Voinov Maxim A, Kirilyuk Igor A, Smirnov Alex I
Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695-8204, USA.
J Phys Chem B. 2009 Mar 19;113(11):3453-60. doi: 10.1021/jp810993s.
The synthesis and characterization of spin-labeled phospholipids (SLP)--derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (PTE)--with pH-reporting nitroxides that are covalently attached to the lipid's polar headgroup are being reported. Two lipids were synthesized by reactions of PTE with thiol-specific, pH-sensitive methanethiosulfonate spin labels methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethylimidazolidin-4-ylmethyl) ester (IMTSL) and S-4-(4-(dimethylamino)-2-ethyl-5,5-dimethyl-1-oxyl-2,5-dihydro-1H-imidazol-2-yl)benzyl methanethiosulfonate (IKMTSL). The pKa values of the IMTSL-PTE lipid measured by EPR titration in aqueous buffer/isopropyl alcohol solutions of various compositions were found to be essentially the same (pKa approximately 2.35), indicating that in mixed aqueous/organic solvents, the amphiphilic lipid molecules could be shielded from changing bulk conditions by a local shell of solvent molecules. To overcome this problem, the spin-labeled lipids were modeled by synthesizing IMTSL- and IKMTSL-2-mercaptoethanol adducts. These model compounds yielded the intrinsic pKa0's for IMTSL-PTE and IKMTSL-PTE in aqueous buffers as 3.33 +/- 0.03 and 5.98 +/- 0.03, respectively. A series of EPR titrations of IMTSL-PTE in mixed water/isopropyl alcohol solution allowed for calibrating the polarity-induced pKa shifts, deltapKapol, vs bulk solvent dielectric permittivity. These calibration data allowed for estimating the local dielectric constant, epsilon(eff), experienced by the reporter nitroxide of the IMTSL-PTE lipid incorporated into the nonionic Triton X-100 micelles as 60 +/- 5 and 57 +/- 5 at 23 and 48 degrees C, respectively. For micelles formed from an anionic surfactant sodium dodecyl sulfate (SDS) the electrostatic-induced pKa shift, deltapKael = 2.06 +/- 0.04 units of pH, was obtained by subtracting the polarity-induced contribution. This shift yields psi = -121 mV electric potential of the SDS micelle surface.
本文报道了自旋标记磷脂（SLP）的合成与表征，SLP是1,2 - 二棕榈酰 - sn - 甘油 - 3 - 硫代磷酸乙醇（PTE）的衍生物，其pH报告型氮氧化物共价连接在脂质的极性头部基团上。通过PTE与硫醇特异性、pH敏感的甲硫基磺酸自旋标记物甲硫基磺酸S - (1 - 氧基 - 2,2,3,5,5 - 五甲基咪唑啉 - 4 - 基甲基)酯（IMTSL）和S - 4 - (4 - (二甲氨基) - 2 - 乙基 - 5,5 - 二甲基 - 1 - 氧基 - 2,5 - 二氢 - 1H - 咪唑 - 2 - 基)苄基甲硫基磺酸酯（IKMTSL）反应合成了两种脂质。通过在各种组成的水性缓冲液/异丙醇溶液中进行EPR滴定测量，发现IMTSL - PTE脂质的pKa值基本相同（pKa约为2.35），这表明在混合水/有机溶剂中，两亲性脂质分子可被溶剂分子的局部壳层屏蔽，免受整体条件变化的影响。为克服此问题，通过合成IMTSL - 和IKMTSL - 2 - 巯基乙醇加合物对自旋标记脂质进行建模。这些模型化合物在水性缓冲液中得到IMTSL - PTE和IKMTSL - PTE的固有pKa0分别为3.33±0.03和5.98±0.03。在水/异丙醇混合溶液中对IMTSL - PTE进行一系列EPR滴定，从而校准极性诱导的pKa变化量ΔpKapol与整体溶剂介电常数的关系。这些校准数据使得能够估计掺入非离子Triton X - 100胶束中的IMTSL - PTE脂质的报告型氮氧化物在23℃和48℃时分别经历的局部介电常数ε(eff)为60±5和57±5。对于由阴离子表面活性剂十二烷基硫酸钠（SDS）形成的胶束，通过减去极性诱导的贡献，得到静电诱导导致的pKa变化量ΔpKael = 2.06±0.04个pH单位。此变化产生SDS胶束表面的ψ = - 121 mV的电势。
