Other Biradicals and Spin Labeling

  • Yuri D. TsvetkovEmail author
  • Michael K. Bowman
  • Yuri A. Grishin


This chapter describes PELDOR or DEER spectroscopic measurements on stable biradicals other than nitroxides and strategies for spin labeling of biomolecules. The most common non-nitroxide biradicals are based on tris-(tetrathiaaryl) methyl radicals, known as trityl or TAM radicals. Their narrow spectral widths can present challenges for measurements, but their slow spin relaxation and intense signals make it possible to measure quite long distances. Metal complexes are also used to construct biradicals, particularly ones having moieties with very different spectral characteristics. Many of these same radicals along with nitroxides are used for site-directed spin labelling of biomolecules and complexes, including proteins and nucleic acids.


  1. 1.
    Reddy TJ, Iwama T, Halpern HJ, Rawal VH (2002) General synthesis of persistent trityl radicals for EPR imaging of biological systems. J Org Chem 67(14):4635–4639CrossRefGoogle Scholar
  2. 2.
    Fielding AJ, Carl PJ, Eaton GR, Eaton SS (2005) Multifrequency EPR of four triarylmethyl radicals. Appl Magn Reson 28(3–4):231–238CrossRefGoogle Scholar
  3. 3.
    Owenius R, Eaton GR, Eaton SS (2005) Frequency (250 MHz to 9.2 GHz) and viscosity dependence of electron spin relaxation of triarylmethyl radicals at room temperature. J Magn Reson 172(1):168–175CrossRefGoogle Scholar
  4. 4.
    Bobko AA, Dhimitruka I, Zweier JL, Khramtsov VV (2007) Trityl radicals as persistent dual function pH and oxygen probes for in vivo electron paramagnetic resonance spectroscopy and imaging: concept and experiment. J Am Chem Soc 129(23):7240–7241. Scholar
  5. 5.
    Liu Y, Villamena FA, Sun J, Xu Y, Dhimitruka I, Zweier JL (2008) Synthesis and characterization of ester-derivatized tetrathiatriarylmethyl radicals as intracellular oxygen probes. J Org Chem 73(4):1490–1497CrossRefGoogle Scholar
  6. 6.
    Bowman MK, Mailer C, Halpern HJ (2005) The solution conformation of triarylmethyl radicals. J Magn Reson 172(2):254–267CrossRefGoogle Scholar
  7. 7.
    Trukhan SN, Yudanov VF, Tormyshev VM, Rogozhnikova OY, Trukhin DV, Bowman MK, Krzyaniak MD, Chen H, Martyanov ON (2013) Hyperfine interactions of narrow-line trityl radical with solvent molecules. J Magn Reson 233:29–36. Scholar
  8. 8.
    Kunjir NC, Reginsson GW, Schiemann O, Sigurdsson ST (2013) Measurements of short distances between trityl spin labels with CW EPR, DQC and PELDOR. Phys Chem Chem Phys 15(45):19673–19685. Scholar
  9. 9.
    Karoui H, Moigne FL, Ouari O, Tordo P (2010) Nitroxide radicals: properties, synthesis and applications. In: Stable Radicals. Wiley, pp 173–229. Scholar
  10. 10.
    Shevelev GY, Krumkacheva OA, Lomzov AA, Kuzhelev AA, Rogozhnikova OY, Trukhin DV, Troitskaya TI, Tormyshev VM, Fedin MV, Pyshnyi DV, Bagryanskaya EG (2014) Physiological-temperature distance measurement in nucleic acid using triarylmethyl-based spin labels and pulsed dipolar EPR spectroscopy. J Am Chem Soc 136(28):9874–9877. Scholar
  11. 11.
    Shevelev GY, Krumkacheva OA, Lomzov AA, Kuzhelev AA, Trukhin DV, Rogozhnikova OY, Tormyshev VM, Pyshnyi DV, Fedin MV, Bagryanskaya EG (2015) Triarylmethyl labels: toward improving the accuracy of EPR nanoscale distance measurements in DNAs. J Phys Chem B 119(43):13641–13648CrossRefGoogle Scholar
  12. 12.
    Krumkacheva O, Bagryanskaya E (2017) EPR-based distance measurements at ambient temperature. J Magn Reson 280:117–126. Scholar
  13. 13.
    Reginsson GW, Kunjir NC, Sigurdsson ST, Schiemann O (2012) Trityl radicals: spin labels for nanometer-distance measurements. Chemistry 18(43):13580–13584. Scholar
  14. 14.
    Akhmetzyanov D, Schops P, Marko A, Kunjir NC, Sigurdsson ST, Prisner TF (2015) Pulsed EPR dipolar spectroscopy at Q- and G-band on a trityl biradical. Phys Chem Chem Phys 17(37):24446–24451CrossRefGoogle Scholar
  15. 15.
    Yang ZY, Liu YP, Borbat P, Zweier JL, Freed JH, Hubbell WL (2012) Pulsed ESR dipolar spectroscopy for distance measurements in immobilized spin labeled proteins in liquid solution. J Am Chem Soc 134(24):9950–9952CrossRefGoogle Scholar
  16. 16.
    Kuzhelev AA, Tormyshev VM, Rogozhnikova OY, Trukhin DV, Troitskaya TI, Strizhakov RK, Krumkacheva OA, Fedin MV, Bagryanskaya EG (2017) Triarylmethyl radicals: EPR study of 13C hyperfine coupling constants. Z Phys Chem 231(4):777–794. Scholar
  17. 17.
    Bode BE, Plackmeyer J, Prisner TF, Schiemann O (2008) PELDOR measurements on a nitroxide-labeled Cu(II) porphyrin: orientation selection, spin-density distribution, and conformational flexibility. J Phys Chem A 112(23):5064–5073. Scholar
  18. 18.
    Bode BE, Plackmeyer J, Bolte M, Prisner TF, Schiemann O (2009) PELDOR on an exchange coupled nitroxide copper(II) spin pair. J Organomet Chem 694(7–8):1172–1179. Scholar
  19. 19.
    Narr E, Godt A, Jeschke G (2002) Selective measurements of a nitroxide-nitroxide separation of 5 nm and a nitroxide-copper separation of 2.5 nm in a terpyridine-based copper(II) complex by pulse EPR spectroscopy. Angew Chem Int Edit 41(20):3907–3910CrossRefGoogle Scholar
  20. 20.
    Lueders P, Jeschke G, Yulikov M (2011) Double electron-electron resonance measured between Gd3+ ions and nitroxide radicals. J Phys Chem Lett 2(6):604–609. Scholar
  21. 21.
    Raitsimring AM, Gunanathan C, Potapov A, Efremenko I, Martin JML, Milstein D, Goldfarb D (2007) Gd3+ complexes as potential spin labels for high field pulsed EPR distance measurements. J Am Chem Soc 129(46):14138–14139. Scholar
  22. 22.
    Potapov A, Song Y, Meade TJ, Goldfarb D, Astashkin AV, Raitsimring A (2010) Distance measurements in model bis-Gd(III) complexes with flexible “bridge”. Emulation of biological molecules having flexible structure with Gd(III) labels attached. J Magn Reson 205(1):38–49CrossRefGoogle Scholar
  23. 23.
    Berliner LJ (ed) (2002) Spin labeling, vol 14. Biological magnetic resonance. Springer US, New York. Scholar
  24. 24.
    Bender C, Berliner LJ (eds) (2004) EPR: instrumental methods, vol 21. biological magnetic resonance, vol 21. Springer, New York. Scholar
  25. 25.
    Hubbell WL, Altenbach C (1994) Site-directed spin labeling of membrane proteins. In: White SH (ed) Membrane protein structure: experimental approaches. methods in physiology. Springer, New York, pp 224–248. Scholar
  26. 26.
    Hubbell WL, Gross A, Langen R, Lietzow MA (1998) Recent advances in site-directed spin labeling of proteins. Curr Opin Struc Biol 8(5):649–656CrossRefGoogle Scholar
  27. 27.
    Berliner LJ, Grunwald J, Hankovszky HO, Hideg K (1982) A novel reversible thiol-specific spin label—papain active-site labeling and inhibition. Anal Biochem 119(2):450–455CrossRefGoogle Scholar
  28. 28.
    Spaltenstein A, Robinson BH, Hopkins PB (1988) A rigid and nonperturbing probe for duplex DNA motion. J Am Chem Soc 110(4):1299–1301CrossRefGoogle Scholar
  29. 29.
    Sale K, Song LK, Liu YS, Perozo E, Fajer P (2005) Explicit treatment of spin labels in modeling of distance constraints from dipolar EPR and DEER. J Am Chem Soc 127(26):9334–9335CrossRefGoogle Scholar
  30. 30.
    Anderson DJ, Hanson P, McNulty J, Millhauser G, Monaco V, Formaggio F, Crisma M, Toniolo C (1999) Solution structures of TOAC-labeled trichogin GA IV peptides from allowed (g ≈ 2) and half-field electron spin resonance. J Am Chem Soc 121(29):6919–6927. Scholar
  31. 31.
    Rassat A, Rey P (1967) Nitroxides. 23. preparation of amino-acid free radicals and their complex salts. B Soc Chim Fr 3:815–817Google Scholar
  32. 32.
    Seidemann R, Dulog L (1986) N-Carboxyanhydride of 4-Amino-4-Carboxy-2,2,6,6-Tetramethylpiperidine-1-Oxyl, a New Paramagnetic Monomer. Makromol Chem 187(11):2545–2551CrossRefGoogle Scholar
  33. 33.
    Toniolo C, Crisma M, Formaggio F (1998) TOAC, a nitroxide spin-labeled, achiral C(alpha)-tetrasubstituted alpha-amino acid, is an excellent tool in material science and biochemistry. Biopolymers 47(2):153–158CrossRefGoogle Scholar
  34. 34.
    Martin L, Ivancich A, Vita C, Formaggio F, Toniolo C (2001) Solid-phase synthesis of peptides containing the spin-labeled 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC). J Pept Res 58(5):424–432CrossRefGoogle Scholar
  35. 35.
    Formaggio E, Broxterman QA, Toniolo C (2003) Synthesis of peptides based on Ca-tetrasubstituted α-amino acids. In: Goodman M, Felix A, Moroder L, Toniolo C (eds) Supplement—synthesis of peptides and peptidomimetics, vol E 22c, 4th edn. Houben-weyl methods of organic chemistry. Thieme, Stuttgart, pp 292–310Google Scholar
  36. 36.
    Salikhov KM, Tsvetkov YD (1979) Electron spin echo studies of spin-spin interactions in solids. In: Kevan L, Schwartz RN (eds) Time domain electron spin resonance. Wiley, New York, pp 231–278Google Scholar
  37. 37.
    Tsvetkov YD, Dzuba SA (1990) Pulsed ESR and molecular motions. Appl Magn Reson 1(2):179–194CrossRefGoogle Scholar
  38. 38.
    Rajca A, Kathirvelu V, Roy SK, Pink M, Rajca S, Sarkar S, Eaton SS, Eaton GR (2010) A spirocyclohexyl nitroxide amino acid spin label for pulsed EPR spectroscopy distance measurements. Chem-Eur J 16(19):5778–5782CrossRefGoogle Scholar
  39. 39.
    Meyer V, Swanson MA, Clouston LJ, Boratynski PJ, Stein RA, McHaourab HS, Rajca A, Eaton SS, Eaton GR (2015) Room-temperature distance measurements of immobilized spin-labeled protein by DEER/PELDOR. Biophys J 108(5):1213–1219. Scholar
  40. 40.
    Shevelev GY, Gulyak EL, Lomzov AA, Kuzhelev AA, Krumkacheva OA, Kupryushkin MS, Tormyshev VM, Fedin MV, Bagryanskaya EG, Pyshnyi DV (2018) A versatile approach to attachment of triarylmethyl labels to DNA for nanoscale structural EPR studies at physiological temperatures. J Phys Chem B 122(1):137–143. Scholar
  41. 41.
    Fielding AJ, Concilio MG, Heaven G, Hollas MA (2014) New developments in spin labels for pulsed dipolar EPR. Molecules 19(10):16998–17025CrossRefGoogle Scholar
  42. 42.
    Rossi R, Carpita A, Bellina F (1995) Palladium-mediated and/or copper-mediated cross-coupling reactions between 1-alkynes and vinyl, aryl, 1-alkynyl, 1,2-propadienyl, propargyl and allylic halides or related-compounds—a review. Org Prep Proced Int 27(2):127–160CrossRefGoogle Scholar
  43. 43.
    Chinchilla R, Najera C (2007) The sonogashira reaction: A booming methodology in synthetic organic chemistry. Chem Rev 107(3):874–922CrossRefGoogle Scholar
  44. 44.
    Piton N, Schiemann O, Mu Y, Stock G, Prisner T, Engels JW (2005) Synthesis of spin-labeled RNAs for long range distance measurements by peldor. Nucleosides, Nucleotides Nucleic Acids 24(5–7):771–775CrossRefGoogle Scholar
  45. 45.
    Gannett PM, Darian E, Powell JH, Johnson EM (2001) A short procedure for synthesis of 4-ethynyl-2,2,6,6-tetramethyl-3,4-dehydro-piperidine-1-oxyl nitroxide. Synthetic Commun 31(14):2137–2141CrossRefGoogle Scholar
  46. 46.
    Gannett PM, Darian E, Powell J, Johnson EM, Mundoma C, Greenbaum NL, Ramsey CM, Dalal NS, Budil DE (2002) Probing triplex formation by EPR spectroscopy using a newly synthesized spin label for oligonucleotides. Nucleic Acids Res 30(23):5328–5337CrossRefGoogle Scholar
  47. 47.
    Frolow O, Bode BE, Angels JW (2007) The synthesis of EPR differentiable spinlabels and their coupling to uridine. Nucleos Nucleot Nucl 26(6–7):655–659CrossRefGoogle Scholar
  48. 48.
    Piton N, Mu YG, Stock G, Prisner TF, Schiemann O, Engels JW (2007) Base-specific spin-labeling of RNA for structure determination. Nucleic Acids Res 35(9):3128–3143CrossRefGoogle Scholar
  49. 49.
    Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Edit 40(11):2004–2021CrossRefGoogle Scholar
  50. 50.
    Ding P, Wunnicke D, Steinhoff HJ, Seela F (2010) Site-directed spin-labeling of DNA by the Azide-Alkyne ‘Click’ reaction: nanometer distance measurements on 7-Deaza-2‘-deoxyadenosine and 2’-deoxyuridine nitroxide conjugates spatially separated or linked to a ‘dA-dT’ base pair. Chem-Eur J 16(48):14385–14396CrossRefGoogle Scholar
  51. 51.
    Jakobsen U, Shelke SA, Vogel S, Sigurdsson ST (2010) Site-directed spin-labeling of nucleic acids by click chemistry: detection of abasic sites in duplex DNA by EPR spectroscopy. J Am Chem Soc 132(30):10424–10428CrossRefGoogle Scholar
  52. 52.
    Barhate N, Cekan P, Massey AP, Sigurdsson ST (2007) A nucleoside that contains a rigid nitroxide spin label: a fluorophore in disguise. Angew Chem Int Edit 46(15):2655–2658CrossRefGoogle Scholar
  53. 53.
    Marko A, Denysenkov V, Margraft D, Cekan P, Schiemann O, Sigurdsson ST, Prisner TF (2011) Conformational flexibility of DNA. J Am Chem Soc 133(34):13375–13379CrossRefGoogle Scholar
  54. 54.
    Tkach I, Pornsuwan S, Hobartner C, Wachowius F, Sigurdsson ST, Baranova TY, Diederichsen U, Sicoli G, Bennati M (2013) Orientation selection in distance measurements between nitroxide spin labels at 94 GHz EPR with variable dual frequency irradiation. Phys Chem Chem Phys 15(10):3433–3437CrossRefGoogle Scholar
  55. 55.
    Stoller S, Sicoli G, Baranova TY, Bennati M, Diederichsen U (2011) TOPP: a novel nitroxide-labeled amino acid for EPR distance measurements. Angew Chem Int Edit 50(41):9743–9746CrossRefGoogle Scholar
  56. 56.
    Fedin MV, Shevelev GY, Pyshnyi DV, Tormyshev VM, Jeschke G, Yulikov M, Bagryanskaya EG (2016) Interaction of triarylmethyl radicals with DNA termini revealed by orientation-selective W-band double electron-electron resonance spectroscopy. Phys Chem Chem Phys 18(42):29549–29554. Scholar
  57. 57.
    Savitsky A, Dubinskii AA, Flores M, Lubitz W, Mobius K (2007) Orientation-resolving pulsed electron dipolar high-field EPR spectroscopy on disordered solids: I. Structure of spin-correlated radical pairs in bacterial photosynthetic reaction centers. J Phys Chem B 111(22):6245–6262. Scholar
  58. 58.
    Denysenkov VP, Prisner TF, Stubbe J, Bennati M (2006) High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase. P Natl Acad Sci USA 103(36):13386–13390. Scholar
  59. 59.
    Denysenkov VP, Biglino D, Lubitz W, Prisner TF, Bennati M (2008) Structure of the tyrosyl biradical in mouse R2 ribonucleotide reductase from high-field PELDOR. Angew Chem Int Edit 47(7):1224–1227CrossRefGoogle Scholar
  60. 60.
    van Amsterdam IMC, Ubbink M, Canters GW, Huber M (2003) Measurement of a Cu-Cu distance of 26 Å by a pulsed EPR method. Angew Chem Int Edit 42(1):62–64CrossRefGoogle Scholar
  61. 61.
    Becker JS, Saxena S (2005) Double quantum coherence electron spin resonance on coupled Cu(II)-Cu(II) electron spins. Chem Phys Lett 414(1–3):248–252CrossRefGoogle Scholar
  62. 62.
    Elsasser C, Brecht M, Bittl R (2002) Pulsed electron-electron double resonance on multinuclear metal clusters: assignment of spin projection factors based on the dipolar interaction. J Am Chem Soc 124(42):12606–12611CrossRefGoogle Scholar
  63. 63.
    Roessler MM, King MS, Robinson AJ, Armstrong FA, Harmer J, Hirst J (2010) Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron-electron resonance. P Natl Acad Sci USA 107(5):1930–1935CrossRefGoogle Scholar
  64. 64.
    Garbuio L, Bordignon E, Brooks EK, Hubbell WL, Jeschke G, Yulikov M (2013) Orthogonal spin labeling and Gd(III)-nitroxide distance measurements on bacteriophage T4-lysozyme. J Phys Chem B 117(11):3145–3153CrossRefGoogle Scholar
  65. 65.
    Potapov A, Yagi H, Huber T, Jergic S, Dixon NE, Otting G, Goldfarb D (2010) Nanometer-Scale distance measurements in proteins using Gd3 + spin labeling. J Am Chem Soc 132(26):9040–9048CrossRefGoogle Scholar
  66. 66.
    Banerjee D, Yagi H, Huber T, Otting G, Goldfarb D (2012) Nanometer-range distance measurement in a protein using Mn2+ tags. J Phys Chem Lett 3(2):157–160CrossRefGoogle Scholar
  67. 67.
    Bagryanskaya EG, Krumkacheva OA, Fedin MV, Marque SRA (2015) Development and application of spin traps, spin probes, and spin labels. Method Enzymol 563:365–396CrossRefGoogle Scholar
  68. 68.
    Goldfarb D (2014) Gd3+ spin labeling for distance measurements by pulse EPR spectroscopy. Phys Chem Chem Phys 16(21):9685–9699. Scholar
  69. 69.
    Akhmetzyanov D, Schop P, Marco A, Kunjir NC, Sigurdsson ST, Prisner TF (2013) Phys Chem Chem Phys 15:619–627CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Yuri D. Tsvetkov
    • 1
    Email author
  • Michael K. Bowman
    • 2
  • Yuri A. Grishin
    • 3
  1. 1.The Voevodsky Institute of Chemical Kinetics and CombustionNovosibirskRussia
  2. 2.Department of Chemistry and BiochemistryUniversity of AlabamaTuscaloosaUSA
  3. 3.The Voevodsky Institute of Chemical Kinetics and CombustionNovosibirskRussia

Personalised recommendations