Real time detection of cell cycle regulator cyclin A on living tumor cells with europium emission

Abstract

Six water-soluble europium complexes (Eu–L₁1–P_nnn and Eu–L₂2–P_nnn, n = 1, 2 and 3) with one antenna chromophore, two different linkers (L₁1 and L₂2) and three proposed cyclin A specific peptides (P₁1: -GAKRRLIF-NH₂; P₂2: -GGAKRRLIF-NH₂; P₃3: -Hex- GAKRRLIF-NH₂) have been synthesized. With structural information available, comparisons of the cyclin grooves of cyclin A and the six europium complexes have been made, and insights have been gained into the determinants for peptide binding and the foundation of differential binding. Experiment-wise, the linear and two-photon induced photophysical properties of these conjugates were monitored in aqueous solution. Numerous in situ/in vitro biological assays have been carried out, such as responsive emission changes in situ/in vitro, Western blot and cellular uptake. As imaging agents, complexes with peptides P₃3: -Hex-GAKRRLIF-NH₂ showed high selectivity to cyclin A in numerous cancer cells. When it comes to responsive optical signal changes, complex Eu–L₂2–P₃3 exhibited a threefold emission enhancement upon binding with cyclin A (100 nM cyclin A, ϕ = 8% to 21%, log K_B = 5.83, detection limit = 5 nM), and this could be initiated by the shortened distance between the antenna and the lanthanide after they bind/get into cyclin A. It is promising that our compounds (especially compound Eu–L₂2–P₃3) could serve as the template for structure-guided efforts to develop potential imaging therapeutics on the basis of selective imaging of CDK2/cyclin A activity.