mechanistic studies of a “declick” reaction kenneth a ... · supporting information s1...
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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
SUPPORTING INFORMATION
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.
SUPPORTING INFORMATION
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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.
SUPPORTING INFORMATION
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0.8
1.0
1.2
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).
Calculated pKa = 6.51.
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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).
Calculated pKa = 6.54.
SUPPORTING INFORMATION
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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).
Calculated pKa = 5.56.
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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).
Calculated pKa = 4.34.
SUPPORTING INFORMATION
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00.20.40.60.8
11.21.41.61.8
2
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).
Calculated pKa = 4.54.
SUPPORTING INFORMATION
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-0.2
0
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Wavelength (nm)
Abs
orba
nce
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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
SUPPORTING INFORMATION
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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.
SUPPORTING INFORMATION
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IV. Time kinetics for 3 and 7
220 240 260 280 300 3200.0
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0.4
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1.0
0 500 1000 1500 2000 25000.0
0.1
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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.
SUPPORTING INFORMATION
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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
SUPPORTING INFORMATION
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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
SUPPORTING INFORMATION
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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.
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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.
SUPPORTING INFORMATION
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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.
SUPPORTING INFORMATION
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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.
SUPPORTING INFORMATION
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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.
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Figure S20. Time-kinetic experiment for reaction between 2Ph-NO2 and DTT with various amounts of concentration.
SUPPORTING INFORMATION
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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.
SUPPORTING INFORMATION
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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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0
200
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600
800
1000
1200
1400
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3800
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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
SUPPORTING INFORMATION
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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)
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
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
80
90
100
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
SUPPORTING INFORMATION
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
SUPPORTING INFORMATION
S26
-2-101234567891011121314f1 (ppm)
0
500
1000
1500
2000
2500
3000
3500
4000
4500Parameter Value
1 Title PROTON_012 Origin Varian3 Instrument vnmrs4 Author
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
15Acquisition Time 2.555916Acquisition Date 2016-06-21T10:09:0917Modification Date 2019-06-05T15:52:0818Spectrometer Frequency400.0919Spectral Width 6410.320Lowest Frequency -804.621Nucleus 1H22Acquired Size 1638423Spectral Size 65536
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
1 Title CARBON_012 Origin Varian3 Instrument vnmrs4 Author
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
15Acquisition Time 1.310716Acquisition Date 2016-09-01T11:03:2017Modification Date 2019-06-05T15:52:0818Spectrometer Frequency100.6119Spectral Width 25000.020Lowest Frequency -1433.821Nucleus 13C22Acquired Size 3276823Spectral Size 65536
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
SUPPORTING INFORMATION
S27
-2-101234567891011121314f1 (ppm)
-100
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300Parameter Value
1 Title PROTON_012 Origin Varian3 Instrument vnmrs4 Author
5 Solvent cd3od6 Temperature 25.07 Pulse Sequence s2pul8 Experiment 1D9 Probe NHB40010Number of Scans 1611Receiver Gain 4212Relaxation Delay 2.000013Pulse Width 2.266714Presaturation Frequency
15Acquisition Time 2.555916Acquisition Date 2016-08-31T09:38:4817Modification Date 2019-06-05T15:52:1118Spectrometer Frequency399.6819Spectral Width 6410.320Lowest Frequency -807.021Nucleus 1H22Acquired Size 1638423Spectral Size 65536
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
1 Title CARBON_012 Origin Varian3 Instrument vnmrs4 Author
5 Solvent cd3od6 Temperature 25.07 Pulse Sequence s2pul8 Experiment 1D9 Probe NHB40010Number of Scans 80011Receiver Gain 3012Relaxation Delay 1.000013Pulse Width 4.000014Presaturation Frequency
15Acquisition Time 1.310716Acquisition Date 2016-08-31T10:39:1717Modification Date 2019-06-05T15:52:1118Spectrometer Frequency100.5119Spectral Width 25000.020Lowest Frequency -1445.121Nucleus 13C22Acquired Size 3276823Spectral Size 65536
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
SUPPORTING INFORMATION
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
1 Title PROTON_012 Origin Varian3 Instrument vnmrs4 Author
5 Solvent cdcl36 Temperature 25.07 Pulse Sequence s2pul8 Experiment 1D9 Probe NHB40010Number of Scans 3211Receiver Gain 4612Relaxation Delay 2.000013Pulse Width 2.266714Presaturation Frequency
15Acquisition Time 2.555916Acquisition Date 2016-07-19T10:40:4817Modification Date 2019-06-21T11:18:2718Spectrometer Frequency399.6819Spectral Width 6410.320Lowest Frequency -807.121Nucleus 1H22Acquired Size 1638423Spectral Size 65536
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
1 Title CARBON_012 Origin Varian3 Instrument vnmrs4 Author
5 Solvent cdcl36 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 2016-07-19T11:30:1617Modification Date 2019-06-21T11:18:2718Spectrometer Frequency100.5119Spectral Width 25000.020Lowest Frequency -1445.221Nucleus 13C22Acquired Size 3276823Spectral Size 65536
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
SUPPORTING INFORMATION
S29
-2-101234567891011121314f1 (ppm)
0
500
1000
1500
2000
2500
3000
3500
4000
4500Parameter 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 4612Relaxation Delay 2.000013Pulse Width 2.266714Presaturation Frequency
15Acquisition Time 2.555916Acquisition Date 2015-12-08T11:02:4417Modification Date 2019-06-05T15:52:1318Spectrometer Frequency399.6819Spectral Width 6410.320Lowest Frequency -800.221Nucleus 1H22Acquired Size 1638423Spectral Size 65536
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
1 Title CARBON_012 Origin Varian3 Instrument vnmrs4 Author
5 Solvent cdcl36 Temperature 25.07 Pulse Sequence s2pul8 Experiment 1D9 Probe NHB40010Number of Scans 200011Receiver Gain 3012Relaxation Delay 1.000013Pulse Width 4.000014Presaturation Frequency
15Acquisition Time 1.310716Acquisition Date 2015-12-09T23:18:4717Modification Date 2019-06-05T15:52:1318Spectrometer Frequency100.5119Spectral Width 25000.020Lowest Frequency -1445.221Nucleus 13C22Acquired Size 3276823Spectral Size 65536
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
SUPPORTING INFORMATION
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
1 Title PROTON_012 Origin Varian3 Instrument vnmrs4 Author
5 Solvent cdcl36 Temperature 25.07 Pulse Sequence s2pul8 Experiment 1D9 Probe NHB40010Number of Scans 3211Receiver Gain 4412Relaxation Delay 2.000013Pulse Width 2.266714Presaturation Frequency
15Acquisition Time 2.555916Acquisition Date 2016-07-15T10:00:1117Modification Date 2019-07-03T09:53:0318Spectrometer Frequency399.6819Spectral Width 6410.320Lowest Frequency -807.121Nucleus 1H22Acquired Size 1638423Spectral Size 65536
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
1 Title CARBON_012 Origin Varian3 Instrument vnmrs4 Author
5 Solvent cdcl36 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 2016-07-15T10:49:5417Modification Date 2019-07-03T09:53:0418Spectrometer Frequency100.5119Spectral Width 25000.020Lowest Frequency -1445.221Nucleus 13C22Acquired Size 3276823Spectral Size 65536
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
SUPPORTING INFORMATION
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.
𝑑[9𝑃ℎ𝑋]𝑑𝑡
= 𝑘𝑐[8𝑃ℎ𝑋]
Plugging in for 8Ph-X
𝑑[9𝑃ℎ𝑋]𝑑𝑡
=𝑘𝑏[10]𝑘𝑐
𝑘𝑐
Plugging in for 10
𝑑[9𝑃ℎ𝑋]𝑑𝑡
=𝑘𝑎𝑘𝑏[2𝑃ℎ𝑋][𝐷𝑇𝑇]
𝑘 ‒ 𝑎+ 𝑘𝑏
Acid dissociation function for 2Ph-X
𝐾𝑎=[𝐻3𝑂+ ][2𝑃ℎ𝑋 ‒ ]
[2𝑃ℎ𝑋]
Final Equation, after pluggin in for 2Ph-X
𝑑[9𝑃ℎ𝑋]𝑑𝑡
=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