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New Spin Probes for Biochemical Applications

This research outlines the application of nitroxides as spin probes in biochemical studies, focusing on pH-sensitive high-stable nitroxides and new spirocyclohexane-substituted nitroxides for PELDOR measurements in proteins. It compares trityl and nitroxide radicals, discusses their structures, functions, and applications. Various methods to overcome the reduction of nitroxides in vivo settings are also presented.

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New Spin Probes for Biochemical Applications

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  1. New Spin Probes for Biochemical Applications N. N.Voroztsov Novosibirsk Institute of Organic Chemistry SB RAS International Tomography Center, SB RAS, Novosibirsk, Russia Elena Bagryanskaya

  2. Outline • - application of nitroxides • pH-sesitive high stable sterically substituted nitroxides • new spirocyclohexane-substituted nitroxides for PELDOR measurements • nitronyl nitroxides as a spin probes for NO

  3. Spin probes - nitroxide and trityl radicals • Structure and function of proteins using EPR and site-directed spin labeling • pH-sensitive probes • spin probes for nitric oxide • oxymetry • redox probes • antioxidants, etc…

  4. Trityl radicals Versus Nitroxide radicals Nitroxide • Moderately broad EPR triplet; • Biostability: easily reduced • EPR resolution: relatively low • Oxygen sensitivity: relatively low; • Multiple use as redox status, pH and ROS probes as well as spin labeling agents and antioxidant, etc Trityl • Sharp EPR Singlet • Biostability: relatively stable – hours • EPR resolution: high, LW < 100 mG • Oxygen sensitivity: High • Main uses for EPR, EPR oximetry and Overhauser-enhanced MRI.

  5. What is the pH- sensitivity of nitroxides? pH 7.06 pH 4.21 pKa + H+ pH 2.56 Observed HFI constants (aN) are pH- dependent Ref.: V.Khramtsov, L. Weiner, I. Grigorjev, Volodarsky, Chem. Phys. Lett. 1982

  6. Spin probes 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 6

  7. NR2 Gramicidin А Incapsulation of spin probes in liposomes NR2in liposome Free NR2 Woldman, Ya.Y.; Semenov, S.V., Bobko, A.A.; Kirilyuk I.A.; Polienko, J.F.; Voinov, M.A.; Bagryanskaya, E.G.; Khramtsov, V.V. The Analyst, 2009, 134, 904 – 910. Reduction of nitroxide in rat homogenate of heart tissue with addition of 10 мМ succinate

  8. Reduction of nitroxide in the presence of cucurbit[7]urile AMP=0.5mM; [Asc ] = 2.5 mМ, NR + CB 1:2 + CB 1:4 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) I. Kirilyuk, D. Polovyanenko, S. Semenov, I. Grigor’ev, O. Gerasko, V. Fedin, E. Bagryanskaya, J. Phys. Chem. B 2010, 114, 1719–1728.

  9. Nitroxide reduction in rat’s blood I.A.Kirilyuk, A.A.Bobko, I.A.Grigor’ev, V.V. Khramtsov, Org.Biomol.Chem., 2004, 2, 1025 Nitroxides radicals with high stability towards reduction The reduction rates L. Marx, R. Chiarelli, T. Guiberteau and A. Rassat, J. Chem. Soc. Perkin Trans. 1, 2000, 1181-1182. 0.027 s-1 0.0009 s-1

  10. Reduction rate constants imidazollidine nitroxides with acrobat KNR NR + Asc- → NR-H + Asc-•

  11. Comparative reduction rate constants of imidazoline and imidazolidine nitroxides with acrobat 0.02 0.005

  12. Imidazolidine nitroxides ATI pK = 6.1 kred = 0.04 pK = 6.3 kred = 0.85

  13. EPR spectra of imidazolidine nitroxides Quantum chemical calculation Gaussian-983B3LYP/6-31G A A Bobko, I A Kirilyuk, N P Gritsan, D N Polovyanenko, I A Grigor’ev, V V Khramtsov, E G Bagryanskaya Applied Magnetic Resonance (2010) 39 (4), 437-451

  14. High stable hydrophilic pH-sensitive spin probe with pK 6.3

  15. PELDOR measurements of distances in proteins π/2 π V(τ) V(T) νA τ τ π T νB PELDORmeasurements are possible only at T<77 K π/2 π V(τ) V(T) νA τ τ τ1 τ1 π T νB

  16. Piperidine nitroxides with spirocyclic moiety in α-carbons Reduction in liver homogenate of mice Okazaki, S.; Mannan, M. A.; Sawai, K.; Masumizu, T.; Miura, Y.; Takeshita, K. Free Rad. Res., 2007, 41(10), 1069-1077. Kinoshita, Yu.; Yamada, K.; Yamasaki, T.; Sadsue, H.; Sakai, K.; Utsumi, H. Free Rad. Res.2009, 43(6), 565-571.

  17. Piperidine nitroxides with spirocyclic moiety as spin labels • Measurements of distances at nitrogen temperatures • Higher stability in reduction media Kathirvelu, V.; Smith, C.; Parks, C.; Mannan, M. A.; Miura, Y.; Takeshita, K.; Eaton S. S.; G. Eaton, R. Chem. Commun.2009, 454–456.

  18. New 2,5-spirocyclohexane-substituted nitroxides

  19. EPR spectra of spirocyclohexane-substituted nitroxides

  20. Comparison of electron spin relaxation times T1 (b) and Tm (a) of spirocyclohexane-substituted nitroxide and MTSSL T2and T1 of new nitroxides allow measurements of distances in proteins at nitrogen temperatures using PELDOR I. Kirilyuk, Y.F.Polienko, O.Krumkacheva, R.Strizhakov, Y. Gatilov, I. Grigorjev, E.Bagryanskaya, J.Org.Chem., 2012, doi org/10.1021/jo301235j.

  21. EPR spectra of spirocyclohexane-substituted spin labels

  22. Reduction of 2,5-spirocyclohexasubstitutednitroxides NR + AscH– → HA + Asc– Time, min 0,1 М carbonate buffer [NR] - 0,5–0,75 mM [AscH–] =100 mМ; [GSH]= 50 mМ

  23. Reduction rate constant of nitroxides by ascorbic acid The stability of 2,5-spirocyclohexane –substituted nitroxides is more that three times higher that their tetramethylated analogs and ~10–15 higher than 2,5-spirocyclohexane piperidine I. Kirilyuk, Y.F.Polienko, O.Krumkacheva, R.Strizhakov, Y. Gatilov, I. Grigorjev, E.Bagryanskaya, J.Org.Chem., 2012, doi org/10.1021/jo301235j.

  24. Role of nitric oxide (II).

  25. Nitric oxide detection using EPR of nitronyl nitroxide Akaike T. et al. Biochemistry,1993, 32, 827. Problems: toxicity and fast reduction in vivo

  26. Nitric oxide detection using NMR INH NNH 19F NMR Bobko A.A., Bagryanskaya E.G. Reznikov V, Kolosova N. Khramtsov V.V. Free Rad. Biol. & Med., 2004, 36 (2), 248–258 , BBRC, 330 (2005) 367–370.

  27. New low toxic hydrophilic nitronylnitroxides • Ration EPR signal intensities s ofNN and IN should reflect nitric oxide concentration in vivo. • EPR tomography could give informnation about NN and IN distribution NN IN If it is possible to use NN1 and NN2 in vivoas nitric oxide spin probesusing EPR tomography?

  28. Stability of nitronylnitroxides in model conditions NN1k= (1,2±0,1)·103 M–1·с–1 NN2k= (1,4±0,1)·103 M–1·с–1 Time, min The reduction rate constants of NN1 and NN2 by ascorbic acid are high and are close to the same for other NNR

  29. Stability of nitronylnitroxides in blood of rats NN1kobs = (4±1)·103 M–1·s–1 NN2kobs = (14,3±0,3)·103 M–1·s–1 Time, min The reduction rate constants of NN1 and NN2 in blood are high at low NNR concentration and are close to the same for other NNR

  30. Penetration of NNR into cells NN1 reduction in blood and it’s component: plasma and erytrocytes kкр = 4·10–3 с–1 kэр = 1.1·10–3 с–1 ≈ 4kкр octanol Coefficient of distribution octanol/water water P(NN1)= 0,85 NN1 penetrate into cells and are reduced in erythrocytes

  31. EPR tomography of mouse Pharmacokinetics of NN1in vivo (mice) Typical EPR spectrum detected during EPR tomography measurements Fast accumulation of NN1 in mice bladder

  32. Nitric oxide detection using EPR tomography of mouse INR NNR Comparison of pharmacokinetics of NN1 in control mice ( ) and with injection of nitroglycerole0,83mg/kg Only EPR spectra of NNR were detected, no contribution of INR Nitric oxide expression invivodecreases NNR concentration, which can be determined by reaction of NNR with NO as well as other physiological processes. INR was not detected, probably due to fast reduction.

  33. Conclusions • Nitroxides are the unique and very promising organic compounds with high potential for biomedical applications in therapy and diagnostics • Sterically substituted imidazoline and imidazolidinenitroxides combine high pH-sensitivity and high stability in reduction media • The new spin labels and spin probes of 7-azadispiro[5.1.5.2]-pendecane and 7-azadispiro[5.1.5.2]pentadeca-14-ene series were synthesized and demonstrated clear advantages over tetramethylpyrrolinenitroxides with respect to electron relaxation rates allowing PELROR distance measurements at liquid nitrogen temperature range and higher stability. • The new low toxic hydrophylicnitronylnitroxides were used as a spin probes for nitric oxide invivo EPR tomography. Nitric oxide expression invivodecreases NNR concentration, which can be determined by reaction of NNR with NO as well as other physiological processes.

  34. Acknowledgement: N.N.Voroztsov Novosibirsk Institute of Organic Chemistry SB RAS IgorKirilyuk Igor Grigor’ev Julia Polienko Denis Komarov International Tomography Center SB RAS Olesya Krumkacheva Sergey Semenov Rodion Strishakov Dmitrii Polovyanenko Victor Ovcharenko Elena Fursova Institute of Cytology and Genetics, Novosibirsk N. Kolosova Ohio State University, Medical Center, USA V. Khramtsov, A. Bobko

  35. Laboratory of Magnetic Resonance International Tomography Center SB RAS, Novosibirsk, Russia D. Polovyanenko S. Semenov O. Krumkacheva M. Fedin

  36. Trityl radicals Versus Nitroxide radicals Nitroxide • Moderately broad EPR triplet; • Biostability: easily reduced • EPR resolution: relatively low • Oxygen sensitivity: relatively low; • Multiple use as redox status, pH and ROS probes as well as spin labeling agents and antioxidant, etc Trityl • Sharp EPR Singlet • Biostability: relatively stable – hours • EPR resolution: high, LW < 100 mG • Oxygen sensitivity: High • Main uses for EPR, EPR oximetry and Overhauser-enhanced MRI.

  37. 0.001 0.01 0.1 1 10 K Oxidative properties of nitroxides Dikanov, S.A.; Grigor’ev, I.A.; Volodarsky, L.B.; Tsvetkov, Yu.D.; Russ. J. Phys. Chem. A, 1982

  38. Nitroxide-hydroxylamine(15N) equilibrium NR CP-15N CPH-15N

  39. Reduction by Ascorbate • DHA = dehydroascorbate • DGA = diketogulonic acid • Asc·– = ascorbate radical NN1k1 = (1.2±0.1)·103M–1·s–1 k–2 = (3.0±0.5)·103 M–1·s–1 NN2 k1 = (1.4±0.1)·103 M–1·s–1 k–2 = (3.5±0.5)·103 M–1·s–1

  40. Обратимость восстановления НР аскорбатом BBO A.A.Bobko, I.A.Kirilyuk, I.A.Grigor’ev, J.L.Zweier, V.V.Khramtsov. Free Radic. Biol. Med., 2007, V. 42, P. 404-412. [BBO] 0.1 mM,pH 7.4

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