Advances in the Synthesis of Bioorthogonal Reagents: s-Tetrazines, 1,2,4-Triazines, Cyclooctynes, Heterocycloheptynes, and trans-Cyclooctenes.

Aligned with the increasing importance of bioorthogonal chemistry has been an increasing demand for more potent, affordable, multifunctional, and programmable bioorthogonal reagents. More advanced synthetic chemistry techniques, including transition-metal-catalyzed cross-coupling reactions, C-H activation, photoinduced chemistry, and continuous flow chemistry, have been employed in synthesizing novel bioorthogonal reagents for universal purposes. We discuss herein recent developments regarding the synthesis of popular bioorthogonal reagents, with a focus on s-tetrazines, 1,2,4-triazines, trans-cyclooctenes, cyclooctynes, hetero-cycloheptynes, and -trans-cycloheptenes. This review aims to summarize and discuss the most representative synthetic approaches of these reagents and their derivatives that are useful in bioorthogonal chemistry. The preparation of these molecules and their derivatives utilizes both classical approaches as well as the latest organic chemistry methodologies.

Minimalist Tetrazine N-Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion.

N-Acetyl muramic acid (NAM) probes containing alkyne or azide groups are commonly used to investigate aspects of cell wall synthesis because of their small size and ability to incorporate into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions are not compatible with live cells, and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction rates are modest and, therefore, not as desirable for tracking the temporal alterations of bacterial cell growth, remodeling, and division. Alternatively, the tetrazine-trans-cyclooctene ligation (Tz-TCO), which is the fastest known bioorthogonal reaction and not cytotoxic, allows for rapid live-cell labeling of PG at biologically relevant time scales and concentrations. Previous work to increase reaction kinetics on the PG surface by using tetrazine probes was limited because of low incorporation of the probe. Described here are new approaches to construct a minimalist tetrazine (Tz)-NAM probe utilizing recent advancements in asymmetric tetrazine synthesis. This minimalist Tz-NAM probe was successfully incorporated into pathogenic and commensal bacterial PG where fixed and rapid live-cell, no-wash labeling was successful in both free bacterial cultures and in coculture with human macrophages. Overall, this probe allows for expeditious labeling of bacterial PG, thereby making it an exceptional tool for monitoring PG biosynthesis for the development of new antibiotic screens. The versatility and selectivity of this probe will allow for real-time interrogation of the interactions of bacterial pathogens in a human host and will serve a broader utility for studying glycans in multiple complex biological systems.

Acute Beetroot Juice Supplementation Has No Effect on Upper- and Lower-Body Maximal Isokinetic Strength and Muscular Endurance in International-Level Male Gymnasts.

Nitrate (NO3-) has properties that can improve muscle function, leading to improvements in metabolic cost of exercise as well as enhance force production. Gymnastics is a whole-body sport, involving events that demand a high level of strength and fatigue resistance. However, the effect of NO3- supplementation on both upper- and lower-body function in gymnasts is unknown. This study examined the effect of acute beetroot juice (BRJ) supplementation on isokinetic strength and endurance of the upper- and lower-body in highly trained international-level male gymnasts. In a double-blind, randomized crossover design, 10 international-level male gymnasts completed two acute supplementation periods, consuming either 2 × 70 ml NO3--rich (∼12.8 mmol/L of NO3-) or NO3--depleted (PLA) BRJ. Maximal strength of the upper-leg and upper-arm at 60°/s, 120°/s, 180°/s, and 300°/s, and muscular endurance (50 repeated isokinetic contractions at 180°/s) were assessed. Plasma NO3- (BRJ: 663 ± 164 µM, PLA: 89 ± 48 µM) and nitrite (NO2-) concentrations (BRJ: 410 ± 137 nmol/L, PLA: 125 ± 36 nmol/L) were elevated following BRJ compared to PLA (both p < .001). Maximal strength of knee and elbow extensors and flexors did not differ between supplements (p > .05 for all velocities). Similarly, fatigue index of knee and elbow extension and flexion was not different between supplements (all p > .05). Acute BRJ supplementation, containing ∼12.8 mmol/L of NO3-, increased plasma NO3- and NO2- concentrations, but did not enhance isokinetic strength or fatigue resistance of either upper or lower extremities in international-level male gymnasts.

Development of 18F-Labeled hydrophilic trans-cyclooctene as a bioorthogonal tool for PET probe construction.

We report the development of a hydrophilic 18F-labeled a-TCO derivative [18F]3 (log P = 0.28) through a readily available precursor and a single-step radiofluorination reaction (RCY up to 52%). We demonstrated that [18F]3 can be used to construct not only multiple small molecule/peptide-based PET agents, but protein/diabody-based imaging probes in parallel.

Gold Nanotapes and Nanopinecones in a Quantitative Lateral Flow Assay for the Cancer Biomarker Carcinoembryonic Antigen.

Colorectal cancer is the third most common malignancy and the second leading cause of cancer death globally. Multiple studies have linked levels of carcinoembryonic antigen in patient serum to poor disease prognosis. Hence, the ability to detect low levels of carcinoembryonic antigen has applications in earlier disease diagnosis, assessment, and recurrence monitoring. Existing carcinoembryonic antigen detection methods often require multiple reagents, trained operators, or complex procedures. A method alleviating these issues is the lateral flow assay, a paper-based platform that allows the detection and quantification of target analytes in complex mixtures. The tests are rapid, are point-of-care, possess a long shelf life, and can be stored at ambient conditions, making them ideal for use in a range of settings. Although lateral flow assays typically use spherical gold nanoparticles to generate the classic red signal, recent literature has shown that alternate morphologies to spheres can improve the limit of detection. In this work, we report the application of alternative gold nanoparticle morphologies, gold nanotapes (∼35 nm in length) and gold nanopinecones (∼90 nm in diameter), in a lateral flow assay for carcinoembryonic antigen. In a comparative assay, gold nanopinecones exhibited a ∼2× improvement in the limit of detection compared to commercially available spherical gold nanoparticles for the same antibody loading and total gold content, whereas the number of gold nanopinecones in each test was ∼3.2× less. In the fully optimized test, a limit of detection of 14.4 pg/mL was obtained using the gold nanopinecones, representing a 24-fold improvement over the previously reported gold-nanoparticle-based carcinoembryonic antigen lateral flow assay.

Modeling the Maturation of the Vocal Fold Lamina Propria Using a Bioorthogonally Tunable Hydrogel Platform.

Toward the goal of establishing an engineered model of the vocal fold lamina propria (LP), mesenchymal stem cells (MSCs) are encapsulated in hyaluronic acid (HA)-based hydrogels employing tetrazine ligation with strained alkenes. To mimic matrix stiffening during LP maturation, diffusion-controlled interfacial bioorthogonal crosslinking is carried out on the soft cellular construct using HA modified with a ferocious dienophile, trans-cyclooctene (TCO). Cultures are maintained in MSC growth media for 14 days to afford a model of a newborn LP that is homogeneously soft (nLP), a homogeneously stiffened construct zero (sLP0) or 7 days (sLP7) post cell encapsulation, and a mature LP model (mLP) with a stiff top layer and a soft bottom layer. Installation of additional HA crosslinks restricts cell spreading. Compared to the nLP controls, sLP7 conditions upregulate the expression of fibrous matrix proteins (Col I, DCN, and FN EDA), classic fibroblastic markers (TNC, FAP, and FSP1), and matrix remodeling enzymes (MMP2, TIMP1, and HAS3). Day 7 stiffening also upregulates the catabolic activities, enhances ECM turnover, and promotes YAP expression. Overall, in situ delayed matrix stiffening promotes a fibroblast transition from MSCs and enhances YAP-regulated mechanosensing.

Thiomethyltetrazines Are Reversible Covalent Cysteine Warheads Whose Dynamic Behavior can be "Switched Off" via Bioorthogonal Chemistry Inside Live Cells.

Electrophilic small molecules that can reversibly modify proteins are of growing interest in drug discovery. However, the ability to study reversible covalent probes in live cells can be limited by their reversible reactivity after cell lysis and in proteomic workflows, leading to scrambling and signal loss. We describe how thiomethyltetrazines function as reversible covalent warheads for cysteine modification, and this dynamic labeling behavior can be "switched off" via bioorthogonal chemistry inside live cells. Simultaneously, the tetrazine serves as a bioorthogonal reporter enabling the introduction of tags for fluorescent imaging or affinity purification. Thiomethyltetrazines can label isolated proteins, proteins in cellular lysates, and proteins in live cells with second-order rate constants spanning 2 orders of magnitude (k2, 1-100 M-1 s-1). Reversible modification by thiomethyltetrazines can be switched off upon the addition of trans-cyclooctene in live cells, converting the dynamic thiomethyltetrazine tag into a Diels-Alder adduct which is stable to lysis and proteomic workflows. Time-course quenching experiments were used to demonstrate temporal control over electrophilic modification. Moreover, it is shown that "locking in" the tag through Diels-Alder chemistry enables the identification of protein targets that are otherwise lost during sample processing. Three probes were further evaluated to identify unique pathways in a live-cell proteomic study. We anticipate that discovery efforts will be enabled by the trifold function of thiomethyltetrazines as electrophilic warheads, bioorthogonal reporters, and switches for "locking in" stability.

Dynamic modulation of matrix adhesiveness induces epithelial-to-mesenchymal transition in prostate cancer cells in 3D.

Synthetic matrices with dynamic presentation of cell guidance cues are needed for the development of physiologically relevant in vitro tumor models. Towards the goal of mimicking prostate cancer progression and metastasis, we engineered a tunable hyaluronic acid-based hydrogel platform with protease degradable and cell adhesive properties employing bioorthogonal tetrazine ligation with strained alkenes. The synthetic matrix was first fabricated via a slow tetrazine-norbornene reaction, then temporally modified via a diffusion-controlled method using trans-cyclooctene, a fierce dienophile that reacts with tetrazine with an unusually fast rate. The encapsulated DU145 prostate cancer single cells spontaneously formed multicellular tumoroids after 7 days of culture. In situ modification of the synthetic matrix via covalent tagging of cell adhesive RGD peptide induced tumoroid decompaction and the development of cellular protrusions. RGD tagging did not compromise the overall cell viability, nor did it induce cell apoptosis. In response to increased matrix adhesiveness, DU145 cells dynamically loosen cell-cell adhesion and strengthen cell-matrix interactions to promote an invasive phenotype. Characterization of the 3D cultures by immunocytochemistry and gene expression analyses demonstrated that cells invaded into the matrix via a mesenchymal like migration, with upregulation of major mesenchymal markers, and down regulation of epithelial markers. The tumoroids formed cortactin positive invadopodia like structures, indicating active matrix remodeling. Overall, the engineered tumor model can be utilized to identify potential molecular targets and test pharmacological inhibitors, thereby accelerating the design of innovative strategies for cancer therapeutics.

Ligand-Directed Photocatalysts and Far-Red Light Enable Catalytic Bioorthogonal Uncaging inside Live Cells.

Described are ligand-directed catalysts for live-cell, photocatalytic activation of bioorthogonal chemistry. Catalytic groups are localized via a tethered ligand either to DNA or to tubulin, and red light (660 nm) photocatalysis is used to initiate a cascade of DHTz oxidation, intramolecular Diels-Alder reaction, and elimination to release phenolic compounds. Silarhodamine (SiR) dyes, more conventionally used as biological fluorophores, serve as photocatalysts that have high cytocompatibility and produce minimal singlet oxygen. Commercially available conjugates of Hoechst dye (SiR-H) and docetaxel (SiR-T) are used to localize SiR to the nucleus and microtubules, respectively. Computation was used to assist the design of a new class of redox-activated photocage to release either phenol or n-CA4, a microtubule-destabilizing agent. In model studies, uncaging is complete within 5 min using only 2 µM SiR and 40 µM photocage. In situ spectroscopic studies support a mechanism involving rapid intramolecular Diels-Alder reaction and a rate-determining elimination step. In cellular studies, this uncaging process is successful at low concentrations of both the photocage (25 nM) and the SiR-H dye (500 nM). Uncaging n-CA4 causes microtubule depolymerization and an accompanying reduction in cell area. Control studies demonstrate that SiR-H catalyzes uncaging inside the cell, and not in the extracellular environment. With SiR-T, the same dye serves as a photocatalyst and the fluorescent reporter for microtubule depolymerization, and with confocal microscopy, it was possible to visualize microtubule depolymerization in real time as the result of photocatalytic uncaging in live cells.

Spectrophotometric Analysis and Optimization of 2D Gold Nanosheet Formation.

Free-standing, 2D gold nanosheets (AuNS) offer broad potential applications from computing to biosensing and healthcare. Such applications, however, require improved control of material growth. We recently reported the synthesis of AuNS only ∼0.47 nm (two atoms) thick, which exhibited very high catalytic activity. The synthesis is a one-pot, seedless procedure in which chloroauric acid is reduced by sodium citrate in the presence of methyl orange (MO). In this study, we use spectrophotometric analysis and TEM imaging to probe AuNS formation and optimize the procedure. Previously, we suggested that MO acted as the confining agent, directing two-dimensional growth of the gold. Here, we provide the first reported analysis of the HAuCl4 and MO reaction. We show that MO is rapidly oxidized to give 4-diazobenzenesulfonic acid, indicating that a complex interplay between HAuCl4, MO, and other reaction products leads to AuNS formation. Time-resolved studies indicate that synthesis time can be significantly reduced from over 12 to 2-3 h. Decreasing the reaction temperature from 20 to 4 °C improved AuNS yield by 16-fold, and the catalytic activity of the optimized material matches that obtained previously. Our elucidation of AuNS formation mechanisms has opened avenues to further improve and tune the synthesis, enhancing the potential applications of ultrathin AuNS.

Matrix Adhesiveness Regulates Myofibroblast Differentiation from Vocal Fold Fibroblasts in a Bio-orthogonally Cross-linked Hydrogel.

Repeated mechanical and chemical insults cause an irreversible alteration of extracellular matrix (ECM) composition and properties, giving rise to vocal fold scarring that is refractory to treatment. Although it is well known that fibroblast activation to myofibroblast is the key to the development of the pathology, the lack of a physiologically relevant in vitro model of vocal folds impedes mechanistic investigations on how ECM cues promote myofibroblast differentiation. Herein, we describe a bio-orthogonally cross-linked hydrogel platform that recapitulates the alteration of matrix adhesiveness due to enhanced fibronectin deposition when vocal fold wound healing is initiated. The synthetic ECM (sECM) was established via the cycloaddition reaction of tetrazine (Tz) with slow (norbornene, Nb)- and fast (trans-cyclooctene, TCO)-reacting dienophiles. The relatively slow Tz-Nb ligation allowed the establishment of the covalent hydrogel network for 3D cell encapsulation, while the rapid and efficient Tz-TCO reaction enabled precise conjugation of the cell-adhesive RGDSP peptide in the hydrogel network. To mimic the dynamic changes of ECM composition during wound healing, RGDSP was conjugated to cell-laden hydrogel constructs via a diffusion-controlled bioorthognal ligation method 3 days post encapsulation. At a low RGDSP concentration (0.2 mM), fibroblasts residing in the hydrogel remained quiescent when maintained in transforming growth factor beta 1 (TGF-ß1)-conditioned media. However, at a high concentration (2 mM), RGDSP potentiated TGF-ß1-induced myofibroblast differentiation, as evidenced by the formation of an actin cytoskeleton network, including F-actin and alpha-smooth muscle actin. The RGDSP-driven fibroblast activation to myofibroblast was accompanied with an increase in the expression of wound healing-related genes, the secretion of profibrotic cytokines, and matrix contraction required for tissue remodeling. This work represents the first step toward the establishment of a 3D hydrogel-based cellular model for studying myofibroblast differentiation in a defined niche associated with vocal fold scarring.

Enhanced Atomic Precision Fabrication by Adsorption of Phosphine into Engineered Dangling Bonds on H-Si Using STM and DFT.

The doping of Si using the scanning probe hydrogen depassivation lithography technique has been shown to enable placing and positioning small numbers of P atoms with nanometer accuracy. Several groups have now used this capability to build devices that exhibit desired quantum behavior determined by their atomistic details. What remains elusive, however, is the ability to control the precise number of atoms placed at a chosen site with 100% yield, thereby limiting the complexity and degree of perfection achievable. As an important step toward precise control of dopant number, we explore the adsorption of the P precursor molecule, phosphine, into atomically perfect dangling bond patches of intentionally varied size consisting of three adjacent Si dimers along a dimer row, two adjacent dimers, and one single dimer. Using low temperature scanning tunneling microscopy, we identify the adsorption products by generating and comparing to a catalog of simulated images, explore atomic manipulation after adsorption in select cases, and follow up with incorporation of P into the substrate. For one-dimer patches, we demonstrate that manipulation of the adsorbed species leads to single P incorporation in 12 out of 12 attempts. Based on the observations made in this study, we propose this one-dimer patch method as a robust approach that can be used to fabricate devices where it is ensured that each site of interest has exactly one P atom.

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.

Tunable Synthesis of Hydrogel Microfibers via Interfacial Tetrazine Ligation.

Crosslinked, degradable, and cell-adhesive hydrogel microfibers were synthesized via interfacial polymerization employing tetrazine ligation, an exceptionally fast bioorthogonal reaction between strained trans-cyclooctene (TCO) and s-tetrazine (Tz). A hydrophobic trisTCO crosslinker and homo-difunctional poly(ethylene glycol) (PEG)-based macromers with the tetrazine group conjugated to PEG via a stable carbamate (PEG-bisTz1) bond or a labile hydrazone (PEG-bisTz2) linkage were synthesized. After laying an ethyl acetate solution of trisTCO over an aqueous solution of bisTz macromers, mechanically robust microfibers were continuously pulled from the oil-water interface. The resultant microfibers exhibited comparable mechanical and thermal properties but different aqueous stability. Combining PEG-bisTz2 and PEG-bisTz3 with a dangling arginine-glycine-aspartic acid (RGD) peptide in the aqueous phase yielded degradable fibers that supported the attachment and growth of primary vocal fold fibroblasts. The degradable and cell-adhesive hydrogel microfibers are expected to find utility in a wide array of tissue engineering applications.

Automated Bone Scan Index to Optimize Prostate Cancer Working Group Radiographic Progression Criteria for Men With Metastatic Castration-Resistant Prostate Cancer.

INTRODUCTION: Radiographic progression-free survival (rPFS) by Prostate Cancer Working Group (PCWG) criteria is a radiographic endpoint. The automated bone scan index (aBSI) quantifies osseous disease burden on bone scintigraphy as a percentage of total skeletal weight. Using the aBSI, we sought to quantify increase in tumor burden represented by PCWG progression criteria, and to determine the interval increase that best associates with overall survival (OS). PATIENT AND METHODS: Retrospective analysis of trials using androgen receptor axis-targeted drugs for metastatic castration resistant prostate cancer patients (mCRPC). aBSI increase in bone disease was assessed from baseline scan to time-to-progression (per PCWG criteria). Threshold for time to aBSI increase were explored and the association between each time-to-threshold and OS was computed. RESULTS: A total of 169 mCPRC patients had bone scans available for aBSI analysis. Of these, 90 (53%) had progression in bone meeting PCWG criteria. Total aBSI increase in patients meeting PCWG criteria was 1.22 (interquartile range [IQR]: 0.65-2.49), with a median relative increase of 109% (IQR: 40%-377%). Median aBSI at baseline was 3.1 (IQR: 1.3-7.1). The best association between OS and time-to-progression occurred with an absolute increase in aBSI equal to 0.6 (Kendall's tau 0.52). CONCLUSION: An absolute increase of 0.6 or more in aBSI from the first follow-up scan results in the highest association with OS in patients with mCRPC. The rPFS by PCWG, identified progression at nearly twice this tumor burden, suggesting that aBSI may be used to further develop the PCWG criteria without degrading its association with OS.

Observation Versus Cast Treatment of Toddler's Fractures.

BACKGROUND: Nondisplaced metaphyseal fractures of the distal tibia, or toddler's fractures, are one of the most common pediatric injuries. Healing typically occurs quickly without sequelae. Treatment ranges from long leg cast immobilization to observation. This study compares short-term clinical and radiographic outcomes of toddler's fractures treated with long leg casting versus observation. METHODS: Patients with toddler's fractures were offered enrollment and randomization at diagnosis. Because many families opposed randomization, a preference arm was added after one year. All subjects were analyzed as a prospective cohort. Radiographs were obtained at diagnosis and 4 weeks. A modified Oxford Ankle Foot Questionnaire for Children (OAFQ-C) and family satisfaction survey were collected at diagnosis, 4 and 8 weeks. Scores were analyzed using mixed effect models. Family satisfaction surveys were compared using a Wilcoxon rank sum test. RESULTS: Forty-four subjects participated in the study, 34 (77%) in the preference arm and 10 (23%) in the randomized cohort. The median patient age was comparable between the cast and the observation groups, 2.0 versus 1.8 years, respectively. Significant improvement in OAFQ-C scores was observed in both groups over 8 weeks (P<0.01). Patients in the observation group had a higher initial play score than the cast group (P=0.03). The observation group trended toward higher physical scores at all time points (P=0.11). There was no significant difference in emotional scores between groups (P=0.77). No displacement was observed in any patient. Casted patients had significantly more minor complications with 4 patients requiring cast change or removal compared with 0 in the observed group (P=0.01). At 8 weeks, 80% of parents in the cast group were likely or very likely to choose the same treatment compared with 95.6% in the observation group. Family satisfaction scores did not differ between groups (P=0.18). They demonstrated differences in perceived normal walking at 4 weeks, with 50% of casted patients walking normally compared with 92% of observed patients. Over 90% of patients in both groups were reportedly walking normally at week 8. CONCLUSION: Observation of toddler's fractures results in equivalent clinical and radiographic outcomes, high family satisfaction and fewer complications compared with treatment with a long leg cast. LEVEL OF EVIDENCE: Level II.

Catalytic Activation of Bioorthogonal Chemistry with Light (CABL) Enables Rapid, Spatiotemporally Controlled Labeling and No-Wash, Subcellular 3D-Patterning in Live Cells Using Long Wavelength Light.

Described is the spatiotemporally controlled labeling and patterning of biomolecules in live cells through the catalytic activation of bioorthogonal chemistry with light, referred to as "CABL". Here, an unreactive dihydrotetrazine (DHTz) is photocatalytically oxidized in the intracellular environment by ambient O2 to produce a tetrazine that immediately reacts with a trans-cyclooctene (TCO) dienophile. 6-(2-Pyridyl)dihydrotetrazine-3-carboxamides were developed as stable, cell permeable DHTz reagents that upon oxidation produce the most reactive tetrazines ever used in live cells with Diels-Alder kinetics exceeding k2 of 106 M-1 s-1. CABL photocatalysts are based on fluorescein or silarhodamine dyes with activation at 470 or 660 nm. Strategies for limiting extracellular production of singlet oxygen are described that increase the cytocompatibility of photocatalysis. The HaloTag self-labeling platform was used to introduce DHTz tags to proteins localized in the nucleus, mitochondria, actin, or cytoplasm, and high-yielding subcellular activation and labeling with a TCO-fluorophore were demonstrated. CABL is light-dose dependent, and two-photon excitation promotes CABL at the suborganelle level to selectively pattern live cells under no-wash conditions. CABL was also applied to spatially resolved live-cell labeling of an endogenous protein target by using TIRF microscopy to selectively activate intracellular monoacylglycerol lipase tagged with DHTz-labeled small molecule covalent inhibitor. Beyond spatiotemporally controlled labeling, CABL also improves the efficiency of "ordinary" tetrazine ligations by rescuing the reactivity of commonly used 3-aryl-6-methyltetrazine reporters that become partially reduced to DHTzs inside cells. The spatiotemporal control and fast rates of photoactivation and labeling of CABL should enable a range of biomolecular labeling applications in living systems.

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.

Enabling In Vivo Photocatalytic Activation of Rapid Bioorthogonal Chemistry by Repurposing Silicon-Rhodamine Fluorophores as Cytocompatible Far-Red Photocatalysts.

Chromophores that absorb in the tissue-penetrant far-red/near-infrared window have long served as photocatalysts to generate singlet oxygen for photodynamic therapy. However, the cytotoxicity and side reactions associated with singlet oxygen sensitization have posed a problem for using long-wavelength photocatalysis to initiate other types of chemical reactions in biological environments. Herein, silicon-Rhodamine compounds (SiRs) are described as photocatalysts for inducing rapid bioorthogonal chemistry using 660 nm light through the oxidation of a dihydrotetrazine to a tetrazine in the presence of trans-cyclooctene dienophiles. SiRs have been commonly used as fluorophores for bioimaging but have not been applied to catalyze chemical reactions. A series of SiR derivatives were evaluated, and the Janelia Fluor-SiR dyes were found to be especially effective in catalyzing photooxidation (typically 3%). A dihydrotetrazine/tetrazine pair is described that displays high stability in both oxidation states. A protein that was site-selectively modified by trans-cyclooctene was quantitatively conjugated upon exposure to 660 nm light and a dihydrotetrazine. By contrast, a previously described methylene blue catalyst was found to rapidly degrade the protein. SiR-red light photocatalysis was used to cross-link hyaluronic acid derivatives functionalized by dihydrotetrazine and trans-cyclooctenes, enabling 3D culture of human prostate cancer cells. Photoinducible hydrogel formation could also be carried out in live mice through subcutaneous injection of a Cy7-labeled hydrogel precursor solution, followed by brief irradiation to produce a stable hydrogel. This cytocompatible method for using red light photocatalysis to activate bioorthogonal chemistry is anticipated to find broad applications where spatiotemporal control is needed in biological environments.

General, Divergent Platform for Diastereoselective Synthesis of trans-Cyclooctenes with High Reactivity and Favorable Physiochemical Properties*.

trans-Cyclooctenes (TCOs) are essential partners in the fastest known bioorthogonal reactions, but current synthetic methods are limited by poor diastereoselectivity. Especially hard to access are hydrophilic TCOs with favorable physicochemical properties for live cell or in vivo experiments. Described is a new class of TCOs, "a-TCOs", prepared in high yield by stereocontrolled 1,2-additions of nucleophiles to trans-cyclooct-4-enone, which itself was prepared on a large scale in two steps from 1,5-cyclooctadiene. Computational transition-state models rationalize the diastereoselectivity of 1,2-additions to deliver a-TCO products, which were also shown to be more reactive than standard TCOs and less hydrophobic than even a trans-oxocene analogue. Illustrating the favorable physicochemical properties of a-TCOs, a fluorescent TAMRA derivative in live HeLa cells was shown to be cell-permeable through intracellular Diels-Alder chemistry and to wash out more rapidly than other TCOs.