Introduction

Fmoc-L-Arg(Pbf)-OH,also kown as Fmoc-Arg(Pbf)-OH, is an arginine derivative containing amine protecting group Fmoc. Fmoc-L-Arg(Pbf)-OH is a building block for the introduction of Arg into Solid-Phase Peptide Synthesis (SPPS). The appearance is White to off-white powder.(Figure 1) Please store Fmoc-L-Arg(Pbf)-OH under the recommended conditions in the Certificate of Analysis. Interesting, the most expensive protected amino acid currently on the market, Fmoc-L-Arg(Pbf)-OH, was one of the exceptions in the enfuvirtide sequence. However, to be fully honest, it is also important to clarify that the synthesis of Fmoc-L-Arg(Pbf)-OH is probably the most complex of all common protected amino acids. Arg is an important residue and has been object of several methodological studies. 23 The relevance of this amino acid lies in its presence in many peptide-based APIs,such as Etelcalcetide, Abaloparatide, and Semaglutide, among others approved by the FDA in recent years. Arg is also key for the development of the so-called Cell-Penetrating Peptides (CPPs). It is important to note that Fmoc-Arg(Pbf)-OH, which is the most used building block for the introduction of Arg into SPPS, is the most expensive of all protected proteinogenic amino acids and, like all Arg derivatives, it undergoes an important side-reaction upon activation.[1]

Highly efficient condensation of fmoc-L-arg(Pbf)-OH and rink amide-AM resin

The coupling reactions of Fmoc-L-Arg(Pbf)-OH and Rink Amide-AM Resin with symmetrical anhydride method, active ester method and 2,6-dichlorobenoyl chloride (DCB) method,respectively, were investigated in a self-designed reactor which congregated the stirring, filtration, and bubbling function together. The effects of reaction strategy, solvent system,reaction time, molar ratio of reactants and stirring method on the yield of condensation reaction were performed. The results indicate that the DIC/HOBt/DMAP strategy is the best method. The optimal reaction conditions are shown as follows: 3:1 of molar ratio of the reactants,3 h of reaction time and DMA/DCM (1:1, V/V) as a solvent using nitrogen-assisted magnetic stir system. The highest yield of the condensation reaction is 93%.[2]

Incorporation of Fmoc-Arg(Pbf)-OH in solid-phase peptide synthesis

NBP has proved an excellent alternative solvent to the hazardous DMF for SPPS. Here researchers studied the incorporation of Fmoc-L-Arg(Pbf)-OH, one of the most problematic amino acids, into a growing peptide chain. The poor performance of this amino acid is attributed to the formation of a fully inactive δ-lactam, which causes a reduction in yield and very often the concomitant formation of the corresponding des-Arg peptides. This problem is exacerbated when NBP is used as solvent, presumably because of its high viscosity, which impairs the penetration of the coupling cocktail into the resin. To tackle this issue, we propose the following strategy for the safe introduction of Fmoc-L-Arg(Pbf)-OH in SPPS at 45°C, keeping excesses to a minimum: 1.75 equiv. of the protected amino acids, 1.8 equiv. of DIC, and 1.5 equiv. of OxymaPure. The cornerstone of the strategy is to carry out in situ activation. In this regard, Fmoc-L-Arg(Pbf)-OH and OxymaPure dissolved in NBP were added to peptidyl-resin, allowed to reach the 45 °C, then half the DIC was added and left for 30 min, followed by the other half and some extra Fmoc-L-Arg(Pbf)-OH. During the entire process, the temperature was kept at 45 °C, with the double purpose of reducing the viscosity of NBP, thus facilitating the penetration of the coupling cocktail into the resin, and speeding up the coupling itself. It is envisaged that this strategy could be widely used to improve the performance of SPPS, including the industrial preparation of peptides using this approach.[2]

Efficient solid-phase synthesis of cyclic RGD peptides

Cyclic RGD peptides are potent antagonists for the αvβ3 integrin receptor. In this study, microwave-assisted solid-phase synthesis of cyclic RGD peptides is described. In a coupling reaction between Fmoc-L-Arg(Pbf)-OH and high-loading H-Gly-Trt(2-Cl) resin, multiple coupling reactions were required for completion under the conventional HBTU activation. They found that the use of COMU, a new coupling reagent, under microwave heating to 50°C accelerated the reaction even inside the resin. This method was applicable to the synthesis of linear pentapeptides, H-Asp(OtBu)-Xxx-Yyy-Arg(Pbf)-Gly-OH (Xxx=d-Phe(p-Br) or d-Tyr, Yyy=Lys(Boc) or MeVal). Cyclization of these peptides followed by deprotection gave the desired cyclic RGD peptides with high purity.[3]

References

[1] Torre B G D L , Kumar A , Alhassan M ,et al.Successful development of a method for the incorporation of Fmoc-Arg(Pbf)-OH in solid-phase peptide synthesis using N-butylpyrrolidinone (NBP) as solvent[J].Green Chemistry, 2020, 22.DOI:10.1039/C9GC03784E.

[2] Zhu LL,et al.Highly efficient condensation of sterically hindered amino acid fmoc-arg( Pbf) -OH and rink amide-AM resin[J].Modern Chemical Industry,2012,32(02):62-65.DOI:10.16606/j.cnki.issn0253-4320.2012.02.017.

[3] Yamada K , Nagashima I , Hachisu M ,et al.Efficient solid-phase synthesis of cyclic RGD peptides under controlled microwave heating[J].Tetrahedron Letters, 2012, 53(9):1066-1070.DOI:10.1016/j.tetlet.2011.12.069.