Conversion and initial decay rate of 4-oxo-TEMPO in aqueous solutions exposed to He plasma. 3. Table S3. Retention times and masses of the ion peaks of the ...
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Y. Gorbanev, N. Stehling, D. O’Connell and V. Chechik Reactions of nitroxide radicals in aqueous solutions exposed to non-thermal plasma: Limitations of spin trapping of the plasma induced species - Supporting information
Table of contents Materials
2
Nitroxides g-factor values analysis
2
Chemical synthesis of 4-oxo-TEMPOH and PTI
2
Table S1. Error assessment (4-oxo-TEMPO and PTIO decay)
2
Figure S1. UV calibration curve of NO2- (a) and LC-MS calibration curves of 4-oxo-TEMP and 4-oxo-TEMPOH (b,c) 3 Table S2. Conversion and initial decay rate of 4-oxo-TEMPO in aqueous solutions exposed to He plasma
3
Table S3. Retention times and masses of the ion peaks of the analysed 4-oxo-TEMPO solutions after plasma exposure
4
Figure S2. Concentrations of 4-oxo-TEMPO and corresponding hydroxylamine (4-oxo-TEMPOH) and piperidine (44 oxo-TEMP) in aqueous solutions exposed to He with 100% H2O vapour saturation Figure S3. The ESI-MS spectrum of a solution of 4-oxo-TEMPO with added glycine exposed to He plasma
4
Figure S4. The UV absorption profiles of 4-oxo-TEMPO solutions with added p-benzoquinone and hydroquinone (pristine and after 60 s exposure to different plasmas) 5 Table S4. The conversion of pre-formed PBN-H radical adduct in aqueous solutions exposed to different plasmas 5 Figure S5. PTIO and PTI decay and PTIO formation in aqueous solutions exposed to different plasmas
6
Table S5. Conversion of PTIO and PTIO pre-treated with PbO2 in aqueous solutions exposed to different plasmas 6 Figure S6. Typical experimental and simulated EPR spectra of (MGD)2-Fe2+-NO radical complex
7
Table S6. Concentration of (MGD)2-Fe2+-NO and PTIO after exposure to He + 0.5% air plasma
7
2 Materials. 4-oxo-TEMPO (>96%), 2,2,6,6-tetramethyl-4-piperidone (95%), hydroquinone (99+%), p-benzoquinone (≥98%), sodium nitrite (≥97%), lead(IV) oxide (97+%), iron(II) sulfate heptahydrate (99+%), hydrogen peroxide (≥30%), sodium hydrosulfite (≥82%), Griess reagent for nitrite, sodium nitrite (≥97%), L-ascorbic acid (99%), deuterium oxide (99.9 atom%) were purchased from Sigma Aldrich. N-tert-butyl-α-phenylnitrone (98%) was from Alfa Aesar. MGD sodium salt monohydrate (≥98%) was supplied by Santa Cruz Biotechnology. PTIO (>98%) was from Tokyo Chemical Industry UK Ltd. Magnesium sulfate, sodium bicarbonate and hydrochloric acid (Reagent grade) were from Fisher Scientific. Glycine (>99%) was obtained from Fisons Analytical Reagent. Methanol and chloroform (Reagent grade) were from VWR Chemicals. De-ionised water was used for the preparation of the solutions. Nitroxides g-factor values. The g-factor values were measured on a JEOL JES-RE1X spectrometer using a DPPH standard (9.43 GHz, 3.17 mW, modulation frequency 100 kHz, modulation amplitude 1 G, time constant 30 ms, sweep time 40 s, number of scans 1, sweep width 100 G). Chemical synthesis. 4-oxo-TEMPOH was prepared by reduction of 4-oxo-TEMPO by ascorbic acid in an aqueous solution (Okazaki et al., J. Phys. Chem. 1985, 89, 4437). 75 mg of L-ascorbic acid were added to a solution of 7 mg of 4-oxo-TEMPO in 100 mL H2O. The resulting mixture was stirred at 800 rpm for 20 min at room temperature. The disappearance of the TEMPO signal was monitored by EPR. PTI synthesis was performed as adapted from literature (Zhao et al., Bioorg. Med. Chem. 2007, 15, 2815). 500 mg of NaNO2 were added to a solution of 233.3 mg PTO in 15 mL methanol with constant stirring. The pH was further adjusted to 5 using 5M aqueous HCl. The reaction mixture was stirred for 1 h at room temperature. The disappearance of the starting material was monitored by thin layer chromatography (CHCl3/CH3OH, 20:1). Then the reaction mixture was evaporated under vacuum to remove most of the solvent, adjusted to pH 7 with a saturated NaHCO3 solution and extracted with CHCl3. Combined CHCl3 phase was dried over anhydrous MgSO4 and solvent was evaporated under vacuum to allow a red syrup (PTI purity ca. 36% by EPR). 29.6 mg of this crude PTI was dissolved in 40 mL H2O and stirred with 500 mg of PbO2 for 30 min at room temperature and then filtered to yield an aqueous solution of PTI (94% purity by EPR). In a control experiment, a solution of PTIO was treated with PbO2. After the removal of lead dioxide, PTIO was exposed to plasma. The results did not reveal differences in decay between untreated PTIO and PTIO solution treated with PbO2 (Table S4). Table S1. The error evaluation. The solutions of 4-oxo-TEMPO and PTIO were exposed to plasma and the remaining concentrations were determined by EPR. The initial concentrations of 4-oxo-TEMPO and PTIO were 200 µM.
Number of experiment 1 2 3 4 5 6 7 8 9 10
4-oxo-TEMPO (He, 30 s) 92.8 99.2 96.6 98.9 86.6 90.6 87.7 107.0 86.9 96.4
st.dev.
6.2
Concentration (µM) 4-oxo-TEMPO PTIO (He + 0.5% O2, 60 s) (He + 0.5% air, 30 s) 23.3 75.0 18.4 56.5 26.3 72.6 24.8 71.2 26.3 67.1 29.4 73.1 28.6 53.3 22.4 71.2 22.1 68.8 27.0 68.5 3.2
6.8
PTIO (He + 0.5% O2, 30 s) 67.8 61.7 63.7 61.7 55.6 63.7 56.7 68.8 69.8 67.8 4.7
3
100
(a)
NO2- concentration / µM
y = a + b*x
Equation
80
?$OP:A=1
Plot
No Weighting
Weight
60
0 ± --
Intercept
54.40848 ± 0.52157
Slope
40
20
Residual Sum of Squares
6.12194
Pearson's r
0.99977
R-Square(COD)
0.99954
Adj. R-Square
0.99945
0 0.0
0.4
0.8
1.2
1.6
2.0
Signal intensity / a.u.
(b)
350 Equation
y = Intercept + B1*x^1 + B2*x^2 + B3*x^3
300
Weight
No Weighting
250
Residual Sum of Squares Adj. R-Square
200
49.89846 0.99956 Value Intercept
150
B1 B2 B3
B
100 50
Standard Error 0
--
4.97985E-9 8.58805E-19 -1.14016E-29
7.68118E-10 1.26078E-19 4.52251E-30
(c)
400
4-oxo-TEMPOH concentration / µM
4-oxo-TEMP concentration / µM
400
350
Equation
y = a + b*x
300
Weight Residual Sum of Squares
No Weighting 83.18453
Pearson's r Adj. R-Square
0.99973 0.99939
250 200
Value
150
?$OP:A=1
Intercept Slope
0 2.58076E-8
Standard Error -2.24821E-10
100 50 0
0 0
5
10
15
Signal intensity / counts x 109
20
0
2
4
6
8
10
12
14
Signal intensity / counts x 109
Figure S1. UV calibration curve of NO2- (a) and LC-MS calibration curves of 4-oxo-TEMP and 4-oxo-TEMPOH (b,c) Table S2. Conversion and initial decay rate of 4-oxo-TEMPO in aqueous solutions exposed to He plasma.
Entry
4-oxo-TEMPO initial concentration (mM) 4-oxo-TEMPO conversiona (%) 0.05 1 99 0.1 2 94 0.2 3 94 2 4 53 20 5 24 50 6 24 a After 60 s of plasma exposure. b Calculated as average for the first 15 s of plasma exposure.
Initial 4-oxo-TEMPO decay rateb (µmol·s-1) 1.2 2.0 3.3 24.7 79.9 314.4
4 Table S3. Retention times and masses of the ion peaks of the analysed 4-oxo-TEMPO solutions after plasma treatment.
Entry
Compound
Isolated ion peak (m/z)
Retention time (min)
1 2
4-oxo-TEMP 4-oxo-TEMPOH
156 172
1.8 6.0
300 He with 100% saturated H2O vapour
Concentration / µM
250
4-oxo-TEMPO 4-oxo-TEMPOH 4-oxo-TEMP
200 150 100 50 0 0
10
20
30
40
50
60
Plasma exposure time / s
Figure S2. Concentrations of 4-oxo-TEMPOH and 4-oxo-TEMP in aqueous solutions exposed to He with 100% H2O vapour saturation as determined by LC-MS. 4-oxo-TEMPO concentrations (dashed line) determined by EPR.
300000
[4-oxo-TEMP-H]+ (m/z 156)
Signal intensity (a.u.)
250000
200000
[4-oxo-TEMPO-aminomethyl-H]+ [4-oxo-TEMPOH-H]+ (m/z 203) not detected (m/z 172)
150000
[4-oxo-TEMPO-glycil-H]+ (m/z 245) not detected
100000
50000
0 160
180
200
220
240
m/z
Figure S3. The ESI-MS spectrum of a 4-oxo-TEMPO solution (200 µM) with added glycine (20 mM) exposed to He plasma.
Here, signals of [4-oxo-TEMP-H]+ and [4-oxo-TEMPOH-H]+ were observed. 4-oxo-TEMPO-glycyl or aminomethyl adducts were not detected. Similar results were obtained when the solution was exposed to He + 0.5% O2 plasma.
5
(a)
pristine after He + 0.5% O2 plasma
after He plasma after He + 0.5% O2 plasma p-hydroquinone 1
UV absorption / a. u.
UV absorption / a.u.
(b)
2.5
pristine after He w/ 100% H2O plasma
2
2.0
after He plasma after He w/ 100% H2O plasma
1.5
p-benzoquinone
1.0
0.5
0 200
0.0 200 300
400
500
300
400
500
Wavelength / nm
Wavelength / nm
Figure S4. The UV absorption profiles of the solutions with added (a) p-benzoquinone and (b) hydroquinone (pristine and after 60 s exposure to different plasmas). Absorption profiles of HQ and BQ, respectively (dashed) are added for comparison. TEMPO concentration was 200 µM, and HQ/BQ concentration was 20 mM in all plasma exposure experiments. All solutions were diluted with H2O 1:50 prior to UV analysis.
Table S4. The conversion of pre-formed PBN-H radical adduct in aqueous solutions exposed to different plasmas. Entry 1 2 3 4 5 6
Plasma feed gas He He + 0.5% O2 He + 100% H2O He, then He + 0.5% O2 He, then He + 100% H2O He, then He (in air)
PBN-H concentration (µM)
PBN-H conversion (%)
17.7 0.9 4.7 5.6 16.9 7.6
68 5 57
6
(a)
PTIO concentration / µM
200
He He + 0.5% O2
150
100
50
0 0
10
20
30
40
50
60
Plasma exposure time / s
(b) PTI decay He He + 0.5% O2 PTIO formation He + 0.5% O2
100
(c) PTIO decay He + 0.5% O2
200
Concentration / µM
Concentration / µM
200
He + 0.5% air
150
100
PTI decay He + 0.5% O2
50
He + 0.5% air
0 0
10
20
30
40
50
60
0 0
10
Plasma exposure time / s
20
30
40
50
60
Plasma exposure time / s
Figure S5. (a) PTIO decay, (b) PTI decay and PTIO formation, (c) PTI + PTIO decay in aqueous solutions exposed to different plasmas. In all experiments initial concentration of each compound was 200 µM.
Table S5. Conversion of PTIO and PTIO pre-treated with PbO2 in aqueous solutions exposed to different plasmas. Entry
Plasma feed gas
Exposure time (s)
PTIO conversion (%)
PTIO solution in H2O 1 2 3 4 5 6 7 8
30 He 60 He 30 He + 0.5% air 60 He + 0.5% air PTIO solution in H2O stirred with PbO2 30 He 60 He 30 He + 0.5% air He + 0.5% air 60
58 94 42 82 60 93 37 78
7
simulated
experiment
3380
3400
3420
3440
3460
3480
3500
Field / G
Figure S6. Typical experimental and simulated EPR spectra of (MGD)2-Fe2+-NO radical complex. aN = 12.7.
Table S6. Concentration of (MGD)2-Fe2+-NO and PTIO after exposure to He + 0.5% air plasma. The (MGD)2-Fe2+ complex was prepared by adding (1:1) solutions of MGD (10 mM) and FeSO4 (2 mM). Entry 1 2 3
Exposure time (s) 0 30 60
(MGD)2-Fe2+-NO concentration (µM) 17.1 47.1
PTIO concentration (mM) 2.3 1.9 1.5
