A photochemically chemically active noncanonical amino acid para-azido-l-phenylalanine widely used in biology was found to be metabolized by Saccharomyces cerevisiae. Contrary to multiple reports, the azide moiety is not reduced to the corresponding amine. The amino acid's concentration was found to decline somewhat with time which was due, at least in part, to modification of the amino acid side chain. The metabolite was found to be photochemically active and further characterization concluded the azide moiety was still intact. This work also goes onto highlight paramount areas of concern with regards to (photo)chemical compatibility, handling, and fidelity in genetically encoding aryl azide amino acids.

The target protein, Hcp1, was first described as part of the bacterial Type VI secretion system from Pseudomonas aeruginosa. The protein first self-assembles into a hexamer and then the hexamers further stack into a nanotubular structure. Hcp1 monomers were targeted for mutagenesis with two widely used photoactivatable amino acids: para-benzoyl phenylalanine or para-azidophenylalanine. The ability of these amino acids to form covalent adducts within the Hcp1 self-assembled system was investigated. Multiple residues, putatively of equal distance between the monomer–monomer interface were targeted. The efficiency of each amino acid to covalently link self-assembled hexamers was determined. The results demonstrate the choice and role of genetically encoded tools applied to complicated biological processes such as self-assembly and also suggested some structural dynamics of the Hcp-1 protein not obvious from crystallographic structures.

Synthesis of a nonnitrogenous phenanthrene-based precursor of oxiranyl carbene yielded a photochemical source for diphenyl ketene after an in situ intramolecular rearrangement. The initial carbene precursor targeted rearranged from a putative spiro-oxiranyl intermediate to a cyclobutanone. Photolysis of the phenanthrene-cyclobutanone generated an infrared signal centered at 2100 cm(-1) consistent with diphenyl ketene formation. Time-resolved infrared spectroscopy followed the reaction progress and measured bimolecular rate constants (k(obs) = 9.1 x 10(6) M-1 s(-1); butyl amine) consistent with diphenyl ketene formation. Quenching experiments combined with Stern-Volmer kinetics suggests a radical pathway is likely involved in the formation of diphenyl ketene from the phenanthrene-based precursor. The generality of the rearrangement is also discussed. (C) 2016 Elsevier Ltd. All rights reserved.

Expanded genetic code approaches are a powerful means to add new and useful chemistry to proteins at defined residues positions. One such use is the introduction of non-biological reactive chemical handles for site-specific biocompatible orthogonal conjugation of proteins. Due to our currently limited information on the impact of non-canonical amino acids (nAAs) on the protein structure-function relationship, rational protein engineering is a "hit and miss" approach to selecting suitable sites. Furthermore, dogma suggests surface exposed native residues should be the primary focus for introducing new conjugation chemistry. Here we describe a directed evolution approach to introduce and select for in-frame codon replacement to facilitate engineering proteins with nAAs. To demonstrate the approach, the commonly reprogrammed amber stop codon (TAG) was randomly introduced in-frame in two different proteins: the bionanotechnologically important cyt b(562) and therapeutic protein KGF. The target protein is linked at the gene level to sfGFP via a TEV protease site. In absence of a nAA, an in-frame TAG will terminate translation resulting in a non-fluorescent cell phenotype. In the presence of a nAA, TAG will encode for nAA incorporation so instilling a green fluorescence phenotype on E. coli. The presence of endogenously expressed TEV proteases separates in vivo target protein from its fusion to sfGFP if expressed as a soluble fusion product. Using this approach, we incorporated an azide reactive handle and identified residue positions amenable to conjugation with a fluorescence dye via strain-promoted azide-alkyne cycloaddition (SPAAC). Interestingly, best positions for efficient conjugation via SPAAC were residues whose native side chain were buried through analysis of their determined 3D structures and thus may not have been chosen through rational protein engineering. Molecular modeling suggests these buried native residues could become partially exposed on substitution to the azide containing nAA.

Genetic code reprogramming allows proteins to sample new chemistry through targeted introduction of non-natural amino acids. By combining with random codon replacement, residues traditionally overlooked can be identified as instilling new properties on a target protein.

Expanding the genetic code opens new avenues to modulate protein function in real time. By genetically incorporating photoreactive phenyl azide, the fluorescent properties of green fluorescent protein (GFP) can be modulated by light. Depending on the residue in GFP programmed to incorporate the phenyl azide, different effects on function and photochemical pathways are observed.

A genetically encoded precursor to an aryl nitrene, para-azidophenylalanine, was introduced site specifically into proteins to deduce if distinct environments were capable of caging a reactive organic intermediate. Following photolysis of mutant T4 lysozyme or green fluorescent proteins, EPR spectra showed, respectively, the presence of a triplet nitrene and an anilino radical.

Introducing new physicochemical properties into proteins through genetically encoded Uaa (unnatural amino acid) incorporation can lead to the generation of proteins with novel properties not normally accessible with the 20 natural amino acids. Phenyl azide chemistry represents one such useful addition to the protein repertoire. Classically used in biochemistry as a non-specific photochemical protein cross-linker, genetically encoding phenyl azide chemistry at selected residues provides more powerful routes to post-translationally modify protein function in situ. The two main routes are modulation by light (optogenetics) and site-specific bio-orthogonal modification (bioconjugation) via Click chemistry. In the present article, we discuss both approaches and their influence on protein function.

GFP and a FRET compatible dye were used to assess the influence of genetically encoded aryl azide positioning on Click chemistry-based protein conjugation. While modification efficiency of the sampled mutants using a strain promoted reaction varied by as much as ∼10 fold, there was no simple correlation with accessibility of the aryl azide on GFP's surface. One labeled GFP mutant (Gln204AzPhe) exhibited high efficiency FRET (∼90%) and an unprecedented pseudo-Stokes shift of 126 nm.

Stabilization of the reaction intermediate eudesmane cation (3) through interaction with Trp 334 during catalysis by aristolochene synthase from Penicillium roqueforti was investigated by site-directed incorporation of proteinogenic and non-canonical aromatic amino acids. The amount of germacrene A (2) generated by the mutant enzymes served as a measure of the stabilization of 3. 2 is a neutral intermediate, from which 3 is formed during PR-AS catalysis by protonation of the C6,C7 double bond. The replacement of Trp 334 with para-substituted phenylalanines of increasing electron-withdrawing properties led to a progressive accumulation of 2 that showed a good correlation with the interaction energies of simple cations such as Na+ with substituted benzenes. These results provide compelling evidence for the stabilizing role played by Trp 334 in aristolochene synthase catalysis for the energetically demanding transformation of 2 to 3.

There are many approaches to introduce non-native functionality into proteins either translationally or post-translationally. When a noncanonical amino acid (NAA) is incorporated translationally, the host organism's existing translational machinery is relied upon to insert the amino acid by the same well-established mechanisms used by the host to achieve high fidelity insertion of its canonical amino acids. Research into the in vivo incorporation of NAAs has typically concentrated on evolving or engineering aminoacyl tRNA synthetases (aaRSs); however, new studies have increasingly focused on other members of the translational apparatus, for example entire ribosomes, in attempts to increase the fidelity and efficiency of incorporation of ever more structurally diverse NAAs. As the biochemical methods of NAA systems increase in complexity, it is informative to ask whether the 'rules' for canonical translation (i.e. aaRSs, tRNA, ribosomes, elongation factors, amino acid uptake, and metabolism) hold for NAA systems, or whether new rules are warranted. Here, recent advances in introducing novel chemical functionality into proteins are highlighted.

The site-selective encoding of noncanonical amino acids (NAAs) is a powerful technique for the installation of novel chemical functional groups in proteins. This is often achieved by recoding a stop codon and requires two additional components: an evolved aminoacyl tRNA synthetase (AARS) and a cognate tRNA. Analysis of the most successful AARSs reveals common characteristics. The highest fidelity NAA systems derived from the Methanocaldococcus jannaschii tyrosyl AARS feature specific mutations to two residues reported to interact with the hydroxyl group of the substrate tyrosine. We demonstrate that the restoration of just one of these determinants for amino acid specificity results in the loss of fidelity as the evolved AARSs become noticeably promiscuous. These results offer a partial explanation of a recently retracted strategy for the synthesis of glycoproteins. Similarly, we reinvestigated a tryptophanyl AARS reported to allow the site-selective incorporation of 5-hydroxy tryptophan within mammalian cells. In multiple experiments, the enzyme displayed elements of promiscuity despite its previous characterization as a high fidelity enzyme. Given the many similarities of the TyrRSs and TrpRSs reevaluated here, our findings can be largely combined, and in doing so they reinforce the long-established central dogma regarding the molecular basis by which these enzymes contribute to the fidelity of translation. Thus, our view is that the central claims of fidelity reported in several NAA systems remain unproven and unprecedented.

BioEssaysVolume 32, Issue 4 p. 314-321 Commentary The juggernauts of biology Isaac T. Yonemoto, Corresponding Author Isaac T. Yonemoto ityonemo@umd.edu Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, 20742, USA Isaac T. Yonemoto, Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA. Eric M. Tippmann, School of Chemistry, Cardiff University, Cardiff CF10 3AT, UK.Search for more papers by this authorEric M. Tippmann, Corresponding Author Eric M. Tippmann tippmann@cardiff.ac.uk School of Chemistry, Cardiff University, Cardiff CF10 3AT, UK Isaac T. Yonemoto, Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA. Eric M. Tippmann, School of Chemistry, Cardiff University, Cardiff CF10 3AT, UK.Search for more papers by this author Isaac T. Yonemoto, Corresponding Author Isaac T. Yonemoto ityonemo@umd.edu Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, 20742, USA Isaac T. Yonemoto, Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA. Eric M. Tippmann, School of Chemistry, Cardiff University, Cardiff CF10 3AT, UK.Search for more papers by this authorEric M. Tippmann, Corresponding Author Eric M. Tippmann tippmann@cardiff.ac.uk School of Chemistry, Cardiff University, Cardiff CF10 3AT, UK Isaac T. Yonemoto, Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA. Eric M. Tippmann, School of Chemistry, Cardiff University, Cardiff CF10 3AT, UK.Search for more papers by this author First published: 26 March 2010 https://doi.org/10.1002/bies.200900142Citations: 3Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume32, Issue4Special Issue: Synthetic BiologyApril 2010Pages 314-321 RelatedInformation

A chloromethylhydroxamiccarbene was generated photochemically in an attempt to form an intramolecularly stabilized carbene. A rapidly formed intermediate at 1645 cm(-1) decayed with an observed rate of 1.99 × 10(6) s(-1). Other intermediates were also observed. These also decayed, albeit much more slowly (k(obs) = 3.47 × 10(3) and 1.98 × 10(4) s(-1)). Multiple intermediates are apparently a function of both the proximal N,O-dimethylhydroxamic ester and multiple conformers of both the carbene and precursor.

The ability to incorporate non-canonical amino acids into proteins by genetic or chemical methods allows one to introduce novel chemical properties into a protein at a defined residue. Such a residue may then be modified using common organic transformations. In this way, the structure or function of the peptide may be altered without perturbing any of the other neighbouring amino acids in the peptide chain. Here, we describe the syntheses and potential applications of multiple para-substituted phenylalanine derivatives comprising an isothiocyanate, α-diazoketone, or nitrone functionality. In all, three novel amino acids were synthesized in good overall yields. These non-canonical amino acids permit the further development of in vitro and in vivo chemoselective and regioselective bioconjugate reactions not possible with other reagents.

The reactivity of two metallated nitrenium ions toward various substrates was examined in the gas phase. The nitrenium ions were generated by a reaction of benzoyl azide with laser-ablated Mg+ or Cu+ in a Fourier transform ion cyclotron resonance mass spectrometer. The two nitrenium ions show drastically different reactivity. While the Mg-nitrenium ion reacts by radical mechanisms (e.g., H atom abstraction), the Cu-nitrenium ion follows non-radical pathways (e.g., metal ion transfer). (C) 2009 Elsevier B.V. All rights reserved.

A directed evolution method has been developed that allows random substitution of a contiguous trinucleotide sequence for TAG throughout a target gene for use in conjunction with an expanded genetic code. Using TEM-1 beta-lactamase and enhanced green fluorescent protein as targets, protein variants were identified whose functional phenotype was rescued in vivo when co-expressed with orthogonal tRNA-aminoacyl-tRNA synthase pairs that insert p-iodophenylalanine in response to UAG. Sequencing of the selected clones that retained the target protein function revealed that >90% of the variants contained in-frame TAG codons distributed throughout the target gene. Such an approach will allow broader sampling of new chemical diversity by proteins, so opening new avenues for studying biological systems and for adapting proteins for biotechnological applications. A common set of reagents allows the method to be used on different protein systems and in combination with an array of different unnatural amino acids, so helping to reveal the true potential for engineering proteins through expanded chemical diversity sampling.

The ability of Escherichia coli to grow on a series of acetylated and glycosylated compounds has been investigated. It is surmised that E. coli maintains low levels of nonspecific esterase activity. This observation may have ramifications for previous reports that relied on nonspecific esterases from E. coli to genetically encode nonnatural amino acids. It had been reported that nonspecific esterases from E. coli deacetylate tri-acetyl O-linked glycosylated serine and threonine in vivo. The glycosylated amino acids were reported to have been genetically encoded into proteins in response to the amber stop codon. However, it is our contention that such amino acids are not utilized in this manner within E. coli. The current results report in vitro analysis of the original enzyme and an in vivo analysis of a glycosylated amino acid. It is concluded that the amber suppression method with nonnatural amino acids may require a caveat for use in certain instances.