Caged reagents are molecules from which biological effectors are released by photolysis. [1,2] In recent studies, caged peptides and proteins have received attention because of their potential importance in investigations of processes such as cell signaling. [3,4] Although methods suited to specific situations have been explored, [5±10] we have focused our attention on general procedures to cage unprotected peptides and proteins in aqueous solutions.Because of the relative ease of nucleophilic substitution on cysteine, this amino acid has been introduced into peptides by chemical synthesis and into proteins by mutagenesis as a site for modification by caging reagents. [3, 11±13] We recognized that most proteins involved in cell signaling are regulated by phosphorylation. Indeed, up to 30 % of proteins in mammalian cells are phosphorylated and well over one percent of mammalian proteins are protein kinases. [14±16] Therefore, we reasoned that thiophosphoryl groups in peptides and proteins might also be utilized as sites for caging. This idea was readily demonstrated with thiophosphorylserine residues in peptides, by reaction with 2-nitrobenzyl bromide (NBB) to generate caged peptides that could be converted back into the thiophosphoryl peptides by near-UV light. [17] Unfortunately, we encountered difficulties with thiophosphoryltyrosine residues, which was especially troublesome because of the pivotal role of tyrosine phosphorylation in cell signaling. [18±23] The initial difficulty centered around the enzymatic thiophosphorylation of model peptides. Herein, we show that tyrosine-containing peptides can be thiophosphorylated with cognate kinases by using divalent transition metal ions instead of Mg II in the reaction buffer. The thiophosphorylated peptides can then be caged by reaction with electrophilic reagents such as NBB or p-hydroxyphenacyl bromide (HPB). The p-hydroxyphenacyl group has been used previously to cage molecules, including amino acids and oligopeptides, on carboxylate groups [24±27] and, recently, a protein phosphatase on a cysteine residue. [28] The mechanism of photorelease of substrates from p-hydroxyphenacyl carboxylate and phos-phate esters has recently been examined. [27,29,30] In the present context, HPB is shown to be superior to the prevailing 2-nitrobenzyl reagents.In attempting to prepare thiophosphotyrosine peptides, we were, in most cases, unable to detect high extents of thiophosphorylation with ATP(g)S by using tyrosine kinases under conditions that work well when ATP itself is used. [31±33] For example, Abl, Src, and EGF and insulin receptor kinases [23] failed to thiophosphorylate peptide substrates. To explore this problem more thoroughly, we focused on EPQYEEIPILG. A related peptide, EPQYEEIPIYL, binds to Src homology (SH2) domains when phosphorylated and phosphorylated EPQYEEIPIA acts as an activator of Src kinase. [34] Divalent metal ions other than Mg II have proved to be useful for promoting thiophosphorylation in other circumstances. For example, Cole and colleague...
