Electron spin relaxation times and rapid scan EPR imaging of pH‐sensitive amino‐substituted trityl radicals

Carboxy‐substituted trityl (triarylmethyl) radicals are valuable in vivo probes because of their stability, narrow lines, and sensitivity of their spectroscopic properties to oxygen. Amino‐substituted trityl radicals have the potential to monitor pH in vivo, and the suitability for this application depends on spectral properties. Electron spin relaxation times T₁ and T₂ were measured at X‐band for the protonated and deprotonated forms of two amino‐substituted triarylmethyl radicals. Comparison with relaxation times for carboxy‐substituted triarylmethyl radicals shows that T₁ exhibits little dependence on protonation or the nature of the substituent, which makes it useful for measuring O₂ concentration, independent of pH. Insensitivity of T₁ to changes in substituents is consistent with the assignment of the dominant contribution to spin lattice relaxation as a local mode that involves primarily atoms in the carbon and sulfur core. Values of T₂ vary substantially with pH and the nature of the aryl group substituent, reflecting a range of dynamic processes. The narrow spectral widths for the amino‐substituted triarylmethyl radicals facilitate spectral‐spatial rapid scan electron paramagnetic resonance imaging, which was demonstrated with a phantom. The dependence of hyperfine splittings patterns on pH is revealed in spectral slices through the image. Copyright © 2014 John Wiley & Sons, Ltd.     

5-(2-Aminoethyl)dithio-2-nitrobenzoate as a more base-stable alternative to Ellman's reagent

[reaction: see text] 5-(2-Aminoethyl)dithio-2-nitrobenzoate (ADNB) reacts with free thiols with kinetics similar to those of Ellman's reagent but has dramatically improved stability under alkaline conditions, making it an excellent alternative to Ellman's reagent for the quantitation of thiol contents and enzymatic assays under basic pH conditions.     

Synthesis and characterization of amino derivatives of persistent trityl radicals as dual function pH and oxygen paramagnetic probes

Triarylmethyl radicals, TAMs, are useful soluble paramagnetic probes for EPR spectroscopic and imaging applications because of their extraordinary stability in living tissues, narrow line width, high analytical resolution at micromolar concentrations and enhanced sensitivity to molecular oxygen. Recently we proposed the concept of dual function pH and oxygen TAM probes based on the incorporation of ionizable groups into the TAM structure (J. Am. Chem. Soc. 2007, 129 (23), 7240-7241). In this paper we report the synthesis of TAM derivatives containing amino groups. The synthesized TAMs combine stability with oxygen and pH sensitivity, in the range of pH from 6.8 to 9.0. To decrease the number of spectral components and improve probe solubility at physiological pH, asymmetric TAM derivatives containing both carboxyl and amino functions were synthesized. The presence of nitrogen and hydrogen atoms in direct proximity to protonatable amino groups resulted in strong pH-induced changes to the corresponding hyperfine splittings, Delta hfs approximately (300-1000) mG, comparable to the values of hfs themselves. Large pH-dependent line shifts of individual spectral components, with narrow linewidths of (160-280) mG, allow for easy discrimination between the pH effect and the observed oxygen-dependent line broadening of about (6 +/- 0.5) mG per % oxygen. The synthesized TAM derivatives represent the first dual function pH and oxygen paramagnetic probes with reasonably valuable properties for biomedical research.     

Synthesis and characterization of ester-derivatized tetrathiatriarylmethyl radicals as intracellular oxygen probes

Electron paramagnetic resonance (EPR) spectroscopy using paramagnetic probes has been employed as an important tool for the accurate determination of oxygen (O2) concentrations in biological systems. However, paramagnetic probes are still limited by their intracellular penetrability. Various esterified trityl derivatives were synthesized and characterized, and an X-ray structure of one of the triyl radicals was determined. The ester-derivatized trityls exhibited higher sensitivity to O2 concentration compared to the trityl tricarboxylate CT-03. Cyclic voltammetry was also carried out to assess the susceptibility of the trityl radicals to oxidation and reduction. Among all of the ester-derivatized trityls studied, facile hydrolysis of the acetoxymethoxy esters to the respective carboxylate was observed using porcine liver esterase. This study demonstrates that cellular permeability of the trityl radicals can be achieved by varying the type and number of ester groups. Therefore, ester-derivatized trityl radicals show great potential as intracellular EPR oximetry probes and imaging agents.     

Proton-Electron Double-Resonance Imaging of pH Using Phosphonated Trityl Probe

Variable radio frequency proton-electron double-resonance imaging (VRF PEDRI) enables extracting a functional map from a limited number of images acquired at pre-selected EPR frequencies using specifically designed paramagnetic probes with high-quality spatial resolution and short acquisition times. In this work we explored the potential of VRF PEDRI for pH mapping of aqueous samples using recently synthesized pH-sensitive phosphonated trityl radical, pTR. The ratio of Overhauser enhancements measured at each pixel at two different excitation frequencies corresponding to the resonances of protonated and deprotonated forms of pTR probe allows for a pH map extraction. Long relaxation times of pTR allow for pH mapping at EPR irradiation power as low as 1.25 W during 130 s acquisition time with spatial resolution of about 1 mm. This is particularly important for in vivo applications enabling one to avoid sample overheating by reducing RF power deposition.     

Fourier Transform EPR Spectroscopy of Trityl Radicals for Multifunctional Assessment of Chemical Microenvironment

Pulse techniques in electron paramagnetic resonance (EPR) allow for a reduction in measurement times and increase in sensitivity but require the synthesis of paramagnetic probes with long relaxation times. Here it is shown that the recently synthesized phosphonated trityl radical possesses long relaxation times that are sensitive to probe the microenvironment, such as oxygenation and acidity of an aqueous solution. In principle, application of Fourier transform EPR (FT‐EPR) spectroscopy makes it possible to acquire the entire EPR spectrum of the trityl probe and assess these microenvironmental parameters within a few microseconds. The performed analysis of the FT‐EPR spectra takes into consideration oxygen‐, proton‐, buffer‐, and concentration‐induced contributions to the spectral shape, therefore enabling quantitative and discriminative assessment of pH, pO₂, and concentrations of the probe and inorganic phosphate.     

Investigation of the Yamaguchi esterification mechanism. Synthesis of a lux-s enzyme inhibitor using an improved esterification method

[reaction: see text] A one-pot procedure for the regioselective synthesis of aliphatic esters is described. This was a result of a study on mixed aliphatic-aromatic anhydrides. The data suggest that during the Yamaguchi esterification reaction, a symmetric aliphatic anhydride is produced in situ, which upon reaction with an alcohol yields the ester. We confirmed that benzoyl chloride could be used instead of the sterically hindered Yamaguchi acid chloride. This method was successfully applied in the synthesis of Lux-S aspartic acid inhibitor.