General Method for the Synthesis of Caged Phosphopeptides:  Tools for the Exploration of Signal Transduction Pathways

Abstract

[reaction: see text] An interassembly approach for the synthesis of peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has been developed. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford the corresponding phosphopeptide. The peptides described herein are based on phosphorylation sites of kinases involved in cell movement or cell cycle regulation and demonstrate the versatility of the method and compatibility with the synthesis of polypeptides, including a variety of encoded amino acids.

Semisynthesis of unnatural amino acid mutants of paxillin: Protein probes for cell migration studies

“…One limitation with these strategies is that they cannot afford information on phosphorylation in ''real time.'' As a complement to these approaches, the synthesis of caged phosphopeptides, which enable the controlled release of specific phosphorylated species upon photolysis, was introduced (Rothman et al 2002). Recently, a general method for the synthesis of these probes has facilitated the application of caged phosphopeptides in cellular studies (Nguyen et al 2004;Humphrey et al 2005).…”

Facile Synthesis of o-Nitrobenzylcarbamate and 1-(2-Nitrophenyl ethyl) Carbamate Protected α,ω-Diamines

“…The required arylmethyl phenyl carbonates were synthesized as shown in Scheme 2 from the corresponding alcohols. 1-(2-Nitrophenyl)ethanol was synthesized in three steps from o-Nitrobenzoic acid via o-Nitroacetophenone [12] according to the published procedure [13]. …”

In Situ Photoactivation of a Caged Phosphotyrosine Peptide Derived from Focal Adhesion Kinase Temporarily Halts Lamellar Extension of Single Migrating Tumor Cells

“…1-(2-Nitrophenyl)ethyl (NPE) was chosen as the caging group because it satisfies key requirements of biologically useful caging groups and those of Fmoc-based solid phase peptide synthesis (19,33). The NPE can be photochemically released with reasonable quantum efficiency at wavelengths around 350 nm (33,43), and the photo-byproduct, nitrosoacetophenone, is less harmful to cells than the corresponding aldehyde released by photolysis of commonly implemented o-nitrobenzyl caging groups (25).…”

“…If the average cell volume of NBT-II cells is 1.5 pl, an additional 1-10% (32) of the total cell volume is added by microinjection. To estimate the concentrations of peptide after uncaging, a quantum efficiency of uncaging for these peptides of 0.3 was used based on characterization of a paxillin peptide with the same caging moiety (33).…”