Tomasz Fekner

A Pyrrolysine Analogue for Protein Click Chemistry

Ignoring the STOP sign: A pyrrolysine analogue bearing a terminal alkyne was site-specifically incorporated into recombinant calmodulin (CaM) through a UAG codon. The resulting protein was labeled with an azide-containing dye using a copper(I)-catalyzed click reaction. Subsequent application of an orthogonal cysteine tagging method yielded a CaM labeled with two distinct fluorophores that enabled its study by FRET spectroscopy.

A Pyrrolysine Analogue for Site‐Specific Protein Ubiquitination

(Scheme 1) to generate a product with a native backbone. Specifically, a reversible transthioesterification in the presence of an exogenous thiol RSH gives an intermediate 3, which in turn undergoes irreversible intramolecular S!N acyl transfer to give the final peptide 4. Herein, we introduce a genetically encoded pyrrolysine analogue that places a ligation handle directly into a recombinant protein. We used NCL at this internal ligation site to generate a semisynthetic ubiquitinated protein. The cellular machinery for the incorporation of pyrrolysine (5, Scheme 2), 4] the 22nd genetically encoded amino acid, is sufficiently flexible to enable a number of other lysine derivatives to read through the amber stop codon. We previously described 6, a stable THF-based analogue of 5. We also introduced 7, which, owing to the presence of a terminal alkyne functionality, can be used as a chemical handle to label proteins through click chemistry. To further expand the range of available pyrrolysine analogues with unique and useful reactivity, we decided to test whether the d-cysteine-based analogue (S,S)-8 (d-Cys-e-Lys) could read through the UAG codon. We focused our attention on this cysteine isomer because our related readthrough studies of simple pyrrolysine analogues for protein click chemistry indicated that the presence of a lysine acyl substituent with the analogous sense of chirality to that found in 5 has a profound and beneficial influence on incorporation efficiency. For comparison purposes, however, we also included in our studies the diastereomeric analogue (R,S)-8 (l-Cys-e-Lys). The target pyrrolysine analogue (S,S)-8 was prepared by coupling the N,S-protected cysteine derivative (S)-9 with BocLys-OtBu (10) to provide amide (S,S)-11 in excellent yield (96 %, d.r.> 99.9:0.1; Scheme 3). Full deprotection with trifluoroacetic acid (TFA)/Et3SiH furnished (S,S)-8 as its TFA salt. The diastereomer (R,S)-8 was prepared in an analogous manner (see the Supporting Information). Scheme 1. Cysteine-based NCL. Scheme 2. Pyrrolysine (5) and analogues 6–8.

The pyrrolysine translational machinery as a genetic-code expansion tool.

The discovery of pyrrolysine not only expanded the set of the known proteinogenic amino acids but also revealed unusual features of its encoding mechanism. The engagement of a canonical stop codon and a unique aminoacyl-tRNA synthetase-tRNA pair that can be used to accommodate a broad range of unnatural amino acids while maintaining strict orthogonality in a variety of prokaryotic and eukaryotic expression systems has proven an invaluable combination. Within a few years since its properties were elucidated, the pyrrolysine translational machinery has become a popular choice for the synthesis of recombinant proteins bearing a wide variety of otherwise hard-to-introduce functional groups. It is also central to the development of new synthetic strategies that rely on stop-codon suppression.

N6‐(2‐(R)‐Propargylglycyl)lysine as a Clickable Pyrrolysine Mimic

The site-specific tagging of proteins with organic fluorophores[2] has been proven to be a powerful method in structural and functional studies of a wide range of biological systems.[3-5] The most challenging aspect of this approach is the regiospecific incorporation of a suitable fluorophore on or nearby a rationally selected amino acid residue within a protein chain. The demonstration that pyrrolysine (1, Figure 1), the 22nd genetically-encoded amino acid, can be incorporated into recombinant proteins in response to the UAG codon,[6,7] prompted us to search for similarly incorporable analogs of 1 harboring reactive functionalities suitable for anchoring small organic fluorophores. We previously reported the synthesis of the THF-containing lysine derivative 2 that reads through the UAG codon,[8] as does its close structural analog 3.[9] The latter compound, thanks to the presence of the terminal alkyne functionality, enables site-specific post-translational modification of the resulting protein with azide-based fluorophores via the CuI-catalyzed azide–alkyne cycloaddition reaction (CuAAC). Our recent work has gone beyond the field of protein click chemistry resulting in the synthesis and application of the cysteine derivative 4 for protein ubiquitination via native chemical ligation.[10]

A click-and-release pyrrolysine analogue.

Whats the catch? A pyrrolysine analogue bearing a terminal alkyne and an ester functionality can be incorporated into recombinant proteins and render them amenable to capture by the click reaction and subsequent release through ester hydrolysis. The utility of this pyrrolysine-inspired technology is demonstrated for the identification of SUMOylation sites.

Pyrrolysine-inspired protein cyclization.

The pyrrolysine translational machinery has been extensively explored for the production of recombinant proteins containing a variety of “site‐specific” non‐canonical amino acids for both in vitro and in vivo biochemical studies. In this study, we report the first use of this technology for the production of branched cyclic proteins with a tadpole‐like topology. As a proof of concept, we fused the well‐studied RGD peptide to the C terminus of an mCherry reporter protein. Previous studies have shown that cyclization of the RGD peptide enhances its internalization into cells compared to its linear counterpart. The cellular uptake efficiencies of mCherry‐cyclo(RGD), mCherry‐linear(RGD), and wild‐type mCherry determined by flow cytometry follow the trends expected, thereby confirming the feasibility and potential utility of this cyclization approach.

Hidden in Plain Sight: The Biosynthetic Source of Pyrrolysine Revealed

ability to convert methylamines into methane. The methylamine methyltransferase genes present in these methanogens contain an in-frame UAG (amber) codon that is translated as 1 by the concerted action of a unique amber suppressor, tRNA, and its cognate pyrrolysyl-tRNA synthetase (PylRS). Although pyrrolysine was discovered almost a decade ago 3] and its translation machinery has been the focal point of intense research ever since, it is only recently that the details of its biosynthesis have become clear. 6] A total of five genes (collectively referred to as pylTSBCD) are linked to pyrrolysine. Two of them, pylT and pylS, encode tRNA and PylRS, respectively, while the remaining three genes are assigned to pyrrolysine biosynthesis. In 2007, it was shown that transfer of the five-gene cluster from Methanosarcina acetivorans is both sufficient and necessary to support biosynthesis and genetic encoding of pyrrolysine in Escherichia coli. This suggested that pyrrolysine must be biosynthetically derived from metabolites common to both Archaea and Bacteria. Considering that pyrrolysine is an acylated lysine, there appeared to be no doubt that lysine itself was one of the substrates from which pyrrolysine is biosynthetically derived. However, the metabolic source of its pyrroline subunit (highlighted in red, Scheme 1) was not so easy to deduce. Based on rudimentary retrosynthetic analysis, d-proline (2), d-isoleucine (3), d-glutamic acid (4), and d-ornithine (5) were suggested as possible candidates. In particular, 3 seemed very appealing as its involvement would not require an additional methylation step, which was predicted to be difficult. An important clue as to the true identity of this elusive metabolite came from the observation that by exogenously supplying d-ornithine, but not amino acids 2–4, it was possible to significantly increase the level of the PylRS–tRNA-mediated UAG suppression in E. coli transformed with pylTSBCD. Based on this finding alone, and despite the fact that it was already becoming apparent that the PylRS–tRNA system accepts close structural analogues of 1, a conclusion was drawn that d-ornithine is the other required biosynthetic precursor of pyrrolysine. Two very recent and independent reports by Krzycki et al. 10] and Geierstanger et al. re-examined the d-ornithine experiments. Both groups confirmed that an exogenous supply of 5 does indeed increase the efficiency of UAG readthrough and, consequently, the production of full-length target proteins in expression systems transformed with pylTSBCD. However, the MS analysis of peptide fragments derived from these proteins showed that, in addition to pyrrolysine, another residue was incorporated at the site corresponding to the UAG codon. Moreover, the full-length target proteins were also produced even when pylB was excluded from the expression systems. It this case, however, the incorporation site contained the new residue exclusively, with no traces of 1 being detected. The mass of the molecular ion corresponding to the new UAG-encoded residue being 14 Da less than that of pyrrolysine suggested that the methylation step necessary to biosynthetically link 5 with 1 did not occur. Based on these observations and taking into account the functional roles of enzymes structurally related to PylC and PylD, the two groups proposed virtually identical biosynthetic schemes that fully account for the experimental observations (Scheme 2). 6] First, in an ATP-dependent process, PylC catalyzes the acylation of lysine (6) with d-ornithine to provide amide 7. Subsequent PylD-mediated oxidation of one its amino groups leads to aldehyde 8, which then undergoes spontaneous intramolecular Schiff base formation resulting in desmethylpyrrolysine (9). During the translation step, the PylRS– tRNA system accepts 9 in lieu of its native substrate 1 and incorporates it into proteins in response to a UAG codon. Scheme 1. Pyrrolysine (1) and the initially proposed potential biosynthetic precursors (2–5) of its pyrroline subunit.