## 2 Results and Discussion  
### Chemical Synthesis  
UMP-VPg1 and FUMP-VPg1 were synthesized following the approach developed by [PERSON] for the preparation of a poliovirus 22 aa long UMP-VPg using sequential solid-phase peptide synthesis (SPPS) and a pre-uridylylated tyrosine building block [23; 24]. We carried out the Fmoc-based SPPS on a Wang resin using the conveniently protected uridylylated or fluororudylylated tyrosine building blocks **14** and **15** (Scheme 1). The synthesis started with the preparation of the pre-uridylylated and pre-fluororudylylated compounds **12** and **13**. Thus, treatment of the Fmoc-Tyr(tBu)-OH **3** with allyl bromide in the presence of Ag2 CO3 gave compound **4** (98% yield) and removal of the tBu Tyr side chain (TFA:CH2 Cl2, 7:3) gave the intermediate **5** in 79% yield (Scheme 1). On the other hand, (fluoro)uridine-5'-phosphoramidites **8** and **9** were prepared upon treatment of corresponding conveniently protected uridine or fluororudine nucleosides (**6** or **7**) [25; 26] with 2-cyanoethyl-\(N\),\(N\)-diisopropylchlorophosphoramidite in the presence of triethylamine [27]. Compounds **8** and **9** were used in the next step without further purification. Thus, coupling of phosphoramidites **8** or **9** with **5** in the presence of 4,5-dicyanoimidazole gave the phosphites **10** and **11**, which, after in situ oxidation with tBuOOH 5.5 M in nonane for 45 min, yielded the fully protected (fluoro)uridylylated tyrosine derivatives **12** and **13**. Finally, palladium(0)-catalyzed  
Figure 2: Fifteen-mer model of FUMP-VPg1 and UMP-VPg1. hydrostannolysis (Pd((PPh\({}_{3}\))4), Bu\({}_{3}\)SnH, AcOH) of protected compounds **12** and **13** gave the target building blocks **14** and **15** in overall yields of 37% and 28% from **6** or **7**, respectively (Scheme 1). Next, the syntheses of the protected nucleopeptides **18** and **19** were performed manually, following standard SPPS protocols, using a Wang polystyrene (1% DVB) resin preloaded with Fmoc-Val-OH and \(N\)-Fmoc/tBu chemistry (Scheme 2). A low load of the resin (0.4 mmol/g) was used to minimize peptide aggregation and unwanted intermolecular side reactions. Thus, after swelling of the resin, removal of the Fmoc protecting group of the Fmoc-Val residue, under basic conditions (DBU/piperidine/DMF) at room temperature, and sequential coupling of the following eleven amino acids was carried out under microwave irradiation at 40 \({}^{\circ}\)C for 10 min, in the presence of HCTU as the coupling reagent and DIEA as the base. A triple coupling protocol (\(3\times 10\) min) was used to give the immobilized 12-mer peptides **16** and **17**. Then, coupling of the next amino acid, the uridyylated or fluororuidylylated tyrosine building blocks (**14** and **15**), was performed in the presence of HCTU/DIEA, following a double coupling protocol (\(2\times 60\) min) at room temperature, to prevent the rupture of the O-P bond (Phosphate bond) and the loss of the nucleotide of **14** or **15**. The basic conditions required for the elimination of the Fmoc group (DBU/piperidine/DMF) also caused the concomitant elimination of the cyanoethyl group. However, the presence of the phosphodieser group did not interfere in the coupling of the last two amino acids that were also performed at room temperature, also following a double coupling protocol (no unwanted side reactions were detected), to yield the immobilized protected 15-mer peptides **18** and **19**. It has been reported in the literature that the presence of the phosphodieser function does not lead to unwanted by-products [28]. Once elongation of the peptide was completed, removal of the Fmoc group of the \(N\)-terminus of the nucleopeptides followed by a standard TFA cleavage (TFA/TIPS/water) led to the simultaneous deprotection of the acid labile side-chains protecting group residues and release of the nucleopeptides from the solid support, to give the partially protected nucleopeptides **20** and **21**.