Boronic Acids as Bioorthogonal Probes for Site‐Selective Labeling of Proteins

Over the past two decades, bioorthogonal chemistry has become a preferred tool to achieve site‐selective modifications of proteins. However, there are only a handful of commonly applied bioorthogonal reactions and they display some limitations, such as slow rates, use of unstable or cytotoxic reagents, and side reactions. Hence, there is significant interest in expanding the bioorthogonal chemistry toolbox. In this regard, boronic acids have recently been introduced in bioorthogonal chemistry and are exploited in three different strategies: 1) boronic ester formation between a boronic acid and a 1,2‐cis diol; 2) iminoboronate formation between 2‐acetyl/formyl‐arylboronic acids and hydrazine/hydroxylamine/semicarbazide derivatives; 3) use of boronic acids as transient groups in a Suzuki–Miyaura cross‐coupling or other reactions that leave the boronyl group off the conjugation product. In this Review, we summarize progress made in the use of boronic acids in bioorthogonal chemistry to enable site‐selective labeling of proteins and compare these methods with the most commonly utilized bioorthogonal reactions.     

Site‐Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines

Dual‐labeled biomolecules constitute a new generation of bioconjugates with promising applications in therapy and diagnosis. Unfortunately, the development of these new families of biologics is hampered by the technical difficulties associated with their construction. In particular, the site specificity of the conjugation is critical as the number and position of payloads can have a dramatic impact on the pharmacokinetics of the bioconjugate. Herein, we introduce dichlorotetrazine as a trivalent platform for the selective double modification of proteins on cysteine residues. This strategy is applied to the dual labeling of albumin with a macrocyclic chelator for nuclear imaging and a fluorescent probe for fluorescence imaging.     

Coumarin‐Based Fluorogenic Probes for No‐Wash Protein Labeling

A fluorescent protein‐labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.     

Modifying the 5′‐Cap for Click Reactions of Eukaryotic mRNA and To Tune Translation Efficiency in Living Cells

The 5′‐cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5′‐cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7‐modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N²‐modified 5′‐caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live‐cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox.     

Protein Organic Chemistry and Applications for Labeling and Engineering in Live‐Cell Systems

The modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins both in vitro and in live cells or in vivo. Herein we review recent progress in chemistry‐based protein modification methods and their application in protein engineering, with particular emphasis on the following four strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test‐tube settings; 2) the bioorthogonal reactions of proteins with non‐natural functional groups; 3) the coupling of recognition and reactive sites using an enzyme or short peptide tag–probe pair for labeling natural amino acids; and 4) ligand‐directed labeling chemistries for the selective labeling of endogenous proteins in living systems. Overall, these techniques represent a useful set of tools for application in chemical biology, with the methods 2–4 in particular being applicable to crude (living) habitats. Although still in its infancy, the use of organic chemistry for the manipulation of endogenous proteins, with subsequent applications in living systems, represents a worthy challenge for many chemists.     

Chromophore-Assisted Proximity Labeling of DNA Reveals Chromosomal Organization in Living Cells

The spatial arrangement of chromosome within the nucleus is linked to genome function and gene expression regulation. Existing genome-wide mapping methods often rely on chemically crosslinking DNA with protein baits, which raises concerns of artifacts being introduced during cell fixation. By genetically targeting a photosensitizer protein to specific subnuclear locations, we achieved blue-light-activated labeling of local DNA with a bioorthogonal functional handle for affinity purification and sequence identification through next-generation sequencing. When applied to the nuclear lamina in human embryonic kidney 293T cells, it revealed lamina-associated domains (LADs) that cover 37.6 % of the genome. These LADs overlap with heterochromatin hallmarks and are depleted with CpG islands. This simple labeling method avoids the harsh treatment of chemical crosslinking and is generally applicable to the genome-wide high-resolution mapping of the spatial chromosome organization in living cells.     

Site‐Selective Cysteine–Cyclooctyne Conjugation

We report a site‐selective cysteine–cyclooctyne conjugation reaction between a seven‐residue peptide tag (DBCO‐tag, Leu‐Cys‐Tyr‐Pro‐Trp‐Val‐Tyr) at the N or C terminus of a peptide or protein and various aza‐dibenzocyclooctyne (DBCO) reagents. Compared to a cysteine peptide control, the DBCO‐tag increases the rate of the thiol–yne reaction 220‐fold, thereby enabling selective conjugation of DBCO‐tag to DBCO‐linked fluorescent probes, affinity tags, and cytotoxic drug molecules. Fusion of DBCO‐tag with the protein of interest enables regioselective cysteine modification on proteins that contain multiple endogenous cysteines; these examples include green fluorescent protein and the antibody trastuzumab. This study demonstrates that short peptide tags can aid in accelerating bond‐forming reactions that are often slow to non‐existent in water.     

A Noncanonical Function of Sortase Enables Site‐Specific Conjugation of Small Molecules to Lysine Residues in Proteins

We provide the first demonstration that isopeptide ligation, a noncanonical activity of the enzyme sortase A, can be used to modify recombinant proteins. This reaction was used in vitro to conjugate small molecules to a peptide, an engineered targeting protein, and a full‐length monoclonal antibody with an exquisite level of control over the site of conjugation. Attachment to the protein substrate occurred exclusively through isopeptide bonds at a lysine ε‐amino group within a specific amino acid sequence. This reaction allows more than one molecule to be site‐specifically conjugated to a protein at internal sites, thereby overcoming significant limitations of the canonical native peptide ligation reaction catalyzed by sortase A. Our method provides a unique chemical ligation procedure that is orthogonal to existing methods, supplying a new method to site‐specifically modify lysine residues that will be a valuable addition to the protein conjugation toolbox.     

Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single‐site labeling of a high‐frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent‐accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys‐selective electrophile connected by a spacer. Consequently, it enables the irreversible single‐site labeling of a Lys residue independent of its place in the reactivity order. The user‐friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site‐selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe‐tagged proteins. Besides, the methodology provides access to antibody‐drug conjugate (ADC), which exhibits highly selective anti‐proliferative activity towards HER‐2 expressing SKBR‐3 breast cancer cells.     

A Minimal,Unstrained S‐Allyl Handle for Pre‐Targeting Diels–Alder Bioorthogonal Labeling in Live Cells

The unstrained S‐allyl cysteine amino acid was site‐specifically installed on apoptosis protein biomarkers and was further used as a chemical handle and ligation partner for 1,2,4,5‐tetrazines by means of an inverse‐electron‐demand Diels–Alder reaction. We demonstrate the utility of this minimal handle for the efficient labeling of apoptotic cells using a fluorogenic tetrazine dye in a pre‐targeting approach. The small size, easy chemical installation, and selective reactivity of the S‐allyl handle towards tetrazines should be readily extendable to other proteins and biomolecules, which could facilitate their labeling within live cells.     

A Modular Method for the High‐Yield Synthesis of Site‐Specific Protein–Polymer Therapeutics

A versatile method is described to engineer precisely defined protein/peptide–polymer therapeutics by a modular approach that consists of three steps: 1) fusion of a protein/peptide of interest with an elastin‐like polypeptide that enables facile purification and high yields; 2) installation of a clickable group at the C terminus of the recombinant protein/peptide with almost complete conversion by enzyme‐mediated ligation; and 3) attachment of a polymer by a click reaction with near‐quantitative conversion. We demonstrate that this modular approach is applicable to various protein/peptide drugs and used it to conjugate them to structurally diverse water‐soluble polymers that prolong the plasma circulation duration of these proteins. The protein/peptide–polymer conjugates exhibited significantly improved pharmacokinetics and therapeutic effects over the native protein/peptide upon administration to mice. The studies reported here provide a facile method for the synthesis of protein/peptide–polymer conjugates for therapeutic use and other applications.     

Debugging Eukaryotic Genetic Code Expansion for Site‐Specific Click‐PAINT Super‐Resolution Microscopy

Super‐resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine‐based GCE system for click chemistry, combined with DNA‐PAINT microscopy, enables the visualization of even low‐abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue‐specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.     

Capture and Recycling of Sortase A through Site‐Specific Labeling with Lithocholic Acid

Enzyme‐mediated protein modification often requires large amounts of biocatalyst, adding significant costs to the process and limiting industrial applications. Herein, we demonstrate a scalable and straightforward strategy for the efficient capture and recycling of enzymes using a small‐molecule affinity tag. A proline variant of an evolved sortase A (SrtA 7M) was N‐terminally labeled with lithocholic acid (LA)—an inexpensive bile acid that exhibits strong binding to β‐cyclodextrin (βCD). Capture and recycling of the LA‐Pro‐SrtA 7M conjugate was achieved using βCD‐modified sepharose resin. The LA‐Pro‐SrtA 7M conjugate retained full enzymatic activity, even after multiple rounds of recycling.     

In‐Membrane Chemical Modification (IMCM) for Site‐Specific Chromophore Labeling of GPCRs

We present in‐membrane chemical modification (IMCM) for obtaining selective chromophore labeling of intracellular surface cysteines in G‐protein‐coupled receptors (GPCRs) with minimal mutagenesis. This method takes advantage of the natural protection of most cysteines by the membrane environment. Practical use of IMCM is illustrated with the site‐specific introduction of chromophores for NMR and fluorescence spectroscopy in the human κ‐opioid receptor (KOR) and the human A_2A adenosine receptor (A_2AAR). IMCM is applicable to a wide range of in vitro studies of GPCRs, including single‐molecule spectroscopy, and is a promising platform for in‐cell spectroscopy experiments.     

Visualization of Protein‐Specific Glycosylation inside Living Cells

Protein glycosylation is a ubiquitous post‐translational modification that is involved in the regulation of many aspects of protein function. In order to uncover the biological roles of this modification, imaging the glycosylation state of specific proteins within living cells would be of fundamental importance. To date, however, this has not been achieved. Herein, we demonstrate protein‐specific detection of the glycosylation of the intracellular proteins OGT, Foxo1, p53, and Akt1 in living cells. Our generally applicable approach relies on Diels–Alder chemistry to fluorescently label intracellular carbohydrates through metabolic engineering. The target proteins are tagged with enhanced green fluorescent protein (EGFP). Förster resonance energy transfer (FRET) between the EGFP and the glycan‐anchored fluorophore is detected with high contrast even in presence of a large excess of acceptor fluorophores by fluorescence lifetime imaging microscopy (FLIM).