Milan Vrabel

A Genetically Encoded Norbornene Amino Acid for the Mild and Selective Modification of Proteins in a Copper‐Free Click Reaction

Methods for the site-specific chemical modification of proteins are currently of immense importance for the synthesis of protein–hybrid compounds for pharmaceutical and diagnostic purposes. Most of the methods rely on the reaction of free protein thiols with maleimides or the reaction of lysine side chains with activated esters. These methods provide only limited specificity, which is prompting researchers to develop alternative strategies that involve the incorporation of special unnatural amino acid into proteins to enable site-specific bioorthogonal functionalization. Among the developed methods, the Cu-catalyzed reaction of a protein containing an alkyne amino acid with azides stands out as the most thoroughly investigated technology. 6] However, the need for Cu salts, which may harm the protein structure, limits the technology. This fuels current interest to develop copperfree coupling reactions that are compatible with fragile protein structures. Here we show that these requirments can be met with a specially encoded norbornene amino acid which reacts selectively with nitrile imines. In order to insert a norbornene amino acid into a protein we used the amber suppression technique based on the pyrrolysyl tRNA/pyrrolysyl-tRNA synthetase (tRNA/ PylRS) pair from Methanosarcina mazei. The main task of the project was to evolve the pyrrolysine synthetase so that it accepts the synthetic norbornene amino acid 1 (Scheme 1 a) for loading onto the pyrrolysyl-tRNA. For the study we synthesized the norbornene-containing Pyl analogue 1 in seven steps from readily available starting materials (see the Supporting Information). To test to what extent 1 is accepted by wild-type (wt) PylRS, we used E. coli cells encoding the full tRNA/PylRS pair and a modified yellow fluorescent protein (YFP) containing one in-frame TAG stop codon. In this system the full-length and hence fluorescent YFP can only be produced when the corresponding Pyl analogue is accepted by the PylRS and successfully loaded onto the tRNA for subsequent incorporation into the protein at the amber stop codon site. The so-prepared E. coli cells were grown in a medium containing 5 mm 1. In addition to the wild-type PylRS we also tested a PylRS mutant (Y384F) previously used by Yanagisawa and co-workers. These initial experiments provided a just faint fluorescence when the mutant PylRS(Y384F) was used. No flurescence and hence no full-length YFP was generated in the presence of wild-type PylRS. In order to increase the PylRS activity we evolved the protein using iterative saturation mutagenesis (ISM) developed by Reetz and co-workers. Based on the co-crystal structure of PylRS in complex with adenylated pyrrolysine (PDB 2Q7H) we selected five residues in the substrate-binding pocket of wt-PylRS for the experiments. After transformation of the plasmid-based PylRS library into E. coli, single colonies were grown in liquid cultures supplemented with 1. The incorporation was monitored by means of the YFP fluorescence intensity of the cells. The most efficient PylRS variants were then sequenced and used in the next round of the saturation mutagenesis. In Scheme 1. a) Structure of norbornene Pyl analogue 1 and b) schematic representation of the click reactions. Top: A nitrile imine is generated by base-promoted HCl elimination from the hydrazonoyl chloride and then used in a cycloaddition reaction with the norbornene. Middle: Alternatively the nitrile imine is generated from a tetrazole in a photochemical reaction. Bottom: The norbornene modification can also react with tetrazines in a reversed-electron-demand Diels–Alder reaction. (Protein representations generated from PDB 3IN5.)

Synthesis of threefold glycosylated proteins using click chemistry and genetically encoded unnatural amino acids.

Eukaryotic proteins and in particular cell-surface proteins are frequently glycosylated, which has fueled the interest of chemist to develop new methods for the synthesis of glycosylated proteins. Protein glycosylation is essential for the proper function of the respective proteins and in the case of glycosylated protein therapeutics, such as erythropoietin, the activity of the protein strongly depends on glycosylation patterns. The synthesis and production of glycosylated proteins to either elucidate their biochemical function or to bring a new therapeutic to the market is a formidable challenge. The current most widely used methods for the production of glycosylated proteins involve over-expression of the protein in particular host systems, such as mammalian or plant cell cultures, moss cultures or tobacco plants, to name a few, that are able to make either partial or even fully glycosylated proteins. Alternatively, solid-phase synthesis of glycosylated peptides and their insertion into the protein in question by native or expressed chemical ligation has emerged as a powerful chemical strategy. We thought that one possibility to produce glycosylated proteins would be to cotranslationally insert unnatural alkyne or alkene amino acids into proteins. These amino acids enable site-specific protein glycosylation by Cu-free or Cu-catalyzed click chemistry methods, for example, with azide modified sugar moieties as reaction partners for the alkynes. The linkage between the protein and the sugar units would be an artificial triazole instead of the naturally occurring O,Oor O,N-acetals (sugar connection via Ser/Thr and Asn, respectively). However, this rather small change of the linker unit is thought to keep the biochemical properties of the glycosylated protein unchanged. The problem associated with a strategy like this is that in the literature mainly methods for the incorporation of a single artificial amino acid into a protein by wellestablished techniques are reported, while glycosylated proteins often possess multiple glycosylation sites. For in vitro incorporation of two different amino acids see refs. [40]–[41] . Here we report the in vivo incorporation of up to three artificial alkyne or alkene amino acids into the yellow fluorescent protein (YFP) and the subsequent glycosylation of these sites with different sugar azides using the Huisgen–Meldal–Sharpless click chemistry method, which was used in our group for the synthesis of multiple sugar modified oligonucleotides. Incorporation of unnatural, mostly aromatic amino acids can be efficiently achieved through the amber suppression technique with the help of a specially evolved, orthogonal tyrosyl-tRNA synthetase/tRNACUA pair. [43] Alternatively, the amber suppression with different Methanosarcina MS pyrrolysyl-tRNA synthetase/tRNACUA pair was reported. [44–52] This particular method was used by Yokoyama et al. and Chin et al. to insert one click site into proteins. 50] Here we show that the system Methanosarcina mazei (Mm) MS pyrrolysyl-tRNA synthetase/tRNACUA pair has the potential to read through more than just one amber stop codon present in the messenger RNA to achieve insertion of up to three artificial alkyne or alkene amino acids. We experimented with various protected Lys derivatives and found that the propargyl-protected Lys derivative 1, also used by Chin, and the commercially available allocLys derivative 2 are efficient substrates for the pyrrolysyl-tRNA synthetase. 53] In order to incorporate them into a protein and to set up an assay that would report efficient stop-codon suppression we transformed E. coli with a vector containing the genes for the M. mazei pyrrolysyl-tRNA synthetase, the M. mazei tRNACUA, a C-terminal StrepII-tag modified YFP with initially one later three amber TAG stop codons at positions 27, 114, and 132 as well as an ampicilline resistance gene. We added the alkyne amino acid 1 and the alkene amino acid 2 to the medium containing the transformed growing E. coli cells. As depicted in Figure 1, the E. coli cells containing the eYFP (Figure 1 A) are strongly fluorescent showing that the YFP protein is efficiently produced by the bacteria. E. coli cells containing the YFP protein with one TAG stop codon in the absence of the alkyne or alkene amino acids 1 or 2 are not fluorescent (Figure 1 B); this shows that in this case the full-length protein cannot be produced. Figure 1 C and D show E. coli cells containing the YFP protein with one and three TAG stop codons, respectively, in the presence of the alkyne amino acid 1. Figure 1 E and F are the corresponding pictures in the presence of the alkene amino acid 2. Here fluorescent cells are clearly detected showing that the stop codons are suppressed in the presence of the artificial amino acids in the medium. The fluorescence in Figure 1 D is clearly weaker; this indicates that although suppression of three stop codons is possible, the total suppression efficiency decreases. In order to quantify the protein yield, we isolated the full-length mutant YFP proteins with the C-terminal StrepII-tag from E. coli cultures (1 L) using a two-step purification procedure (Strep-tag affinity chromatography and anion exchange chromatography). The yield was determined to be about 0.3 mg for the YFP proteins comprising one alkyne or alkene amino acid (1 mut). For comparison we also quantified [a] E. Kaya, K. Gutsmiedl, Dr. M. Vrabel, Dr. M. M ller, P. Thumbs, Prof. Dr. T. Carell Center for Integrated Protein Science (CiPS) Department of Chemistry and Biochemistry, LMU Munich Butenandtstrasse 5–13, 81377 Munich (Germany) Fax: (+ 49) 89-2180-77756 E-mail : thomas.carell@cup.uni-muenchen.de [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.200900625.

Norbornenes in Inverse Electron‐Demand Diels–Alder Reactions

Significant differences in the reactivity of norbornene derivatives in the inverse electron-demand Diels-Alder reaction with tetrazines were revealed by kinetic studies. Substantial rate enhancement for the exo norbornene isomers was observed. Quantum-chemical calculations were used to rationalize and support the observed experimental data.

Structural Insights into Incorporation of Norbornene Amino Acids for Click Modification of Proteins

Three for two: by using a Methanosarcina mazei PylRS triple mutant (Y306G, Y384F, I405R) the incorporation of two new exo-norbornene-containing pyrrolysine analogues was achieved. X-ray crystallographic analysis led to the identification of the crucial structural elements involved in substrate recognition by the evolved synthetase.

Orchestrating the Biosynthesis of an Unnatural Pyrrolysine Amino Acid for Its Direct Incorporation into Proteins Inside Living Cells

We here report the construction of an E. coli expression system able to manufacture an unnatural amino acid by artificial biosynthesis. This can be orchestrated with incorporation into protein by amber stop codon suppression inside a living cell. In our case an alkyne-bearing pyrrolysine amino acid was biosynthesized and incorporated site-specifically allowing orthogonal double protein labeling.

Synthesis of epsilon-N-propionyl-, epsilon-N-butyryl-, and epsilon-N-crotonyl-lysine containing histone H3 using the pyrrolysine system

Recently new lysine modifications were detected in histones and other proteins. Using the pyrrolysine amber suppression system we genetically inserted three of the new amino acids ε-N-propionyl-, ε-N-butyryl-, and ε-N-crotonyl-lysine site specifically into histone H3. The lysine at position 9 (H3 K9), which is known to be highly modified in chromatin, was replaced by these unnatural amino acids.

Azidopropylvinylsulfonamide as a New Bifunctional Click Reagent for Bioorthogonal Conjugations: Application for DNA-Protein Cross-Linking

N-(3-Azidopropyl)vinylsulfonamide was developed as a new bifunctional bioconjugation reagent suitable for the cross-linking of biomolecules through copper(I)-catalyzed azide-alkyne cycloaddition and thiol Michael addition reactions under biorthogonal conditions. The reagent is easily clicked to an acetylene-containing DNA or protein and then reacts with cysteine-containing peptides or proteins to form covalent cross-links. Several examples of bioconjugations of ethynyl- or octadiynyl-modified DNA with peptides, p53 protein, or alkyne-modified human carbonic anhydrase with peptides are given.

Mechanism-Based Fluorogenic trans-Cyclooctene–Tetrazine Cycloaddition

Abstract The development of fluorogenic reactions which lead to the formation of fluorescent products from two nonfluorescent starting materials is highly desirable, but challenging. Reported herein is a new concept of fluorescent product formation upon the inverse electron‐demand Diels–Alder reaction of 1,2,4,5‐tetrazines with particular trans‐cyclooctene (TCO) isomers. In sharp contrast to known fluorogenic reagents the presented chemistry leads to the rapid formation of unprecedented fluorescent 1,4‐dihydropyridazines so that the fluorophore is built directly upon the chemical reaction. Attachment of an extra fluorophore moiety is therefore not needed. The photochemical properties of the resulting dyes can be easily tuned by changing the substitution pattern of the starting 1,2,4,5‐tetrazine. We support the claim with NMR measurements and rationalize the data by computational study. Cell‐labeling experiments were performed to demonstrate the potential of the fluorogenic reaction for bioimaging.

Cycloadditions in Bioorthogonal Chemistry

The series Topics in Current Chemistry Collections presents critical reviews from the journal Topics in Current Chemistry organized in topical volumes. The scope of coverage is all areas of chemical science including the interfaces with related disciplines such as biology, medicine and materials science. The goal of each thematic volume is to give the non-specialist reader, whether in academia or industry, a comprehensive insight into an area where new research is emerging which is of interest to a larger scientific audience. Each review within the volume critically surveys one aspect of that topic and places it within the context of the volume as a whole. The most significant developments of the last 5 to 10 years are presented using selected examples to illustrate the principles discussed. The coverage is not intended to be an exhaustive summary of the field or include large quantities of data, but should rather be conceptual, concentrating on the methodological thinking that will allow the non-specialist reader to understand the information presented. Contributions also offer an outlook on potential future developments in the field

Bioorthogonal Chemistry—Introduction and Overview

Abstract Bioorthogonal chemistry has emerged as a new powerful tool that facilitates the study of structure and function of biomolecules in their native environment. A wide variety of bioorthogonal reactions that can proceed selectively and efficiently under physiologically relevant conditions are now available. The common features of these chemical reactions include: fast kinetics, tolerance to aqueous environment, high selectivity and compatibility with naturally occurring functional groups. The design and development of new chemical transformations in this direction is an important step to meet the growing demands of chemical biology. This chapter aims to introduce the reader to the field by providing an overview on general principles and strategies used in bioorthogonal chemistry. Special emphasis is given to cycloaddition reactions, namely to 1,3-dipolar cycloadditions and Diels–Alder reactions, as chemical transformations that play a predominant role in modern bioconjugation chemistry. The recent advances have established these reactions as an invaluable tool in modern bioorthogonal chemistry. The key aspects of the methodology as well as future outlooks in the field are discussed.