Facile Synthesis of N-Acyl-aminoacyl-pCpA for Preparation of Mischarged Fully Ribo tRNA

Abstract

Chemical synthesis of N-acyl-aminoacyl-pdCpA and its ligation to tRNA(minus CA) is widely used for the preparation of unnatural aminoacyl-tRNA substrates for ribosomal translation. However, the presence of the unnatural deoxyribose can decrease incorporation yield in translation and there is no straightforward method for chemical synthesis of the natural ribo version. Here, we show that pCpA is surprisingly stable to treatment with strong organic bases provided that anhydrous conditions are used. This allowed development of a facile method for chemical aminoacylation of pCpA. Preparative synthesis of pCpA was also simplified by using t-butyl-dithiomethyl protecting group methodology, and a more reliable pCpA postpurification treatment method was developed. Such aminoacyl-pCpA analogues ligated to tRNA(minus CA) transcripts are highly active in a purified translation system, demonstrating utility of our synthetic method.

Emerging Methods for Efficient and Extensive Incorporation of Non Canonical Amino Acids using Cell-free Systems

“…Thus, orthogonality evolution of aminoacylation factors can be skipped to save cost, while orthogonal tRNA is needed to www.videleaf.com achieve accurate ncAAs incorporation. Recently, several novel ncAAs were successfully incorporated into protein by such in vitro aminoacylation methods, including D-α-amino acids [34], β-amino acids [35], γ-amino acids [117], N-alkylated-α-amino acids [118], N-acylated-α-amino acids [119], α-hydroxy acids [33], α-benzoic acids and even foldamers [120,121] (Figure 3). These novel ncAAs widely expand the ability to engineer artificial proteins with novel features, which may increase the proteolytic stability, membrane permeability, and conformational rigidity of the peptide.…”

“…Chemical aminoacylation is a versatile classical strategy for aminoacyl-tRNA synthesis in vitro [132] (Figure 4B). Although several improvements have been made, the general process remained consistent [116,119,[133][134][135]. The amino acid with appropriate protecting groups is chemically charged to the 3'-OH of pCpA or pdCpA, which was chemical synthesized.…”

Emerging Methods for Efficient and Extensive Incorporation of Non-canonical Amino Acids Using Cell-Free Systems

“…Chemical aminoacylation is a versatile classical strategy for aminoacyl-tRNA synthesis in vitro ( Hecht et al, 1978 ; Figure 4B ). Although several improvements have been made, the general process remained consistent ( Heckler et al, 1984 ; Noren et al, 1989 ; Ninomiya et al, 2003 ; Lodder et al, 2005 ; Kwiatkowski et al, 2014 ). The amino acid with appropriate protecting groups is chemically charged to the 3′-OH of pCpA or pdCpA, which was chemical synthesized.…”

“…Thus, orthogonality evolution of aminoacylation factors can be skipped to save cost, while orthogonal tRNA is needed to achieve accurate ncAAs incorporation. Recently, several novel ncAAs were successfully incorporated into protein by such in vitro aminoacylation methods, including D- α-amino acids ( Katoh et al, 2017b ), β-amino acids ( Katoh and Suga, 2018 ), γ-amino acids ( Ohshiro et al, 2011 ), N-alkylated-α-amino acids ( Kawakami et al, 2013 ), N-acylated-α-amino acids ( Kwiatkowski et al, 2014 ), α-hydroxy acids ( Ohta et al, 2008 ), α-benzoic acids and even foldamers ( Kawakami et al, 2016 ; Rogers et al, 2018 ; Figure 3 ).…”

“…The only two limitations imposed on the amino acids are the feasibility to activate the carboxyl with a certain group and chemical stability during the aminoacylation process. In fact, different ncAAs have been utilized even with multiple incorporations, including D- α-amino acids ( Katoh et al, 2017b ), β-amino acids ( Katoh and Suga, 2018 ), γ-amino acids ( Ohshiro et al, 2011 ), α-hydroxy acids ( Ohta et al, 2008 ), α-benzoic acids ( Kawakami et al, 2016 ), N-alkyl- L -α-amino acids ( Kawakami et al, 2013 ), N-methyl- L -α-amino acids ( Kwiatkowski et al, 2014 ), and even foldamers ( Rogers et al, 2018 ). However, the efficiency of flexizyme aminoacylation varies depending on the ncAAs with a wide range of 17–91%, which can only be optimized empirically, since there are no established design rules ( Goto et al, 2011 ).…”