Bioorthogonal Functionalities Research Articles

Chemical Synthesis of Modified RNA.

Ribonucleic acids (RNAs) play a vital role in living organisms. Many of their cellular functions depend critically on chemical modification. Methods to modify RNA in a controlled manner-both in vitro and in vivo-are thus essential to evaluate and understand RNA biology at the molecular and mechanistic levels. The diversity of modifications, combined with the size and uniformity of RNA (made up of only 4 nucleotides) makes its site-specific modification a challenging task that needs to be addressed by complementary approaches. One such approach is solid-phase RNA synthesis. We discuss recent developments in this field, starting with new protection concepts in the ongoing effort to overcome current size limitations. We continue with selected modifications that have posed significant challenges for their incorporation into RNA. These include deazapurine bases required for atomic mutagenesis to elucidate mechanistic aspects of catalytic RNAs, and RNA containing xanthosine, N4-acetylcytidine, 5-hydroxymethylcytidine, 3-methylcytidine, 2'-OCF3, and 2'-N3 ribose modifications. We also discuss the all-chemical synthesis of 5'-capped mRNAs and the enzymatic ligation of chemically synthesized oligoribonucleotides to obtain long RNA with multiple distinct modifications, such as those needed for single-molecule FRET studies. Finally, we highlight promising developments in RNA-catalyzed RNA modification using cofactors that transfer bioorthogonal functionalities.

Chemical Synthesis of Modified RNA

AbstractRibonucleic acids (RNAs) play a vital role in living organisms. Many of their cellular functions depend critically on chemical modification. Methods to modify RNA in a controlled manner—both in vitro and in vivo—are thus essential to evaluate and understand RNA biology at the molecular and mechanistic levels. The diversity of modifications, combined with the size and uniformity of RNA (made up of only 4 nucleotides) makes its site‐specific modification a challenging task that needs to be addressed by complementary approaches. One such approach is solid‐phase RNA synthesis. We discuss recent developments in this field, starting with new protection concepts in the ongoing effort to overcome current size limitations. We continue with selected modifications that have posed significant challenges for their incorporation into RNA. These include deazapurine bases required for atomic mutagenesis to elucidate mechanistic aspects of catalytic RNAs, and RNA containing xanthosine, N4‐acetylcytidine, 5‐hydroxymethylcytidine, 3‐methylcytidine, 2′‐OCF3, and 2′‐N3 ribose modifications. We also discuss the all‐chemical synthesis of 5′‐capped mRNAs and the enzymatic ligation of chemically synthesized oligoribonucleotides to obtain long RNA with multiple distinct modifications, such as those needed for single‐molecule FRET studies. Finally, we highlight promising developments in RNA‐catalyzed RNA modification using cofactors that transfer bioorthogonal functionalities.

Isonitrile biosynthesis by non-heme iron(II)-dependent oxidases/decarboxylases.

The isonitrile group is a compact, electron-rich moiety coveted for its commonplace as a building block and bioorthogonal functionality in synthetic chemistry and chemical biology. Hundreds of natural products containing an isonitrile group with intriguing bioactive properties have been isolated from diverse organisms. Our recent discovery of a conserved biosynthetic gene cluster in some Actinobacteria species highlighted a novel enzymatic pathway to isonitrile formation involving a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase. Here, we focus this chapter on recent advances in understanding and probing the biosynthetic machinery for isonitrile synthesis by non-heme iron(II) and α-ketoglutarate-dependent dioxygenases. We will begin by describing how to harness isonitrile enzymatic machinery through heterologous expression, purification, synthetic strategies, and in vitro biochemical/kinetic characterization. We will then describe a generalizable strategy to probe the mechanism for isonitrile formation by combining various spectroscopic methods. The chapter will also cover strategies to study other enzyme homologs by implementing coupled assays using biosynthetic pathway enzymes. We will conclude this chapter by addressing current challenges and future directions in understanding and engineering isonitrile synthesis.

Enzymatische Peptid‐ und Proteinbromierung: Der BromoTrp Tag

AbstractBioorthogonale Reaktionen von Proteinen und ungeschützten Peptiden sind von hohem Wert für die chemische Biologie. Die Kombination aus enzymatischer Halogenierung und übergangsmetallkatalysierter Kreuzkupplung bietet einen praktikablen Ansatz zur Modifizierung von Proteinen und ungeschützten Peptiden. Durch semirationales Protein Engineering, wurden Varianten der Tryptophan 6‐Halogenase Thal identifiziert, welche die Bromierung von Peptiden mit einem C‐terminalen Tryptophanrest ermöglichen. Das Substratspektrum wurde anhand von Di‐, Tri‐ und Tetrapeptid‐Bibliotheken charakterisiert, und ein optimierter Peptid‐Tag identifiziert, dem wir den Namen BromoTrp‐Tag gegeben haben. Dieser Tag wurde in drei Modellproteine eingeführt. Deren präparative, posttranslationale Bromierung mit nur einem Kultivierungs‐ und Aufreinigungsschritt wurde durch das bromierende E. coli Koexpressionssystem Brocoli ermöglicht. Pd‐katalysierte Suzuki–Miyaura Kreuzkupplung war mittels Pd‐Nanopartikeln als Katalysator bei 37 °C möglich, was das vielversprechende Potential dieser Strategie zum Zweck bioorthogonaler Funktionalisierung und Konjugation demonstriert.

Enzymatic Peptide and Protein Bromination: The BromoTrp Tag.

Bio-orthogonal reactions for modification of proteins and unprotected peptides are of high value in chemical biology. The combination of enzymatic halogenation with transition metal-catalyzed cross-coupling provides a feasible approach for the modification of proteins and unprotected peptides. By a semirational protein engineering approach, variants of the tryptophan 6-halogenase Thal were identified that enable efficient bromination of peptides with a C-terminal tryptophan residue. The substrate scope was explored using di-, tri-, and tetrapeptide arrays, leading to the identification of an optimized peptide tag we named BromoTrp tag. This tag was introduced into three model proteins. Preparative scale post-translational bromination was possible with only a single cultivation and purification step using the brominating E. coli coexpression system Brocoli. Palladium-catalyzed Suzuki-Miyaura cross-coupling of the bromoarene was achieved with Pd nanoparticle catalysts at 37 °C, highlighting the rich potential of this strategy for bio-orthogonal functionalization and conjugation.

Semiconducting Polymer Dots Directly Stabilized with Serum Albumin: Preparation, Characterization, and Cellular Immunolabeling.

Semiconducting polymer dots (Pdots) are brightly fluorescent nanoparticles of growing interest for bioanalysis and imaging. A recurring challenge with these materials is obtaining robust physical and colloidal stability and low nonspecific binding. Here, we prepared and characterized Pdots with bovine serum albumin (BSA) as the stabilizing agent (BSA-Pdots) instead of a more conventionally used amphiphilic polymer, both without and with cross-linking of the protein using glutaraldehyde (BSA(GA)-Pdots) or disuccinimidyl glutarate. Characterization included fluorescence properties; colloidal stability as a function of pH, ionic strength, and solvent perturbation; shape retention and hardness; and nonspecific binding with common assay substrates, fixed cells, and live cells. These properties were contrasted with the same properties for amphiphilic polymer-stabilized Pdots and silica-coated Pdots. On balance, the BSA-stabilized Pdots were similar or more favorable in their properties, with BSA(GA)-Pdots being especially advantageous. Bioconjugation of the BSA-stabilized Pdots was possible using amine-reactive active-ester chemistry, including biotinylation and bioorthogonal functionalization for immunoconjugation via tetrazine-strained-alkene click chemistry. These approaches were used for selective fluorescent labeling of cells based on ligand-receptor and antibody-antigen binding, respectively. Overall, direct BSA stabilization is a very promising strategy for preparing Pdots with improved physical and colloidal stability, reduced nonspecific interactions, and utility for in vitro diagnostics and other bioanalyses and imaging.

Regulating Chemokine-Receptor Interactions through the Site-Specific Bioorthogonal Conjugation of Photoresponsive DNA Strands.

Oligonucleotide conjugation has emerged as a versatile molecular tool for regulating protein activity. A state-of-the-art labeling strategy includes the site-specific conjugation of DNA, by employing bioorthogonal groups genetically incorporated in proteins through unnatural amino acids (UAAs). The incorporation of UAAs in chemokines has to date, however, remained underexplored, probably due to their sometimes poor stability following recombinant expression. In this work, we designed a fluorescent stromal-derived factor-1β (SDF-1β) chemokine fusion protein with a bioorthogonal functionality amenable for click reactions. Using amber stop codon suppression, p-azido-L-phenylalanine was site-specifically incorporated in the fluorescent N-terminal fusion partner, superfolder green fluorescent protein (sfGFP). Conjugation to single-stranded DNAs (ssDNA), modified with a photocleavable spacer and a reactive bicyclononyne moiety, was performed to create a DNA-caged species that blocked the receptor binding ability. This inhibition was completely reversible by means of photocleavage of the ssDNA strands. The results described herein provide a versatile new direction for spatiotemporally regulating chemokine-receptor interactions, which is promising for tissue engineering purposes.

Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium.

Residue-specific incorporation of non-canonical amino acids (ncAAs) introduces bio-orthogonal functionalities into proteins. As such, this technique is applied in protein characterization and quantification. Here, we studied protein expression with three methionine analogs, namely photo-methionine (pMet), azidohomoalanine (Aha) and homopropargylglycine (Hpg), in prototrophic E. coli BL-21 and auxotrophic E. coli B834 to maximize ncAA content, thereby assessing the effect of ncAAs on bacterial growth and the expression of cytochrome b5 (b5M46), green fluorescence protein (MBP-GFP) and phage shock protein A. In auxotrophic E. coli, ncAA incorporation ranged from 50 to 70% for pMet and reached approximately 50% for Aha, after 26 h expression, with medium and low expression levels of MBP-GFP and b5M46, respectively. In the prototrophic strain, by contrast, the protein expression levels were higher, albeit with a sharp decrease in the ncAA content after the first hours of expression. Similar expression levels and 70-80% incorporation rates were achieved in both bacterial strains with Hpg. Our findings provide guidance for expressing proteins with a high content of ncAAs, highlight pitfalls in determining the levels of methionine replacement by ncAAs by MALDI-TOF mass spectrometry and indicate a possible systematic bias in metabolic labeling techniques using Aha or Hpg.

Bone-Targeting Exosome Mimetics Engineered by Bioorthogonal Surface Functionalization for Bone Tissue Engineering.

Extracellular vesicles have received a great interest as safe biocarriers in biomedical engineering. There is a need to develop more efficient delivery strategies to improve localized therapeutic efficacy and minimize off-target adverse effects. Here, exosome mimetics (EMs) are reported for bone targeting involving the introduction of hydroxyapatite-binding moieties through bioorthogonal functionalization. Bone-binding ability of the engineered EMs is verified with hydroxyapatite-coated scaffolds and an ex vivo bone-binding assay. The EM-bound construct provided a biocompatible substrate for cell adhesion, proliferation, and osteogenic differentiation. Particularly, the incorporation of Smoothened agonist (SAG) into EMs greatly increased the osteogenic capacity through the activation of hedgehog signaling. Furthermore, the scaffold integrated with EM/SAG significantly improved in vivo reossification. Lastly, biodistribution studies confirmed the accumulation of systemically administered EMs in bone tissue. This facile engineering strategy could be a versatile tool to promote bone regeneration, offering a promising nanomedicine approach to the sophisticated treatment of bone diseases.

Genetically Encoded 1,2-Aminothiol for Site-Specific Modification of a Cellular Membrane Protein via TAMM Condensation.

Site-specific modification of proteins has wide applications in probing and perturbing biological systems. A popular means to achieve such a modification on a target protein is through a reaction between bioorthogonal functionalities. Indeed, various bioorthogonal reactions have been developed, including a recently reported reaction between 1,2-aminothiol and ((alkylthio)(aryl)methylene)malononitrile (TAMM). Here, we describe the procedure that combines genetic code expansion and TAMM condensation for site-specific modification of cellular membrane proteins. The 1,2-aminothiol functionality is introduced through a genetically incorporated noncanonical amino acid to a model membrane protein on mammalian cells. Treatment of the cells with a fluorophore-TAMM conjugate leads to fluorescent labeling of the target protein. This method can be applied to modify different membrane proteins on live mammalian cells.

Chemoenzymatic Probes Reveal Peptidoglycan Recognition and Uptake Mechanisms in Candida albicans.

Candida albicans, the major fungal pathogen in humans, is under the strong influence of bacterial peptidoglycan fragments to undergo the yeast-to-hyphae transition, a key virulent step in C. albicans pathogenesis and infections. However, due to the synthetic difficulties of obtaining peptidoglycan fragments for biological studies, mechanistic details of how C. albicans recognizes and uptakes these peptidoglycan fragments have not been well elucidated. Notably, previous works have solely focused on the synthetic peptidoglycan ligand, muramyl dipeptide (MDP), despite its poor hyphal-inducing activity in C. albicans. In this work, we isolated and purified natural peptidoglycan fragments via enzymatic degradation of bacteria cell wall sacculi and chemoenzymatically installed a series of functional d-amino acids into the natural muropeptide, creating peptidoglycan probes that bear photoaffinity, bio-orthogonal, or fluorescent functionality. Using these chemoenzymatic peptidoglycan probes, we established that natural peptidoglycan fragments, which are potent hyphal-inducers, interact with the C. albicans Cyr1 sensor protein in the in-gel fluorescence assay as well as in in vitro pulldown studies. Moreover, we established that bacterial peptidoglycan probes enter C. albicans cells via an energy-dependent endocytic process.

Azide-Modified Nucleosides as Versatile Tools for Bioorthogonal Labeling and Functionalization.

Azide-modified nucleosides are important building blocks for RNA and DNA functionalization by click chemistry based on azide-alkyne cycloaddition. This has put demand on synthetic chemistry to develop approaches for the preparation of azide-modified nucleoside derivatives. We review here the available methods for the synthesis of various nucleosides decorated with azido groups at the sugar residue or nucleobase, their incorporation into oligonucleotides and cellular RNAs, and their application in azide-alkyne cycloadditions for labelling and functionalization.

Engineered biosynthesis of alkyne-tagged polyketides.

Polyketides have demonstrated their significance as therapeutics, industrial products, pesticides, and biological probes following intense study over the past decades. Tagging polyketides with a bioorthogonal functionality enables various applications such as diversification, quantification, visualization and mode-of-action elucidation. The terminal alkyne moiety, as a small, stable and highly selective clickable functionality, is widely adopted in tagging natural products. De novo biosynthesis of alkyne-tagged polyketides offers the unique advantage of reducing the background from feeding the biorthogonal moiety itself, leading to the accomplishment of in situ generation of a clickable functionality for bioorthogonal reactions. Here, we introduce several engineering strategies to apply terminal alkyne biosynthetic machinery, represented by JamABC, which produces a short terminal alkyne-bearing fatty acyl chain on a carrier protein, to functions with different downstream polyketide synthases (PKSs). Successful results in engineering type III and type I PKSs provide engineering guidelines and strategies that are applicable to additional PKSs to produce targeted alkyne-tagged metabolites for chemical and biological applications.

4-Deoxy-4-fluoro-GalNAz (4FGalNAz) Is a Metabolic Chemical Reporter of O-GlcNAc Modifications, Highlighting the Notable Substrate Flexibility of O-GlcNAc Transferase.

Bio-orthogonal chemistries have revolutionized many fields. For example, metabolic chemical reporters (MCRs) of glycosylation are analogues of monosaccharides that contain a bio-orthogonal functionality, such as azides or alkynes. MCRs are metabolically incorporated into glycoproteins by living systems, and bio-orthogonal reactions can be subsequently employed to install visualization and enrichment tags. Unfortunately, most MCRs are not selective for one class of glycosylation (e.g., N-linked vs O-linked), complicating the types of information that can be gleaned. We and others have successfully created MCRs that are selective for intracellular O-GlcNAc modification by altering the structure of the MCR and thus biasing it to certain metabolic pathways and/or O-GlcNAc transferase (OGT). Here, we attempt to do the same for the core GalNAc residue of mucin O-linked glycosylation. The most widely applied MCR for mucin O-linked glycosylation, GalNAz, can be enzymatically epimerized at the 4-hydroxyl to give GlcNAz. This results in a mixture of cell-surface and O-GlcNAc labeling. We reasoned that replacing the 4-hydroxyl of GalNAz with a fluorine would lock the stereochemistry of this position in place, causing the MCR to be more selective. After synthesis, we found that 4FGalNAz labels a variety of proteins in mammalian cells and does not perturb endogenous glycosylation pathways unlike 4FGalNAc. However, through subsequent proteomic and biochemical characterization, we found that 4FGalNAz does not widely label cell-surface glycoproteins but instead is primarily a substrate for OGT. Although these results are somewhat unexpected, they once again highlight the large substrate flexibility of OGT, with interesting and important implications for intracellular protein modification by a potential range of abiotic and native monosaccharides.

Site-selective tyrosine bioconjugation via photoredox catalysis for native-to-bioorthogonal protein transformation.

The growing prevalence of synthetically modified proteins in pharmaceuticals and materials has exposed the need for efficient strategies to enable chemical modifications with high site-selectivity. While genetic engineering can incorporate non-natural amino acids into recombinant proteins, regioselective chemical modification of wild-type proteins remains a challenge. Herein, we use photoredox catalysis to develop a site-selective tyrosine bioconjugation pathway that incorporates bioorthogonal formyl groups, which subsequently allows for the synthesis of structurally defined fluorescent conjugates from native proteins. A water-soluble photocatalyst, lumiflavin, has been shown to induce oxidative coupling between a previously unreported phenoxazine dialdehyde tag and a single tyrosine site, even in the presence of multiple tyrosyl side chains, through the formation of a covalent C-N bond. A variety of native proteins, including those with multiple tyrosines, can successfully undergo both tyrosine-specific and single-site-selective labelling. This technology directly introduces aldehyde moieties onto native proteins, enabling rapid product diversification using an array of well-established bioorthogonal functionalization protocols including the alkyne-azide click reaction.

N3 -Kethoxal-Based Bioorthogonal Intracellular RNA Labeling.

There is growing interest in developing intracellular RNA tools. Herein, we describe a strategy for N3 -kethoxal (N3 K)-based bioorthogonal intracellular RNA functionalization. With N3 K labeling followed by an in vivo click reaction with DBCO derivatives, RNA can be modified with fluorescent or phenol groups. This strategy provides a new way of labeling RNA inside cells.

Bioorthogonal protein labelling enables the study of antigen processing of citrullinated and carbamylated auto-antigens.

Proteolysis is fundamental to many biological processes. In the immune system, it underpins the activation of the adaptive immune response: degradation of antigenic material into short peptides and presentation thereof on major histocompatibility complexes, leads to activation of T-cells. This initiates the adaptive immune response against many pathogens. Studying proteolysis is difficult, as the oft-used polypeptide reporters are susceptible to proteolytic sequestration themselves. Here we present a new approach that allows the imaging of antigen proteolysis throughout the processing pathway in an unbiased manner. By incorporating bioorthogonal functionalities into the protein in place of methionines, antigens can be followed during degradation, whilst leaving reactive sidechains open to templated and non-templated post-translational modifications, such as citrullination and carbamylation. Using this approach, we followed and imaged the post-uptake fate of the commonly used antigen ovalbumin, as well as the post-translationally citrullinated and/or carbamylated auto-antigen vinculin in rheumatoid arthritis, revealing differences in antigen processing and presentation.

O-Acetylated Chemical Reporters of Glycosylation Can Display Metabolism-Dependent Background Labeling of Proteins but Are Generally Reliable Tools for the Identification of Glycoproteins.

Monosaccharide analogs bearing bioorthogonal functionalities, or metabolic chemical reporters (MCRs) of glycosylation, have been used for approximately two decades for the visualization and identification of different glycoproteins. More recently, proteomics analyses have shown that per-O-acetylated MCRs can directly and chemically react with cysteine residues in lysates and potentially cells, drawing into question the physiological relevance of the labeling. Here, we report robust metabolism-dependent labeling by Ac42AzMan but not the structurally similar Ac44AzGal. However, the levels of background chemical-labeling of cell lysates by both reporters are low and identical. We then characterized Ac42AzMan labeling and found that the vast majority of the labeling occurs on intracellular proteins but that this MCR is not converted to previously characterized reporters of intracellular O-GlcNAc modification. Additionally, we used isotope targeted glycoproteomics (IsoTaG) proteomics to show that essentially all of the Ac42AzMan labeling is on cysteine residues. Given the implications this result has for the identification of intracellular O-GlcNAc modifications using MCRs, we then performed a meta-analysis of the potential O-GlcNAcylated proteins identified by different techniques. We found that many of the proteins identified by MCRs have also been found by other methods. Finally, we randomly selected four proteins that had only been identified as O-GlcNAcylated by MCRs and showed that half of them were indeed modified. Together, these data indicate that the selective metabolism of certain MCRs is responsible for S-glycosylation of proteins in the cytosol and nucleus. However, these results also show that MCRs are still good tools for unbiased identification of glycosylated proteins, as long as complementary methods are employed for confirmation.

The Unique Bioorthogonal Chemistry of Isonitriles

The isocyano group is the structurally most compact bioorthogonal group known. It reacts with tetrazines under physiological conditions and has great potential for widespread use in the biosciences. In this account, we highlight the unique properties of the isocyano group as a bioorthogonal functionality. Protecting group chemistry based on the reaction of isonitriles and tetrazines that allows releasing payloads is a particular focus of the article. We further discuss the atypical steric attractions that take place in the transition state of the reaction between isonitriles and tetrazines, which result in an increase in the rate of the reaction with steric bulk of the tetrazine substituents. These findings will open up new possibilities in bioorthogonal chemistry where reactivity and stability are simultaneously desired.1 Introduction2 The Isocyano Group: A Structurally Compact Group for Bioorthogonal Chemistry3 Bioorthogonal Protecting Group Chemistry4 Steric Attractions in the Transition State Accelerate the Cycloaddition of Isonitriles and Tetrazines5 Reactions of Tetrazines and Isonitriles are Compatible with Biomolecules and Living Organisms6 Conclusions

Conformational landscape of substituted prolines.

The cyclic side chain of the amino acid proline confers unique conformational restraints on its backbone and side chain dihedral angles. This affects two equilibria-one at the backbone (cis/trans) and the other at the side chain (endo/exo). Substitutions on the proline ring impose additional steric and stereoelectronic effects that can further modulate both these equilibria, which in turn can also affect the backbone dihedral angle (ϕ, ψ) preferences. In this review, we have explored the conformational landscape of several termini capped mono-(2-, 3-, 4-, and 5-) substituted proline derivatives in the Cambridge Structural Database, correlating observed conformations with the nature of substituents and deciphering the underlying interactions for the observed structural biases. The impact of incorporating these derivatives within model peptides and proteins are also discussed for selected cases. Several of these substituents have been used to introduce bioorthogonal functionality and modulate structure-specific ligand recognition or used as spectroscopic probes. The incorporation of these diversely applicable functional groups, coupled with their ability to define an amino acid conformation via stereoelectronic effects, have a broad appeal among chemical biologists, molecular biophysicists, and medicinal chemists.