Spin-labeled pH-sensitive phospholipids for interfacial pKa determination: synthesis and characterization in aqueous and micellar solutions

摘要

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 head group is 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-1 H-imidazol-2-yl)benzyl methanethiosulfonate (IKMTSL). The pKa value of the IMTSL-PTE lipid measured by EPR titration in aqueous buffer/iso-propanol solutions of various compositions was found to be essentially the same – pKa≈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/iso-propanol solution allowed for calibrating the polarity-induced pKa shifts, ΔpKapol, vs. bulk solvent dielectric permittivity. These calibration data allowed for estimating the local dielectric constant, εeff, experienced by the reporter nitroxide of the IMTSL-PTE lipid incorporated into the non-ionic Triton^® X-100 micelles as 60±5 and 57±5 at 23 and 48 °C respectively. For micelles formed from an anionic surfactant sodium dodecyl sulfate (SDS) the electrostatic-induced pKa shift, ΔpKael=2.06±0.04 units of pH, was obtained by subtracting the polarity-induced contribution. This shift yields Ψ= –121 mV electric potential of the SDS micelle surface.