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Electron paramagnetic resonance (EPR) with the use of exogenous spin probes is an indispensable tool to measure pH in vitro and in vivo. Monophosphonated tetrathiatriarylmethyl (p1TAM) radicals are so far the most promising EPR probes for pH measurement. However, their biological applications were limited by extremely low efficiency in synthesis and potential albumin binding, such that they could not be administered systematically. In the present work, an efficient procedure for p1TAM synthesis was reported, with a total yield of 25% from the corresponding triarylmethanol (compared to 1.6% with the commonly used procedure). Further PEGylation prevented the interaction between p1TAM and albumin. The EPR spectra of nondeuterated (POP) and deuterated (dPOP) PEGylated derivatives of p1TAM were also recorded at different pH values. The two probes had similar pH sensitivities with pKa values of 6.80 and 6.79, respectively. POP had a much more complex EPR spectrum than dPOP, with the latter always exhibiting simple EPR doublet signals regardless of the pH. These results suggested that dPOP is more advantageous for pH measurement, relative to POP and p1TAM. Moreover, dPOP was shown to have excellent biological stability and high O2 sensitivity, and thus might have the potential to be used as the probe for simultaneous pH and O2 measurements in biological system.

In order to deeply understand calix[4]arene bridge mononitration, four representative tetrasubstituted p‐tert‐butylcalix[4]arenes were selected as substrates to theoretically explore calix[4]arene structural effects. Based on our proposed mechanism, the four corresponding calix[4]arene bridge radicals were artificially constructed. Then, three different levels of density functional theory (DFT) were used to calculate their pertinent species energies in radical generations and radical spin densities. The nitration differences of these four substrates were tentatively interpreted through comparison of their reaction activation energies in radical generations and radical stability. As a result, Gibbs activation free energies in radical generations cannot rationally interpret their nitration behaviors while radical stability can, which are characterized with radical stability energies (RSEs) and center carbon spin densities (SDcs). From radical structural analysis, it can be concluded that aromatic ring inversion is remarkably beneficial to enhance calix[4]arene bridge radical stability through strengthening spin delocalization onto their connecting aromatic rings, whereas a substituent with large steric hindrance is unbeneficial due to weakening spin delocalization. The theoretical analysis of radical intrinsic structural effects on their spin delocalizations provides guidance in designing new efficient calix[4]arene‐based substrates. [ABSTRACT FROM AUTHOR]