Synthesis and Electron Spin Relaxation of Tetracarboxylate Pyrroline Nitroxides

We report the design, synthesis, and electron spin relaxation properties of hydrophilic tetracarboxylate ester pyrroline nitroxides 1 and 2, which serve as models in the search for new spin labels for DEER distance measurement at room temperature. The nitroxides are designed to have the methyl groups further away from the N–O spin site to decrease the inequivalent couplings of the unpaired electron to the methyl protons that shorten Tm at T > 70 K in currently used labels. The key step in the synthesis of 1 and 2 is the reaction of the dianion of pyrrole-1,2,5-tricarboxylic acid tert-butyl ester dimethyl ester with electrophiles such as methyl chloroformate and methyl bromoacetate. Structures of 1 and 2 are confirmed by X-ray crystallography. Studies of electron spin relaxation rates in rigid trehalose/sucrose matrices reveal approximately temperature independent values of 1/Tm for 1 and 2 up to about 160 K and modest temperature dependence up to 295 K, demonstrating that increasing the distance between the nitroxide moiety and methyl groups is effective in lengthening Tm at T > 70 K.