified with 1 and were then hydrolyzed as we describe. Such a modification would not be removed by DTT.

b. Reaction of acIAHRFK(1)DLGE with VVKLKC The peptides acIAHRFK(1)DLGE/acAHRFK(1)DLGE (1.9 mg, 1.15 µmol, 1 eq) and VVKLKC (2.7 mg, 2.62 µmol, 2.3 eq) were dissolved in 10 mM carbonate buffer, pH 9.2. The




buffer capacity was exceeded, so 1 M NaOH (aq) was added to adjust the pH to 7.5. The total reaction volume at the end of the adjustment was 1 mL. The reaction was allowed to stir at room temperature for one week in an open Eppendorf tube. The reaction was periodically replenished with H2O as it evaporated. After this time, the solution was filtered and analyzed by LC-MS. The desired products were present. LRMS (ESI): 1034.7 ((M+1)/2), 978.2 ((M+1)/2), 690.3 ((M+1)/3), 652.6 ((M+1)/3), 518.0 ((M+1)/4), 489.7 ((M+1)/4).

Supplementary Figure 44. MS analysis (pos. mode) of acIAHRFK(1)DLGE and acAHRFK(1)DLGE crosslinked to VVKLKC

c. Reaction of acIAHRFK(1)DLGE with MEEEE

The peptides acIAHRFK(1)DLGE/acAHRFK(1)DLGE (1.29 mg, 0.78 µmol, 1 eq) were dissolved in 250 µL of water. The pH of the solution was adjusted to 7 with 10 mM NaOH (aq), resulting in a total volume of 1 mL. MEEEE (2 µL, 10.4 µM, 13.3 eq) was added to the solution, and the reaction was stirred for 20 h in an open vial. LRMS (ESI): 795.4 ((M+1)/2), 738.9 ((M+1)/2).




Supplementary Figure 45. MS analysis (pos. mode) of acIAHRFK(1)DLGE and acAHRFK(1)DLGE crosslinked to MEEEE

d. Mass spectrometry analysis of modified peptides

The 1-modified peptide samples were first diluted to a concentration of 1 µM in 49/50/1 (water/acetonitrile/ formic acid) solution and then analyzed via direct infusion electrospray ionization on a Thermo Velos Pro dual linear ion trap mass spectrometer (Thermo Scientific; Waltham, MA, USA). Collision induced dissociation (CID) was used to characterize the peptides, and the normalized collision energy was set to an NCE value of 35. For each modified sample two stages of MS/MS analysis were performed as CID of each peptide resulted in a predominant neutral loss of 102 Da corresponding to elimination of part of the 1 modification as shown in Figure S32. The product of this neutral loss was then isolated and subjected to a second stage of CID, resulting in formation of diagnostic b and y ions that allowed the peptide to be sequenced.




Supplementary Figure 46. A) MS3 spectrum obtained for doubly charged Ac-IAHRFKDLGE crosslinked to VVKLKC through an 1 modification (1035 → 984 → ); B) list of m/z values and assignments of possible crosslinked fragments. Cells highlighted in yellow indicate the fragment ions which are observed in the spectrum from part A.




Supplementary Figure 47. CID of doubly protonated 1-modified peptide (2+) results in the dominant neutral loss of 102 Da. The peptide is acetyl-AHRFKDLGE modified with both 1 and MEEEE. The precursor ion is marked with an asterisk.

e. Decoupling of acIAHRFKDLGE and VVKLKC by DTT

To the solution of acIAHRFK(1)DLGE(1.15 µmol, 1 eq) was added DTT (1 mg, 6.5 µmol, 5.7 eq). The reaction was stirred at room temperature in an open container for three days. MS analysis indicated that the reaction had occurred. LRMS (ESI): VVKLKC- 230.7 ((M+1)/3), 345.2 ((M+1)/2), 689.4 (M+1); acAHRFKDLGE- 372.3 ((M+1)/3), acIAHRFKDLGE- 410.0 ((M+1)/3).




Supplementary Figure 48. MS analysis (pos. mode) of crosslinked peptide decoupling reaction

f. Crosslinking of myogloblin and MEEEE with 1

Equine skeletal muscle myoglobin (14 mg, 0.82 µmol, 1 eq) was dissolved in 4 mL of 0.1 M phosphate buffer, 0.15 M NaCl, H2O, pH 7.2. 1 (8.2 mg, 33 µmol, 40 eq) was dissolved in 0.5 mL acetonitrile and was added to the myoglobin solution. The reaction was stirred in an open container for 24 h. The protein was filtered and purified against 0.1 M phosphate buffer, 0.15 M NaCl, H2O, pH 7.2 with 10 kDa MWCO centrifugal filter units. Then the MEEEE (16 µL, 84 µmol, 102 eq) was added, and the reaction was stirred for another 24 h. The protein was filtered and purified against 0.1 M phosphate buffer, 0.15 M NaCl, H2O, pH 7.2 with 10 kDa MWCO centrifugal filter units. A sample was taken of MyoEvaMEEEE and submitted for LC-ESI-MS3 analysis. g. Mass spectrometry analysis of modified myoglobin

After modification of myoglobin with 1 and MEEEE, 50 µg of the sample were digested using GluC (Promega). GluC was added at a 10:1 ratio of protein to enzyme and incubated at 37 °C for 24 hours. After digestion the sample was cleaned up using a C18 SPE cartridge and then dried using a Savant DNA120 Speedvac concentrator (Thermo Electron, Waltham, MA, USA). Once dried the modified digests were reconstituted in appropriate LC solvents. HPLC separation was carried out using a Dionex NSLC 3000 nanoLC system (Thermo Scientific; Waltham, MA, USA) which was interfaced to the Velos Pro mass spectrometer. The LC gradient consisted of mobile phase A: water with 0.1% formic acid and mobile phase B: acetonitrile with 0.1% formic acid. The loading solvent used was 98% water/2% acetonitrile with 0.1% formic acid. For each run 1 µL of the sample was injected, loaded on to a trap column prior to separation, and then eluted from the analytical column. The column was packed in-house using a New Objective PicoFrit analytical column (Woburn, MA) (75 µm x 15 cm). 3 µm Michrom Magic C18 was used to pack the column. The gradient started at 3% B and increased to 40% B over 60 min at a flow rate of 300 nL/min. For all runs source fragmentation was applied (35 V) to promote the loss of 102 Da, prior to isolation and activation of the resulting peptides for sequencing by CID. CID was again performed using a NCE of 35.




SEQUEST and Percolator from Proteome Discoverer v. 1.4 (ThermoFisher Scientific, San Jose, CA) were used for the analysis of all LCMS data for the myoglobin digests. Three custom modifications were added into the search parameters: 1 (+97.990 Da), 1-OH (+68.00 Da), and 1-MEEEE (+260.080 Da), all for which the neutral loss of 102 Da is already accounted. Each dataset was searched with the inclusion of all three modifications in order to determine the sites modified by solely 1 versus those also modified by the addition of MEEEE. All results were searched against the Uniprot myoglobin horse heart FASTA. h. Decoupling of myoglobin and MEEEE by DTT

To the remainder was added DTT (12.8 mg, 83 µmol, 101 eq), and the reaction was stirred for 36 h. The protein was filtered and purified against 0.1 M phosphate buffer, 0.15 M NaCl, H2O, pH 7.2 with 10 kDa MWCO centrifugal filter units. The protein sample was submitted for intact ESI-MS (Figure S35), while the flow-through was collected and analyzed by LC-MS. MEEEE and 5 were observed (Figure S37).

Supplementary Figure 49. Deconvoluted mass spectra for analysis of intact myoglobin A) modified with 1 and MEEEE B) modified with 1 and MEEEE and exposed to DTT. The large shift observed from the exposer to DTT indicates that 1 modification removed.




Supplementary Figure 50. LC-MS analysis of flow-through from decoupling reaction of myo-1-MEEEE and DTT to yield free MEEEE and 5 (top: pos. mode, bottom: neg. mode).

10. Oligomers

a. Synthesis of 13

82.37 mg (604.82 µmol, 1 equivalent) of p-xylylenediamine (XDA) were suspended in a 2:1 buffer/acetonitrile mixture. 301.5 mg (1.214 µmol, 2 equivalents) of 1 were added and the solution was allowed to stir overnight at room temperature. The resulting off white precipitate was then collected via suction filtration and dried overnight in a vacuum dessicator, yielding 13, as confirmed by 1H-NMR (400 MHz, CDCl3):




Supplementary Figure 51. 1-XDA-1 (compound 13) spectrum taken in CDCl3.

b. Synthesis of oligomers

To a solution of 101.6 mg (189.34 µmol) of 13 in 750 µL of chloroform were added sequentially 30.4 µL (186.77 µmol) of 2,2’-(ethylenedioxy)diethanethiol (EDDT) and 32.5 µL (186.58 µmol) of Hünig’s base. The vial was sealed and heated at 50 °C for 24 h. This solution was then added to about 50 mL methanol, precipitating a white solid, which was centrifuged/collected. 1H-NMR (400 MHz, CDCl3) and HRMS were then used to confirm oligomer formation:

NH H

N S

S

OO

O O

OO

OO

A

B

D

EE

EE

D

A

B

C

C




Supplementary Figure 52. Oligomer spectrum taken in CDCl3.

Supplementary Figure 53. HRMS of CYCLIC 1-XDA-1-EDDT (structure shown above)

HNS

OO

O

ONHS

OO

O

O

OO

Chemical Formula: C28H34N2O10S2Exact Mass: 622.17




Supplementary Figure 54. HRMS of LINEAR 1-XDA-1-EDDT (structure shown above). NOTE: Further structures will not be shown, due to size constraints. Instead, they will simply be abbreviated as (1)n(XDA)n(EDDT)n

Supplementary Figure 55. HRMS of LINEAR (1)4(XDA)2(EDDT)1

HNS

O O

O O

NH

S

OO

O O

OO

SH





Supplementary Figure 56. HRMS of CYCLIC (1)4(XDA)2(EDDT)2 (structure shown above)


HNS

OO

O

ONHS

OO

O

O

O O

O

S

O

SNH HN

OO O

O

O

OO

O






Supplementary Figure 59. HRMS of CYCLIC (1)6(XDA)3(EDDT)3




Supplementary Figure 62. HRMS of CYCLIC (1)4(XDA)4(EDDT)4






c. Reaction of oligomers with DTT

5.7 mg of the solid oligomers were dissolved in 0.5 mL of chloroform and 1.2 mg (7.779 µmol, an arbitrary amount because no equivalents of oligomers were known) of DTT were added. The reaction was stirred at room temperature and monitored by LC/MS (a drop of the reaction mixture was taken and diluted with excess acetonitrile for these experiments). After 12 h of reaction time the extracted chromatogram at 254 nm (below) already showed an abundant presence of declicked products.




Supplementary Figure 71. UV trace at 254 nm of the DTT reaction with the oligomers in chloroform after 12 h @ RT.

Supplementary Figure 72. LC/MS analysis (neg. mode) of the DTT reaction with the oligomers in chloroform after 12 h @ RT. The LC/MS trace corresponding to the above UV trace at 254 nm was scanned over the range of 8.7-11.5 mins.

min2 4 6 8 10 12 14

mAU

0

5

10

15

20

DAD1 A, Sig=254,4 Ref=360,100 (M:\NHB_534...-16\KOLESNICHENKO\180116-LC12MIN_MULTI-IVK_2_191_MECN_MS21.D)

0.27

6

2.57

5

3.83

5

5.38

6

5.92

7 6.

172

6.50

3

7.04

8 7.

225

7.43

1

8.00

9 8.

116

8.38

1

8.94

0

9.42

9

9.77

1

10.17

3

m/z0 200 400 600 800 1000 1200 1400

0

20

40

60

80

100

*MSD2 SPC, time=8.751:11.479 of M:\NHB_5342_LCMS\01-16\KOLESNICHENKO\180116-LC12MIN_MULTI-IVK_2_191_MECN_MS21.D MM-E

Max: 20078

327.0

958.9 30

7.0

203.0

635.9 93

8.9

634.9 95

6.9 135.1

954.9

305.0

632.9


suementary inrmatin10.1038/nchem.2601... · meeee) at room temperature - lc-ms i. reaction of 1...

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