## 2 Results  
### Cap Analogs Synthesis  
Cap analogs bearing propargyl moiety on the guanosine at N1 position (**1-5**) were derived from N1-propargylguanosine (**16**), which was synthesized from unprotected nucleoside as previously described (Scheme 1) [27]. Guanosine (**15**), sodium hydride (NaH), and tetrabutylammonium iodide (TBAI) were suspended in anhydrous dimethylformamide (DMF) and propargyl bromide was added to generate N1-propargylguanosine (**16**) in 71% yield. Compound **16** was phosphorylated to N1-propargylguanosine 5\({}^{\prime}\)-O-monophosphate (**17**) with POCl\({}_{3}\) in trimethyl phosphate [28]. After purification by ion exchange chromatography, compound **17** was converted to compound **18** by reacting it with imidazole in the presence of 2,2\({}^{\prime}\)-dithiodipyridine (DTDP), triethylamine (TEA), and triphenylphosphine (PPh\({}_{3}\)) followed by precipitation with sodium perchlorate (NaClO\({}_{4}\)) in acetone [29]. The respective yields were 54% and 95%. A cap analog bearing a propargyl moiety and an unmodified phosphate bridge (**1**) was obtained by coupling compound **18** with N7-methylguanosine 5\({}^{\prime}\)-O-diphosphate (m\({}^{7}\)GDP) (**19**) in DMF in the presence of ZnCl\({}_{2}\). Cap analogs modified at the \(\beta\),\(\gamma\) position (**2, 3**) were obtained in the similar manner, except that the m\({}^{7}\)GDP analog (m\({}^{7}\)GpCH\({}_{2}\)p (**20**) or m\({}^{7}\)GpNHp (**21**), respectively) was used in the reaction. To obtain cap analogs bearing a modification within phosphate bridge at the \(\alpha\),\(\beta\)-position (**4-5**), compound **16** was treated with either CH\({}_{2}\)(POCl\({}_{2}\))\({}_{2}\) or Cl\({}_{3}\)PNP(O)Cl\({}_{2}\) in trimethyl phosphate to obtain N1-propargylguanosine 5\({}^{\prime}\)-O-(1,2-methylenphosphate) (**22**) or N1-propargylguanosine 5\({}^{\prime}\)-O-(1,2-imidazolphosphate) (**23**) in 75% and 63% yield, respectively [28].  
Figure 1: Synthesis of N1 functionalized dinucleotides (**6-14**) from N1-propargylated cap precursors (**1-5**) via a CuAAC reaction.  
Compounds **22** or **23** were coupled with N7-methylguanosine-5'-O-monophosphate-P-imidazolide (m7 GMP-Im) (**24**) in DMF in the presence of ZnCl\({}_{2}\) to obtain cap analogs **4** and **5**, respectively. The final products (**1-5**) were purified by ion exchange chromatography and the yields were 36-58%. The products were re-purified by semipreparative RP-HPLC, isolated as ammonium salts, and used for labeling in the CuAAC reaction and biophysical studies. The structures and purities of all new compounds were confirmed by HRMS, \({}^{1}\)H NMR, and \({}^{31}\)P NMR (Supplementary Information).  
Reactivity of the cap analogs containing N1-propargylguanosine in CuAAC was evaluated. To this end, probes (**6-14**) were obtained by click chemistry in the reaction between commercially available fluorescent tags azide analogs (**25-28**) or biotin azide (**29**) [14] and cap analogs bearing the propargyl moiety **(1-5)** (Figure 1). An aqueous solution of cap analog (**1-5**) was mixed with a tag (**25-29**) dissolved in dimethyl sulfoxide (DMSO) at room temperature (RT) followed by the addition of aqueous solutions of CuSO\({}_{4}\) and sodium ascorbate. The concentrations of the tags used for labeling ranged from 100-190 mM. HPLC analysis revealed that under these conditions, the conversion rates were in the range of 51-100%. The reactions were quenched by the addition of sodium ethylenediaminetetraacetate/sodium bicarbonate solution (Na\({}_{2}\)EDTA/NaHCO\({}_{3}\)), and the probes were purified by HPLC and isolated as ammonium salts. Using this approach, nine new probes were synthesized (Figure 1). To assess how substitution at the N1-position of guanosine influences mRNA cap recognition by specific proteins and enzymes, we performed a series of experiments with unlabeled cap analogs (**1-5**) and fluorescently labeled probes (**6-14**).