A novel molecular probe for the detection of phosphorylated proteins

Phosphorylation of proteins is an important feature for human health. The process of phosphorylation has some direct or indirect relationship with the biological processes in the human body [1], [2]. The traditional methods for detection of phosphorylated proteins have problems connected with sensitivity, selectivity and price. New detection methods that are simple, fast, sensitive, accurate, specific and especially those that can be used at home are being developed [3], [4]. Organic conjugated small molecules with donor-π-acceptor (D-π-A) structures, that are widely used in organic nonlinear optical materials and show photoinduced nonlinear optical effects, can also be used for the detection of phosphorylated proteins [5], [6], [7], [8]. A new probe of the D-π-A type (Scheme 1) for the detection of phosphorylated proteins is the subject of this report.

Scheme 1

Synthesis of chromophore ZKY-1.

In the first step, compound 1 was obtained by the modification of a published procedure [9]. Thus, 5-bromoindole was allowed to react with pyrrolidine under basic conditions in the presence of 2-dicyclohexylphosphino-2′,6′-diisopropoxy-1,1′-biphenyl (RuPhos) and Pd(OAc)₂. The use of the Pd(OAc)₂/RuPhos system furnished product 1 in a 54% yield, even under aerobic conditions. By comparison, the reaction conducted under classical conditions in the presence of PdCl₂/triphenylphosphine gave product 1 in the yield of 14%. After alkylation of compound 1, the resultant product 2 was subjected to the Vilsmeier reaction to furnish compound 3. The final reaction of 3 with 2-(3-cyano-4-methyl-5-oxo-1,5-dihydro-2H-pyrrol-2-ylidene)malononitrile (TCP) furnished the desired probe ZKY-1.

The thermal stability of compound ZKY-1 was investigated by using thermogravimetric analysis (TGA) under the protective atmosphere of nitrogen (Figure 1). As can be seen, the onset of decomposition starts above 210°C. The optical stability of ZKY-1 was studied by direct laser irradiation with a visible light of 30 nanowatt per square millimeter. After irradiation for 48 h, the absorption was reduced by a mere 5%. Unfortunately, the stability of ZKY-1 in acidic or alkaline solution is low. After the addition of ZKY-1 to 3 m hydrochloric acid or 3 m sodium hydroxide, the color of the solution changed immediately.

Figure 1

TGA profile of compound ZKY-1.

The application of ZKY-1 as a probe for phosphorylated protein requires the presence of tetravalent zirconium ion which is complexed by ZKY-1. The UV-visible absorption peak of ZKY-1 changes from 656 nm (blue) to 741 nm (green) after the complex formation. The coordination ability of zirconium ion is stronger for phosphoric acid than for ZKY-1 which undergoes a weak complexation with zirconium(IV). As a result, after the addition of phosphorylated protein to the solution, the zirconium ion is removed from the complex Zr(IV)-ZKY-1 by phosphorylated protein. The resultant free molecule ZKY-1 shows again the UV-visible absorption peak at 656 nm and the color of the solution changes from green to blue.

The optical response of chromophore ZKY-1 to phosphorylated cAMP-response element binding protein (CREB) was studied using UV-vis spectra in the methanol-water solution (1:3, v/v, PBS buffer, pH 7.1) at room temperature. Before the addition of phosphorylated protein, there is a strong absorption at 741 nm for the Zr(IV)-ZKY-1 complex. Upon the gradual addition of the phosphorylated protein, the UV-visible absorption peak at 741 nm is reduced, and the UV-visible absorption peak at 565 nm for free molecule ZKY-1 increases in intensity. These spectral changes are accompanied by visible color changes from green to blue, which can be observed by the naked eye.

The relationship between the concentration of phosphorylated protein and the ratio of absorption strength between 741 nm and 565 nm maxima is shown in Figure 2. Along with the increase in concentration of phosphorylated protein, the absorption peak at 741 nm gradually decreases and the absorption peak at 565 nm increases and saturation is reached after the addition of about 1 equiv. of phosphorylated protein. The relationship between the ratio of the intensity of absorption and concentration of phosphorylated protein is linear in the range of 0–4.5 μm. The detection limit thus can be estimated to be about 100 nm.

Figure 2

The linear relationship between the UV-VIS absorption coefficient ratio (I₅₆₅/I₇₄₁) and the concentration of phosphorylated CREB protein.

It is known that pregnant women with high level of phosphorylated CREB protein are at risk of pre-eclampsia [10]. Accordingly, timely monitoring of the level of phosphorylated CREB protein is necessary for preventing pre-eclampsia. For this work, tissue samples of human placenta were collected from the Obstetrics Department of Aviation General Hospital of China. After processing, the protein solution was used for analysis by this new method. The difference of the phosphorylated level of protein for different groups was easily detected by using the ZKY-1 probe. The normal pregnancy group showed the lowest phosphorylated level; severe pre-eclampsia groups showed the highest phosphorylated level [11].