A spirocyclohexyl nitroxide amino acid spin label for pulsed EPR spectroscopy distance measurements.

Site-directed spin labeling and EPR spectroscopy offer accurate, sensitive tools for the characterization of structure and function of macromolecules and their assemblies. A new rigid spin label, spirocyclohexyl nitroxide alpha-amino acid and its N-(9-fluorenylmethoxycarbonyl) derivative, have been synthesized, which exhibit slow enough spin-echo dephasing to permit accurate distance measurements by pulsed EPR spectroscopy at temperatures up to 125 K in 1:1 water/glycerol and at higher temperatures in matrices with higher glass transition temperatures. Distance measurements in the liquid nitrogen temperature range are less expensive than those that require liquid helium, which will greatly facilitate applications of pulsed EPR spectroscopy to the study of structure and conformation of peptides and proteins.