Supporting Information

Transcription

1 Supporting Information Wiley-VCH Weinheim, Germany

2 page S1 Relative orientation of rigid nitroxides by pulsed EPR spectroscopy: Beyond distance measurements in nucleic acids Olav Schiemann 1,2 *, Pavol Cekan 3, Dominik Margraf 2, Thomas Prisner 2 and Snorri Th. Sigurdsson 3 * 1 University of St Andrews, Centre for Biomolecular Sciences, orth Haugh, St Andrews, KY16 9ST, UK 2 J. W. Goethe-University, Institute of Physical and Theoretical Chemistry, Max-von Laue-Str. 7, Frankfurt am Main, Germany 3 University of Iceland, Science Institute, Dunhaga 3, 107 Reykjavik, Iceland Table of Contents General synthetic procedures page S2 List of abbreviations page S2 Organic synthesis procedures page S3 Sample Preparation for PELDOR experiments page S18 DA modelling page S18 PELDOR page S18 Simulations page S19 References page S23

3 page S2 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. CH 2 Cl 2 was freshly distilled over calcium hydride under nitrogen. Water was purified on a MILLI-Q water purification system. eutral silica gel ( mesh, 60 Å) was purchased from Silicycle. Analytical TLC was performed on glass plates (Silicycle, ultra pure silica gel, 60 Å, F 254 ). MR spectra for all organic compounds were recorded on an Avance 400 MHz Bruker MR spectrometer and the chemical shifts were reported in parts per million (ppm) relative to the deuterated MR solvent used [ 1 H-MR: CDCl 3 (7.26 ppm), MeOH-d 4 (4.84 and 3.31 ppm); 13 C-MR: CDCl 3 (77.00 ppm), MeOH-d 4 (49.10 ppm)]. Commercial grade CDCl 3 was passed over basic alumina, immediately prior to use. Molecular weight (MW) of organic compounds was determined by HR-ESI-MS (Bruker, MicroTof-Q). List of abbreviations. TLC MCPBA HR-ESI-MS thin layer chromatography meta-chloroperbenzoic acid high resolution electrospray ion trap mass spectrometer

4 page S3 Organic synthesis procedures. H 2 2 H 1. Ac 2 O AcH 2a Ac 2. HO 3 65% 2 steps O 2 AcH 3 Ac -(2-acetyl-1,1,3,3-tetramethyl-6-nitroisoindolin-5-yl)acetamide (3). A solution of compound 2 (244 mg, mmol) in acetic anhydride (7 ml) was stirred for 12 h at 22 C. TLC showed complete conversion to 2a. A pre-cooled fumic HO 3 (100%, 15 ml, 0 C) was slowly added to pre-cooled solution of 2a and the reaction mixture was stirred at 0 o C for 2 h. After completion of the reaction, ph was adjusted to 12 by addition of aoh (aq) (40% w/v). The aqueous layer was extracted with CH 2 Cl 2 (3 30 ml) and the combined organic phases washed sequentially with saturated ahco 3 (aq) (2 20 ml), brine (aq) (2 20 ml) and water (2 20 ml) and dried over MgSO 4. The solvent was removed in vacuo and the product was purified by column chromatography using neutral silica gel (gradient 100:0 to 93:07, CH 2 Cl 2 :MeOH) to yield 3 (270 mg, 65%, two steps) as a dark yellow solid. The product has 2 different conformations, as judged by MR spectroscopy. TLC (silica gel 7 % MeOH/CH 2 Cl 2 ): Rf (2) = 0.05, Rf (2a) = TLC (silica gel 5 % MeOH/CH 2 Cl 2 ): Rf (2a) = 0.35, Rf (3) = H-MR (CDCl 3 ): δ 1.74 (m, 12H, 4 CH 3 ), 2.72 (s, 6H, 2 COCH 3 ), 7.97 (s, 1H, ArH), 8.57 (0.5H, ArH), 8.60 (0.5H, ArH), (s, 1H, H). 13 C-MR (CDCl 3 ): 25.21, 25.52, 27.73, 28.26, 30.35, 30.83, 64.85, 65.62, 68.15, 68.96, , , , , , , , , , , , , , , HR-ESI-MS (M+H + ): calcd. for C 16 H 21 3 O 4 H , found

5 page S4 1 H-MR (400 MHz, CDCl 3 ) spectrum of C-MR (400 MHz, CDCl 3 ) spectrum of 3.

6 page S5 O 2 AcH 3 Ac 70% H 2 SO 4 70% O 2 H 2 4 H 1,1,3,3-tetramethyl-6-nitroisoindolin-5-amine (4). A solution of 3 (270 mg, mmol) in 70% H 2 SO 4 (v/v, 10 ml) was refluxed for 60 min after which the solution was poured into water-ice slush (100 ml) and ph adjusted to 12 by addition of aoh (aq) (40% w/v). The aq layer was extracted with CH 2 Cl 2 (3 30 ml), the combined organic phases washed sequentially with saturated ahco 3 (aq) (2 20 ml), brine (aq) (2 20 ml) and water (2 20 ml). The organic phase was dried over MgSO 4, concentrated in vacuo and the product was purified by column chromatography using neutral silica gel (gradient 100:0 to 80:20, CH 2 Cl 2 :MeOH) to yield 4 (140 mg, 70%) as a dark yellow solid. TLC (silica gel 20 % MeOH/CH 2 Cl 2 ): Rf (3) = 0.90, Rf (4) = H-MR (CDCl 3 ): δ 1.42 (d, J = 2.0 Hz, 12H, 4 CH 3 ), 1.99 (bs, 1H, H), 6.09 (bs, 2H, H 2 ), 6.51 (s, 1H, ArH), 7.85 (s, 1H, ArH). 13 C-MR (CDCl 3 ): 31.40, 31.91, 61.99, 62.53, , , , , , HR-ESI-MS (M+H + ): calcd. for C 12 H 17 3 O 2 H , found

7 page S6 1 H-MR (400 MHz, CDCl 3 ) spectrum of C-MR (400 MHz, CDCl 3 ) spectrum of 4.

8 page S7 O 2 H 2 4 H H 2, Pd/C 100% H 2 H 2 5 H 1,1,3,3-tetramethylisoindoline-5,6-diamine (5). A solution of 4 (110 mg, mmol) in MeOH (30 ml) containing 10% Pd/C (11 mg) was hydrogenated at 55 psi for 2 h. The reaction mixture was filtered through a pad of celite and the filtrate was concentrated in vacuo to yield 5 (96 mg, 100%) as a white solid. TLC (silica gel 30 % MeOH/CH 2 Cl 2 ): Rf (4) = 0.85, Rf (5) = H-MR (CDCl 3 ): δ 1.44 (s, 12H, 4 CH 3 ), 3.36 (bs, 4H, 2 H 2 ), 6.46 (s, 2H, ArH). 13 C-MR (CDCl 3 ): 31.81, 63.01, , , HR-ESI-MS (M+H + ): calcd. for C 12 H 19 3 H , found

9 page S8 1 H-MR (400 MHz, CDCl 3 ) spectrum of C-MR (400 MHz, CDCl 3 ) spectrum of 5.

10 page S9 O O O O 5 EtOH 97% O O H 6 7 Diketone 7. A saturated solution of 6 (420 mg, mmol) in EtOH (100 ml) was treated with an ethanolic solution of 5 (131 mg, mmol, 70 ml of EtOH). After stirring at 22 C for 14 h, the solvent was removed in vacuo. The product was purified by column chromatography using neutral silica gel (gradient 100:0 to 96:04, CH 2 Cl 2 :MeOH) to yield 7 (335 mg, 97%) as light yellow crystals. TLC (silica gel 10% MeOH/CH 2 Cl 2 ): Rf (5) = 0.05, Rf (6) = 0.95, Rf (7) = H-MR (CDCl 3 ): δ 1.58 (d, J = 2.3 Hz, 18H, 6 CH 3 ), 1.73 (s, 12H, 4 CH 3 ) 8.12 (s, 2H, 2 ArH), 8.60 (d, J = 2.3 Hz, 2H, 2 ArH), 9.65 (d, J = 2.3 Hz, 2H, 2 ArH). 13 C-MR (CDCl 3 ): 31.20, 31.83, 35.53, 62.91, , , , , , , , , , , HR-ESI-MS (M+H + ): calcd. for C 36 H 37 3 O 2 H , found

11 page S10 1 H-MR (400 MHz, CDCl 3 ) spectrum of C-MR (400 MHz, CDCl 3 ) spectrum of 7.

12 page S11 O O H H 2 OH BaCO 3 EtOH 63% O HO H H 7 8 Compound 8. A mixture of 7 (120 mg, mmol), H 2 OH HCl (54 mg, mmol) and BaCO 3 (65 mg, mmol) in EtOH (10 ml) was refluxed for 4 d. After removal of the solvent, the residue was treated with 0.2 M HCl (10 ml), stirred for 30 min and filtered. The solid was washed with water and the product was purified by column chromatography using neutral silica gel (gradient 100:0 to 94:06, CH 2 Cl 2 :MeOH) to yield 8 (80 mg, 63%) as a yellow solid. TLC (silica gel 10% MeOH/CH 2 Cl 2 ): Rf (7) = 0.50, Rf (8) = H-MR (30% MeOH-d 4 /CDCl 3 ): δ 1.37 (d, J = 3.5 Hz, 18H, 6 CH 3 ), 1.51 (s, 12H, 4 CH 3 ), 7.93 (s, 2H, 2 ArH), 8.47 (d, J = 1.7 Hz, 1H, ArH), 9.25 (d, J = 1.7 Hz, 1H, ArH), 9.35 (d, J = 1.7 Hz, 1H, ArH), 9.49 (d, J = 1.7 Hz, 1H, ArH). 13 C-MR: was not recorded due to poor solubility of 8. HR-ESI-MS (M+H + ): calcd. for C 36 H 39 5 O 2 H , found

13 page S12 1 H-MR (400 MHz, 30% MeOH-d 4 /CDCl 3 ) spectrum of 8.

14 page S13 O HO H H 2 H 4, Pd/C EtOH 98% H 2 H 2 H 8 9 Amine 9. 2 H 4 H 2 O (0.5 ml) was added dropwise (ca. 5 min) to a slurry of 8 (50 mg, mmol) and Pd/C (10%, 50 mg) in abs. EtOH (10 ml). The resulting mixture was refluxed for 3 h under argon, cooled to 22 C, filtered through a bed of celite and ca. half of the solvent removed in vacuo. The resulting solution containing 9 (47 mg, 98%) was used directly in the next reaction. For MR and MS analysis, a small aliquot was removed. TLC (silica gel 10% MeOH/CH 2 Cl 2 ): Rf (8) = 0.45, Rf (9) = H-MR (CDCl 3 ): δ 1.69 (s, 18H, 6 CH 3 ), 1.86 (s, 12H, 4 CH 3 ), 1.87 (bs, 4H, 2 H 2 ), 8.18 (s, 2H, 2 ArH), 8.21 (bs, 2H, 2 ArH), 9.56 (bs, 2H, 2 ArH). 13 C-MR (CDCl 3 ): 29.74, 32.03, 35.82, 58.53, , , , , , , , , , , HR-ESI-MS (M+H + ): calcd. for C 36 H 41 5 H , found

15 page S14 1 H-MR (400 MHz, CDCl 3 ) spectrum of C-MR (400 MHz, CDCl 3 ) spectrum of 9.

16 page S15 H 2 H 2 H 7 EtOH 30% H H 9 10 Compound 10. A solution of 7 (46 mg, mmol) and 9 (42 mg, mmol) in EtOH (50 ml) was refluxed for 55 h. The precipitate was collected, isolated, washed 3 times with EtOH (25 ml, 10 ml, 5 ml) and dried under vacuum to yield 10 (30 mg, 37 %) as an orange-brown solid. TLC (silica gel 10% MeOH/CH 2 Cl 2 ): Rf (7) = 0.50, Rf (9) = 0.40, Rf (10) = H-MR (CDCl 3 /dichloroacetic acid): δ 1.83 (s, 36H, 12 CH 3 ), 2.10 (s, 24H, 8 CH 3 ), 8.36 (s, 4H, 4 ArH), 9.82 (d, J = 1.9 Hz, 4H, 4 ArH), (s, J = 1.9 Hz, 4H, 4 ArH). 13 C-MR: was not recorded due to poor solubility of 10. HR-ESI-MS (M+H + ): calcd. for C 72 H 74 8 H , found

17 page S16 1 H-MR (400 MHz, CDCl 3 /dichloroacetic acid) spectrum of 10.

18 page S17 H H MCPBA Toluene 60% 10 O O 1 Biradical 1. A suspension of 10 (15 mg, mmol) in toluene (5 ml) was treated with MCPBA (65 mg, mmol, 77%) and the reaction stirred at 22 C for 4 h, after which the solvent was removed in vacuo. The crude product was recrystallized in 10% H 2 O/MeOH and dried under vacuum to yield 1 (9 mg) as an orange solid, however, the product contained some monoradical. 1 was purified by dissolving 3 mg in molten ortho-terphenyl (230 mg, 210 µl) at 56 C, adding CH 2 Cl 2 (3 drops) and cooling to 22 C. The undissolved solid was removed by filtration and discarded. The filtrate was placed in a test tube inside a Schlenck-tube containing n-pentane, yielding an amorphous yellow precipitate within 48 h, which was collected by filtration. This procedure was repeated to give an analytically pure 1. TLC (silica gel 10% MeOH/CH 2 Cl 2 ): Rf (10) = 0.30, Rf (1) = H-MR and 13 C-MR: was not recorded due to limited solubility of 1. HR-ESI-MS (M+H + ): calcd. for C 72 H 72 8 O 2 H , found

19 page S18 Sample Preparation for PELDOR experiments. Compound 1 was dissolved in molten orthoterphenyl (100 µl, final conc. 100 µm). Spin-labeled DA oligomers were synthesized and purified as previously described. [1] The spin-labeled DA duplexes 5?-d(GTÇAGTCGCGCGCGCGCATC) 5?- d(gatgcgçgcgcgcgactgac) (I) and 5?-d(GTÇAGTGCGCGCGCGCGATC) 5?- d(gatcgcgcgcgcgçactgac) (II), were prepared by annealing the appropriate spin-labeled, single-stranded DA oligomers (10 nmol each strand) in PE buffer (100 µl, final conc. 100 µm of duplex). [1] The water was evaporated in vacuo and the residue redissolved in 20% ethyleneglycol/h 2 O (100 µl, final conc. 100 µm of duplex). All samples were rapidly frozen and stored in liquid nitrogen until they were measured. DA modelling. DA duplexes were modelled on a Silicon graphics workstation using the modelling program Sybyl 7.3. Simple unmodified B-DA double helices were built in the bioplymer module and then the Ç-modification was fused onto a C using the sketching-function. The constructs were minimized using the Tripos force field with 20 iterations (a minimal number of the iterations). This enabled minimization of the site of modification without affecting the rest of the DA helix. PELDOR. PELDOR spectra were recorded on a Bruker ELEXSYS E580 pulsed X-band EPR spectrometer containing a standard flex line probe head housing a dielectric ring resonator (MD5 W1) equipped with a continuous flow helium cryostat (CF935) and temperature control system (ITC 502) both purchased from Oxford instruments. The second microwave frequency was coupled into the microwave bridge by a commercially available setup (E U) from Bruker. All pulses were amplified via a pulsed travelling wave tube amplifier from Applied Systems Engineering (117X) and the resonator was over-coupled to a quality factor Q of about Pulse ELDOR experiments were recorded with the pulse sequence p/2(? A )-t 1 -p (? A )-t 1 +t-p(? B )-(t 2 -t)-p(? A )-t 2 -echo. The pump pulse was set to 12 ns and applied to the maximum of the nitroxide spectrum, selecting the central m I = 0 transition of A zz and the m I = 0, ±1 transitions of A xx and A yy, at the resonance frequency of the resonator. Its amplitude was optimized on the maximum inversion of a Hahn-echo on? B. Detection pulses (? A ) were set to 32 ns and applied at 40 to 80 MHz higher frequency for 1 and 40 to 90 MHz for DAs resulting in an increasing selectivity towards A zz, with an amplitude chosen to optimize the refocused echo.?? = 90 MHz is experimentally not accessible in 1 due to the solvent dependent smaller width of the EPR spectrum. The p/2-pulse was phase-cycled in order to eliminate receiver offsets. Proton modulation surpression was achieved via addition of 8 spectra of variable t 1 starting from t 1 = 136 ns

20 page S19 with?t 1 of 8 ns. Time increments?t = 12 ns were applied. All spectra were recorded at 40 K and divided by a decay function and normalized to the point t = 0. Compound 1 was measured with t 2 = 2500 ns, an experiment repetition time of 4.5 ms, a video amplifier bandwidth of 25 MHz and an amplifier gain of 57 db. For DA I and DA II t 2 = 2400 ns, an experiment repetition time of 7.5 ms, a video amplifier bandwidth of 25 MHz and an amplifier gain of 63 db were chosen. Usually, 500 scans with 204 data points were accumulated resulting in an average measuring time of 1.5 hours for 1. In case of DAs mostly 3200 scans with 198 data points were recorded giving an approximate experiment run time of 8 hours. Simulations. Simulations were performed based on equation S1 taking the effect of the distribution function P(??,?) of dipolar angles on the PELDOR time domain signal V(t) explicitly into account. V 0 describes the spin echo intensity of the detected spin for time t = 0,? d is the dipolar coupling,?? the frequency offset, and? the angle between the spin-spin distance vector and the external magnetic field. [1] π /2 Vt () = V0 1 P(?, θ) [ 1 cos( ωdt)dθ] (Eq. S1) 0 Geometry and spin Hamiltonian parameters were implemented into the simulations as described in previous papers. [2] Only one conformer was used for the simulations shown on the next pages. It should be noted that the error in f is estimated from a visual comparison of the simulated with the experimental time traces (see below). A more detailed error analysis is prevented by neglecting conformational flexibility, leading to non-damped oscillations. A more elaborate error analysis will be presented in the future including dynamics.

21 page S20 Figure S1: Experimental PELDOR time traces of molecule 1 with?? equal 40 (orange), 50 (magenta), 60 (red), 70 (dark green), 80 MHz (blue) compared to simulations obtained with f -values of a) f = 90 (dark blue) as shown in the main text and f = 75 (green) and b) with f = 90 and f = 70 (pink). Decreasing f from 90 to 70 shows systematic deviations between simulation and experiment. The parallel component is not pronounced enough for?? of 40 MHz and too pronounced for?? 50 MHz and the modulation depth does not fit for any??. Thus the upper limit for the error in phi for molecule 1 is 15.

22 page S21 Figure S2: Experimental PELDOR time traces of DA I with?? equals 40 (orange), 50 (magenta), 60 (red), 70 (dark green), 80 (blue) and 90 MHz (black) compared to simulations with f -values of a) f = 5 (dark blue) as shown in the main text and f = 20 (green) and b) f = 5 and f = 25 (pink). Increasing f from 5 to 25 starts to show deviations in the decay down to the first minimum for all frequency off-sets. Furthermore, deviations with respect to the depth of the minima and maxima become apparent for?? 70 MHz. These deviations increase with increasing f even further. Thus, the error for DA I is ±20.

23 page S22 Figure S3: Experimental PELDOR time traces of DA II with?? equal 40 (orange), 50 (magenta), 60 (red), 70 (dark green), 80 (blue) and 90 MHz (black) compared to simulations obtained with f - values of a) f = 45 (dark blue) as shown in the main text and f = 30 (green) and b) f = 45 (dark blue) and f = 25 (pink). In this case deviations become apparent for f = 25 or 30 especially for?? = 80 MHz. At this frequency offset the parallel component is too dominant. Overall the minima and maxima are too pronounced for these angles. Thus, the error for DA II has an upper limit of ±20.

24 page S23 References [1] Cekan, P., Smith, A. L., Barhate,., Robinson, B. H., and Sigurdsson, S.T., ucleic Acid Res. 2008, 36, [2] D. Margraf, B. E. Bode, A. Marko, O. Schiemann, T. F. Prisner, Mol. Phys. 2007, 105, b) B.E. Bode, J. Plackmeyer, T.F. Prisner, O. Schiemann, J. Phys. Chem. A 2008, 112,