Genetic Encoding of Cyanopyridylalanine for In-Cell Protein Macrocyclization by the Nitrile-Aminothiol Click Reaction

Abstract

Cyanopyridylalanines are non-canonical amino acids that react with aminothiol compounds under physiological conditions in a biocompatible manner without requiring added catalyst. Here we present newly developed aminoacyl-tRNA synthetases for genetic encoding of meta- and para-cyanopyridylalanine to enable the site-specific attachment of a wide range of different functionalities. The outstanding utility of the cyanopyridine moiety is demonstrated by examples of (i) post-translational functionalization of proteins, (ii) in-cell macrocyclization of peptides and proteins, and (iii) protein stapling. The biocompatible nature of the protein ligation chemistry enabled by the cyanopyridylalanine amino acid opens a new path to specific in vivo protein modifications in complex biological environments.

An N-terminal selective thiazoline peptide macrocyclisation compatible with mRNA display and efficient SPPS

“…21 This unexpected result may reflect either a difference in reactivity preference for such a C-terminal chloroacetamide (with an N-terminal reactive group benefiting from cotranslational macrocyclisation increasing selectivity for the first cysteine incorporated into the peptide) or a partial blocking of the internal cysteine by transient reaction with the proximal mCNP, driving reaction of the chloroacetamide with the otherwise disfavoured distal N-terminal cysteine. In the setting of reprogrammed in vitro translation, the mCNP macrocyclization reaction thus proved to be robust, selective, and efficient, consistent with previous literature on cyclisation following SPPS 21 and bacterial translation using an orthogonal aaRS enzyme, 27 and its orthogonality with other thiol-based cyclisations allows spontaneous generation of libraries with more complicated bicyclic architectures.…”

Through-Space Scalar 19F–19F Couplings between Fluorinated Noncanonical Amino Acids for the Detection of Specific Contacts in Proteins

“…An aminoacyl-tRNA synthetase for genetic encoding of TFA-Lys has been published previously, 43 but the protein expression yields reported were low. We therefore selected a new synthetase for TFA-Lys from our library based on the pyrrolysyl-tRNA synthetase derived from the methanogenic archaeon ISO4-G1, 44 using a recently established fluorescence activated cell sorting (FACS) protocol (see SI section 3 for details). 30 The enzyme identified from this selection incorporates TFA-Lys with high specificity in response to amber stop codons, provided that the amino acid is present in high concentration.…”

Site-Specific Incorporation of 7-Fluoro-L-tryptophan into Proteins by Genetic Encoding to Monitor Ligand Binding by 19F NMR Spectroscopy

“…Using our previously reported library screening approach based on the pyrrolysyl-tRNA synthetase derived from the methanogenic archaeon ISO4-G1 (G1PylRS) and fluorescence-activated cell sorting (FACS), 13,36,37 we identified G1PylRS enzymes that specifically recognize 7FTrp (Figure 1a and c). In contrast, selection for G1PylRS enzymes specific for 4-fluorotryptophan (4FTrp; Figure 1b) carried out in parallel was unsuccessful (Figure 1d).…”

“…The selection of 7FTrp-specific RS enzymes was conducted in E. coli DH10B cells cotransformed with the selection plasmid pBAD-H6RFP, which harbors the gene of mCherry red fluorescent protein (RFP) preceded by an amber stop codon and an N-terminal His 6 tag (His 6 -TAG-RFP), and the plasmid pBK-G1RS containing the G1PylRS library (Leu124, Tyr125, Ala221, and Trp237 fully randomized to the 20 natural amino acids; Asn165 selectively mutated to Gly/Ala/ Val/Ser/Asn/Thr/Ile/Asp; Val167 selectively mutated to Gly/ Ala/Val/Ser/Cys/Leu/Phe; Tyr204 selectively mutated to Phe/Tyr/Trp). 37 The cells were cultured under positive and negative selection conditions in subsequent screening rounds, with independent selections targeting 7FTrp or 4FTrp carried out in parallel (Figures 1, S1, and S2). The positive growth condition included 2.5 mM of FTrp supplied to LB medium.…”