Fmoc-POAC: [(9-fluorenylmethyloxycarbonyl)-2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-carboxylic acid]: A novel protected spin labeled beta-amino acid for peptide and protein chemistry

The stable free radical 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carb oxylic acid (TOAC) is the only spin labeled amino acid that has been used t o date to successfully label peptide sequences for structural studies. Howe ver, severe difficulty in coupling the subsequent amino acid has been the m ost serious shortcoming of this paramagnetic marker. This problem stems fro m the low nucleophilicity of TOAC's amine group towards the acylation react ion during peptide chain elongation. The present report introduces the alte rnative beta -amino acid 2,2,5,5-tetramethylpyrrolidine-N-oxyl-3-amino-4-ca rboxylic acid (POAC), potentially useful in peptide and protein chemistry. Investigations aimed at addressing the stereochemistry of this cyclic molec ule through X-ray diffraction measurements of crystalline and bulk samples revealed that it consists only of the trans conformer. The 9-fluorenylmethy loxyearbonyl group (Fmoc) was chosen for temporary protection of the POAC a mine function, allowing insertion of the probe at any position in a peptide sequence. The vasoactive octapeptide angiotensin II (AII, DRVYIHPF) was sy nthesized by replacing Pro(7) with POAC. The reaction of Fmoc-POAC with the peptidyl-resin occurred smoothly, and the coupling of the subsequent amino acid showed a much faster reaction when compared with TOAC. POAC(7)-AII wa s obtained in good yield, demonstrating that, in addition to TOAC, POAC is a convenient amino acid for the synthesis of spin labeled peptide analogues . The present findings open the possibility of a wide range of chemical and biological applications for this novel beta -amino acid derivative, includ ing structural investigations involving its differentiated bend-inducing ch aracteristics.