Two‐fold Bioorthogonal Derivatization by Different Formylglycine‐Generating Enzymes

Formylglycine‐generating enzymes are of increasing interest in the field of bioconjugation chemistry. They catalyze the site‐specific oxidation of a cysteine residue to the aldehyde‐containing amino acid C^α‐formylglycine (FGly). This non‐canonical residue can be generated within any desired target protein and can subsequently be used for bioorthogonal conjugation reactions. The prototypic formylglycine‐generating enzyme (FGE) and the iron‐sulfur protein AtsB display slight variations in their recognition sequences. We designed specific tags in peptides and proteins that were selectively converted by the different enzymes. Combination of the different tag motifs within a single peptide or recombinant protein enabled the independent and consecutive introduction of two formylglycine residues and the generation of heterobifunctionalized protein conjugates.     

Selective two-step labeling of proteins with an off/on fluorescent probe

We present a novel design strategy for off/on fluorescent probes suitable for selective two-step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1-Ni(2+), which targets a cysteine-modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)-Ni(2+) as the His-tag recognition site and a 2,4-dinitrophenyl ether moiety, which quenches the probe's fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4-dinitrophenyl ether (donor-excited PeT; d-PeT) and also has reactivity with cysteine. His-tag recognition by the NTA-Ni(2+) moiety is followed by removal of the 2,4-dinitrophenyl ether quencher by proximity-enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His-tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20-fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light-excitable fluorescent probe for a His-based peptide tag. Further, we successfully visualized CysHis(6)-peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His(6)Cys-tagged enhanced blue fluorescent protein (EBFP), but not His(6)-tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags.     

Kinetic Assay of the Michael Addition‐Like Thiol–Ene Reaction and Insight into Protein Bioconjugation

The chemical modification of proteins is a valuable technique in understanding the functions, interactions, and dynamics of proteins. Reactivity and selectivity are key issues in current chemical modification of proteins. The Michael addition‐like thiol–ene reaction is a useful tool that can be used to tag proteins with high selectivity for the solvent‐exposed thiol groups of proteins. To obtain insight into the bioconjugation of proteins with this method, a kinetic analysis was performed. New vinyl‐substituted pyridine derivatives were designed and synthesized. The reactivity of these vinyl tags with L ‐cysteine was evaluated by UV absorption and high‐resolution NMR spectroscopy. The results show that protonation of pyridine plays a key role in the overall reaction rates. The kinetic parameters were assessed in protein modification. The different reactivities of these vinyl tags with solvent‐exposed cysteine is valuable information in the selective labeling of proteins with multiple functional groups.     

Manipulating the Click Reactivity of Dibenzoazacyclooctynes: From Azide Click Component to Caged Acylation Reagent by Silver Catalysis

Strain-promoted azide–alkyne cycloaddition using dibenzoazacyclooctyne (DBCO) is widely applied in copper-free bioorthogonal reactions. Reported here is the efficient acid-promoted rearrangement and silver-catalyzed amidation of DBCO, which alters its click reactivity robustly. In the switched click reaction, DBCO, as a caged acylation reagent, enables rapid peptide/protein modification after decaging facilitated by silver catalysts, rendering site-specific conjugation of an IgG antibody by a Fc-targeting peptide.     

Separation efficiency of free‐solution conjugated electrophoresis with drag‐tags incorporating a synthetic amino acid

DNA sequencing or separation by conventional capillary electrophoresis with a polymer matrix has some inherent drawbacks, such as the expense of polymer matrix and limitations in sequencing read length. As DNA fragments have a linear charge‐to‐friction ratio in free solution, DNA fragments cannot be separated by size. However, size‐based separation of DNA is possible in free‐solution conjugate electrophoresis (FSCE) if a “drag‐tag” is attached to DNA fragments because the tag breaks the linear charge‐to‐friction scaling. Although several previous studies have demonstrated the feasibility of DNA separation by free‐solution conjugated electrophoresis, generation of a monodisperse drag‐tag and identification of a strong, site‐specific conjugation method between a DNA fragment and a drag‐tag are challenges that still remain. In this study, we demonstrate an efficient FSCE method by conjugating a biologically synthesized elastin‐like polypeptide (ELP) and green fluorescent protein (GFP) to DNA fragments. In addition, to produce strong and site‐specific conjugation, a methionine residue in drag‐tags is replaced with homopropargylglycine (Hpg), which can be conjugated specifically to a DNA fragment with an azide site.     

A Native‐Chemical‐Ligation‐Mechanism‐Based Ratiometric Fluorescent Probe for Aminothiols

Thiol‐containing amino acids (aminothiols) such as cysteine (Cys) and homocysteine (Hcy) play a key role in various biological processes including maintaining the homeostasis of biological thiols. However, abnormal levels of aminothiols are associated with a variety of diseases. The native chemical ligation (NCL) reaction has attracted great attention in the fields of chemistry and biology. NCL of peptide segments involves cascade reactions between a peptide‐α‐thioester and an N‐terminal cysteine peptide. In this work, we employed the NCL reaction mechanism to formulate a Förster resonance energy transfer (FRET) strategy for the design of ratiometric fluorescent probes that were selective toward aminothiols. On the basis of this new strategy, the ratiometric fluorescent probe 1 for aminothiols was judiciously designed. The new probe is highly selective toward aminothiols over other thiols and exhibits a very large variation (up to 160‐fold) in its fluorescence ratio (I₄₅₈/I₆₀₃). The new fluorescent probe is capable of ratiometric detection of aminothiols in newborn calf and human serum samples and is also suitable for ratiometric fluorescent imaging of aminothiols in living cells.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

Bioinspired Nitroalkylation for Selective Protein Modification and Peptide Stapling

Nitroalkanes react specifically with aldehydes, providing rapid, stable, and chemoselective protein bioconjugation. These nitroalkylated proteins mimic key post‐translational modifications (PTMs) of proteins and can be used to understand the role of these PTMs in cellular processes. Demonstrated here is the substrate scope of this bioconjugation by attaching a variety of tags, such as NMR tags, fluorescent tags, affinity tags, and alkyne tags, to proteins. The structure and enzymatic activity of modified proteins remain conserved after labeling. Notably, the nitroalkane group leads to easy characterization of proteins by mass spectrometry because of its distinct fingerprint pattern. Importantly, the nitro‐alkylated peptides provide a new handle for site‐selective fluorination of peptides, thus installing a specific probe to study peptide–protein interactions by ¹⁹F NMR spectroscopy. Furthermore, nitroalkane reagents can be used for the late‐stage diversification of peptides and for the synthesis of peptide staples.     

A Genetically Encoded,Phage‐Displayed Cyclic‐Peptide Library

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic‐peptide ligands for therapeutic targets, phage‐displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage‐display technique in which its displayed peptides are cyclized through a proximity‐driven Michael addition reaction between a cysteine and an amber‐codon‐encoded N^?‐acryloyl‐lysine (AcrK). Using a randomized 6‐mer library in which peptides were cyclized at two ends through a cysteine–AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4‐ to 6‐fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.     

Three‐Dimensional Arrays Using GlycoPEG Tags: Glycan Synthesis,Purification and Immobilisation

Glycan arrays have become the premier tool for rapidly establishing the binding or substrate specificities of lectins and carbohydrate‐processing enzymes. New approaches for accelerating carbohydrate synthesis to address the enormous complexity of natural glycan structures are necessary. Moreover, optimising glycan immobilisation is key for the development of selective, sensitive and reproducible array‐based assays. We present a tag‐based approach that accelerates the preparation of glycan arrays on all levels by improving the synthesis, the purification and immobilisation of oligosaccharides. Glycan primers were chemically attached to bifunctional polyethyleneglycol (PEG) tags, extended enzymatically with the help of recombinant glycosyltransferases and finally purified by ultrafiltration. When printed directly onto activated glass slides, these glycoPEG tags afforded arrays with exceptionally high sensitivity, low background and excellent spot morphology. Likewise, the conjugation of glycoPEG tags to latex nanoparticles yielded multivalent scaffolds for carbohydrate‐binding assays with very low non‐specific binding.     

Detection of Protein S‐Sulfhydration by a Tag‐Switch Technique

Protein S‐sulfhydration (forming ‐S‐SH adducts from cysteine residues) is a newly defined oxidative posttranslational modification and plays an important role in H₂S‐mediated signaling pathways. In this study we report the first selective, “tag‐switch” method which can directly label protein S‐sulfhydrated residues by forming stable thioether conjugates. Furthermore we demonstrate that H₂S alone cannot lead to S‐sulfhydration and that the two possible physiological mechanisms include reaction with protein sulfenic acids (P‐SOH) or the involvement of metal centers which would facilitate the oxidation of H₂S to HS^..     

Recognition‐then‐Reaction Enables Site‐Selective Bioconjugation to Proteins on Live‐Cell Surfaces

Site‐selective protein modification is a key step in facilitating protein functionalization and manipulation. To accomplish this, genetically engineered proteins were previously required, but the procedure was laborious, complex, and technically challenging. Herein we report the development of aptamer‐based recognition‐then‐reaction to guide site‐selective protein/DNA conjugation in a single step with outstanding selectivity and efficiency. As models, several proteins, including human thrombin, PDGF‐BB, Avidin, and His‐tagged recombinant protein, were studied, and the results showed excellent selectivity under mild reaction conditions. Taking advantage of aptamers as recognition elements with extraordinary selectivity and affinity, this simple preparation method can tag a protein in a complex milieu. Thus, with the aptamer obtained from cell‐SELEX, real‐time modification of live‐cell membrane proteins can be achieved in one step without any pre‐treatment.     

Design and synthesis of a solid-phase fluorescent mass tag

In this study, we demonstrate the design of a new solid-phase fluorescent mass tag (FMT) that contains the following features: (1) the FMT is synthesized using Fmoc chemistry which is simple, rapid, and cost-effective; (2) lysine is used as a uniformly labeled amino acid (using stable isotopes) to allow 8 Da difference between "heavy" and "light" tags; (3) a fluorescent molecule is coupled to the isotope tag that allows a tagged peptide to be detected by online fluorescence; and (4) an iodoacetyl reactive group provides cysteine reactivity. Using MALDI-TOF MS and HPLC, we show that the FMT reagent can be used to label standard cysteine-containing peptides as well as cysteine-containing peptides from a BSA tryptic digest.     

Second-Generation Covalent TMP-Tag for Live Cell Imaging

Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology.     

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR,EPR and Time-Resolved Luminescence Studies

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral Ln^III tag, referred to as C12 , is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with Tb^III or Tm^III ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron–electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the Tb^III complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.     

Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se–S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se–S cleavage, analogous to the S–S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se–S of the tag to the S–S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se–S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.     

A Double‐Clicking Bis‐Azide Fluorogenic Dye for Bioorthogonal Self‐Labeling Peptide Tags

Herein, we give the very first example for the development of a fluorogenic molecular probe that combines the two‐point binding specificity of biarsenical‐based dyes with the robustness of bioorthogonal click‐chemistry. This proof‐of‐principle study reports on the synthesis and fluorogenic characterization of a new, double‐quenched, bis‐azide fluorogenic probe suitable for bioorthogonal two‐point tagging of small peptide tags by double strain‐promoted azide–alkyne cycloaddition. The presented probe exhibits remarkable increase in fluorescence intensity when reacted with bis‐cyclooctynylated peptide sequences, which could also serve as possible self‐labeling small peptide tag motifs.     

Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells.     

Cysteine‐Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations

We describe a new technique in protein synthesis that extends the existing repertoire of methods for protein modification: A chemoselective reaction that induces reactivity for a subsequent bioconjugation. An azide‐modified building block reacts first with an ethynylphosphonite through a Staudinger‐phosphonite reaction (SPhR) to give an ethynylphosphonamidate. The resulting electron‐deficient triple bond subsequently undergoes a cysteine‐selective reaction with proteins or antibodies. We demonstrate that ethynylphosphonamidates display excellent cysteine‐selective reactivity combined with superior stability of the thiol adducts, when compared to classical maleimide linkages. This turns our technique into a versatile and powerful tool for the facile construction of stable functional protein conjugates.     

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.