ПРИМЕНЕНИЕ МЕТОДА ЭПР ДЛЯ ИССЛЕДОВАНИЯ СУПРАМОЛЕКУЛЯРНЫХ КОМПЛЕКСОВ НИТРОКСИЛЬНЫХ

1. ПРИМЕНЕНИЕ МЕТОДА ЭПР ДЛЯ ИССЛЕДОВАНИЯ СУПРАМОЛЕКУЛЯРНЫХ КОМПЛЕКСОВ НИТРОКСИЛЬНЫХ РАДИКАЛОВ И СПИНОВЫХ ЛОВУШЕК C НАНОКОНТЕЙНЕРАМИ Елена Багрянская Международный томографический центр СО РАН

2. Spin labels pH-sensitive probes NO detection O2 concentration measurement EPR - imaging Nitroxide radicals Main problem for in vivo application: reduction of nitroxides to diamagnetic (EPR-silent) compounds The ways to overcome problem: Synthesis of sterically substituted nitroxides with low reduction rate Incapsulation of nitroxide radicals into nanocapsules and liposomes - “host-guest” systems. 2

3. pH-sensitivity of nitroxide radicals pK=6.26 ∆aN=0.08 mТ *, Observed aN depends on pH value Khramtsov V., Weiner L., Grigorev I., Volodarsky V. Chem.Phys. Lett.1982 3

4. pKa + H+ EPR on EPR off subtraction Application of NR with two pKa constants for the measurements of acidity in rats stomach in vivo. Techniques: RF-ESR LODESR FC-DNP PEDRI Ref. D.I. Potapenko, M. A. Foster, D.J. Lurie, I. A. Kirilyuk, J. M.S. Hutchison, I.A. Grigor’ev, E.G. Bagryanskaya and V.V. Khramtsov, J. Magn. Reson. 182 (2006) 1-11.

5. Nitroxide application for nitric oxide detection INR NNR Akaike, T.; Yoshida, M. et al. Biochemistry, 1993, 32, 827–832 NNR kinetics in mice Reduction of INR is too fast to measure EPR image. Nitroglycerole Strishakov R., et.al.

6. Host-guest complexes and supramolecules G + H → [GH] G – nitroxide radicals,H – nanocapsules Cyclodextrins Calixarenes Cucurbiturils Liposomes How encapsulation affect: stability of nitroxides towards reduction functional properties of pH-sensitive nitroxides 6

7. Методы исследования комплексов: спектры оптического поглощения спектры ЭПР спектры ЯМР метод модуляций ЭСЭ Измерение констант комплексования – Зависимость от концентрации гостя и хозяина Температурная зависимость. Использование молекул конкурентов. [NR@M] K= [NR]x[M]

8. Objects - - • stability of the complexes of nitroxides with cucurbit[7]uril • persistency of encapsulated nitroxide radicals to reduction • pH-sensitivity of nitroxide radicals in complexes ? 8 Kirilyuk et al. J. Phys. Chem. B., 2010, 114

9. ComplexHMP@CB7 exchange HMP + CB7HMP@CB7 giso HMP giso=2.0057, aN=(1.626 ±0.005) mT, tc=10 ps. aN HMP CB7 (1:2) HMP@CB7 giso=2.0060, aN=(1.500±0.008) mT, tc=68ps. ΔaN = 0.126 mT Complex formation slow exchange on EPR timescale (k<< 1010Hz) change of g-factor, aNи tс 9

10. Formation of the complexes NR@CB7ESEEM in D2O at 77K pH=6.8 ATI ATI@CB pH=6.8 AMP AMP@CB pH=6.8 ATI ATI@CB pH=6.8 AMP AMP@CB V.Chechik, D.Goldfarb et al. A.Milov, Yu. Tsvetkov

11. Комплексы гидроксиламинов с CB7 по данным ЯМР CB7 ATI-H ATI-H / CB7 = 1 : 1 Δδ=0.21 and 0.72ppm ATI-H / CB7 = 1 : 5 ATI-H / CB7 = 5 : 1 • формирование комплекса ATIH+-H@CB7 • различное Δδ для метильных групп –> различная глубина • погружения в полость CB7 11

12. Binding constants K NR + CB7NR@CB7 [NR@CB7] = K [CB7] [NR] 50 mM ATIH+ [NR@CB7] K= K= (1.8 ±0.2)×103 M-1 forATIH+ [NR]x[CB7] K= (2.3 ±0.3)×103 M-1forAMP K= (6.4 ±1.2)×104M-1forAMPH+ Binging constants of charged forms of nitroxides higher than for neutral forms 12

13. pH-sensitivity of complexes NR + CB 1:2 + CB 1:4 MTI@CB7 for pH 0.5 – 3.0 ATI@CB7 for pH 4.0 – 9.0 AMP@CB7 for pH 8.0 – 14.0 13

14. Persistency of complexes to reduction NR + Asc- → NR-H + Asc-• AMP@CB7 + Asc- → AMP-H@CB7 + Asc-• pH 3.4 pH 3.0 ATI (kATIH+= 26 ±1 M-1s-1) ATI/CB7 = 1:2 (kobs= 16 ±1 M-1s-1) ATI/CB7 = 1:10 (kobs= 5.6 ±0.4 M-1s-1) ATI/CB7 = 1:40 (kobs= 0.44 ±0.03 M-1s-1) AMP (kAMPH+= 0.320 ±0.020 M-1s-1;) AMP/CB7 = 1:1 (kobs= 0.097 ±0.008 M-1s-1) AMP/CB7 = 1:2 (kobs= 0.040 ±0.006 M-1s-1) AMP/CB7 = 1:10 (kobs= 0.020 ±0.004 M-1s-1) Lifetime of AMPH+@CB7 by a factor of 16 higher than for AMPH+. Lifetimeof ATIH+@CB7 by a factor of 50 higher than for ATIH+.

15. Complex stability in stabilized mouse blood ATI@CB7 Blood of rats Wistar pH ~ 7.0 AMP@CB7 AMPgiso=2.0059,aN=(1.605±0.006) mT,tc=12 ps. AMP@CB7 giso=2.0059, aN=(1.600 ±0.005) mT, tc=150ps. Lifetime - ~hours Complexes are not stable in mouse blood

16. Influence of metal cations K1 =1.7×105M-1 K2=600 M-1 * K3=53 M-1 * K5the value 3.1×104 M-1 *Lucarini et al., Chem. Eur. J. 2007, 13, 7223 0.25 mM AMP, 0.5 mM CB7, pH 3.5 Metal cations form complexes with CB7 and lead to destruction of nitroxide@CB7 complexes

17. ComplexTAMP@CB7 HMP@CB7 – complex with nitroxide inside the CB7 cavity (ΔaN = 0.126 mT) giso aN TAMP giso=2.0057, aN=(1.600 ±0.006) mT, tc=12 ps. TAMP CB7 (1:2) TAMP@CB7 giso=2.0057, aN=(1.607 ±0.007) mT, tc=110ps. ΔaN~ 0 мТ Complex formation no change of g-factor, aN 17

18. ComplexTAMP@CB7: reduction rate Ascorbic acid (vitamin С) kTAMP=0.48±0.05 M-1s-1 Reduction rate constants the same for TAMP и TAMP@CB7. 18

19. New complexes nitroxide@CB7 … НИОХ СО РАН ? Positive charged nitroxide Synthesis in the process…

20. H+ Gramicidin A Encapsulation of nitroxides in polysomes (SG is a glutathione residue). Pure nitroxide: Encapsulated nitroxide: Kinetics of the NR2 (0.1 mM) reduction by sodium ascorbate: 0.4 mM (Δ), 0.75 mM (○) and 1.5 mM (■). 10 mM (●) of sodium ascorbate (100th –fold excess). Add. of K3Fe(CN)6 Liposomes are impermeable for large charged molecules like K3Fe(CN)6 Encapsulation in semipermeable liposomes doesn’t affect the pH- sensitivity of nitroxides Woldman, Y. Y., Semenov, S. V., Bobko, A. A., Kirilyuk, I. A., Polienko, J. F., Voinov, M. A., Bagryanskaya E. G. and Khramtsov V.V. Analyst, 2009, 134, 904–910. .

21. Nitroxides in Octa Acid nanocapsules EPR methods pH-probes Reduction of encapsulated nitroxide by sodium ascorbate +AscH- probe for nitric oxide Octa Acid capsules increase the guest resistance to reduction Ref. C. Gibb, B.Gibb, JACS 126 (2004)11408-11409.

22. Mobility of nitroxide in CB7 (solid state) AMP@CB7 AMP/aniline=1/15 in CB7 tc=500 ns tc=170 ns Nitroxides in CB7 reveal low mobility due to interaction of NH2-group with CB-portals Correlation time of motion in liquids is determined by rotation motion of complex

23. Inclusion complexes nitroxides with calixarene complexes isolated and characterized Ref. G. Ananchenko, K.Udachin, A.Coleman, D.Polovyanenko, E. Bagryanskaya and J. Ripmeester, Crystalline inclusion complex of a calixarene with a nitroxide, Chem. Commun., 2008, 223–225.

24. Does encapsulation protect nitroxide from reduction? Test for reduction of encapsulated nitroxides by ascorbic acid. T1 relaxation of H2O Encapsulated nitroxides do not affect nuclear spin relaxation times of water EPR spectra of MeOTEMPO in C6 (buffer solution pH 7.4) Calixarene works as a supramolecular protector of nitroxide Polovyanenko, DN; Bagryanskaya, EG; Schnegg, A, A.Savitsky,K.Moebius, A.Coleman, G. Ananchenko and J.Ripmeester,Phys.Chem.Chem.Phys. V.10, 5299-5307, 2008.

25. Comparison of the shapes of EPR spectra in solid state and in solution The shapes of EPR spectra are the same for encapsulated nitroxide in solid state and in solution What is the mobility of nitroxide in C6? What interactions determine the shape of EPR spectra? Which processes determine the reorientation motion correlation times?

26. Temperature dependence of EPR spectra MeTEMPO MeTEMPO:DBK 1:30 dibenzylketon To study the mobility of nitroxide one should use the diluted samples

27. 360 GHz EPR reveals two forms of MeTEMPO in C6 Exit of ethanol molecule from C6 4% MEtOH= 4%(2MC6+MTEMPO) Ref. D.Polovyanenko, E. Bagryanskaya, A.Schnegg, K.Möbius, A.W. Coleman, G.S. Ananchenko, K. A. Udachin and J.A. Ripmeester, Phys. Chem. Chem. Phys. 2008., 10, 5299-5307.

28. Mobility of nitroxides in nanocapsules – CW-EPR Temperature dependence of EPR spectra of thedilutedMT@2C6OH. X-band W-band 360 GHz Poor agreement of the simulations using the isotropic spin-probe motion clearly demonstrates the necessity to improve this model.

29. Model of motion – simulations 1. Fast restricted motion model (solid lines) 2. Microscopic-Order Macroscopic-Disorder (MOMD) Schnieder D. J., Freed J. H., Biol.Magn.Reson, 1989, 8, p.1-76 Orientational potential Y2,0, motion anisotropy parameter l2,0=-1.5. tc=0.05 ns NRs inside calix(6)arenes reveal anisotropic motion with activation energy of reorientation about 1.5-3 kcal/M. Ref. E.Bagryanskaya, D.Polovyanenko, M.Fedin,L.Kulik, A.Schnegg,A.Savitsky,K.Moebius, A.Coleman, G. Ananchenko and J.Ripmeester, Phys. Chem. Chem. Phys., 2009, 11, 6700–6707

30. ESE detected EPR spectra Libration motions without preferential orientation are observed ESE-detected EPR spectra reveal the existence of immobilized nitroxides in defects

31. Nitroxide attached to cyclodextrin gAiso=2.00591, (A) aANiso=1.54 mT, aAHiso=0.26 mT, tcorrA=3.5·10-10 s. gBiso=2.00563, (B) aBNiso=1.57 mT, aBHiso=0.26 mT, tcorrB=2.7·10-10 s. W-band EPR ESEEM in D2O X-band Bagryanskaya, E.G., et. al, Appl. Magn. Reson,, (2009). Polovyanenko ett al J.Phys. Chem. 2008 Krumkacheva O.et al. Lamgmur 2010 Krumkacheva et al. Appl.Magn.Reson. 2011

32. pH-sensitive NR attached to CD Attaching of NR to CD keeps pH-sensitivity with shifting pKa, but does not improve NR stability against reduction by ascorbic acid due to fast equilibrium between weak and loose complexes

33. Influence of β-cyclodextrins on EPR spin trapping of glutathiyl radicals by PBN, DMPO and DEPMPO PBN t1/2 < 40 s PBN + RAMEB t1/2 180 s 1 mT ST+ •R ST/R•diamagnetic products main properties of spin trap: • rate constant of scavenging • adduct lifetime PBN + superoxide PBN/superoxide H. Karoui et al.Chem.Comm.2002, 3030 Increase the stability of the PBN-OOH spin adduct in the presence of Randomly Methylated β-cyclodextrin

34. Objectives • Influence on the rate constant of scavengingand adduct lifetime • presence of β-cyclodextins GS• + PBN + • attaching of PBN to β-cyclodextins GS• + 34

35. Measurement of ksc kGSNO = (1.5±0.3)∙109 M-1s-1 kSCPBN=(6.7±1.5)∙107M-1s-1 35 Polovyanenko et.al., J.Phys.Chem. B, 2008, 112.

36. PBN/β-cyclodextrins: adduct decay PBN/GS• decay (PBN:CD 1:2) RAMEB and DIMEB demonstrate the ability to strikingly (factor of 7) increase the lifetime of the adduct PBN/GS• 36

37. Spin traps attached to cyclodextrines (PBN) kIPBN=1.7 ±0.2s-1 kIPBN-DIMEB= kIPBN /4.9=(0.35 ±0.03)s-1 kIPBN-TRIMEB= kIPBN /2.4=(0.71±0.04)s-1 Lifetimes of PBN-DIMEB/GS● and PBN-TRIMEB/GS● adduct are by a factor of 4.9 and 2.4 longer than PBN/GS● adduct lifetime.

38. Заключение • Методом ЭПР и лазерного показано, что спиновые ловушки и нитроксильные радикалы образуют комплексы с супрамолекулами, такими как циклодекстрины, каликсарены и кукурбитурилы. • На основе комплексов спиновых ловушек с циклодекстринами можно получать более эффективные ловушки для детектирования биологически важных радикалов, в том числе в живых системах. • Комплексы нитроксильных радикалов с каликсаренами и кукурбитурилами обладают повышенной устойчивостью к восстановлению аскорбиновой кислотой и чувствительностью к pH среды (для кукурбитурилов). • Супрамолекулярные комплексы спиновых ловушек и нитроксильных радикалов являются перспективными объектами для детектирования короткоживущих радикалов и использования в качестве pH-чувствительных зондов с применением ЭПР и ЭПР томографии, в том числе для живых систем.

39. International Tomography Center SB RAS, Novosibirsk, Russia D. Polovyanenko S. Semenov O. Krumkacheva M. Fedin

40. Acknowledgement: Novosibirsk Institute of Organic Chemistry I. Kirilyuk I. Grigor’ev M.Voinov J.Polienko Novosibirsk Institute of Inorganic Chemistry V.Fedin O.Gerasko • University of Provence, Marseille • Sylvain Marque • Paul Tordo • Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa • Gennady Ananchenko • John Ripmeester Free University of Berlin, Germany A.Schnegg A.Savitsky K.Moebius Ohio State University, Medical Center, USA V. Khramtsov, A. Bobko J. Woldman

41. THANK YOU FOR YOUR ATTENTION !

42. Binding constants • K is controlled by: • hydrophobic interactions with CB7 cavity • ion-dipole interactions of cationic groups with portals