Predicting how polyphenol antioxidants prevent DNA damage by binding to iron

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1 Predicting how polyphenol antioxidants prevent DNA damage by binding to iron Nathan R. Perron, James N. Hodges, Michael Jenkins, and Julia L. Brumaghim Department of Chemistry, Clemson University, Clemson, SC SUPPORTING INFORMATION Gel electrophoresis results for phenolic compounds. Gel electrophoresis experiments of DNA damage inhibition by the twelve compounds shown in Figure are shown in Figure S with average tabulated values for the DNA band intensities observed in the electrophoresis experiments given in Tables S-S. Dose-response curves showing the percent inhibition of DNA damage vs. log concentration of phenolic compound (in µm) are provided in Figure S. Reaction components for gel electrophoresis lanes. In order to obtain results for the entire concentration range, several electrophoresis experiments had to be performed for each compound. As a result, different control lanes with iron and H O (lanes marked A, B, C, etc.) were used for different concentration ranges of each polyphenol compound. Only one of these lanes is shown in the gel images (Figures S and S), while all are shown in the tables (Tables S-S3). Additional control lanes (p-p3) were needed to ensure that DNA damage did not occur with the added reaction components indicated. The reaction lanes correspond to their specific Fe + /H O control lane. Control lanes: MW = kb DNA ladder; p = plasmid DNA, 3 mm NaCl, mm ethanol, and mm MES buffer; p = same as p with 5 µm H O ; p3 = same as p with 5 µm polyphenol compound; A, B, C, etc. = same as p with µm Fe + (or µm [Fe(EDTA)] - ). Reaction lanes: #A, #B, #C, etc. = same as lanes A, B, C, etc. with indicated amount of polyphenol compound given in tables S-S3.

2 MW p p p3 A A A 3A A 5A B C MW p p p3 A A A 3A B A 5A 6A 7A MW p p p3 A A A 3A B A 5A 6A 7A [EC] [ECG] [EGC] Figure Sa. Lanes A-5A, -, C: Increasing concentration EC:. µm, µm, µm, µm, 5 µm, µm, µm, 3 µm, µm, 5 µm and µm, respectively. Lanes A-5A, -, C correspond to tabulation of data for EC. Figure Sb. Lanes A-3A, -, A-7A: Increasing concentration ECG:. µm,. µm,. µm,. µm, µm, µm, µm, 5 µm, µm, µm, µm and µm, respectively. Lanes A-3A, -, A-7A correspond to tabulation of data for ECG. Figure Sc. Lanes A-3A, -, A-7A: Increasing concentration EGC:. µm,. µm,. µm,. µm, µm, µm, µm, 5 µm, µm, µm, 5 µm and µm, respectively. Lanes A-3A, -, A-7A correspond to tabulation of data for EGC. MW p p p3 A A A B C C3C C 5C D D 3D D 5D MW p p p3 A A A 3A A 5A B MW p p p3 A A A 3A B A 5A 6A [EGCG] [GA] [MEGA] Figure Sd. Lanes A-A, -, C-5C, D-5D: Increasing concentration EGCG:. µm,.5 µm,. µm,. µm,. µm,.5 µm,. µm,. µm, µm, µm, µm, 5 µm, µm, µm, 3 µm, µm and 5 µm, respectively. Lanes A-A, -, C-5C, D-5D correspond to tabulation of data for EGCG. Figure Se. Lanes A-5A, -: Increasing concentration GA:. µm, µm, µm, µm, 5 µm, µm, µm, 3 µm, µm and 5 µm, respectively. Lanes A-5A, - correspond to tabulation of data for GA. Figure Sf. Lanes A-3A, -, A-6A: Increasing concentration MEGA:. µm,. µm,. µm,. µm, µm, µm, µm, 5 µm, µm, µm and 5 µm, respectively. Lanes A-3A, -, A-6A correspond to tabulation of data for MEGA. Figure S. Gel electrophoresis images of polyphenols under Fenton reaction conditions ( µm Fe µm H O ) in mm MES buffer (ph = 6.): a = ( )-epicatechin (EC), b = ( )-epicatechin-3-gallate (ECG), c = ( )-epigallocatechin (EGC), d = ( )-epigallocatechin-3-gallate(egcg), e = gallic acid (GA), f = methyl-3,,5- trihydroxybenzoate (MEGA) % Agarose gel imaged with ethidium bromide. Control lanes: MW = kb MW ladder, p = plasmid DNA (p), p = p + 5 µm H O, p3 = p + largest concentration of phenolic compound + 5 µm H O, A = p + µm Fe µm H O.

3 MW p p p3 A A A B 3A A 5A C MW p p p3 A A A 3A A 5A B C C 3C C 5C MW p p p3 A A A B C C 3C C D D 3D [MEPCA] [Myr] [PrEGA] Figure Sg. Lanes A-A, -, 3A-5A, C: Increasing concentration MEPCA:. µm,. µm,. µm, µm, µm, µm, 5 µm, µm, µm, 5 µm and µm, respectively. Lanes A- A, -, 3A-5A, C correspond to tabulation of data for MEPCA. Figure Sh. Lanes A-5A, -, C-5C: Increasing concentration Myr:. µm,. µm,. µm,.5 µm,. µm,. µm, µm, µm, µm, 5 µm, µm, µm, 3 µm, µm and 5 µm, respectively. Lanes A-5A, -, C-5C correspond to tabulation of data for Myr. Figure Si. Lanes A-A, -B, C-C, D-3D: Increasing concentration PrEGA:. µm,. µm,. µm,. µm, µm, µm, µm, 5 µm, µm, µm and 5 µm, respectively. Lanes A-A, -B, C-C, D-3D correspond to tabulation of data for PrEGA. MW p p p3 A A A 3A A 5A B C [PCA] MW p p p3 A A A 3A A B C C 3C C 5C [Q] MW p p p3 A A A 3A A 5A B 6B [VA] Figure Sj. Lanes A-5A, -, C: Increasing concentration PCA:. µm, µm, µm, µm, 5 µm, µm, µm, 3 µm, µm, 5 µm and µm, respectively. Lanes A-5A, -, C correspond to tabulation of data for PCA. Figure Sk. Lanes A-5A, -, C-5C: Increasing concentration Q:. µm,. µm,. µm,.5 µm,. µm,. µm, µm, µm, µm, 5 µm, µm, µm, 3 µm, µm and 5 µm, respectively. Lanes A-5A, -, C-5C correspond to tabulation of data for Q. Figure Sl. Lanes A-5A, -6B: Increasing concentration VA:. µm, µm, µm, µm, 5 µm, µm, µm, 3 µm, µm, 5 µm and µm, respectively. Lanes A-5A, -6B correspond to tabulation of data for VA. Figure S (continued). Gel electrophoresis images of polyphenols under Fenton reaction conditions ( µm Fe µm H O ) in mm MES buffer (ph = 6.): g = methyl-3,-dihydroxybenzoate (MEPCA), h = myricetin (Myr), i = n-propyl gallate (PrEGA), j = protocatechuic acid (PCA), k = quercetin (Q), l = vanillic acid (VA). % Agarose gel imaged with ethidium bromide. Control lanes: MW = kb MW ladder, p = plasmid DNA (p), p = p + 5 µm H O, p3 = p + largest concentration of phenolic compound + 5 µm H O, A = p + µm Fe µm H O.

4 Table S: Tabulation of gel electrophoresis results for ( )-epicatechin (EC) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration EC, µm Fe + + H O A None 7. ± ± 3.55 A A 3A A b 5A Fe + + H O B. 5 None 5. ± ± ± ± ± ± ± ± ± ± ± ±.6 -. ±.5 -. ± ±.9 -. ±.5 3. ± 5. B Fe + + H O C 3 5 None 7.9 ± ± ± ±.6 9. ± ± c 8. ±.5.9 ±.99.8 ± ± ±.6 7. ± c 7. ± ± ± ± ±.83 C 87. ±.37.6 ±.37. ±.95 a All data are reported as the average of three trials; calculated standard deviations are shown. b Lane A was calculated based on one additional replacement Fe + + H O lane and µm EC lane (not shown), which causes the raw data of % Supercoiled and to appear to have an incorrect value. However, the % Damage Inhibition has been corrected for this and represents the true for all three trials. c Standard deviation for this gel lane could not be calculated because all three trials of µm EC were on the same gel, and thus the calculations were based on only one Fe + + H O lane. Table S: Tabulation of gel electrophoresis results for ( )-epicatechin-3-gallate (ECG) with µm Fe + and 5 µm H O in mm MES (ph = 6.). a Concentration ECG, µm Fe + + H O A A None.. ± ± ± ±.8 -. ±.75 A 3A Fe + + H O B.. None. ± ± ± ± ± ±.8. ±.3. ±.6 B b A 5A 6A b ± ± ± ± ± ± ± ± ± ±.6 3. ±.7. ± ± ±.3 7. ± ±.6. ± ± ± ± ±.5.3 ± ± ±.87 7A 8.8 ±. 7. ± ±.69 a All data are reported as the average of three trials; calculated standard deviations are shown. b Lanes and 6A were calculated based on one additional replacement Fe + + H O lane and µm ECG or 5 µm ECG lane (not shown), respectively which causes the raw data of % Supercoiled and to appear to have an incorrect value. However, the has been corrected for this and represents the true for all three trials.

5 Table S3: Tabulation of gel electrophoresis results for ( )-epigallocatechin (EGC) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration EGC, µm Fe + + H O A None 3. ± ±.96 A..8 ± ± ±.3 A. 3. ± ±.6.3 ±.8 3A. 3.3 ± ± 3.6. ±. Fe + + H O B None 7.5 ± ± ± ± ±.6 B 9.3 ± ± ± ± ± ±. 5.7 ± ± ± ±.. ±. 87. ±.86 A 76. ±. 3.8 ±. 9.7 ±. 5A 83. ± ±.58.3 ± A ±. 7.7 ± ± 3. 7A 8.7 ± ± ±.8 a All data are reported as the average of three trials; calculated standard deviations are shown.

6 Table S: Tabulation of gel electrophoresis results for ( )-epigallocatechin-3-gallate (EGCG) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration EGCG, µm Fe + + H O A None. ± ± 7.6 A A Fe + + H O B..5 None 9.6 ± ± ±.7 9. ± ± ± ±.75. ±.3 B Fe + + H O C None 6.8 ± ±. 9.8 ± ±.5 5. ± ± ± ±. 9. ± ± ± ± 7..5 ±.3.3 ± ± ± ±.6 C b C 3C C 5C ± ± ± ±.9 9. ± ± ± ±.69.8 ± ± ±. 65. ±. 8. ± ± ±.9 Fe + + H O D None 6.5 ± ± 3.9 D D 3D D 5D ± ± ± ± ± ± ± ± ±.6 5. ± ± ± ± ±.7.5 ±.6 a All data are reported as the average of three trials; calculated standard deviations are shown. b Lane C was calculated based on one additional replacement Fe + + H O lane and. µm EGCG lane (not shown), which causes the raw data of % Supercoiled and to appear to have an incorrect value. However, the % Damage Inhibition has been corrected for this and represents the true for all three trials.

7 Table S5: Tabulation of gel electrophoresis results for gallic acid (GA) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration GA, µm Fe + + H O A None. ± ±.3 A A 3A A 5A Fe + + H O B. 5 None.9 ±.5 3. ± ±. 37. ± ± ± ± ± ±. 63. ±.93.6 ± ± ± ± ± ± ±.3 B ± ± ± ± ±.9. ± ±.6 6. ±.9. ±.37. ± ± ± ±.33.8 ±.9.7 ±.68 a All data are reported as the average of three trials; calculated standard deviations are shown. Table S6: Tabulation of gel electrophoresis results for methyl-3,,5-trihydroxybenzoate (MEGA) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration MEGA, µm Fe + + H O A None.9 ± ±.9 A A 3A Fe + + H O B... None 3.8 ±.. ± ±.83.3 ± ± ± ± ±.53. ± ±.9 7. ±.3 B A 5A 6A ± ± ± ± ±. 87. ± ± ± ± ± ±.5. ± ±..6 ±.6 3. ±.5.8 ±.77.6 ± ± ± ±.95. ±.3. ± ± ±.6 a All data are reported as the average of three trials; calculated standard deviations are shown.

8 Table S7: Tabulation of gel electrophoresis results for methyl-3,-dihydroxybenzoate (MEPCA) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration MEPCA, µm Fe + + H O A None 8.7 ± ± 8.95 A A b Fe + + H O B.. None 7.3 ±. 3.6 ± 6..8 ± ±. 69. ± ±.7 -. ± ±. B 3A A 5A Fe + + H O C. 5 5 None.7 ±.. ± ±.9.9 ± ± ± ± ± ± c 87.3 ± ± ± ±.6. ±.9 8. ±.5 5. ±.8.7 ± ± c -. ±.96.5 ± ± ±.6 8. ± ± ± ±. C 87.6 ±.6. ± ±. a All data are reported as the average of three trials; calculated standard deviations are shown. b Lane A was calculated based on one additional replacement Fe + + H O lane and. µm MEPCA lane (not shown), which causes the raw data of % Supercoiled and to appear to have an incorrect value. However, the % Damage Inhibition has been corrected for this and represents the true for all three trials. c Standard deviation for this gel lane could not be calculated because all three trials of µm MEPCA were on the same gel, and thus the calculations were based on only one Fe + + H O lane.

9 Table S8: Tabulation of gel electrophoresis results for myricetin (Myr) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration Myr, µm Fe + + H O A None. ± ±.56 A A 3A A 5A Fe + + H O B None.7 ±.6.3 ±.9 3. ±.9.8 ±.8. ±..7 ± ± ± ± ± ± ± ±. -. ±.7.9 ±. -.7 ±.88. ±.7 B Fe + + H O C. 5 None.5 ± ± ±. 9.7 ± ±. 5.8 ± ± 8.7. ± ±. 7.3 ± ±. 8. ± ±. 5.7 ± ± ±.55.9 ±.33 C 93. ±. 7. ± ±.3 C 3C C 5C ± ± ± ± ± ± ± ± ±.5. ±.5.3 ±.9. ±.37 a All data are reported as the average of three trials; calculated standard deviations are shown.

10 Table S9: Tabulation of gel electrophoresis results for protocatechuic acid (PCA) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration PCA, µm Fe + + H O A None. ± ±. A A 3A A 5A Fe + + H O B. 5 None.3 ±.5.5 ± ± ± ± ± ± ± ± ± ± ±.6. ±.. ±. 3.6 ± ± ±.33 B Fe + + H O C 3 5 None 79.7 ±.7 9. ± ± ±. 96. ± ± b.3 ± ±.7 7. ±.6 5. ±.. ±.66.7 ± b ± ± ±.6.5 ±.77.9 ±.58 C 87. ±.37.6 ±.37.7 ±.99 a All data are reported as the average of three trials; calculated standard deviations are shown. b Standard deviation for this gel lane could not be calculated because all three trials of µm PCA were on the same gel, and thus the calculations were based on only one Fe + + H O lane. Table S: Tabulation of gel electrophoresis results for n-propyl gallate (PrEGA) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration PrEGA, µm Fe + + H O A None 6.8 ± b 93. ± b A. 7. ± ±.9.3 ±.8 A. 7.3 ± ±.76.7 ±.5 Fe + + H O B None.7 ± b 89.3 ± b..3 ± ±.5.8 ±.7 B. 3. ± ± ±.66 Fe + + H O C None 6.7 ± ±.56 C 5. ± ±.78.7 ±.76 C 39.9 ± ±.33.5 ±.39 3C 68. ± ± ±. C ±.3 3. ± ±.77 Fe + + H O D None 5. ± ± 3. D 8.7 ± ± ±.65 D 85.9 ±.57. ±.57.7 ±.9 3D ± ± ±.35 a All data are reported as the average of three trials; calculated standard deviations are shown. b Standard deviation for these gel lanes could not be calculated because all three trials of.,.,., and. µm PrEGA were on single gels, and thus the calculations were based on only one Fe + + H O lane.

11 Table S: Tabulation of gel electrophoresis results for quercetin (Q) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration Q, µm Fe + + H O A None 6.9 ± ±.68 A A 3A A Fe + + H O B....5 None 6.9 ± ±.68.3 ±.96. ±.7 3. ± ±.6 8. ± ± ± ±.78. ±.8 -. ±. 5.7 ±.8.5 ±.97 B Fe + + H O C. 5 None 3.6 ± ±.. ± ±. 8. ±.3. ± ±. 6. ± ±.98. ±. 9.9 ± ±.9.8 ± ± ± ± ±.9 C 9.8 ±.8 9. ± ±.97 C 3C C 5C ±.3 9. ±. 9.5 ± ± ± ±. 7.5 ± ± ± ± ±.7 3. ±.9 a All data are reported as the average of three trials; calculated standard deviations are shown. Table S: Tabulation of gel electrophoresis results for vanillic acid (VA) with µm Fe + and 5 µm H O in mm MES buffer (ph = 6.). a Concentration VA, µm Fe + + H O A None 6.8 ± ±.3 A A 3A A 5A Fe + + H O B. 5 None 6.5 ± ± ± ± ±.3.8 ± ± ± ± ± ± ± ±.88.6 ±.66.7 ±.5.9 ± ±. B 6B ± 3..5 ± ±. 55. ± ± ± ± ±.6.9 ±..6 ±.33.3 ± ± ±. 5. ± ± ± ± ±.7 a All data are reported as the average of three trials; calculated standard deviations are shown.

12 Figure S. Gel electrophoresis images of polyphenols under Fenton reaction conditions ( µm Fe µm H O ) in mm MES buffer (ph = 6.): a = ( )-epicatechin (EC), b = ( )-epicatechin-3-gallate (ECG), c = ( )-epigallocatechin (EGC), d = ( )-epigallocatechin-3-gallate (EGCG), e = gallic acid (GA), f = methyl-3,,5- trihydroxybenzoate (MEGA) % Agarose gel imaged with ethidium bromide. Control lanes: Lane : kb MW ladder, Lane : plasmid DNA (p), Lane 3: p + 5 µm H O, Lane : p + 5 µm EGCG + 5 µm H O, Lane 5: p + µm Fe µm H O. log Concentration of EGCG (µm) log Concentration of GA (µm) log Concentration of MEGA (µm) d e f log Concentration of EC (µm) log Concentration of ECG (µm) log Concentration of EGC (µm) a b c

13 Figure S (continued). Gel electrophoresis images of polyphenols under Fenton reaction conditions ( µm Fe µm H O ) in mm MES buffer (ph = 6.): g = methyl-3,-dihydroxybenzoate (MEPCA), h = myricetin (Myr), i = protocatechuic acid (PCA), j = n-propyl gallate (PrEGA), k = quercetin (Q), l = vanillic acid (VA). % Agarose gel imaged with ethidium bromide. Control lanes: Lane : kb MW ladder, Lane : plasmid DNA (p), Lane 3: p + 5 µm H O, Lane : p + 5 µm EGCG + 5 µm H O, Lane 5: p + µm Fe µm H O. log Concentration of PrEGA (µm) log Concentration of Q (µm) log Concentration VA (µm) j 6 8 k 6 8 l log Concentration of MEPCA (µm) log Concentration of Myr (µm) log Concentration of PCA (µm) g 6 8 h 6 8 i

14 Absorbance mm FeSO, ph 6. mm FeSO, ph Figure S3. UV-Vis spectra of mm FeSO solutions in 9 mm MES buffer, ph 6. (grey) or 9 mm MOPS buffer, ph 7. (black). At ph 6., iron(ii) is relatively stable compared to ph 7., where iron(ii) is readily oxidized and forms iron(iii) oxide and hydroxide precipitates. IC 5 (µm) Gallates Catecholates pk a Most Acidic Phenolic Hydrogen Figure S. Graph of IC 5 vs. pk a (first phenolic hydrogen) showing the best-fit linear correlation to the data for catecholate polyphenols (solid line, y = 8.5x , R =.89). The data point for quercetin was omitted from the catecholate data set because of its non-catechol binding site. Error bars for IC 5 values are within the size of the data points. Correlation of polyphenol oxidation and reduction potentials to IC 5 values. Oxidation potentials for all polyphenol compounds were observed in the cyclic voltammetry (CV) scans (Figure S), but reduction potentials were observed only for catecholate compounds as previously observed for some of these compounds by Kondo, et al. (See Kondo, K.; Kurihara, M.; Fukuhara, K. Mechanism of Antioxidant Effect of Catechins Methods Enzymol., 335, 3-7). The IC 5 values showed a weak linear correlation with the reduction potentials (Ep c ) of the catecholate compounds (Figure S5, y = -3.68x , R =.79). A similarly weak, though opposite, linear correlation with the catecholate oxidation potentials (Ep a ) was also observed (Figure S6, y = 35.3x , R =.76). No correlation of IC 5 to Ep a was observed for gallate compounds (R =.39), although these were generally more potent antioxidants than the catecholate compounds. IC 5 (µm) Reduction Potential, E p c vs. Ag/AgCl (V) Figure S5. Graph of IC 5 vs. reduction potential (Ep c ) showing the best linear fit for the catecholate compounds, including vanillic acid (R =.79). Gallate compounds did not display a reduction wave in the cyclic voltammetry experiments. Values were taken from Table. IC 5 (µm) Oxidation Potential, E p a vs. Ag/AgCl (V) Figure S6. Graph of IC 5 vs. oxidation potential (Ep a ) showing the best linear fit for the catecholate compounds, including vanillic acid (R =.76). Values were taken from Table.

15 IC 5 (µm) Octanol/Water Partition Coefficient (C logp ) Figure S7. IC 5 vs. ClogP for phenolic compounds. Octanol/water partition coefficient (ClogP) is a measure of lipophilicity of a compound. ClogP > implies greater solubility in -octanol than in water. IC 5 was defined as the concentration of phenolic compound (in µm) required to inhibit 5% of DNA damage observed in gel electrophoresis experiments. Values are taken from Table. No dependence upon lipophilicity was observed for IC 5 values obtained in this research. ClogP values were calculated using ChemDraw Ultra IC 5 (µm) k obs (min - ) Figure S8. A graph of IC 5 vs. initial rate of iron oxidation upon binding of polyphenols. For these experiments, a 3: polyphenol to iron(ii) molar ratio was used. Initial rates were obtained by fitting the linear portion of the absorbance versus time kinetics data. The slope of the best-fit line is reported as the rate of iron oxidation (k obs ) for each polyphenol compound. The solid line in the graph represents the best-fit exponential function (y = 6.856x , R =.79) and shows a weak correlation between IC 5 and iron oxidation rate. Values of IC 5 are taken from Table.

16 Tabulation of DNA band intensities from iron(ii)edta electrophoresis experiments with EGCG. The lane numbering for Table S3 follows similarly to those for free Fe + experiments. Table S3: Tabulation of gel electrophoresis results for ( )-epigallocatechin-3-gallate (EGCG) with µm iron(ii)edta and 5 µm H O in mm MES buffer (ph = 6.). a Conc. EGCG (µm) p None 89.7 ±.9.3 ±.9 N/A p p3 None ± ± ±.3 9. ±.73 N/A N/A Iron(II)EDTA + H O A None 37.6 ± ±.83 A A 3A. 37. ±.98.6 ±.9.6 ± ± ± ± ±.9.7 ±.9.83 ±.56 A.6 ± ±.6.6 ±.3 5A ± ±. -.8 ±.6 Iron(II)EDTA + H O B None 38. ± ± ± ±.95. ±.69 B 3. ± ± ± ± ±.7. ± ± ± ± ±.6 a Data are reported as the average of three trials; calculated standard deviations are shown. -. ±. -. ±. MW p p p3 A A A 3A A 5A B [EGCG] Figure S9. Gel electrophoresis image of ( )- epigallocatechin-3-gallate (EGCG), under µm iron(ii)edta + 5 µm H O reaction conditions in mm MES buffer (ph = 6.): % agarose gel imaged with ethidium bromide. Control lanes: MW = kb MW ladder, p = plasmid DNA (p), p = p + 5 µm H O, p3 = p + 5 µm EGCG + 5 µm H O, A = p + µm iron(ii)edta + 5 µm H O. Lanes A 5A, : Increasing concentration of EGCG:.,,,, 5,,, 3,, and 5 µm, respectively. Lanes A 5A, correspond to tabulation of data for EGCG/iron(II)EDTA (Table S).

17 Figure S. Cyclic voltammetry (CV) scans for polyphenols in phosphate buffer (6 mm final concentration, ph = 6.) containing KNO 3 (6 mm final concentration) as supporting electrolyte. Polyphenol solutions (375 µm final concentration) were cycled between -35 mv and 65 mv vs. Ag/AgCl/3M KCl using a glassy carbon working electrode and platinum wire counter electrode. The scan rate was mv/s. All solutions were prepared with deoxygenated ddh O. a = ( )-epicatechin (EC), b = ( )-epicatechin-3-gallate (ECG), c = ( )-epigallocatechin (EGC), d = ( )-epigallocatechin3-gallate (EGCG), e = gallic acid (GA), f = methyl-3,,5-trihydroxy benzoate (MEGA). -.E E E E-5 d -.E-5 e -8.E-6 f -8.E-6-8.E-6-6.E-6-6.E-6 Ep a =.93-6.E-6 -.E-6 -.E-6 Ep a =.33 Ep a =.93 -.E-6 -.E-6 -.E-6 -.E-6.E+.E+.E+.E-6.E-6.E-6 -.E E E E-5 a -8.E-6 b -.E-5 c -8.E-6 -.E-5-6.E-6-6.E-6 -.E-6 Ep a = E-6 -.E-6.E+ Ep a =.36 -.E-5-8.E-6-6.E-6 -.E-6 Ep a =.55 -.E-6.E-6 -.E-6.E+.E-6 Ep c =.6 Ep c =..E+ 6.E-6.E-6.E-6

18 Figure S (continued). Cyclic voltammetry (CV) scans for polyphenols in phosphate buffer (6 mm final concentration, ph = 6.) containing KNO 3 (6 mm final concentration) as supporting electrolyte. Polyphenol solutions (375 µm final concentration) were cycled between -35 mv and 65 mv vs. Ag/AgCl/3M KCl using a glassy carbon working electrode and platinum wire counter electrode. The scan rate was mv/s. All solutions were prepared with deoxygenated ddh O. g = methyl-3,-dihydroxybenzoate (MEPCA), h = myricetin (Myr), i = protocatechuic acid (PCA), j = n-propyl gallate (PrEGA), k = quercetin (Q), l = vanillic acid (VA). -.6E E E E-5 j -5.E-6 k -.E-5 -.E-6 Ep a =.5 -.E-5 l -8.E-6 -.E-5-8.E-6 Ep a =.88-3.E-6 -.E-6-6.E-6 -.E-6 -.E-6 Ep a =.5-6.E-6 -.E-6 -.E-6.E+ Ep a =.77 -.E-6.E-6 Ep c =.87.E+.E+.E-6.E-6.E-6 3.E-6 Ep c = -.93 Ep c = -.3.E-6 -.5E E E g -.E-5 h -8.E-6 i -.E-5-8.E-6-6.E-6-5.E-6 Ep a =.38-6.E-6 -.E-6.E+ -.E-6 Ep c =.9 -.E-6 Ep a =.69 Ep a = E-6.E+.E+.E-6 Ep c =.8 5.E-6.E-6.E-6.E-5.E-6 6.E-6

19 Absorbance a EC EC + 8 EGCG d EGCG b ECG ECG + 8 e GA GA + 8 Absorbance Absorbance Absorbance Absorbance c EGC EGC + 8 f MEGA MEGA + 8 Figure S. UV-vis spectra for 9 µm phenolic compounds + 5 µm Fe+: a = ( )-epicatechin (EC), b = ( )-epicatechin-3-gallate (ECG), c = ( )-epigallocatechin (EGC), d = ( )epigallocatechin3-gallate (EGCG), e = gallic acid (GA), f = methyl-3,,5-trihydroxy benzoate (MEGA). All solutions were prepared in 5 mm MES buffer (ph = 6.); the blank MES spectrum was subtracted from each sample spectrum. Absorbance

20 Absorbance g MEPCA MEPCA + 8 j PCA PCA h Myr Myr + 8 k Q Q + 8 Absorbance Absorbance Absorbance Absorbance i 85 l VA VA + 8 PrEGA PrEGA + 75 Figure S (continued). UV-vis spectra for 9 µm phenolic compounds + 5 µm Fe+: g = methyl-3,-dihydroxybenzoate (MEPCA), h = myricetin (Myr), i = n-propyl gallate (PrEGA), j = protocatechuic acid (PCA), k = quercetin (Q), l = vanillic acid (VA). All solutions were prepared in 5 mm MES buffer (ph = 6.); the blank MES spectrum was subtracted from each sample spectrum. Absorbance