Genetic Incorporation of Twelve meta-Substituted Phenylalanine Derivatives Using a Single Pyrrolysyl-tRNA Synthetase Mutant

Abstract

When coexpressed with its cognate amber suppressing tRNACUAPyl, a pyrrolysyl-tRNA synthetase mutant N346A/C348A is able to genetically incorporate twelve meta-substituted phenylalanine derivatives into proteins site-specifically at amber mutation sites in Escherichia coli. These genetically encoded noncanonical amino acids resemble phenylalanine in size and contain diverse bioorthogonal functional groups such as halide, trifluoromethyl, nitrile, nitro, ketone, alkyne, and azide moieties. The genetic installation of these functional groups in proteins provides multiple ways to site-selectively label proteins with biophysical and biochemical probes for their functional investigations. We demonstrate that a genetically incorporated trifluoromethyl group can be used as a sensitive 19F NMR probe to study protein folding/unfolding, and that genetically incorporated reactive functional groups such as ketone, alkyne, and azide moieties can be applied to site-specifically label proteins with florescent probes. This critical discovery allows the synthesis of proteins with diverse bioorthogonal functional groups for a variety of basic studies and biotechnology development using a single recombinant expression system.

Expanding the chemical diversity of lasso peptide MccJ25 with genetically encoded noncanonical amino acids

“…33 Trends could also be observed with regards to the ncAAs; variants with the nitro substitution were consistently the most poorly produced (Table 1, Figure S1). Of the four amino acids examined here, m -NO 2 Phe exhibited the lowest protein yields in previous experiments on ncAA incorporation into GFP, 22 potentially indicating that m -NO 2 Phe is simply a poorer substrate for the PylRS than the other ncAAs tested here.…”

“…21 A specific variant of Methanosarcina mazei PylRS that includes two amino acid substitutions, N346A and C348A, has extensive substrate polyspecificity and can incorporate a growing list of aromatic amino acids into recombinant proteins in E. coli . 22–26 For this study, we chose a panel of four meta -substituted Phe derivatives, m -ClPhe, m -BrPhe, m -NO 2 Phe, and m -CF 3 Phe (Figure 1B). This panel of Phe residues includes both polar (nitro) and non-polar (trifluoromethyl) substituents as well as halides, which introduce steric bulk to the meta -position…”

Synthesis and Explosion Hazards of 4-Azido-l-phenylalanine

“…For example, previously reported optical rotation values for 1 are −20°, −10.6°, −9.1°, −6.2°, and +20°. 3a,7,36 We note that variation in both sign and magnitude is reported, despite each value being measured apparently from the same stereoisomer. The literature characterization data of 4 is similarly poor.…”

“…2 Combined with carefully chosen tRNA/synthase pairs, organisms with an expanded genetic code can be trained to explicitly incorporate unnatural amino acids with high fidelity alongside their natural counterparts during ribosomal protein synthesis. 3 The identity of the unnatural amino acid included in the expanded genetic code is selectable, and its position inside a target protein can be programmed at the genetic level. Thus, the expanded genetic code represents an enormously powerful tool for site-specific introduction of exotic functional groups into proteins.…”

“…4 For example, incorporation of 4-iodo-L-phenylalanine ( 1 ) into proteins allows synthetic post-translational modification by Suzuki-Miyaura reactions, 5 and has been used for site-selective introduction of polyethylene glycol 6 and various fluorescent dyes. 3a,7 The phenyl azide functional group in 4-azido-L-phenylalanine ( 4 ) makes it one of the most versatile unnatural amino acids in common use. This wide usage reflects two important properties.…”