Affinity Bioorthogonal Chemistry (ABC) Tags for Site-Selective Conjugation, On-Resin Protein-Protein Coupling, and Purification of Protein Conjugates.

The site-selective functionalization of proteins has broad application in chemical biology, but can be limited when mixtures result from incomplete conversion or the formation of protein containing side products. It is shown here that when proteins are covalently tagged with pyridyl-tetrazines, the nickel-iminodiacetate (Ni-IDA) resins commonly used for His-tags can be directly used for protein affinity purification. These Affinity Bioorthogonal Chemistry (ABC) tags serve a dual role by enabling affinity-based protein purification while maintaining rapid kinetics in bioorthogonal reactions. ABC-tagging works with a range of site-selective bioconjugation methods with proteins tagged at the C-terminus, N-terminus or at internal positions. ABC-tagged proteins can also be purified from complex mixtures including cell lysate. The combination of site-selective conjugation and clean-up with ABC-tagged proteins also allows for facile on-resin reactions to provide protein-protein conjugates.

Bioorthogonal chemistry.

Bioorthogonal chemistry represents a class of high-yielding chemical reactions that proceed rapidly and selectively in biological environments without side reactions towards endogenous functional groups. Rooted in the principles of physical organic chemistry, bioorthogonal reactions are intrinsically selective transformations not commonly found in biology. Key reactions include native chemical ligation and the Staudinger ligation, copper-catalysed azide-alkyne cycloaddition, strain-promoted [3 + 2] reactions, tetrazine ligation, metal-catalysed coupling reactions, oxime and hydrazone ligations as well as photoinducible bioorthogonal reactions. Bioorthogonal chemistry has significant overlap with the broader field of 'click chemistry' - high-yielding reactions that are wide in scope and simple to perform, as recently exemplified by sulfuryl fluoride exchange chemistry. The underlying mechanisms of these transformations and their optimal conditions are described in this Primer, followed by discussion of how bioorthogonal chemistry has become essential to the fields of biomedical imaging, medicinal chemistry, protein synthesis, polymer science, materials science and surface science. The applications of bioorthogonal chemistry are diverse and include genetic code expansion and metabolic engineering, drug target identification, antibody-drug conjugation and drug delivery. This Primer describes standards for reproducibility and data deposition, outlines how current limitations are driving new research directions and discusses new opportunities for applying bioorthogonal chemistry to emerging problems in biology and biomedicine.

Dual-Reactivity trans-Cyclooctenol Probes for Sulfenylation in Live Cells Enable Temporal Control via Bioorthogonal Quenching.

Sulfenylation (RSH → RSOH) is a post-translational protein modification associated with cellular mechanisms for signal transduction and the regulation of reactive oxygen species. Protein sulfenic acids are challenging to identify and study due to their electrophilic and transient nature. Described here are sulfenic acid modifying trans-cycloocten-5-ol (SAM-TCO) probes for labeling sulfenic acid functionality in live cells. These probes enable a new mode of capturing sulfenic acids via transannular thioetherification, whereas "ordinary" trans-cyclooctenes react only slowly with sulfenic acids. SAM-TCOs combine with sulfenic acid forms of a model peptide and proteins to form stable adducts. Analogously, SAM-TCO with the selenenic acid form of a model protein leads to a selenoetherification product. Control experiments illustrate the need for the transannulation process coupled with the activated trans-cycloalkene functionality. Bioorthogonal quenching of excess unreacted SAM-TCOs with tetrazines in live cells provides both temporal control and a means of preventing artifacts caused by cellular-lysis. A SAM-TCO biotin conjugate was used to label protein sulfenic acids in live cells, and subsequent quenching by tetrazine prevented further labeling even under harshly oxidizing conditions. A cell-based proteomic study validates the ability of SAM-TCO probes to identify and quantify known sulfenic acid redox proteins as well as targets not captured by dimedone-based probes.

Spatial Patterning of Molecular Cues and Vascular Cells in Fully Integrated Hydrogel Channels via Interfacial Bioorthogonal Cross-Linking.

Fully integrated hydrogel channels were fabricated via interfacial bioorthogonal cross-linking, a diffusion-controlled method for the creation and patterning of synthetic matrices based on the rapid bioorthogonal reaction between s-tetrazines (Tz) and trans-cyclooctene (TCO) dienophiles. Injecting an aqueous solution of a bisTCO cross-linker into a reservoir of tetrazine-modified hyaluronic acid (HA-Tz), while simultaneously drawing the syringe needle through the reservoir, yielded a cross-linked hydrogel channel that was mechanically robust. Fluorescent tags and biochemical signals were spatially patterned into the channel wall through time-dependent perfusion of TCO-conjugated molecules into the lumen of the channel. Different cell populations were spatially encapsulated in the channel wall via temporal alteration of cells in the HA-Tz reservoir. The interfacial approach enabled the spatial patterning of vascular cells, including human abdominal aorta endothelial cells, aortic vascular smooth muscle cells, and aortic adventitial fibroblasts, into the hydrogel channels with high viability and proper morphology in the anatomical order found in human arteries. The bioorthogonal platform does not rely on external triggers and represents the first step toward the engineering of functional and implantable arteries.

Cellular interactions with hydrogel microfibers synthesized via interfacial tetrazine ligation.

Fibrous proteins found in the natural extracellular matrix (ECM) function as host substrates for migration and growth of endogenous cells during wound healing and tissue repair processes. Although various fibrous scaffolds have been developed to recapitulate the microstructures of the native ECM, facile synthesis of hydrogel microfibers that are mechanically robust and biologically active have been elusive. Described herein is the use of interfacial bioorthogonal polymerization to create hydrogel-based microfibrous scaffolds via tetrazine ligation. Combination of a trifunctional strained trans-cyclooctene monomer and a difunctional s-tetrazine monomer at the oil-water interface led to the formation of microfibers that were stable under cell culture conditions. The bioorthogonal nature of the synthesis allows for direct incorporation of tetrazine-conjugated peptides or proteins with site-selectively, genetically encoded tetrazines. The microfibers provide physical guidance and biochemical signals to promote the attachment, division and migration of fibroblasts. Mechanistic investigations revealed that fiber-guided cell migration was both F-actin and microtubule-dependent, confirming contact guidance by the microfibers. Prolonged culture of fibroblasts in the presence of an isolated microfiber resulted in the formation of a multilayered cell sheet wrapping around the fiber core. A fibrous mesh provided a 3D template to promote cell infiltration and tissue-like growth. Overall, the bioorthogonal approach led to the straightforward synthesis of crosslinked hydrogel microfibers that can potentially be used as instructive materials for tissue repair and regeneration.

Synthesis of Functionalized N-Acetyl Muramic Acids To Probe Bacterial Cell Wall Recycling and Biosynthesis.

Uridine diphosphate N-acetyl muramic acid (UDP NAM) is a critical intermediate in bacterial peptidoglycan (PG) biosynthesis. As the primary source of muramic acid that shapes the PG backbone, modifications installed at the UDP NAM intermediate can be used to selectively tag and manipulate this polymer via metabolic incorporation. However, synthetic and purification strategies to access large quantities of these PG building blocks, as well as their derivatives, are challenging. A robust chemoenzymatic synthesis was developed using an expanded NAM library to produce a variety of 2 -N-functionalized UDP NAMs. In addition, a synthetic strategy to access bio-orthogonal 3-lactic acid NAM derivatives was developed. The chemoenzymatic UDP synthesis revealed that the bacterial cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1 phosphate uridylyl transferase (MurU) were permissive to permutations at the two and three positions of the sugar donor. We further explored the utility of these derivatives in the fluorescent labeling of both Gram (-) and Gram (+) PG in whole cells using a variety of bio-orthogonal chemistries including the tetrazine ligation. This report allows for rapid and scalable access to a variety of functionalized NAMs and UDP NAMs, which now can be used in tandem with other complementary bio-orthogonal labeling strategies to address fundamental questions surrounding PG's role in immunology and microbiology.

Photochemical syntheses, transformations, and bioorthogonal chemistry of trans-cycloheptene and sila trans-cycloheptene Ag(i) complexes.

A photochemical synthesis of AgNO3 complexes of trans-cycloheptene (TCH) and trans-1-sila-4-cycloheptene (Si-TCH) derivatives is described. A low temperature flow photoreactor was designed to enable the synthesis of carbocyclic TCH derivatives due to their thermal sensitivity in the absence of metal coordination. Unlike the free carbocycles, TCH·AgNO3 complexes can be handled at rt and stored for weeks in the freezer (-18 °C). Si-TCH·AgNO3 complexes are especially robust, and are bench stable for days at rt, and for months in the freezer. X-ray crystallography was used to characterize a Si-TCH·AgNO3 complex for the first time. With decomplexation of AgNO3in situ, metal-free TCO and Si-TCH derivatives can engage in a range of cycloaddition reactions as well as dihydroxylation reactions. Computation was used to predict that Si-TCH would engage in bioorthogonal reactions that are more rapid than the most reactive trans-cyclooctenes. Metal-free Si-TCH derivatives were shown to display good stability in solution, and to engage in the fastest bioorthogonal reaction reported to date (k2 1.14 × 107 M-1 s-1 in 9 : 1 H2O : MeOH). Utility in bioorthogonal protein labeling in live cells is described, including labeling of GFP with an unnatural tetrazine-containing amino acid. The reactivity and specificity of the Si-TCH reagents with tetrazines in live mammalian cells was also evaluated using the HaloTag platform. The cell labeling experiments show that Si-TCH derivatives are best suited as probe molecules in the cellular environment.

Correction: Computationally guided discovery of a reactive, hydrophilic trans-5-oxocene dienophile for bioorthogonal labeling.

Correction for 'Computationally guided discovery of a reactive, hydrophilic trans-5-oxocene dienophile for bioorthogonal labeling' by William D. Lambert et al., Org. Biomol. Chem., 2017, 15, 6640-6644.

Computationally guided discovery of a reactive, hydrophilic trans-5-oxocene dienophile for bioorthogonal labeling.

The use of organic chemistry principles and prediction techniques has enabled the development of new bioorthogonal reactions. As this "toolbox" expands to include new reaction manifolds and orthogonal reaction pairings, the continued development of existing reactions remains an important objective. This is particularly important in cellular imaging, where non-specific background fluorescence has been linked to the hydrophobicity of the bioorthogonal moiety. Here we report that trans-5-oxocene (oxoTCO) displays enhanced reactivity and hydrophilicity compared to trans-cyclooctene (TCO) in the tetrazine ligation reaction. Aided by ab initio calculations we show that the insertion of a single oxygen atom into the trans-cyclooctene (TCO) ring system is sufficient to impart aqueous solubility and also results in significant rate acceleration by increasing angle strain. We demonstrate the rapid and quantitative cycloaddition of oxoTCO using a water-soluble tetrazine derivative and a protein substrate containing a site-specific genetically encoded tetrazine moiety both in vitro and in vivo. We anticipate that oxoTCO will find use in studies where hydrophilicity and fast bioconjugation kinetics are paramount.

Rapid Bioorthogonal Chemistry Turn-on through Enzymatic or Long Wavelength Photocatalytic Activation of Tetrazine Ligation.

Rapid bioorthogonal reactivity can be induced by controllable, catalytic stimuli using air as the oxidant. Methylene blue (4 µM) irradiated with red light (660 nm) catalyzes the rapid oxidation of a dihydrotetrazine to a tetrazine thereby turning on reactivity toward trans-cyclooctene dienophiles. Alternately, the aerial oxidation of dihydrotetrazines can be efficiently catalyzed by nanomolar levels of horseradish peroxidase under peroxide-free conditions. Selection of dihydrotetrazine/tetrazine pairs of sufficient kinetic stability in aerobic aqueous solutions is key to the success of these approaches. In this work, polymer fibers carrying latent dihydrotetrazines were catalytically activated and covalently modified by trans-cyclooctene conjugates of small molecules, peptides, and proteins. In addition to visualization with fluorophores, fibers conjugated to a cell adhesive peptide exhibited a dramatically increased ability to mediate contact guidance of cells.