## 3 Discussion  
In the present study, we aimed to develop new mRNA cap analogs amenable to click chemistry and convertible into potential binding or activity probes. We synthesized m\({}^{7}\)GP\({}_{3}\)G dinucleotides bearing a \"clickable\" propargyl group handle at the N1 position of guanosine. Certain compounds were further modified within the triphosphate chain to modulate their susceptibility to enzymatic degradation. Utility of the probes in the investigation of cap-binding proteins and cap-specific enzymes was evaluated with eIF4E and DcpS, respectively. The N1-propargyl group did not significantly decrease the binding affinity for eIF4E significantly. Moreover, HPLC-monitored enzymatic degradation experiments showed that the N1 modification only slightly impaired recognition by DcpS. The N1-propargyl compounds were then converted into fluorescent probes with CuAAC. Characterization of the probes by emission spectroscopy revealed that the labels attached to the N1 position are comparatively insensitive to fluorescence intensity changes in response to protein binding or cleavage. Therefore, the labels are useful for experiments monitored by fluorescence intensity changes and for biophysical binding assays based on FP measurements or MST. To demonstrate this, we performed MST binding  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multirow{2}{*}{**Compound**} & \multicolumn{2}{c}{**meIF4E**} & \multicolumn{2}{c}{**hDcpS**} \\ \cline{2-5}  & \(K_{\text{D}}\) **[nM]** & **Confidence Interval** & \(K_{\text{D}}\) **[nM]** & **Confidence Interval** \\ \hline
**6** & 50 & (43, 58) & n.d. & n.d. \\
**11** & 201 & (184, 220) & 38 & (28, 51) \\
**12** & 53 & (46, 62) & 14 & (7, 24) \\ \hline \hline \end{tabular}
\end{table}
Table 2: \(K_{\text{D}}\) values of the binding affinities for murine eIF4E and human DcpS determined by MST. \(K_{\text{D}}\) values were calculated from triplicate experiments.  
Figure 5: Direct binding microscale thermophoresis (MST) experiments with eIF4E and DcpS. (**a**) MST binding curves for probes **6, 11, 12,** and meIF4E. Probe **6, 11**, or **12** (50 nM) was mixed with a half-log dilution series of an meIF4E solution (10 \(\upmu\)M–0.30 nM). (**b**) MST binding curves for probes **11** and **12** and DcpS. A solution of probe **11** or **12** (50 nM) was mixed with a dilution series of hDcpS solution (2.0 \(\upmu\)M–0.061 nM for **11;** 700–1.6 nM for **12**). MST traces for each experiment are shown in Figures S5 and S6. Data were analyzed in Palmist v. 1.4.4 using 1:1 binding model. The \(K_{\text{D}}\) values were calculated from triplicate experiments and are shown in Table 2.  
experiments using both eIF4E and DcpS in which we plotted reproducible binding curves and determined \(K_{\mathrm{D}}\). For most compounds, the \(K_{\mathrm{D}}\) were in the low nanomolar range (Table 2). To the best of our knowledge, the present study is the first example of the application of MST in the analysis of ligand binding to DcpS enzyme. Although compounds **1**-**5** do not possess extraordinary inhibitory properties in vitro experiments, the probe **12** tightly bound DcpS (\(K_{\mathrm{D}}\) = 14 nM) and is, therefore, the preferred candidate for the development of an MST-based competitive binding assay to be used in the discovery and further in vitro evaluation of DcpS inhibitors. Studies on the specificity of N1-propagyl containing analogs for other cap binding proteins and decapping enzymes or possibility to incorporate them into RNA are underway.