## 2 Results
### Enzymatic Degradation Studies  
Susceptibility of the new cap analogs to hydrolysis by human decapping scavenger enzyme (hDcpS) was investigated to assess the influence of N1 substitution on cap analogs recognition by DcpS. Caps **1**-**5** or m\({}^{7}\)Gp\({}_{3}\)G (30 \(\upmu\)M each) were incubated with hDcpS (30 nM) at 30 \({}^{\circ}\)C. Then, reaction samples were thermally deactivated and analyzed by RP-HPLC. Under these conditions, cap analog **1** was efficiently cleaved by DcpS but more slowly than m\({}^{7}\)Gp\({}_{3}\)G. All analogs modified within the phosphate bridge (**2**-**5**) were resistant to hDcpS under the applied conditions (Figure 3). The propargyl moiety at the N1 position of a second nucleobase slightly decreased the susceptibility of the new cap analogs to hDcpS cleavage but prevented neither the recognition nor the regioselectivity of the cleavage (Table S2).  
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Compound** & **IC\({}_{\text{50}}\) (\(\upmu\)M)** & **\(K_{\text{D}}\) (nM)** \\ \hline
**1** & 3.57 \(\pm\) 0.25 & 305 \(\pm\) 45 \\
**2** & 6.37 \(\pm\) 0.73 & 548 \(\pm\) 95 \\
**3** & 3.48 \(\pm\) 0.22 & 297 \(\pm\) 43 \\
**4** & 6.04 \(\pm\) 0.46 & 520 \(\pm\) 78 \\
**5** & 2.16 \(\pm\) 0.20 & 182 \(\pm\) 29 \\ m\({}^{7}\)Gp\({}_{3}\)G & 3.46 \(\pm\) 0.30 & 294 \(\pm\) 46 \\ \hline \hline \end{tabular}
\end{table}
Table 1: IC\({}_{\text{50}}\) and \(K_{\text{D}}\) values determined at 30 \({}^{\circ}\)C for the binding affinity for murine eIF4E. The IC\({}_{\text{50}}\) values were determined from the data shown in Figure 2. The \(K_{\text{D}}\) values were calculated from the IC\({}_{\text{50}}\) values using a previously reported equation [33].  
Figure 2: Dose–response curves for compounds **1**–**5** and m\({}^{7}\)Gp\({}_{3}\)G plotted from a competition binding assay for melF4E [16]. eIF4E (75 nM) and a pyrene-labeled fluorescent probe (10 nM) were incubated at 30 \({}^{\circ}\)C with the indicated concentrations of the investigated compounds (**1**–**5** or m\({}^{7}\)Gp\({}_{3}\)G as a reference). IC\({}_{\text{50}}\) and \(K_{\text{D}}\) values from triplicate experiments are shown in Table 1.  
Fluorescently labeled cap analogs that serve as DcpS substrates can be considered as activity probes. We performed an enzymatic cleavage test with 30 nM hDcpS and 30 uM probe to evaluate whether fluorescently labeled derivatives of cap analog **1** (probes **6-9**) can be used to monitor enzymatic activity via changes in fluorescence emission. For probes **6-8**, the fluorescence intensity at the emission maximum did not significantly change following complete cleavage by DcpS (Figure 4 and Figure S2). For the Py-labeled probe **9**, the fluorescence intensity at the emission maximum increased by 50% after complete DcpS cleavage (Figure 4c and Figure S3). Nevertheless, certain previously developed probes provided higher sensitivity than the one formulated here [15; 16]. Modest changes in fluorescence emission upon the enzymatic cleavage of compounds **6-9** were independently confirmed with the nonspecific pyrophosphatase PDE-I (Figure S4).  
The lack of fluorescence changes upon enzymatic cleavage suggests that the fluorescent labels attached to the N1 position of guanosine are relatively insensitive to alterations in the local environment. This characteristic is desirable in fluorescence polarization (FP) and microscale thermophoresis (MST) binding assays. Therefore, in the next step, we tested whether compounds **6-14** could serve as binding probes for either eIF4E or DcpS.