Sequential activation and deactivation of protein function using spectrally differentiated caged phosphoamino acids

Abstract

Photolabile caging groups, including the 1-(2-nitrophenyl)ethyl (NPE) group, have been applied to probe many biological processes, including protein phosphorylation. Although studies with NPE-caged phosphoamino acids have provided valuable information, these investigations have been limited to the use of only one caged species in a single experiment. To expand the scope of these tools, we have developed an approach for sequentially uncaging two different phosphopeptides in one system, enabling interrogation of multiple phosphorylation events. We present the synthesis of [7-(diethylamino)coumarin-4-yl]methyl (DEACM)-caged phosphorylated serine, threonine, and tyrosine building blocks for Fmoc-based solid-phase peptide synthesis to allow convenient incorporation of these residues into peptides and proteins. Exposure of DEACM- and NPE-caged phosphopeptides to 420 nm light selectively releases the DEACM group without affecting the NPE-caged peptide. This then enables a subsequent irradiation event at 365 nm to remove the NPE group and liberate a second phosphopeptide. We demonstrate the versatility of this general sequential uncaging approach by applying it to control Wip1 phosphatase with two wavelengths of light.

Figure 1

Sequential uncaging to control activity of Wip1 phosphatase. The DEACM-caged phosphopeptide substrate enables the first irradiation at 420 nm to initiate the Wip1 reaction. A subsequent irradiation at 365 nm releases the NPE-caged phosphopeptide inhibitor to attenuate Wip1 activity.

Figure 2

DEACM-caged phosphoserine, threonine, and tyrosine building blocks for SPPS.

Figure 3. Uncaging time course of DEACM-caged substrate and NPE-caged inhibitor peptides at 420 nm and then 365 nm

a) A solution of the DEACM- and NPE-caged peptides (113 μM each) in 10 mM HEPES (pH 7.1) with 70 μM inosine was irradiated at 420 nm and then at 365 nm. The reactions were followed by analytical RP-HPLC at 228 nm. Peptides were quantified based on HPLC standard curves of the caged and non-caged derivatives. The amount of each peptide was normalized to the amount of inosine present, and the amounts of the uncaged peptides are plotted relative to the initial amounts of the corresponding caged species. b) HPLC traces of the DEACM- and NPE-caged peptides (2 and 3, respectively) before (t = 0 min) and following irradiation at 420 nm and 365 nm. The released substrate peptide, DEACM-OH byproduct of uncaging, and released inhibitor peptide are denoted by 4, 5, and 6, respectively. The inset shows the inosine peak (1), which was used to normalize for the HPLC injection volume, on the same scale as the peptide traces.

Figure 4. Wip1 Phosphatase Activity

Wip1 was incubated with the DEACM-caged substrate (30 μM) and the NPE-caged inhibitor (5 μM). No activity occurred before irradiation (Region A), but exposure to 420 nm (orange bar) initiated Wip1 activity (Region B). A subsequent irradiation at 365 nm (yellow bar) completely abolished activity (Region C). If the assay was irradiated only at 420 nm and was not subjected to the 365 nm irradiation, the reaction continued.

Scheme 1

Synthesis of DEACM-caged phosphoserine.^{a a}Reagents and conditions. (a) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, triethylamine, DCM (quantitative); (b) NaHCO₃/H₂O, Aliquat 336, allyl bromide, CH₂Cl₂ (91%); (c) 5, 4,5-dicyanoimidazole, THF; (d) tert-butyl hydroperoxide, 0 °C (75% over 2 steps); (e) Wilkinson’s catalyst, EtOH:H₂O (9:1) (73%).