Supporting Information

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1 Supporting Information Wiley-VCH Weinheim, Germany

2 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 1 ucleoside Containing a Rigid itroxide Spin Label: A Fluorophore in Disguise ivrutti Barhate, Pavol Cekan, Archna P. Massey, and Snorri Th. Sigurdsson* Department of Chemistry, University of Iceland, Science Institute, Dunhaga 3, 107 Reykjavik, Iceland Table of Contents General synthetic procedures... page S2 List of abbreviations... page S2 rganic synthesis procedures... page S3 DA synthesis and purification... page S13 Reduction of spin-labeled DA... page S13 EPR measurements... page S14 Steady-state fluorescence and absorption spectra... page S14 Quantum yield determination... page S16 Simulation of EPR spectrum... page S16 References... page S17

3 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 2 General synthetic procedures. All air or water sensitive reactions were performed in flame-dried glassware under a positive pressure of argon. All commercial reagents were used without further purification. Dichloromethane, acetonitrile and pyridine were freshly distilled over calcium hydride under nitrogen. Water was purified on a MILLI-Q water purification system. eutral silica gel was purchased from Silicycle. Analytical thin layer chromatography (TLC) was performed on glass plates (Merck Kieselgel 60 F 254 ). MR spectra for all organic compounds were recorded on DRX 500 MHz Bruker MR spectrometer and the chemical shifts were reported relative to the deuterated MR solvent used [ 1 H-MR: CDCl 3 (7.26 ppm), MeH-d 4 (4.84 and 3.31 ppm), DMS-d 6 (2.50 ppm); 13 C-MR: CDCl 3 (77.0 ppm), MeH-d 4 (49.1 ppm), DMS-d 6 (39.7 ppm)]. Commercial grade CDCl 3 was passed over basic alumina shortly before use. Mass spectrometric analyses of all organic compounds were performed on an Esquire-LC electrospray ion trap mass spectrometer (Bruker/Hewlett-Packard) in positive or negative mode. High resolution (HRMS), fast atom bombardment (FAB) mass spectra were obtained on a JEL HX-110 mass spectrometer. UV-Vis spectra were recorded on Perkin Elmer Lambda 25 UV/VIS spectrometer. Fluorescence spectra were recorded on SPEX FluoroMax spectrometer. EPR spectra were recorded on a 9.4 GHz Bruker EMX spectrometer. List of abbreviations. DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene TEA Triethyl amine DMAP 4-Dimethylaminopyridine DMTrCl 4,4 -Dimethoxytrityl chloride DTT 1,4-Dithio-DL-threitol TE buffer 250 mm acl, 10 mm Tris, 1 mm a 2 EDTA, ph 7.5 PE buffer 10 mm Phosphate, 100 mm acl, 0.1 mm a 2 EDTA, ph 7.0 DPAGE Denaturing polyacrylamide gel electrophoresis

4 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 3 rganic synthesis procedures. H % H 2 S 4 H 2. a 2 H 3. urea, reflux 3 100% 4 H 5-Hydroxy-1,1,3,3-tetramethylisoindoline (4). 5-Amino-1,1,3,3-tetramethylisoindoline 3 (25.0 g, mol) was dissolved in H 2 S 4 (35% w/v, 63 ml) and cooled to -10 C. An ice-cold solution of a 2 (10.9 g, mol) in water (109 ml) was slowly added and the resultant solution was stirred at -10 C for 2 h. The progress of diazo-intermediate formation was monitored by removing a small aliquot and recording its 1 H-MR spectrum in D 2. After completion of the diazotization reaction, urea (1.6 g, mol) was added, the reaction mixture was stirred for 15 min at -10 C, allowed to warm to 22 C and then heated at reflux for 4 h. After cooling to -5 C and neutralizing (to ph ) with ah (aq) (30% w/v), the salts were filtered, washed with MeH (2 250 ml) and discarded. The filtrate was concentrated in vacuo and the product was extracted from the crude mixture with 20% MeH/CH 2 Cl 2 (2 500 ml) and 40% MeH/CH 2 Cl 2 (2 500 ml). The solvent was removed in vacuo to yield a white solid (28.1 g) that was used in the next step without further purification. 1 H-MR (MeH-d 4 ): δ 1.76 (s, 12H, 4 CH 3 ), 6.70 (s, 1H, ArH), 6.88 (d, J = 8 Hz, 1H, ArH), 7.16 (d, J = 8 Hz, 1H, ArH) ppm. 13 C-MR (MeH-d 4 ): δ 28.02, 28.36, 68.28, 68.39, , , , , , ppm. MS (ESI, +ve mode): (M+1). HRMS-FAB (M+H + ): calcd. for C 12 H , found

5 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 4 H 2 H 70% H 3-10 to -3 o C H 45% 4 5 H 5-Hydroxy-6-nitro-1,1,3,3-tetramethylisoindoline (5). A pre-cooled H 3 solution (70%, 75 ml, -5 C) was slowly added to 4 (18.0 g) at -5 o C (± 2 C) and the reaction mixture was stirred at -2 to 0 C for 2 h. The progress of reaction was monitored by removing a small aliquot and recording its 1 H-MR spectrum in D 2. After completion of reaction, the ph was adjusted to 7.0 by addition of ah (aq) (40% w/v). The inorganic salts were filtered, washed with MeH (2 x 300 ml) and discarded. The filtrate was concentrated in vacuo to give mixture of regioisomers. The desired product was purified by column chromatography using neutral silica gel (gradient 100:0 to 75:25, CH 2 Cl 2 :MeH) to yield 5 as a dark yellow solid (10.2 g, 45%, three steps). 1 H-MR (CDCl 3 ): δ 1.47 (s, 12H, 4 CH 3 ), 6.87 (s, 1H, ArH), 7.83 (s, 1H, ArH) ppm. 13 C-MR (CDCl 3 ): δ 31.31, 31.85, 62.51, 63.16, , , , , , ppm. MS (ESI, -ve mode): 235.8, (M+23). HRMS-FAB (M+H + ): calcd. for C 12 H , found

6 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 5 2 H H 2, Pd/C H 2 H MeH 100% H 5 6 H 5-Hydroxy-6-amino-1,1,3,3-tetramethylisoindoline (6). A solution of compound 5 (4.0 g, mol) in MeH (200 ml) containing 10% Pd/C (400 mg) was hydrogenated at 55 psi for 6 h. The reaction mixture was filtered through a pad of celite and the filtrate was concentrated in vacuo to yield 6 as a white solid (3.5 g, 100 %). 1 H-MR (CDCl 3 ): δ 1.40 (s, 6H, 2 CH 3 ), 1.41 (s, 6H, 2 CH 3 ), 4.10 (bs, 2H, H 2 ), 6.50 (s, 1H, ArH), 6.52 (s, 1H, ArH) ppm. 13 C-MR (CDCl 3 ): 31.64, (d, J = 18 Hz), , , , , , ppm. MS (ESI, -ve mode): HRMS-FAB (M+H + ): calcd. for C 12 H , found

7 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 6 Ac Br Ac H PPh 3, CCl 4 CH 2 Cl 2 6 DBU 52% H Ac Br H H Ac 7 3,5 -Diacetyl-5-bromo- 4 -(6-hydroxy-1,1,3,3-tetramethylisoindolinyl)-2 -deoxycytidine (7). PPh 3 (8.91 g, mmol) was added to a solution of 50% CCl 4 /CH 2 Cl 2 (120 ml) and the solution was stirred for 15 min at 22 C, after which 3,5 -diacetyl-5-bromo-2 -deoxyuridine (6.95 g, mmol) was added. The resulting solution was refluxed for 4 h, cooled to 22 C and treated with a solution of freshly prepared 6 (3.49 g, mmol) and DBU (2.70 g, mmol, 2.68 ml) in 50% CCl 4 /CH 2 Cl 2 (30 ml). After stirring at 22 C for 16 h, the reaction mixture was concentrated in vacuo and purified by column chromatography using neutral silica gel (hexane (1000 ml); 50% hexane/ethyl acetate (1000 ml); gradient 100:0 to 90:10, CH 2 Cl 2 :MeH). Upon trituration with acetonitrile (45 ml) the product solidified. Filtration yielded 7 as a white solid (5.08 g, 52%). 1 H-MR (MeH-d 4 ): δ 1.73 (s, 6H, 2 CH 3 ), 1.78 (s, 6H, 2 CH 3 ), 2.12, 2.15 (2 s, 6H, 2 CCH 3 ), 2.36 (m, 1H, H2 ), 2.66 (m, 1H, H2 ), 4.39 (m, 3H, H5 and H3 ), 5.29 (m, 1H, H4 ), 6.25 (t, J = 6 Hz, 1H, H1 ), 6.77 (s, 1H, ArH), 8.18 (s, 1H, ArH), 8.60 (s, 1H, H6) ppm. 13 C-MR (MeH-d 4 ): δ 19.90, 19.95, 28.13, 28.20, 38.22, 63.93, 68.35, 68.94, 74.80, 83.42, 87.71, 89.80, , , , , , , , , , ppm. MS (ESI, -ve mode): 577.6, 247.2, HRMS-FAB (M+H + ): calcd. for C 25 H Br , found ; calcd. C 25 H Br , found for

8 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 7 H H H Ac Br H KF/EtH 70% H H Ac H 7 8 Phenoxazine derived nucleoside (8). KF (2.50 g, mmol) was added to a solution of 7 (2.50 g, 4.31 mmol) in abs. EtH (120 ml) and the reaction mixture was refluxed for 8 d. The salts were filtered and discarded, the filtrate was concentrated in vacuo and the product was purified by column chromatography using neutral silica gel (gradient 100:0 to 65:35, CH 2 Cl 2 :MeH) to give 8 as an off-white solid (1.25 g, 70%). 1 H-MR (MeH-d 4 ): δ 1.50 (s, 12H, 4 CH 3 ), 2.15 (m, 1H, H2 ), 2.33 (m, 1H, H2 ), 3.78 (m, 2H, H5 ) 3.94 (q, J = 3 Hz, 1H, H3 ), 4.38 (m, 1H, H4 ), 6.23 (t, J = 7 Hz, 1H, H1 ), 6.60 (s, 1H, ArH), 6.66 (s, 1H, ArH), 7.72 (s, 1H, C 6 H) ppm. 13 C-MR (MeH-d 4 ): (d, J = 18 Hz), 41.84, 62.73, 65.44, 72.13, 87.55, 89.01, , , , , , , , , , ppm. MS (ESI, +ve mode): (M+H + +23), 415 (M+1), 300.9, 153. HRMS-FAB (M+H + ): calcd. for C 21 H , found UV (abs. EtH): λ max = 359 nm, ε = 7486 ± 122 M -1 cm -1.

9 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 8 H H H H H 2 2 a 2 W 4 MeH 80% H H 8 9 H Spin-labeled nucleoside (9). To a solution of 8 (1.50 g, 3.62 mmol) was added ahc 3 (305 mg, 3.62 mmol) in MeH (100 ml). This mixture was treated dropwise with H 2 2 (50% w/v, 1.18 g/ml, 1.44 ml, mmol). After 5 min, a 2 W 4 (0.18 g, 0.54 mmol) was added and resulting mixture was stirred for 30 h at 22 C. The salts were filtered and discarded, the filtrate was concentrated in vacuo and the residue purified by column chromatography using neutral silica gel (gradient 100:0 to 83:17, CH 2 Cl 2 :MeH) to yield 9 as a bright yellow solid (1.25 g, 80%). 1 H MR (MeH-d 4 ): 1.30 (s, 12H, low intensity), 2.17 (bs, 1H), 2.34 (bs, 1H), 3.77 (bs, 1H), 3.83 (bs, 1H), 3.94 (bs, 1H), 4.41 (bs, 1H), 6.30 (bs, 1H), 7.59 (bs, 1H) ppm. 13 C MR (DMS-d 6 ): 32.83, 38.45, 50.91, 62.48, 71.42, 85.67, 88.40, , , , , , , , , ppm. MS (ESI, +ve mode): (M+23), 430 (M+1), , , and HRMS-FAB (M+H + ): calcd. for C 21 H , , found EPR (PE buffer, 20 C): three lines having spectral width G, center line width 1.24 G, Tc = 0.07 ns. UV (abs. EtH) λ max = 360 nm, ε = 7454 ± 148 M -1 cm -1.

10 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 9 H DMTrCl, Pyr. H H DMAP 95% DMTr H H 9 9a 5 -Dimethoxytritylated, spin-labeled nucleoside (9a). Spin-labeled nucleoside 9 (0.60 g, 1.40 mmol), DMTrCl (0.57 g, 1.68 mmol) and DMAP (8.60 mg, 0.07 mmol) were added to a round bottomed flask and kept under vacuum for 16 h. Anhydrous pyridine (15 ml) was added and the solution stirred for 6 h, after which MeH (0.50 ml) was added and the solvent removed in vacuo. The residue was purified by column chromatography using neutral silica gel (gradient, 1:99:0 to 1:95.5:3.5, TEA:CH 2 Cl 2 :MeH) to yield 9a as a yellow solid (1.04 g, 95%). 1 H MR (CDCl 3 ): 1.29 (s, 12H, low intensity), 2.36 (bs, 1H), 3.37 (m, 3H), 3.73 (bs, 6H), 3.77 (bs, 1H), 4.05 (bs, 1H), 4.62 (bs, 1H), 6.21 (bs, 1H), 6.88 (bm, 4 H), 7.28 (m, 12H) ppm. 13 C MR (CDCl 3 ): Low intensity peaks (58.79, 61.00, 68.90, 70.45, 73.25, 74.95, 75.58, 78.5, 81.91, 92.46, 92.55), , , , , , , , , , , , , ppm. MS (ESI, -ve mode): HRMS-FAB (M+H + ): calcd. for C 42 H , found

11 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 10 DMTr H H Phosphitylation DMTr 85% P H 9a C 10 Spin-labeled phosphoramidite (10). Diisopropyl ammonium tetrazolide (84 mg, mmol) and compound 9a (300 mg, mmol) were dissolved in pyridine, the pyridine removed in vacuo and the residue kept under reduced pressure for 16 h. CH 2 Cl 2 (15 ml) was added, along with 2-cyanoethyl,,, -tetraisopropylphosphane (170 µl, mmol). The resulting solution was stirred for 11 h at 22 C. CH 2 Cl 2 (50 ml) was added and the organic phase washed with saturated aq. ahc 3 (3 50 ml). The organic phase was washed with saturated acl (50 ml), dried over a 2 S 4 and concentrated in vacuo. The residue was dissolved in a minimum amount of ether (7-9 ml), followed by a slow addition of hexane (40-50 ml) at 22 C. The solvent was decanted and discarded. This procedure was repeated four times and yielded 10 as a yellowish solid (320 mg, 85%). 1 H-MR (CDCl 3 ): δ 1.30 (m, 24H), 2.65 (m, 6H), 3.54 (bs, 10H), 4.32 (bs, 2H), 4.82 (bs, 1H), 6.52 (bs, 1H), 7.00 (s, 4H), 7.57 (m, 11H) ppm. 31 P-MR (CDCl 3 ): , ppm. MS (ESI -ve mode): (M-1), , , HRMS-FAB (M+H + ) calcd. for C 51 H P, found EPR (CH 3 C, 20 C): three lines having spectral width G, center line width 3.16 G, Tc = 0.20 ns.

12 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 11 H H H Reduction DTT H H H H 9 11 Hydroxyl amine nucleoside (11). A solution of spin-labeled nucleoside 9 (3.5 mg, mmol) in abs. EtH (0.5 ml) was treated with DTT (40.0 mg, mmol) and the reaction mixture stirred for 7 d at 22 C. The completion of the reaction was determined by TLC and EPR spectroscopy. ucleoside 11 was analyzed by MR spectroscopy after performing reduction of 9 (7.0 mg, mmol) with DTT (58.0 mg, mmol) in MeH-d 4 (0.7 ml). 1 H-MR (MeH-d 4 ): δ 1.40 (s, 12H, 4 CH 3 ), (m, 1H, H2 ), (m, 1H, H2 ), (m, 2H, H5 ) (q, J = 3 Hz, 1H, H3 ), (m, 1H, H4 ), (t, J = 7 Hz, 1H, H1 ), 6.64 (s, 1H, ArH), 6.67 (s, 1H, ArH), 7.74 (s, 1H, H6), peaks corresponding to DTT and oxidized DTT: (d, 2H, J = 2 Hz), (d, 2H, J = 1 Hz), (m, 2H) ppm. 13 C-MR (MeH-d 4 ): (d, J = 7 Hz), 41.66, 62.57, (d, J = 9 Hz), 71.84, 87.32, 88.65, , , , , , , , , , , peaks corresponding to DTT and oxidized DTT: 28.19, ppm.

13 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 12 S H H H a 2 S 2 4 MeH/H 2 27% H H H H 9 12 Sulfurous ester nucleoside (12). A solution of spin-labeled nucleoside 9 (10.0 mg, mmol) in MeH (1 ml) was treated with a solution of a 2 S 2 4 (16.2 mg, mmol) in H 2 (0.5 ml). The resultant solution was stirred at 22 C for 4 h, after which the solvent was removed in vacuo. The residue dissolved in 30% MeH/CH 2 Cl 2 and filtered through short pad of silica gel to yield 12 as a yellowish solid (3.1 mg, 27%). 1 H MR (MeH- d 4 ): 1.69 (s, 12H, 4 CH 3 ), 2.20 (m, 1H, H2 ), 2.35 (m, 1H, H2 ), 3.78 (m, 2H, H5 ), 3.94 (m, 1H, H3 ), 4.40 (m, 1H, H4 ), 6.23 (t, J= 6Hz, 1H, H1 ), 6.55 (s, 1H, ArH), 6.60 (s, 1H, ArH), 7.70 (s, 1H, H6) ppm. MS (ESI, -ve mode): (M-1).

14 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 13 DA synthesis and purification. Modified and unmodified oligonucleotides were synthesized on 1.0 µmol scale (1000 Å CPG columns) with Applied Biosystems 394 DA/RA synthesizer and trityl-off syntheses using phosphoramidites with standard base protection. All phosphoramidites, columns and solutions were purchased from Glen Research. For spin-labeled DA, the spin-labeled phosphoramidite 10 was site-specifically incorporated into the oligonucleotides by manual coupling. The DA was deprotected at 55 C for 8 h and purified by 20% DPAGE. The oligonucleotides were visualized by UV shadowing, the bands were excised from the gel, crushed and extracted with TE buffer. The resulting solution was filtered (0.45 µm polyethersulfone membrane, disposable filter device from Whatman) and desalted using Sep-Pak cartridge (Waters Corporation, USA, MA). The resulting solutions were freeze-dried and dissolved in sterile H 2 (100 µl). Concentrations of oligonucleotides were calculated from Beer s law based on measurements of absorbance at 260 nm. The MW of unmodified oligonucleotide 5 -d(gacctcgcatcgtg) and spinlabeled oligonucleotide 5 -d(gacctcgçatcgtg) were determined via MALDI-TF MS. The mass (m/z) of the unmodified DA and spin-labeled DA was observed to be (calcd ) and (calcd ), respectively. Reduction of spin-labeled DA. An aqueous solution of the spin-labeled oligomer 5 -d(gacctcgçatcgtg) in 0.1 M a 2 S 2 4 (w/v, 10 µl) was heated at 45 C for 5 h. The completion of the reaction was verified by EPR spectroscopy. The DA residue was EtH-precipitated and analyzed by analytical gel electrophoresis (Figure S1). A B C Figure S1. Analysis of DA oligomers by DPAGE. A) 5 d(gacctcgcatcgtg), B) 5 -d(gacctcgçatcgtg) and C) dithionite-reduced 5 -d(gacctcgçatcgtg).

15 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 14 EPR measurements. Spin-labeled nucleoside 9 and DA oligomer 5 -d(gacctcgçatcgtg) were dissolved in PE buffer (10 µl, final conc. 200 µm). For spin-labeled DA duplex, DA oligomer 5 -d(gacctcgçatcgtg) (2.0 nmol) was annealed to its complementary strand 5 -d(cacgatgcgaggtc) (2.4 nmol) in PE buffer (final conc. 200 µm of duplex). Samples (10 µl) were placed in quartz capillary for EPR measurements. Continuous wave (CW) EPR spectra were recorded on a commercial 9.4 GHz Bruker EMX spectrometer. The CW measurements were taken with 100 khz modulation frequency, 1.0 G modulation amplitude, and 2.0 mw microwave power. Temperature was regulated to ±0.2 K. Steady-state fluorescence and absorption spectra. The molar extinction coefficients (ε) for nucleoside 8 and spin-labeled nucleoside 9 were determined to be 7486 ± 122 M -1 cm -1 and 7454 ± 148 M -1 cm -1 at 360 nm, respectively, which are comparable to ε of deoxycytidine (7500 M -1 cm -1 ). Steady-state fluorescence and absorbance measurements were carried out at 22 C in a macro fluorescence cell with a path length of 1.0 cm (Hellma GmbH & Co KG, Mullheim, Germany) on SPEX FluoroMax Spectrometer and Perkin Elmer Lambda 25 UV/VIS Spectrometer, respectively. All nucleoside samples were measured at M in abs. EtH using an excitation wavelength of nm. Fluorescence spectra were averaged over three scans (Figure S2).

16 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 15 A 0,16 0,16 0,12 Absorption (a.u.) 0,08 0,04 0,12 ormalized Emission (a.u.) 0,08 0, Wavelength (nm) B 0,16 0,16 0,12 Absorption (a.u.) 0,08 0,04 0,12 ormalized Emission (a.u.) 0,08 0, Wavelength (nm) C 0,16 0,16 0,12 Absorption (a.u.) 0,08 0,04 0,12 ormalized Emission (a.u.) 0,08 0, Wavelength (nm) Figure S2. Absorption and emission spectra of 8 (A), 11 (B) and 12 (C).

17 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 16 Quantum yield determination. Quantum yields were determined using anthracene as a standard [1] for nucleosides 8, 11 and 12 in abs. EtH (Table S1). Quantum yields of all nucleosides were determined using the following equation: Φ F(x) = (A s /A x ) (F x /F s ) (n x /n s ) 2 Φ F(s), where S is the standard, X is the unknown, A is the absorbance at the excitation wavelength (365.5 nm), F is the area under the emission curve, n is the refractive index of the solvent and Φ is the quantum yield [2]. Table S1: Spectroscopic properties of nucleosides 8, 9, 11 and 12. Compound λ a max (nm) [a] λ f max (nm) [b] Φ F [c] ± n.d. n.d ± ± 0.02 [a] absorption maxima; [b] fluorescence emission maxima; [c] fluorescence quantum yield Simulation of EPR spectrum. Figure S3 illustrates an experimental CW-EPR spectrum (solid line) of a spin-labeled DA 14-mer and a simulated spectrum (dotted line), which is based on a model of Brownian diffusion for a cylindrical object, of the same width and length of the DA 14-mer molecule [3,4]. The simulation was done using the tensors in Table S2, which are determined from a fit to the rigid-limit spectra for this sample at 273 K in 50% sucrose solution [5]. The Lorentzian (homogeneous) linewidths in the simulation were equal, all 0.85 G, convoluted with a 0.58 G inhomogeneous broadening function. Figure S3. CW-EPR spectum (solid line) of a spin-labeled DA 14- mer in PE buffer, at 20 C and the simulated spectrum (dotted line).

18 Supporting information for Barhate, Cekan, Massey and Sigurdsson, Angewandte Chemie page S 17 A diffusion tilt angle of the Euler angle β equal to 20, as measured from the principle helix axis was used, and the static field was assumed to be 3353 G, equal to the center field value of the measurement. The simulation employs the Lanczos algorithm, and was run for 33 steps, which was sufficient for convergence. Table S2: Tensors used for the simulation. Element x y z A g References. [1] H. Du, R. A. Fuh, J. Li, A. Corkan, J. S. Lindsey, Photochem. and Photobiol. 1998, 68, [2] D. Lavabre, S. Fery-Forgues, J. Chem. Educ. 1999, 76, [3] J. H. Freed and G. K. Fraenkel, J. Chem. Phys. 1963, 39, [4] M. M. Tirado and J. Garcia de la Torre, J. Chem. Phys , [5] T. M. konogi, A. W. Reese, S. C. Alley, P. B. Hopkins, and B. H. Robinson, Biophys. J. 1999, 77,