title: IKMTSL-PTE, a Phospholipid-Based EPR Probe for Surface Electrostatic Potential of Biological Interfaces at Neutral pH: Effects of Temperature and Effective Dielectric Constant of the Solvent.
abstract: The synthesis and characterization of a lipidlike electrostatic spin probe, (S)-2,3-bis(palmitoyloxy)propyl 2-((4-(4-(dimethylamino)-2-ethyl-1-oxyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-2-yl)benzyl)disulfanyl)ethyl phosphate (IKMTSL-PTE), are being reported. The intrinsic pKa0of IKMTSL-PTE was determined by X-band (9.5 GHz) electron paramagnetic resonance (EPR) titration of a water-soluble model compound, 4-(dimethylamino)-2-ethyl-2-(4-(((2-hydroxyethyl)disulfanyl)methyl)phenyl)-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl (IKMTSL-ME), an adduct of methanethiosulfonate spin label IKMTSL and 2-mercaptoethanol. The pKa0of IKMTSL-ME in bulk aqueous solutions was found to be significantly higher than that of 4-(((2-hydroxyethyl)disulfanyl)methyl)-2,2,3,5,5-pentamethylimidazolidin-1-oxyl (IMTSL-ME), an adduct of the corresponding methanethiosulfonate spin label IMTSL and 2-mercaptoethanol (17 °C, pKa0= 6.16 ± 0.03 vs 20 °C, pKa0= 3.33 ± 0.03, respectively). A series of EPR titration experiments with IKMTSL-ME in aqueous solutions containing 0-60% v/v isopropanol have been carried out at 17 and 48 °C to determine the effects of temperature and bulk dielectric permittivity constant, ε, on the probe pKa. A linear relationship between the probe pKaand ε has been established and found to be essentially the same at 17 and 48 °C. The polarity term contributing to the pKaof IKMTSL-PTE at an uncharged lipidlike interface was determined by incorporating the probe into electrically neutral micelles formed from nonionic detergent Triton X-100, and it was found, similar to IMTSL-PTE, to be negative. In negatively charged DMPG lipid bilayers, IKMTSL-PTE exhibits ionization transitions with significantly higher pKavalues than those previously reported for IMTSL-PTE (e.g., at 17 °C, pKai= 7.80 ± 0.03 vs pKa0= 5.70 ± 0.05). The surface electrostatic potentials of DMPG lipid bilayers calculated using IKMTSL-PTE titration data were found to be somewhat lower than those calculated using IMTSL-PTE. The lower values measured by IKMTSL-PTE are the likely consequences of the structure of the linker that positions the reporter nitroxide further away from the bilayer plane into aqueous phase. Overall, the ionization transitions of IKMTSL-PTE with pKavalues close to the neutral pH range make this lipidlike molecule a valuable spectroscopic EPR probe for studying the electrostatic phenomena at biological interfaces, including lipid bilayer/membrane protein systems, that could be unstable in the acidic pH range accessible by the previously available probes.
authors.affiliation: Department of Chemistry, North Carolina State University , 2620 Yarbrough Drive, Raleigh, North Carolina 27695-8204, United States.
authors.forename: Maxim A
authors.initials: MA
authors.lastname: Voinov
authors.affiliation: Department of Chemistry, North Carolina State University , 2620 Yarbrough Drive, Raleigh, North Carolina 27695-8204, United States.
authors.forename: Christina T
authors.initials: CT
authors.lastname: Scheid
authors.affiliation: N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, SB RAS , Lavrentiev Avenue 9, Novosibirsk 630090, Russia.|Novosibirsk State University , Pirogova Street 2, Novosibirsk 630090, Russia.
authors.forename: Igor A
authors.initials: IA
authors.lastname: Kirilyuk
authors.affiliation: N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, SB RAS , Lavrentiev Avenue 9, Novosibirsk 630090, Russia.
authors.forename: Dmitrii G
authors.initials: DG
authors.lastname: Trofimov
authors.affiliation: Department of Chemistry, North Carolina State University , 2620 Yarbrough Drive, Raleigh, North Carolina 27695-8204, United States.
authors.forename: Alex I
authors.identifier: 0000-0002-0037-2555
authors.initials: AI
authors.lastname: Smirnov
mesh_terms: D019840:2-Propanol / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D020404:Glycerophospholipids / Q000138:chemical synthesis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000138:chemical synthesis / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry; D008823:Micelles; D017830:Octoxynol / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D012997:Solvents / Q000737:chemistry*; D013113:Spin Labels / Q000138:chemical synthesis; D055672:Static Electricity; D013696:Temperature; D014867:Water / Q000737:chemistry
chemical_list: D020404:Glycerophospholipids; D007093:Imidazoles; D008051:Lipid Bilayers; D008823:Micelles; D010715:Phosphatidylglycerols; D012997:Solvents; D013113:Spin Labels; D014867:Water; D017830:Octoxynol; C002773:dimyristoylphosphatidylglycerol; D019840:2-Propanol