mechanistic studies of a “declick” reaction kenneth a ... · supporting information s1...

SUPPORTING INFORMATION

S1

Mechanistic Studies of a “Declick” ReactionMargaret K. Meadows,a‡ Xiaolong Sun,b‡ Igor V. Kolesnichenko,c Caroline M. Hinson,d

Kenneth A. Johnson,e,* and Eric V. Anslynd,*

a Department of Chemistry, Mercer University, 1501 Mercer University Dr., Macon, Georgia 31207, United States of America;b The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an, 710049, P.R. China;c Photovoltaics & Materials Technology Department, Sandia National Laboratories, PO Box 5800, MS 0734, Albuquerque, New Mexico 87185, United States of America;d Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States of America;e Institute for Cellular and Molecular Biology, Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, United States of America.

Email: [email protected]; [email protected]

I. General procedure ..................................................................................................2

II. pH dependence titration .........................................................................................3

III. Reaction between 2Ph-X and β-mercaptoethanol....................................................8

IV. ...........................................................................................Time kinetics for 3 and 7

9

V. Stability of 5 ..........................................................................................................10

VI. 1H NMR time kinetics.............................................................................................11

VII. Kinetic Analysis in Detail………………………………………………………………………………………12

VIII. Time kinetics for 2Ph-X with DTT ...........................................................................17

IX. Synthetic procedure ..............................................................................................20

X. NMR spectra..........................................................................................................22

XI. Derivation of Eq. 3………………………………………………………………………………………………..30

Electronic Supplementary Material (ESI) for Chemical Science.This journal is © The Royal Society of Chemistry 2019

mailto:[email protected]

mailto:[email protected]


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S3

I. General procedure

Materials

All materials used in the synthesis of compounds and related tests, were purchased from Sigma-Aldrich Chemical Co., Acros Organics, Tokyo Chemical Industry, etc. and used without further purification. The reactions were performed in standard glassware. Evaporation was done by rotatory evaporation using a standard rotovap equipped with dry ice condenser. NMR solvents were purchased from Cambridge Isotope Laboratories.

In the experiments described in this paper, the unpleasantness of the thiol 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 or in sealed cuvettes for spectra collection.

All cuvettes made by fused quartz were purchased from Starna Cells with standard screw and septum top.

Methods and instruments

Column chromatography

Column chromatography was performed using silica gel 60 (230 ± 400 mesh, 0.040 ± 0.063 mm) from Dynamic Adsorbents.

Nuclear magnetic resonance (NMR)1H and 13C NMR spectra were recorded on Varian DirectDrive or Varian INOVA 400 MHz NMR spectrometers. The NMR spectra were referenced to solvent and the spectroscopic solvents (CDCl3, CD3CN, D2O, DMSO-d6, etc.) were purchased from Cambridge Isotope Laboratories and stored over 3 Å molecular sieves.

Liquid chromatography–mass spectrometry (LC-MS)

Finnigan MAT-VSQ 700 and DSQ spectrometers were used to obtain mass spectra. HR electrospray ionization (ESI) mass spectra were recorded using either Agilent 6530 Accurate-Mass Q-TOF LC/MS or MALDI-TOF (Vogayer, PerSeptive Biosystem).

High-resolution mass spectrometry (HRMS)

High-resolution mass spec (HRMS) analysis was conducted by the University of Texas Mass Spectrometry Facility using Agilent Technologies 6530 Accurate Mass Q-TOF LC/MS system. MALDI-TOF MS analysis was performed using an AB-Sciex Voyager-DE PRO MALDI-TOF equipped with a 337 nm nitrogen laser in linear mode using CHCA as a matrix.

UV-Vis spectroscopy

The UV-Vis absorbance spectra and kinetics were obtained in Cary 100 UV-Vis spectrophotometer from Agilent Technology. The spectra were run in Cary WinUV software: Scan, Kinetics and Scanning Kinetics, respectively.


S4

II. pH dependence titration

Representative procedure for pH titrations: 2Ph-X (500 μM) was dissolved in 2:1

water:methanol and divided into two solutions. One solution was acidified with a drop of 12

M HCl and the other was basified with a drop of 7 M NaOH solution. The absorbance cuvette

was charged with the acidic solution. The pH was measured and the absorbance was then

taken. Basic solution was titrated in and absorbance spectra taken at varying pH (every 0.3-

1.0 pH units). Once the pH of the solution stopped increasing, acidic solution was titrated back

in to measure additional pH values in order to more accurately determine the pKa. (Note the

“bump” visible in the spectra at 345 nm is due to lamp switchover in the instrument.)

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00.5

11.5

22.5

33.5

44.5

pH Titration of 2Ph-H

Wavelength

Abs

orba

nce

Figure S1. pH titration of 2Ph-H. Red indicates lowest pH’s (0.65), purple indicates highest (13.52).

Calculated pKa = 6.02.


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pH Titration of 2Ph-CH3

Wavelength

Abs

orba

nce

Figure S2. pH titration of 2Ph-CH3. Red indicates lowest pH’s (2.41), purple indicates highest (12.7).


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00.20.40.60.8

11.21.41.61.8

2

pH Titration of 2Ph-OCH3

Wavelength

Abs

orba

nce

Figure S3. pH titration of 2Ph-OCH3. Red indicates lowest pH’s (0.87), purple indicates highest (11.64).



S6

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pH Titration of 2Ph-Br

Wavelength

Abs

orba

nce

Figure S4. pH titration of 2Ph-Br. Red indicates lowest pH’s (0.68), purple indicates highest (10.51).


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00.20.40.60.8

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pH Titration of 2Ph-NO2

Wavelength

Abs

orba

nce

Figure S5. pH titration of 2Ph-NO2. Red indicates lowest pH’s (1.21), purple indicates highest (11.11).



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00.20.40.60.8

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pH Titration of 2Ph-CN

Wavelength

Abs

orba

nce

Figure S6. pH titration of 2Ph-CN. Red indicates lowest pH’s (0.85), purple indicates highest (11.33).



S8

220 240 260 280 300 320 340

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

Wavelength (nm)

Abs

orba

nce

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-0.1

0

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Wavelength (nm)

Abs

orba

nce

Figure S7. Acid/base induced reversibility of 3 and 7 (50 µM). (A) adjusted to pH 7.5 through adding

aqueous sodium hydroxide (2 N) and then measured every 4 mins. (B) adjusted to pH 2.3 through

adding aqueous hydrogen chloride (2 N) and then measured every 20 mins. UV-Vis time kinetics were

carried out in (2:1) H2O/MeOH solvent mixture.

B

A


S9

III. Reaction between 2Ph-X and β-mercaptoethanol

225 250 275 300 325 350 375 400 425 450 475 5000.00

0.25

0.50

0.75

1.00

1.25

1.50O O

O O

SHN+ HO

SHO O

O O

O S

1 eq. :100 eq.

H2O:MeOH= 2:1

Abso

rban

ce

Wavelength (nm)

0 min 20 min 40 min 60 min 80 min 100 min 120 min 140 min 160 min 180 min

Figure S8. Time kinetics for UV-Vis absorbance of 2Ph-H (50 µM) with 100 eq. β-mercaptoethanol in 2:1 H2O:MeOH mixture.


S10

IV. Time kinetics for 3 and 7

220 240 260 280 300 3200.0

0.2

0.4

0.6

0.8

1.0

0 500 1000 1500 2000 25000.0

0.1

0.2

0.3

0.4

0.5

0.6

Abso

rban

ce

Wavelength (nm)

O O

O O

O S

HO

HS

O O

O O

OS

HO

S

A 290

nm

Time (second)

(A) (B)

Figure S9. (A) Time kinetics for UV-Vis absorbance spectra of 3 (50 µM) (B) Time-kinetic curve for absorbance at 290 nm, referring to A. The detection was carried out in 2:1 H2O:MeOH mixture.


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V. Stability of 5

200 250 300 350 4000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

O O

O O

O S

CABMEAbso

rban

ce

Wavelength (nm)

Figure S10. UV-Vis absorbance and stability test (0 – 120 mins) of 5 (50 µM) in 2:1 H2O:MeOH mixture. Compound 5 is referred to as CABME.

5


S12

VI. 1H NMR time kinetics

O O

O O

HN S

NO2

SHHS

OH

OH O O

O O

SS

HO

O

O O

O O

SS

HO OH

Intermediate

Rearrange

NH2

NO2

+

Figure S11. Time kinetics for 1H-NMR spectra between 2Ph-NO2 and DTT in 2:1 D2O/MeOD mixture. Postulated the indicated resonances correspond to intermediate 6.

0 mins

30 mins

90 mins

150 mins

600 mins

3 days

6 days

6 days + p-nitroanaline

DTT

6


S13

VII. Kinetic Analysis in Detail

Although there are software packages that will do numerical integration of rate equations (DynaFit, Berkeley Madonna, Copasi) there are none that include SVD analysis of time resolved spectra. Applied Photophysics and OLIS offer software for SVD analysis based on using analytic solutions of rate equations and cannot be used to globally fit spectra collected at multiple concentrations, and suffer from the errors inherent in fitting data to multiple exponentials. It does not seem appropriate to reference alternative software that falls short of the needs for this study. Note that KinTek Explorer was modified to enable global fitting of data collected at multiple concentrations of DTT specifically for this study, and this extensive modification was paid for by KAJ through KinTek Corp.

Here we provide a summary of the data supporting the Hammett plot (Figure 6). We monitored the reaction of each derivative with DTT by measuring spectra as a function of time, as described in the main text for 2Ph-H. The time-dependent spectra were resolved by singular value decomposition (SVD) analysis (1,2). The SVD amplitudes were then fit to a minimal model by nonlinear regression analysis based on numerical integration of the rate equations using KinTek Explorer (KinTek Corporation, Austin, TX).

2Ph-X + DTTk1 9Ph-X 6

+ An-X

7k2 k3

Experiments performed at 1, 2, 5, and 10 mM DTT were fit simultaneously with the known spectra of the aniline derivatives (products of the reaction) included. However, unlike the case for 2Ph-H, we did not include data to define the spectra of the products of the reaction. The value of k3 was not well defined, so it was locked at 0.0002 s-1. The purpose of this analysis was only to derive estimates for the values of k1 and k2 for each derivative. In achieving the best fit possible, we fit the SVD amplitude data, but also fit data directly to the spectra by deriving an extinction coefficient at each wavelength for each species. Alternating between the two methods helped to find the solution for the best fit to data. In each case, below we show the results of fitting the SVD amplitude vectors, which are weighted by their significance values.

By globally fitting the time-dependent spectra over a range of concentrations, the data provided good definition of the two rate constants, k1 and k2. However, there may be some limitations to the data because the absorption spectrum of DTT was subtracted from each data set and the high levels of absorbance at lower wavelengths may have exceeded the linear response range of the spectrophotometer. Thus, we were unable to provide reliable spectra for 6 and 7 and in some cases the best-fit predicted negative absorbance. Again because we are only using these data to obtained estimates for k1 and and k2, this was not considered to be a serious problem.

For each 2Ph-X derivative below, we present the results of fitting the time-resolved spectra at 5 mM DTT as representative of data over the range of concentrations; that is, we show the fit to the SVD amplitude vectors, the predicted time dependence of species and the reconstituted spectra at 5 mM DTT. The predicted spectra of individual species are derived from the global fit to data at all concentrations.


S14

Figure S12. Fitting time-resolved spectra for 2Ph-OH. A. The reconstituted time-dependent spectra are shown (smooth lines) superimposed on the data (points). B. The reaction sequence with color-coding for each species in panels D and E. C. The fit to the SVD amplitude vectors at 5 mM DTT; note the colors do not correspond to any species. D. Predicted spectra of each species color coded at shown in B. E. Time dependence of each species, color-coded as in B. Note the matrix for the reconstituted spectra are a product of vectors in panels D and E.


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Figure S13. Fitting time-resolved spectra for 2Ph-OMe. A. The reconstituted time-dependent spectra are shown (smooth lines) superimposed on the data (points). B. The reaction sequence with color-coding for each species in panels D and E. C. The fit to the SVD amplitude vectors at 5 mM DTT; note the colors do not correspond to any species. D. Predicted spectra of each species color coded at shown in B. E. Time dependence of each species, color-coded as in B. Note the matrix for the reconstituted spectra are a product of vectors in panels D and E.


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Figure S14. Fitting time-resolved spectra for 2Ph-Br. A. The reconstituted time-dependent spectra are shown (smooth lines) superimposed on the data (points). B. The reaction sequence with color-coding for each species in panels D and E. C. The fit to the SVD amplitude vectors at 5 mM DTT; note the colors do not correspond to any species. D. Predicted spectra of each species color coded at shown in B. E. Time dependence of each species, color-coded as in B. Note the matrix for the reconstituted spectra are a product of vectors in panels D and E.


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Figure S15. Fitting time-resolved spectra for 2Ph-NO2. A. The reconstituted time-dependent spectra are shown (smooth lines) superimposed on the data (points). B. The reaction sequence with color-coding for each species in panels D and E. C. The fit to the SVD amplitude vectors at 5 mM DTT; note the colors do not correspond to any species. D. Predicted spectra of each species color coded at shown in B. E. Time dependence of each species, color-coded as in B. Note the matrix for the reconstituted spectra are a product of vectors in panels D and E.


S18

VIII. Time kinetics for 2Ph-X with DTT

Representative procedure for kinetics experiments: 2Ph-X was dissolved in 2:1

water:methanol (50 μM). DTT was added to the solution and the vial sealed. The solution was

scanned every 30 min for 240 min then every 60 min until 2400 total minutes had passed,

unless otherwise specified. In each spectra t=0 is red.

2Ph-H:

Figure S16. Time-kinetic experiment for reaction between 2Ph-H and DTT with various amounts of concentration.

2Ph-OCH3:

Figure S17. Time-kinetic experiment for reaction between 2Ph-OCH3 and DTT with various amounts of concentration.


S19

2Ph-OH:

Figure S18. Time-kinetic experiment for reaction between 2Ph-OH and DTT with various amounts of concentration.

2Ph-Br: 2Ph-Br did not show enough product formation at 2400 min for kinetic analysis so experiment was extended to 4320 min. Additionally the experiment with 10 mM DTT showed too much background absorbance from the DTT for clean analysis and was omitted from final analysis.

Figure S19. Time-kinetic experiment for reaction between 2Ph-Br and DTT with various amounts of concentration.

2Ph-NO2: 2Ph-NO2 did not show enough product formation with 1 mM DTT for kinetic analysis so the 1 mM DTT experiment was excluded.


S20

Figure S20. Time-kinetic experiment for reaction between 2Ph-NO2 and DTT with various amounts of concentration.


S21

IX. Synthetic procedure

Representative procedure for the synthesis of compounds 2Ph-X: 1 (0.0805 mmol, 20 mg) is

dissolved in 9.6 mL 5:1 pH 7.2 phosphate buffer:acetonitrile. Aniline (0.0805 mmol) was added

to the reaction mixture and a nitrogen sparged through for 16 hours. The reaction mixture

was purified by reverse phase chromatography (acetonitrile:water).

2Ph-H: 1H-NMR (400 MHz, CDCl3): δ = 7.45 (m, 2H), 7.37 (m, 1H), 7.32 (m, 2H), 2.28 (s, 3H),

1.77 (s, 6H). HRMS (ESI): m/z calculated for C11H16NO4 [M+Na]+: 244.1118, found 244.1116.

2Ph-CH3: 1H-NMR (400 MHz, CDCl3): δ = 7.24 (d, 2H, J = 8.12 Hz), 7.18 (m, 2H), 2.39 (s, 3H),

2.29 (s, 3H), 1.76 (s, 6H). 13C-NMR (400 MHz, CDCl3): δ = 178.11, 138.25, 134.56, 130.05,

125.19, 103.07, 85.83, 26.35, 21.13, 18.89. HRMS (ESI): m/z calculated for C15H17NO4S [M-

H]-: 306.08060, found 306.0801.

2Ph-OCH3: 1H-NMR (400 MHz, CDCl3): δ = 7.20 (m, 2H), 6.93 (m, 2H), 3.83 (s, 3H), 2.30 (s, 3H),

1.75 (s, 6H). 13C-NMR (400 MHz, CDCl3): δ = 178.35, 159.19, 129.81, 126.81, 114.57, 103.05,

85.49, 55.52, 26.34, 18.87. HRMS (ESI): m/z calculated for C15H17NO5S [M+Na]+: 346.07200,

found 346.0721.

2Ph-Br: 1H-NMR (400 MHz, CD3CN): δ = 7.32 (d, 2H, J = 8.52 Hz), 6.81 (d, 2H, J = 8.02 Hz), 2.35

(s, 3H), 1.38 (s, 6H). 13C-NMR (400 MHz, CD3CN): δ = 132.70, 126.91, 121.72, 103.32, 86.78,

26.41, 19.07, 8.59. HRMS (ESI): m/z calculated for C14H14NO4SBr [M-H]-: 369.97540 and

371.97340, found 369.9753 and 371.9734.

2Ph-NO2: 1H-NMR (400 MHz, CD3OD): δ = 8.01 (m, 2H), 6.93 (m, 2H), 2.31 (m, 3H), 1.31 (s, 6H). 13C-NMR (400 MHz, CD3OD): δ = 164.99, 143.41, 123.71, 121.38, 102.10, 24.50, 13.71. HRMS

(ESI): m/z calculated for C14H14N2O6S [M-H]-: 337.05000, found 337.0500.

2Ph-CO2H: 1H-NMR (400 MHz, CD3OD): δ = 8.52 (m, 2H), 7.55 (m, 2H), 3.05 (s, 3H), 2.04 (s, 6H). 13C-NMR (400 MHz, CD3OD): δ = 175.03, 165.73, 131.68, 130.66, 129.68, 120.16, 114.94,

103.67, 78.73, 14.07. HRMS (ESI): m/z calculated for C15H15NO6S [M-H]-: 336.05470, found

336.0538.

2Ph-N(CH3)2: 1H-NMR (400 MHz, CD3OD): δ = 7.13 (m, 2H), 6.79 (m, 2H), 2.97 (s, 6H), 2.35 (s,

3H), 1.71 (s, 6H). 13C-NMR (400 MHz, CD3OD): δ = 164.29, 125.59, 112.10, 109.99, 102.82,

39.24, 29.25, 24.92. HRMS (ESI): m/z calculated for C16H20N2O4S [M-H]-: 335.10710, found

335.1069.


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2Ph-CN: 1H-NMR (400 MHz, CDCl3): δ = 7.70 (d, 2H, J = 8.44 Hz), 7.41 (d, 2H, J = 8.21 Hz), 2.29

(s, 3H), 1.72 (s, 6H). 13C-NMR (400 MHz, CDCl3): δ = 192.66, 159.93, 133.43, 125.16, 103.50,

103.21, 103.01, 26.80, 26.45, 21.45, 19.02. HRMS (ESI): m/z calculated for C15H14N2O4S [M-

H]-: 317.06020, found 317.0604.

2Ph-OH: HRMS (ESI): m/z calculated for C14H15NO5S [M-H]-: 308.05980, found 308.0595.


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X. NMR spectra

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)

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3400

3600

3800

6.00

1.00

2.51

2.13

1.03

1.01

-0.0

01.

711.

711.

931.

951.

972.

922.

922.

932.

932.

942.

942.

952.

962.

962.

972.

983.

353.

373.

383.

403.

433.

453.

463.

483.

923.

933.

933.

943.

953.

96

5.22

5.23

5.24

5.24

5.25

5.25

5.26

5.27

7.26

1H NMR (400 MHz, Chloroform-d) δ5.24 (ddd, J= 9.7, 7.5, 3.7 Hz, 1H), 3.94 (td,J= 5.9, 3.7 Hz, 1H),3.73 – 3.07 (m, 2H), 3.03 – 2.67 (m, 5H), 1.95 (t,J= 8.9 Hz, 1H), 1.71 (d,J= 2.0 Hz, 6H).

Parameter Value

1 Data File Name C:/ Users/ Shawn/ Desktop/ sxl-EVA-DTT_20170505_01/ PROTON_01.fid/ fid

2 Title PROTON_01

3 Comment sxl-EVA-DTT

4 Origin Varian

5 Instrument vnmrs

6 Author

7 Solvent cdcl3

8 Temperature 25.0

9 Pulse Sequence s2pul

10 Experiment 1D

11 Probe NHB400

12 Number of Scans 32

13 Receiver Gain 58

14 Relaxation Delay 2.0000

15 Pulse Width 2.2667

16 Presaturation Frequency

17 Acquisition Time 2.5559

18 Acquisition Date 2017-05-05T09:58:26

19 Modification Date 2017-05-05T10:38:15

20 Spectrometer Frequency 399.68

21 Spectral Width 6410.3

22 Lowest Frequency -800.3

23 Nucleus 1H

24 Acquired Size 16384

25 Spectral Size 65536

Figure S21. 1H NMR for compound 3 in CDCl3.

-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

27.0

228

.28

31.1

0

71.2

9

90.5

991

.47

104.

25

159.

02

163.

89

189.

48

76.577.077.578.0f1 (ppm)

0

50

100

150

200

77.1

9

13C NMR (101 MHz, Chloroform-d) δ189.48, 163.89, 159.02,104.25, 91.47, 90.59, 77.19, 71.29, 31.10, 28.28, 27.02.

Parameter Value

1 Data File Name C:/ Users/ Shawn/ Desktop/ sxl-EVA-DTT-C_20170505_01/ CARBON_01.fid/ fid

2 Title CARBON_01

3 Comment sxl-EVA-DTT-C

4 Origin Varian

5 Instrument vnmrs

6 Author

7 Solvent cdcl3

8 Temperature 25.0

9 Pulse Sequence s2pul

10 Experiment 1D

11 Probe NHB400

12 Number of Scans 10000

13 Receiver Gain 30

14 Relaxation Delay 2.0000

15 Pulse Width 4.0000

16 Presaturation Frequency

17 Acquisition Time 1.3107

18 Acquisition Date 2017-05-05T21:26:11

19 Modification Date 2017-05-06T15:51:37

20 Spectrometer Frequency 100.51

21 Spectral Width 25000.0

22 Lowest Frequency -1445.2

23 Nucleus 13C

24 Acquired Size 32768

25 Spectral Size 65536

Figure S22. 13C NMR for compound 3 in CDCl3.

O O

S

OO

OH

HS

3

O

O O

S

OO

OH

HS

3

O


S24

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.012.513.013.5f1 (ppm)

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3800

4000Parameter Value

1 Title PROTON_012 Origin Varian3 Instrument vnmrs4 Author

5 Solvent cdcl36 Temperature 25.07 Pulse Sequence s2pul8 Experiment 1D9 Probe NHB40010Number of Scans 1611Receiver Gain 4812Relaxation Delay 2.000013Pulse Width 2.266714Presaturation Frequency

15Acquisition Time 2.555916Acquisition Date 2016-08-17T11:58:4917Modification Date 2019-06-05T15:52:0918Spectrometer Frequency399.6819Spectral Width 6410.320Lowest Frequency -807.121Nucleus 1H22Acquired Size 1638423Spectral Size 65536

Figure S23. 1H NMR for compound 2PH-H in CDCl3.

-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)

-10

0

10

20

30

40

50

60

70

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90

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110

120

130

140

150Parameter Value

1 Title CARBON_012 Origin Varian3 Instrument vnmrs4 Author

5 Solvent cd3od6 Temperature 25.07 Pulse Sequence s2pul8 Experiment 1D9 Probe NHB40010Number of Scans 120011Receiver Gain 3012Relaxation Delay 1.000013Pulse Width 4.000014Presaturation Frequency

15Acquisition Time 1.310716Acquisition Date 2016-06-10T10:29:4017Modification Date 2019-06-22T18:10:5018Spectrometer Frequency100.5119Spectral Width 25000.020Lowest Frequency -1445.121Nucleus 13C22Acquired Size 3276823Spectral Size 65536

Figure S24. 13C NMR for compound 2Ph-H in CD3OD.

O O

S

OO

NCH3

H

2PH-H

H

O O

S

OO

NCH3

H

2PH-H

H


S25

Figure S25. 1H NMR for compound 2PH-OCH3 in CDCl3.

Figure S26. 13C NMR for compound 2Ph-OCH3 in CDCl3.

O O

S

OO

NCH3

OCH3

2PH-OCH3

H

O O

S

OO

NCH3

OCH3

2PH-OCH3

H


S26

-2-101234567891011121314f1 (ppm)

0

500

1000

1500

2000

2500

3000

3500

4000

4500Parameter Value


5 Solvent cd3od6 Temperature 25.07 Pulse Sequence s2pul8 Experiment 1D9 Probe robo_2dec201410Number of Scans 3211Receiver Gain 5012Relaxation Delay 2.000013Pulse Width 2.066714Presaturation Frequency


Figure S27. 1H NMR for compound 2PH-Br in CD3OD.

-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70Parameter Value


5 Solvent cdcl36 Temperature 25.07 Pulse Sequence s2pul8 Experiment 1D9 Probe robo_2dec201410Number of Scans 80011Receiver Gain 3012Relaxation Delay 1.000013Pulse Width 2.233314Presaturation Frequency


Figure S28. 13C NMR for compound 2Ph-Br in CDCl3.

O O

S

OO

NCH3

Br

2PH-Br

H

O O

S

OO

NCH3

Br

2PH-Br

H


S27

-2-101234567891011121314f1 (ppm)

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300Parameter Value




Figure S29. 1H NMR for compound 2PH-NO2 in CD3OD.

-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)

-5

0

5

10

15

20

25

30

35

40

45

50

55

60

65

Parameter Value




Figure S30. 13C NMR for compound 2Ph-NO2 in CD3OD.

O O

S

OO

NCH3

NO2

2PH-NO2

H

O O

S

OO

NCH3

NO2

2PH-NO2

H


S28

-2-101234567891011121314f1 (ppm)

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

4000

4200Parameter Value




Figure S31. 1H NMR for compound 2PH-CNin CDCl3.

-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)

-10

0

10

20

30

40

50

60

70

80

90

100

110

Parameter Value




Figure S32. 13C NMR for compound 2Ph-CN in CDCl3.

O O

S

OO

NCH3

CN

2PH-CN

H

O O

S

OO

NCH3

CN

2PH-CN

H


S29

-2-101234567891011121314f1 (ppm)

0

500

1000

1500

2000

2500

3000

3500

4000

4500Parameter Value




Figure S33. 1H NMR for compound 2PH-OH in CDCl3.

-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

230

240Parameter Value




Figure S34. 13C NMR for compound 2Ph-OH in CDCl3.

O O

S

OO

NCH3

OH

H

2PH-OH

O O

S

OO

NCH3

OH

H

2PH-OH


S30

-2-101234567891011121314f1 (ppm)

-500

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

5500

6000

6500

7000

7500

8000

8500

9000Parameter Value




Figure S35. 1H NMR for compound 2PH-CH3 in CDCl3.

-100102030405060708090100110120130140150160170180190200210220230f1 (ppm)

-10

0

10

20

30

40

50

60

70

80

90

Parameter Value




Figure S36. 13C NMR for compound 2Ph-CH3 in CDCl3.

O O

S

OO

NCH3

CH3

2PH-CH3

H

O O

S

OO

NCH3

CH3

2PH-CH3

H


S31

XI. Derivation of Eq. 3

Assuming SSA on 10.𝑑[10]𝑑𝑡

= 0 = 𝑘𝑎[2𝑃ℎ𝑋][𝐷𝑇𝑇] + 𝑘 ‒ 𝑏[8𝑃ℎ𝑋][𝑀𝑒𝑆𝐻] ‒ 𝑘 ‒ 𝑎[10] ‒ 𝑘𝑏[10]

Solving for 10, and making the MeSH step irreversible.

[10] =𝑘𝑎[2𝑃ℎ𝑋][𝐷𝑇𝑇] + 𝑘 ‒ 𝑏[8𝑃ℎ𝑋][𝑀𝑒𝑆𝐻]

𝑘 ‒ 𝑎+ 𝑘𝑏

Assuming SSA on 8Ph-X

𝑑[8𝑃ℎ𝑋]𝑑𝑡

= 0 = 𝑘𝑏[10] + 𝑘 ‒ 𝑐[9𝑃ℎ𝑋] ‒ 𝑘 ‒ 𝑏[8𝑃ℎ𝑋][𝑀𝑒𝑆𝐻] ‒ 𝑘𝑐[8𝑃ℎ𝑋]

Solving for 8Ph-X, and making the MeSH step irreversible

[8𝑃ℎ𝑋] =𝑘𝑏[10] + 𝑘 ‒ 𝑐[9𝑃ℎ𝑋]

𝑘 ‒ 𝑏[𝑀𝑒𝑆𝐻]+ 𝑘𝑐

The rate of formation of 9Ph-X.


= 𝑘𝑐[8𝑃ℎ𝑋]

Plugging in for 8Ph-X


=𝑘𝑏[10]𝑘𝑐

𝑘𝑐

Plugging in for 10


=𝑘𝑎𝑘𝑏[2𝑃ℎ𝑋][𝐷𝑇𝑇]

𝑘 ‒ 𝑎+ 𝑘𝑏

Acid dissociation function for 2Ph-X

𝐾𝑎=[𝐻3𝑂+ ][2𝑃ℎ𝑋 ‒ ]

[2𝑃ℎ𝑋]

Final Equation, after pluggin in for 2Ph-X


=𝑘𝑎𝑘𝑏[2𝑃ℎ𝑋 ‒ ][𝐷𝑇𝑇][𝐻3𝑂

+ ]

𝐾𝑎(𝑘 ‒ 𝑎+ 𝑘𝑏)

1. Hendler, R. W., and Shrager, R. I. (1994) Deconvolutions based on singular value decomposition and the pseudoinverse: a guide for beginners. J. Biochem. and Biophys. Methods 28, 1-33

2. Henry, E. R., and Hofrichter, J. (1992) Singular Value Decomposition: Application to Analysis of Experimental Data. Methods Enzymol. 210, 129-192


S32

mechanistic studies of a “declick” reaction kenneth a ... · supporting information s1...

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