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

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

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

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

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

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

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. K=K= [NR]x[M]

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

Complex 9 Complex formation slow exchange on EPR timescale (k

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

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

Binding constants Binding constants 12 Binging constants of charged forms of nitroxides higher than for neutral forms NR + CB7 K K=K= [NR]x[CB7] = K [CB7] [NR] K= (2.3 ±0.3)×10 3 M -1 for AMP K= (6.4 ±1.2)×10 4 M -1 for AMPH+ 50 M ATIH + K= (1.8 ±0.2)×10 3 M -1 for ATIH+

pH-sensitivity of complexes 13 for pH 0.5 – 3.0 for pH 4.0 – 9.0 for pH 8.0 – 14.0 NR + CB 1:2 + CB 1:4

Persistency of complexes to reduction AMP (k AMPH+ = ±0.020 M -1 s -1 ;) AMP/CB7 = 1:1 (k obs = ±0.008 M -1 s -1 ) AMP/CB7 = 1:2 (k obs = ±0.006 M -1 s -1 ) AMP/CB7 = 1:10 (k obs = ±0.004 M -1 s -1 ) pH 3.4 NR + Asc- NR-H + Asc- + Asc- + Asc- Lifetime of by a factor of 16 higher than for AMPH+. Lifetime of by a factor of 50 higher than for ATIH+. ATI (k ATIH+ = 26 ±1 M -1 s -1 ) ATI/CB7 = 1:2 (k obs = 16 ±1 M -1 s -1 ) ATI/CB7 = 1:10 (k obs = 5.6 ±0.4 M -1 s -1 ) ATI/CB7 = 1:40 (k obs = 0.44 ±0.03 M -1 s -1 ) pH 3.0

Complex stability in stabilized mouse blood Complexes are not stable in mouse blood Blood of rats Wistar pH ~ 7.0 AMP g iso =2.0059, a N =(1.605 ±0.006) mT, c =12 ps. g iso =2.0059, a N = (1.600 ±0.005) mT, c =150 ps. Lifetime - ~hours

Influence of metal cations *Lucarini et al., Chem. Eur. J. 2007, 13, mM AMP, 0.5 mM CB7, pH 3.5 Metal cations form complexes with CB7 and lead to destruction of complexes K 1 =1.7×10 5 M -1 K 2 =600 M -1 * K 3 =53 M -1 * K 5 the value 3.1×10 4 M -1

Complex 17 Complex formation no change of g-factor, a N TAMP g iso =2.0057, a N =(1.600 ±0.006) mT, c =12 ps. g iso =2.0057, a N = (1.607 ±0.007) mT, c =110 ps. TAMP CB7 (1:2) Δa N ~ 0 мТ aNaN g iso – complex with nitroxide inside the CB7 cavity (Δa N = mT)

Complex reduction rate 18 Reduction rate constants the same for TAMP и k TAMP =0.48 ±0.05 M -1 s -1 Ascorbic acid (vitamin С)

New complexes … New complexes … ? Positive charged nitroxide НИОХ СО РАН Synthesis in the process…

(SG is a glutathione residue). Gramicidin A H+H+ 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.. Add. of K 3 Fe(CN) 6 Liposomes are impermeable for large charged molecules like K3Fe(CN)6 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 (100 th –fold excess). Encapsulation in semipermeable liposomes doesnt affect the pH- sensitivity of nitroxides Encapsulation of nitroxides in polysomes Pure nitroxide:Encapsulated nitroxide:

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

Mobility of nitroxide in CB7 (solid state) c =170 ns Nitroxides in CB7 reveal low mobility due to interaction of NH 2 -group with CB-portals Correlation time of motion in liquids is determined by rotation motion of complex in CB7 c =500 ns

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.

Does encapsulation protect nitroxide from reduction? Test for reduction of encapsulated nitroxides by ascorbic acid. Encapsulated nitroxides do not affect nuclear spin relaxation times of water Calixarene works as a supramolecular protector of nitroxide T 1 relaxation of H 2 O EPR spectra of MeOTEMPO in C6 (buffer solution pH 7.4) Polovyanenko, DN; Bagryanskaya, EG; Schnegg, A, A.Savitsky,K.Moebius, A.Coleman, G. Ananchenko and J.Ripmeester, Phys.Chem.Chem.Phys. V.10, , 2008.

Comparison of the shapes of EPR spectra 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? The shapes of EPR spectra are the same for encapsulated nitroxide in solid state and in solution

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

360 GHz EPR reveals two forms of MeTEMPO in C6 4%4% Exit of ethanol molecule from C6 M EtOH = 4%(2M C6 +M TEMPO ) 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 , 10,

Mobility of nitroxides in nanocapsules – CW-EPR X-bandW-band360 GHz Poor agreement of the simulations using the isotropic spin-probe motion clearly demonstrates the necessity to improve this model. Temperature dependence of EPR spectra of the diluted

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 Y 2,0, motion anisotropy parameter 2,0 Model of motion – simulations NRs inside calix(6)arenes reveal anisotropic motion with activation energy of reorientation about kcal/M. t c =0.05 ns 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

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

Nitroxide attached to cyclodextrin g Biso = , (B) a BNiso =1.57 mT, a BHiso =0.26 mT, t corrB =2.7· s. g Aiso = , (A) a ANiso =1.54 mT, a AHiso =0.26 mT, t corrA =3.5· s. W-band EPRESEEM in D2O X-band Bagryanskaya, E.G., et. al, Appl. Magn. Reson,, (2009). Polovyanenko ett al J.Phys. Chem Krumkacheva O.et al. Lamgmur 2010 Krumkacheva et al. Appl.Magn.Reson. 2011

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

ST + R ST/R diamagnetic products main properties of spin trap: rate constant of scavenging adduct lifetime Influence of β-cyclodextrins on EPR spin trapping of glutathiyl radicals by PBN, DMPO and DEPMPO t 1/2 < 40 s t 1/2 180 s PBN PBN + RAMEB 1 mT 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

Objectives Influence on the rate constant of scavenging and adduct lifetime presence of β-cyclodextins GS + PBN + attaching of PBN to β-cyclodextins GS + 34

Measurement of k sc k GSNO = (1.5±0.3)10 9 M -1 s Polovyanenko et.al., J.Phys.Chem. B, 2008, 112. k SC PBN =(6.7±1.5)10 7 M -1 s -1

PBN/β-cyclodextrins: adduct decay RAMEB and DIMEB demonstrate the ability to strikingly (factor of 7) increase the lifetime of the adduct PBN/GS Cyclodextrink 1 obs, s -1 () none (blank)1.42 ±0.06 () RAMEB0.26 ±0.05 () DIMEB0.21 ±0.07 (Δ) TRIMEB1.26 ±0.08 PBN/GS decay (PBN:CD 1:2) 36

Spin Trapa N, mTa H, mT corr, ns k 1 obs, s -1 PBN

Заключение 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.

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

Acknowledgement: Novosibirsk Institute of Organic Chemistry I. Kirilyuk I. Grigorev 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

THANK YOU FOR YOUR ATTENTION ! THANK YOU FOR YOUR ATTENTION !

NR pK a K, M -1 H, a.u. HMP-(2.0 ±0.3)× ±0.03 AMP9.68(2.9 ±0.3)×10 3 (pH 13)0.18 ±0.05 AMPH + -(2.7 ±0.2)×10 5 (pH 5.8)8.50 ±1.40 ATI6.26N/I0.98 ±0.14 ATIH + -(1.8 ±0.4)×10 3 (pH 3.3)2.92 ±0.45 MTI1.16N/I0.02 ±0.003 MTIH + -(2.6 ±0.3)× ±0.01 Binding constants K is controlled by: - hydrophobic interactions with CB7 cavity - ion-dipole interactions of cationic groups with portals