New Phototriggers 9: p-Hydroxyphenacyl as a C-Terminal Photoremovable Protecting Group for Oligopeptides

In our search for a more versatile protecting group that would exhibit fast release rates for peptides, we have designed and developed the p-hydroxyphenacyl (pHP) group as a new photoremovable protecting group. We report the application of this protecting group for the dipeptide Ala-Ala (1) and for the nonapeptide bradykinin (2), two representative peptides that demonstrate C-terminus “caging” and photorelease. The synthesis of these p-hydroxyphenacyl esters was accomplished in good yields by DBU-catalyzed displacement of bromide from p-hydroxyphenacyl bromide. As in the case of caged γ-amino acids 11 (pHP glu) and 12 (pHP GABA) and caged nucleotide 17 (pHP ATP) reported earlier,1,2 irradiations of the p-hydroxyphenacyl esters of 1 and 2 actuate the release of the peptides with rate constants that are consistently greater than 108 s-1 and appearance efficiencies (Φapp) that range from 0.1 to 0.3. Release of the substrate is accompanied by a deep-seated rearrangement of the protecting group into the near-UV silent p-hydroxyphenylacetic acid (6). Quenching studies of pHP Ala-Ala (7) with either sodium 2-naphthalenesulfonate or potassium sorbate gave good SternVolmer kinetics yielding a rate constant for release of 1.82 × 108 s-1. Quenching of the phosphorescence emission from pHP Ala-Ala (7, ET ) 70.1 kcal/mol) and pHP GABA (12, ET ) 68.9 kcal/mol) were also observed. The biological efficacy of bradykinin released from pHP bradykinin (9) was examined on single rat sensory neurons grown in tissue culture. A single 337 nm flash (<1 ns) released sufficient bradykinin from the p-hydroxyphenacyl protected nonapeptide to activate cell-surface bradykinin receptors as indicated by a rapid increase in the intracellular calcium concentration. A selective antagonist of type 2 bradykinin receptors blocked the biological response. From these results, it is apparent that flash photolysis of p-hydroxyphenacyl protected peptides provides a powerful tool for the rapid and localized activation of biological receptors.