Highly Sensitive Fluorescence Molecular Switch for the Ratio Monitoring of Trace Change of Mitochondrial Membrane Potential Caixia Wang, Ge Wang, Xiang Li, Kui Wang, Jing Fan, Kai Jiang,, Yuming Guo, Hua Zhang *, Key Laboratory of Green Chemical Media and Reactions, Ministry of Education; Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals; School of Chemistry and Chemical Engineering, Henan Normal University; Xinxiang Medical University; Key Laboratory for Yellow River and Huai River Water Environment and Pollution Control, Ministry of Education; School of Environment, Henan Normal University; Xinxiang, 453, P.R. China. Corresponding Author Email: zhh116@htu.edu.cn, zhanghua116@163.com, Tel/Fax: +86-373-33293. Contents 1. Synthetic procedures of probe and intermediates 2. Spectral properties of probe to sodium-dodecyl sulphate 3. Quantum calculations 4. Specific selectivity of probe for sodium-dodecyl sulphate 5. The photostability of probe and MitoTracker Red CMXRos 6. Cell viability of probe 7. The spectrum extraction of different concentrition with catechol in living cancer cells 8. ph effect on the emission spectra of the TPP-CY/TPP-SP 9. Incubation and staining of living cells with probe Attached Figure: 1 H and 13 C NMR spectra of probe and intermediates. Supporting Information-Video 1: The dynamic imaging of HepG2 cells that were treated with the membrane-potential uncoupler 3-chlorophenylhydrazone (CCCP,.5mM) with AVT (*. avi). S-1
1. Synthetic procedures of probe and intermediates Figure S1. Synthetic procedures of probe and intermediates. (1) 2,3,3-Trimethylindolenine, iodoethane, reflux, 85%; (2) HNO 3, CH 3 COOH, room temperature, 96%; (3) Compound a, 5-nitrosalicylaldehyde, reflux, 74%; (4) Compound SP, triphenylphosphine, 4%; Synthesis of Compound SP Compound a was prepared by the literature methods Figure S1. To a solution of (2.4 mmol) in anhydrous ethanol (1 ml) was stirred at 8 C for 4 h, and the Compound a in 1 ml was dropwise added into, simultaneously. The mixture was cooled to room temperature, and precipitate solids, washed with little anhydrous ethanol. Yield: 74 %; mp: 111 C; 1 H NMR (6 MHz, DMSO) δ 8.67 (s, 1H), 8.1 (d, J = 8.8 Hz, 1H), 7.99 (d, J = 16.4 Hz, 1H), 7.56-7.43 (m, 3H), 7.32 (t, J = 7.5 Hz, 1H), 7.23 (t, J = 7.3 Hz, 1H), 7.7 (d, J = 9. Hz, 1H), 1.41 (s, 6H), 1.23 (s, 4H). 13 C NMR (1 MHz, DMSO) δ: 183.9, 154., 147.1, 131.5, 128., 126.4, 125.9, 124.3, 123.8 122., 12.6, 52.4, 4., 39.6, 39.4, 37.4, 23.2. ESI-MS: m/z calcd for C 2 H 19 BrN 2 O 3 : 414.579, found: 414.585. Synthesis of the probe Compound TPP (1. mmol), Compound SP (3.5 mmol) and K 2 CO 3 (1.2 mol) in THF (1 ml) were refluxed under nitrogen for 8 h. After the mixture was cooled to room temperature, concentrated hydrochloric acid (45 ml) was added dropwise under nitrogen. The solvent was evaporated and the crude product was purified by column chromatography using CH 2 Cl 2 / petroleum ether (3:5-1:9) as the eluent to give Compound probe. Yield: 53 %; mp: 167 C; 1 H NMR (6 MHz, DMSO) δ: 8.42-7.42 (m, 18H), 7.41-6.77 (m, 3H), 6.76-6.47 (m, 1H), 5.18 (d, J = 174.7 Hz, 1H), 4.18-4.13 (m, 2H), 3.93 (d, J = 5.4 Hz, 2H), 3.51 (s, 1H), 2.27 (t, J = 6.8 Hz, 4H), 2.6-1.91 (m, 2H). 13 C NMR (1 MHz, DMSO) δ: 183.9, 154.1, 147.1, 135.8, 135.3, 134.9, 131.4, 13.8, 13.5, 13., 128.2, 125.9, 124.3, 123.8 122., 121.7, 117.8, 117.6, 117.2, 52.4, 4., 39.4, 38.7, 23.2. ESI-MS: m/z calcd for C 38 H 34 N 2 O 3 P + : 597.237, found: 597.2299. S-2
2. Spectral properties of probe to sodium-dodecyl sulphate (SDS) 3 a 25 2 15 1 5 3 2 1 d 6 65 7 75 55 6 65 7 75 I 563 12 g 1 8 6 4 SDS(mM) 4.2 5.1 6. 6.9 7.8 8.7 9.6 16 s 2 15 1 5 1 b SDS(mM).8 1.7 2.4 3.3 4.2 64 66 68 7 72 74 3 e SDS(mM) 4.2 25 5.1 6. 2 6.9 7.8 15 8.7 9.6 5 66 68 7 72 74 76 78 Absorbance.8.6.4.2 h I 563 I 663 25 c 2 15 1 2 15 1 5 5 I 563 = 3.98+7.95C SDS R 2 =.99..5 1. 1.5 2. 2.5 3. C SDS /mm f I 563 = 2.81+.78C SDS R 2 =.99 1 2 3 4 5 6 CSDS/mM TPP-SP+SDS (6. mm) TPP-CY TPP-SP 2 2 4 6 8 1 12 Time / s. 46 48 5 52 54 56 58 Figure S2. Probe (3.3 µm) recongnize the different concentrations of SDS. (a) Probe (C = 3.3 µm, ε = 14375) appears a low fluorescence (Φfree state =.3) of black line, and its fluorescence quantum yield Φ =.11 with 8.82 mm SDS of red line. (b) Fluorescence intensity of probe response to SDS (-4.2 mm). (c) The linear fluorescent response of probe to SDS (-4.2 mm) in secondary water, I 663 = 3.98 + 7.95C SDS (R 2 =.99). (d) Emission spectra of 3.3 µm probe mixtures with different concentrations of SDS (4.2-9.6 mm); (e) Emission spectra of 3.3 µm probe mixtures with different concentrations of SDS (4.2-9.6 mm); (f) The linear fluorescent response of probe to SDS (-5.1 mm) in secondary water. I 563 = 2.81 +.78 C SDS (R 2 =.99). (g) Reponse time of probe to SDS (4.2 mm). (h) the absorbance spectre of probe to SDS (6. mm). S-3
3. Quantum calculations The Gaussian 9 suite was used to obtain the data of quantum chemical calculations. The density functional theory (DFT) with Becke s three-parameter hybrid exchange function used to perform the geometry optimizations of probe and SP. C(9)-O(12): length:1.435; angles: 17.94 11.5. C(9)-O(15):Length: 1.429; angles: 15.952 11.5. Figure S3. Geometry optimizations of the probe and SP. 4. Specific selectivity of probe for sodium-dodecyl sulphate (SDS) I 563 /I 663 7 a 6 5 1 2 3 4 5 6 7 8 9 111121314151617 4 3 2 1 5 1 15 2 SDS/Zeph 5 b ZEPH SDS Zeta Potential/mv -14 c -12-1 -8-6 -4-2 Zeta Potential = -1.97-1.41C SDS R 2 = -.99 1 2 3 4 5 6 7 8 CSDS/mM Figure S4. (a) The influence of common coexisting substances for probe under 54nm. 1, Glycyl-DL-phenylalanine (.1 mm); 2, Lysine (.1 mm); 3, DL-Threonine (.1 mm); 4, Glutamine (Gln,.1 mm); 5, Cystine (.1 mm); 6, Arginine (.1 mm); 7, Glycyl-DL- tyrosine (.1 mm); 8, DL-methionine (.1 mm); 9, D-cysteine (.1 mm); 1, Glycine (.1 mm); 11, DL-leucine (.1 mm); 12, Serine (Ser,.1 mm); 13, Homocysteine (.1 mm); 14, Dithiothreitol (DTT,.1 mm); 15, Glutamic acid (.1 mm); 16, 6-Hydroxypurine (.1 mm); 17, L-aspartic acid (.1 mm); 18, SDS (.1 mm). (b) The fluorescence intensity of probe respond to cationic surfactant (Benzalkonium Chloride, Zeph) and anion surfactant (SDS). (c) The linear fluorescent response of zeta potential to SDS (R 2 = -.99). S-4
5. The photostability of probe and MitoTracker Red CMXRos 55 5 45 4 35 3 5 1 15 2 Time/s Figure S5. The green line is probe and the red line is MitoTracker Red CMXRos. The fluorecence intencity were detected by LS55. 6. Cell viability of probe HepG2, MCF-7, CHO, 293T, L929 and HeLa cell were prepared for cell viability studies in 96 well plates (1 1 5 cells per well that were incubated in 1 µl). The cells were incubated for an additional 24 h in different concentrations of probe. Subsequently, 2 µl of 5 mg/ml 3 - (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma Chemical Co. USA) was added into each well, followed by further incubation for 4 h at 37 C. After DMEM was removed, DMSO (15 µl /well) was added to dissolve the reddish blue crystals of formazan. Optical density was determined on a Microplate Reader (Spectra Max M5, Molecular Devices) at 57 nm with subtraction of the absorbance of the cell free blank volume at 63 nm. The results from the six individual experiments were averaged. The relative cell viability (%) was calculated using the following equation: cells viability (%) = (OD dye ODK dye ) / (OD contral -ODK contral ) 1 Survival Rate / % 1..5 MCF-7 CHO HePG2 HeLa 293T L929. 1X 1X Figure S6. Cell viability. Data were obtained from replicate experiments (n = 5). S-5
7. The spectrum extraction of different concentrition with catechol in living cancer cells. 1 a 25 b 8 6 4 2 2 15 1 5 55 6 65 7 55 6 65 7 Figure S7. The spectrum extraction of living cells that were stained by catechol with the same time (36 h), but different concentration (a:.1 mm and b: 2. mm) in the apoptotic model. 8. ph effect on the emission spectra of the TPP-CY/TPP-SP 8 6 4 2 ph 11.7 8.8 7.8 7. 6.5 5. -2 56 58 6 62 64 66 68 7 Figure S8. The fluorescence intensities in PBS buffer as a function of ph (5. - 11.7). Solution of ph 5.:.2 mol/l KH 2 PO 3, ph was adjusted to 5. by.2 mol/l NaOH; Solution of ph 6.5: KH 2 PO 3.68 g, ph was adjusted to 5. by.1 mol/l NaOH; Solution of ph 7.: KH 2 PO 3.68 g, ph was adjusted to 5. by.1 mol/l NaOH; Solution of ph 7.8: (1) Na 2 HPO 3 35.9 g was diluted by H 2 O (.5 L), ph was adjusted to 5. by.1 mol/l NaOH; (2) NaH 2 PO 3 2.76 g was diluted by H 2 O (.1 L), then, 91.5ml of solution; (3) and 8.5 1-3 L of solution; (4) Solution of ph 8.8:.2 mol/l KH 2 PO 3, ph was adjusted to 8.8 by.1 mol/l NaOH; Solution of ph 1.3:.1 mol/l KH 2 PO 3, ph was adjusted to 1.3 by.1 mol/l NaOH; Solution of ph 11.7:.1mol/L KH 2 PO 3, ph was adjusted to 11.7 by.1 mol/l NaOH. S-6
9. Incubation and staining of living cells with probe Figure S9. The cells were stained by different concentration (.5,.1,.23 and 3.3 µm) of probe at 5. min. S-7
Attached Figure: 1 H and 13 C NMR spectra of probe and intermediates S-8
S-9
Length and angles of probe: C(1)-C(2) 1.4 1.395 C(1)-C(6) 1.397 1.392 C(1)-H(76) 1.12 1.1 C(2)-C(3) 1.395 1.39 C(2)-H(75) 1.12 1.1 C(3)-C(4) 1.43 1.41 C(3)-H(78) 1.99 1.1 C(4)-C(5) 1.397 1.399 C(4)-N(7) 1.391 1.392 C(5)-C(6) 1.396 1.392 C(5)-C(8) 1.513 1.497 C(6)-H(77) 1.11 1.1 N(7)-C(9) 1.49 1.47 N(7)-C(25) 1.471 1.47 C(8)-C(9) 1.566 1.514 C(8)-C(1) 1.544 1.523 C(8)-C(11) 1.54 1.523 C(9)-O(12) 1.43 1.42 C(9)-C(16) 1.513 1.497 C(1)-H(48) 1.112 1.113 C(1)-H(49) 1.114 1.113 C(1)-H(5) 1.113 1.113 C(11)-H(45) 1.113 1.113 C(11)-H(46) 1.113 1.113 C(11)-H(47) 1.113 1.113 O(12)-C(13) 1.372 1.355 O(12)-Lp(79).6.6 O(12)-Lp(8).596.6 C(13)-C(14) 1.511 1.51 C(13)-C(17) 1.57 1.495 C(14)-C(15) 1.5 1.52 C(14)-C(2) 1.456 1.454 C(15)-C(16) 1.5 1.53 C(15)-H(7) 1.1 1.1 C(16)-H(72) 1.12 1.1 C(17)-C(18) 1.359 1.356 C(17)-H(71) 1.13 1.1 C(18)-C(19) 1.58 1.491 C(18)-H(68) 1.13 1.1 C(19)-C(2) 1.521 1.51 C(19)-N(21) 2.198 1.496 C(2)-H(69) 1.1 1.1 N(21)-O(22) 1.172 N(21)-O(23) 1.539 P(24)-C(26) 1.839 1.856 P(24)-C(27) 1.847 1.856 P(24)-C(28) 1.846 1.856 P(24)-C(29) 1.848 1.856 C(25)-C(26) 1.534 1.523 C(25)-H(73) 1.115 1.113 C(25)-H(74) 1.113 1.113 C(26)-H(66) 1.116 1.113 C(26)-H(67) 1.113 1.113 S-1
C(27)-C(35) 1.398 1.392 C(27)-C(39) 1.399 1.392 C(28)-C(3) 1.399 1.392 C(28)-C(34) 1.399 1.392 C(29)-C(4) 1.398 1.392 C(29)-C(44) 1.399 1.392 C(3)-C(31) 1.396 1.392 C(3)-H(65) 1.1 1.1 C(31)-C(32) 1.396 1.392 C(31)-H(63) 1.12 1.1 C(32)-C(33) 1.396 1.392 C(32)-H(62) 1.12 1.1 C(33)-C(34) 1.397 1.392 C(33)-H(61) 1.12 1.1 C(34)-H(64) 1.12 1.1 C(35)-C(36) 1.397 1.392 C(35)-H(6) 1.12 1.1 C(36)-C(37) 1.396 1.392 C(36)-H(58) 1.12 1.1 C(37)-C(38) 1.396 1.392 C(37)-H(57) 1.12 1.1 C(38)-C(39) 1.396 1.392 C(38)-H(56) 1.12 1.1 C(39)-H(59) 1.12 1.1 C(4)-C(41) 1.397 1.392 C(4)-H(55) 1.11 1.1 C(41)-C(42) 1.396 1.392 C(41)-H(53) 1.12 1.1 C(42)-C(43) 1.396 1.392 C(42)-H(52) 1.12 1.1 C(43)-C(44) 1.396 1.392 C(43)-H(51) 1.12 1.1 C(44)-H(54) 1.12 1.1 C(1)-C(2) 1.4 1.395 C(1)-C(6) 1.397 1.392 C(1)-H(76) 1.12 1.1 C(2)-C(3) 1.395 1.39 C(2)-H(75) 1.12 1.1 C(3)-C(4) 1.43 1.41 C(3)-H(78) 1.99 1.1 C(4)-C(5) 1.397 1.399 C(4)-N(7) 1.391 1.392 C(5)-C(6) 1.396 1.392 C(5)-C(8) 1.513 1.497 C(6)-H(77) 1.11 1.1 N(7)-C(9) 1.49 1.47 N(7)-C(25) 1.471 1.47 C(8)-C(9) 1.566 1.514 C(8)-C(1) 1.544 1.523 C(8)-C(11) 1.54 1.523 C(9)-O(12) 1.43 1.42 C(9)-C(16) 1.513 1.497 C(1)-H(48) 1.112 1.113 C(1)-H(49) 1.114 1.113 C(1)-H(5) 1.113 1.113 S-11
C(11)-H(45) 1.113 1.113 C(11)-H(46) 1.113 1.113 C(11)-H(47) 1.113 1.113 O(12)-C(13) 1.372 1.355 O(12)-Lp(79).6.6 O(12)-Lp(8).596.6 C(13)-C(14) 1.511 1.51 C(13)-C(17) 1.57 1.495 C(14)-C(15) 1.5 1.52 C(14)-C(2) 1.456 1.454 C(15)-C(16) 1.5 1.53 C(15)-H(7) 1.1 1.1 C(16)-H(72) 1.12 1.1 C(17)-C(18) 1.359 1.356 C(17)-H(71) 1.13 1.1 C(18)-C(19) 1.58 1.491 C(18)-H(68) 1.13 1.1 C(19)-C(2) 1.521 1.51 C(19)-N(21) 2.198 1.496 C(2)-H(69) 1.1 1.1 N(21)-O(22) 1.172 N(21)-O(23) 1.539 P(24)-C(26) 1.839 1.856 P(24)-C(27) 1.847 1.856 P(24)-C(28) 1.846 1.856 P(24)-C(29) 1.848 1.856 C(25)-C(26) 1.534 1.523 C(25)-H(73) 1.115 1.113 C(25)-H(74) 1.113 1.113 C(26)-H(66) 1.116 1.113 C(26)-H(67) 1.113 1.113 C(27)-C(35) 1.398 1.392 C(27)-C(39) 1.399 1.392 C(28)-C(3) 1.399 1.392 C(28)-C(34) 1.399 1.392 C(29)-C(4) 1.398 1.392 C(29)-C(44) 1.399 1.392 C(3)-C(31) 1.396 1.392 C(3)-H(65) 1.1 1.1 C(31)-C(32) 1.396 1.392 C(31)-H(63) 1.12 1.1 C(32)-C(33) 1.396 1.392 C(32)-H(62) 1.12 1.1 C(33)-C(34) 1.397 1.392 C(33)-H(61) 1.12 1.1 C(34)-H(64) 1.12 1.1 C(35)-C(36) 1.397 1.392 C(35)-H(6) 1.12 1.1 C(36)-C(37) 1.396 1.392 C(36)-H(58) 1.12 1.1 C(37)-C(38) 1.396 1.392 C(37)-H(57) 1.12 1.1 C(38)-C(39) 1.396 1.392 C(38)-H(56) 1.12 1.1 C(39)-H(59) 1.12 1.1 S-12
C(4)-C(41) 1.397 1.392 C(4)-H(55) 1.11 1.1 C(41)-C(42) 1.396 1.392 C(41)-H(53) 1.12 1.1 C(42)-C(43) 1.396 1.392 C(42)-H(52) 1.12 1.1 C(43)-C(44) 1.396 1.392 C(43)-H(51) 1.12 1.1 C(44)-H(54) 1.12 1.1 C(2)-C(1)-C(6) 12.471 12. C(2)-C(1)-H(76) 119.778 12. C(6)-C(1)-H(76) 119.745 12. C(1)-C(2)-C(3) 12.463 12. C(1)-C(2)-H(75) 119.744 12. C(3)-C(2)-H(75) 119.792 12. C(2)-C(3)-C(4) 118.938 12. C(2)-C(3)-H(78) 119.525 12. C(4)-C(3)-H(78) 121.535 12. C(3)-C(4)-C(5) 12.577 12. C(3)-C(4)-N(7) 13.281 12. C(5)-C(4)-N(7) 19.137 12. C(4)-C(5)-C(6) 12.288 12. C(4)-C(5)-C(8) 112.559 121.4 C(6)-C(5)-C(8) 127.78 121.4 C(1)-C(6)-C(5) 119.255 12. C(1)-C(6)-H(77) 119.799 12. C(5)-C(6)-H(77) 12.938 12. C(4)-N(7)-C(9) 11.236 18. C(4)-N(7)-C(25) 121.14 18. C(9)-N(7)-C(25) 128.568 C(5)-C(8)-C(9) 1.752 19.47 C(5)-C(8)-C(1) 16.86 19.47 C(5)-C(8)-C(11) 11.67 19.47 C(9)-C(8)-C(1) 114.261 19.47 C(9)-C(8)-C(11) 115.97 19.47 C(1)-C(8)-C(11) 19.21 19.47 N(7)-C(9)-C(8) 15.654 11.74 N(7)-C(9)-O(12) 17.94 11.5 N(7)-C(9)-C(16) 17.494 11.3 C(8)-C(9)-O(12) 111.787 17.5 C(8)-C(9)-C(16) 112.419 19.47 O(12)-C(9)-C(16) 111.197 19.5 C(8)-C(1)-H(48) 111.354 11. C(8)-C(1)-H(49) 112.78 11. C(8)-C(1)-H(5) 111.766 11. H(48)-C(1)-H(49) 18.566 19. H(48)-C(1)-H(5) 15.995 19. H(49)-C(1)-H(5) 16.777 19. C(8)-C(11)-H(45) 111.195 11. C(8)-C(11)-H(46) 111.831 11. C(8)-C(11)-H(47) 112.139 11. H(45)-C(11)-H(46) 16.66 19. H(45)-C(11)-H(47) 18.83 19. H(46)-C(11)-H(47) 16.477 19. C(9)-O(12)-C(13) 118.972 11.8 S-13
C(9)-O(12)-Lp(79) 13.288 15.36 C(9)-O(12)-Lp(8) 14.75 15.36 C(13)-O(12)-Lp(79) 1.8 13.26 C(13)-O(12)-Lp(8) 13.422 13.26 Lp(79)-O(12)-Lp(8) 127.12 131. O(12)-C(13)-C(14) 122.899 124.3 O(12)-C(13)-C(17) 119.174 124.3 C(14)-C(13)-C(17) 112.789 12. C(13)-C(14)-C(15) 117.697 12. C(13)-C(14)-C(2) 12.72 12. C(15)-C(14)-C(2) 121.433 12. C(14)-C(15)-C(16) 113.71 12. C(14)-C(15)-H(7) 121.354 12. C(16)-C(15)-H(7) 12.159 12. C(9)-C(16)-C(15) 119.74 122. C(9)-C(16)-H(72) 119.129 118.2 C(15)-C(16)-H(72) 118.185 12. C(13)-C(17)-C(18) 122.476 12. C(13)-C(17)-H(71) 118.338 12. C(18)-C(17)-H(71) 118.675 12. C(17)-C(18)-C(19) 123.482 12. C(17)-C(18)-H(68) 119.98 12. C(19)-C(18)-H(68) 117.359 12. C(18)-C(19)-C(2) 18.239 12. C(18)-C(19)-N(21) 12.874 12. C(2)-C(19)-N(21) 17.168 12. C(14)-C(2)-C(19) 12.412 12. C(14)-C(2)-H(69) 119.549 12. C(19)-C(2)-H(69) 12.29 12. C(19)-N(21)-O(22) 9.74 C(19)-N(21)-O(23) 97.13 O(22)-N(21)-O(23) 83.866 C(26)-P(24)-C(27) 17.33 12.6 C(26)-P(24)-C(28) 111.94 12.6 C(26)-P(24)-C(29) 11.998 12.6 C(27)-P(24)-C(28) 111.334 12.6 C(27)-P(24)-C(29) 18.376 12.6 C(28)-P(24)-C(29) 16.98 12.6 N(7)-C(25)-C(26) 112.8 11.74 N(7)-C(25)-H(73) 17.24 17.5 N(7)-C(25)-H(74) 18.781 17.5 C(26)-C(25)-H(73) 19.976 19.41 C(26)-C(25)-H(74) 112.138 19.41 H(73)-C(25)-H(74) 16.646 19.4 P(24)-C(26)-C(25) 113.552 111.5 P(24)-C(26)-H(66) 18.531 111. P(24)-C(26)-H(67) 19.528 111. C(25)-C(26)-H(66) 18.598 19.41 C(25)-C(26)-H(67) 19.566 19.41 H(66)-C(26)-H(67) 16.839 19.4 P(24)-C(27)-C(35) 121.21 12. P(24)-C(27)-C(39) 119.62 12. C(35)-C(27)-C(39) 119.72 12. P(24)-C(28)-C(3) 12.78 12. P(24)-C(28)-C(34) 12.528 12. S-14
C(3)-C(28)-C(34) 118.631 12. P(24)-C(29)-C(4) 121.775 12. P(24)-C(29)-C(44) 119.238 12. C(4)-C(29)-C(44) 118.985 12. C(28)-C(3)-C(31) 12.829 12. C(28)-C(3)-H(65) 12.614 12. C(31)-C(3)-H(65) 118.556 12. C(3)-C(31)-C(32) 12.31 12. C(3)-C(31)-H(63) 12.3 12. C(32)-C(31)-H(63) 119.935 12. C(31)-C(32)-C(33) 119.583 12. C(31)-C(32)-H(62) 12.215 12. C(33)-C(32)-H(62) 12.191 12. C(32)-C(33)-C(34) 12.129 12. C(32)-C(33)-H(61) 119.855 12. C(34)-C(33)-H(61) 12.14 12. C(28)-C(34)-C(33) 12.717 12. C(28)-C(34)-H(64) 12.663 12. C(33)-C(34)-H(64) 118.619 12. C(27)-C(35)-C(36) 12.466 12. C(27)-C(35)-H(6) 12.643 12. C(36)-C(35)-H(6) 118.891 12. C(35)-C(36)-C(37) 12.95 12. C(35)-C(36)-H(58) 12.12 12. C(37)-C(36)-H(58) 119.891 12. C(36)-C(37)-C(38) 119.718 12. C(36)-C(37)-H(57) 12.154 12. C(38)-C(37)-H(57) 12.121 12. C(37)-C(38)-C(39) 12.48 12. C(37)-C(38)-H(56) 119.943 12. C(39)-C(38)-H(56) 12.8 12. C(27)-C(39)-C(38) 12.524 12. C(27)-C(39)-H(59) 12.583 12. C(38)-C(39)-H(59) 118.893 12. C(29)-C(4)-C(41) 12.526 12. C(29)-C(4)-H(55) 12.976 12. C(41)-C(4)-H(55) 118.498 12. C(4)-C(41)-C(42) 12.145 12. C(4)-C(41)-H(53) 119.973 12. C(42)-C(41)-H(53) 119.882 12. C(41)-C(42)-C(43) 119.681 12. C(41)-C(42)-H(52) 12.164 12. C(43)-C(42)-H(52) 12.155 12. C(42)-C(43)-C(44) 12.36 12. C(42)-C(43)-H(51) 119.936 12. C(44)-C(43)-H(51) 12.27 12. C(29)-C(44)-C(43) 12.627 12. C(29)-C(44)-H(54) 12.574 12. C(43)-C(44)-H(54) 118.8 12. S-15