Caged Thiophosphotyrosine Peptides

Abstract

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...

Applications of p-hydroxyphenacyl (pHP) and coumarin-4-ylmethyl photoremovable protecting groups

“…15,16 Assorted applications of p HP thio derivatives that take advantage of the high nucleophilicity of thiol for direct incorporation of the protecting group using p HP Br have been reported. 25,26 …”

“…Bayley et al 26 compared the protection–deprotection effectiveness of o NB and p HP for thiophosphorylated tyrosine. Both protection sequences were accomplished in high yield (75% and 90%, respectively) by treatment of the thiophosphorylated tyrosyl peptide EPOYEEIPILG with o NB Br and p HP Br.…”

“…Bayley et al 26 extended this approach to the protection–deprotection of thiophosphorylated threonine, Thr-197, in the catalytic site of a cell-signalling protein kinase A found in the catalytic C α subunit by phosphorylation. The phosphothreonine unit plays a key role in stabilizing the active conformation of the C α catalytic subunit.…”

Light-mediated remote control of signaling pathways

“…This backbone caging strategy should be applicable to a variety of peptides and has been used to cage peptide substrates for PKA as well as chymotrypsin. Two other SH2 domain-targeted peptides have been developed which are caged at the phosphotyrosine group [76, 77]. …”

Molecular tools for cell and systems biology

“…An alternative to cysteines are thiophosphorylated serines. This unnatural The increased nucleophilicity of the thiophosphate then permits specific introduction of the cage (Zou et al, 2002(Zou et al, , 2001.…”