Trans-cyclooctenes Research Articles

A Targeted Click-to-Release Activation of the Last-Resort Antibiotic Colistin Reduces its Renal Cell Toxicity.

The use of highly potent but very toxic antibiotics such as colistin has become inevitable due to the rise of antimicrobial resistance. We aimed for a chemically-triggered, controlled release of colistin at the infection site to lower its systemic toxicity by harnessing the power of click-to-release reactions. Kinetic experiments with nine tetrazines and three dienophiles demonstrated a fast release via an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene (TCO) and the amine-functionalised tetrazine Tz7. The antibiotic activity of colistin against Escherichia coli was masked by TCO units, but restored upon reaction with d-Ubi-Tz, a tetrazine functionalised with the bacterial binding peptide d-Ubi29-41. While standard TCO did not improve toxicity against human proximal tubular kidney HK-2 cells, the installation of an aspartic acid-modified TCO masking group reduced the overall charge of the peptide and entry to the kidney cells, thereby dramatically lowering its toxicity. The analog Col-(TCO-Asp)1 had favourable pharmacokinetic properties in mice and was successfully activated locally in the lung by d-Ubi-Tz in an in vivo infection model, whereas it remained inactive and non-harmful without the chemical trigger. This study constitutes the first example of a systemically acting two-component antibiotic with improved drug tolerability.

Imaging of fibrogenesis in the liver by [18F]TZ-Z09591, an Affibody molecule targeting platelet derived growth factor receptor β

BackgroundPlatelet-derived growth factor receptor beta (PDGFRβ) is a receptor overexpressed on activated hepatic stellate cells (aHSCs). Positron emission tomography (PET) imaging of PDGFRβ could potentially allow the quantification of fibrogenesis in fibrotic livers. This study aims to evaluate a fluorine-18 radiolabeled Affibody molecule ([18F]TZ-Z09591) as a PET tracer for imaging liver fibrogenesis.ResultsIn vitro specificity studies demonstrated that the trans-Cyclooctenes (TCO) conjugated Z09591 Affibody molecule had a picomolar affinity for human PDGFRβ. Biodistribution performed on healthy rats showed rapid clearance of [18F]TZ-Z09591 through the kidneys and low liver background uptake. Autoradiography (ARG) studies on fibrotic livers from mice or humans correlated with histopathology results. Ex vivo biodistribution and ARG revealed that [18F]TZ-Z09591 binding in the liver was increased in fibrotic livers (p = 0.02) and corresponded to binding in fibrotic scars.ConclusionsOur study highlights [18F]TZ-Z09591 as a specific tracer for fibrogenic cells in the fibrotic liver, thus offering the potential to assess fibrogenesis clearly.Graphical abstract

Evaluation of Tetrazine Tracers for Pretargeted Imaging within the Central Nervous System.

The pretargeting approach separates the biological half-life of an antibody from the physical half-life of the radioisotope label, providing a strategy for reducing the radiation burden. A widely explored pretargeting approach makes use of the bioorthogonal click reaction between tetrazines (Tzs) and trans-cyclooctenes (TCOs), combining the targeting specificity of monoclonal antibodies (mAbs) with the rapid clearance and precise reaction of Tzs and TCOs. Such a strategy can allow for the targeting and imaging (e.g., by positron emission tomography (PET)) of molecular markers, which cannot be addressed by solely relying on small molecules. Tz derivatives that undergo inverse electron-demand Diels-Alder (IEDDA) reactions with an antibody bearing TCO moieties have been investigated. This study describes the synthesis and characterization of 11 cold Tz imaging agent candidates. These molecules have the potential to be radiolabeled with 18F or 3H, and with the former label, they could be of use as imaging tracers for positron emission tomography studies. Selection was made using a multiparameter optimization score for the central nervous system (CNS) PET tracers. Novel tetrazines were tested for their pH-dependent chemical stability. Those which turned out to be stable in a pH range of 6.5-8 were further characterized in in vitro assays with regard to their passive permeability, microsomal stability, and P-glycoprotein transport. Furthermore, selected Tzs were examined for their systemic clearance and CNS penetration in a single-dose pharmacokinetic study in rats. Two tetrazines were successfully labeled with 18F, one of which showed brain penetration in a biodistribution study in mice. Another Tz was successfully tritium-labeled and used to demonstrate a bioorthogonal click reaction on a TCO-modified antibody. As a result, we identified one Tz as a potential fluorine-18-labeled CNS-PET agent and a second as a 3H-radioligand for an IEDDA-based reaction with a modified brain-penetrating antibody.

A Concise Synthetic Approach to Highly Reactive Click-to-Release Trans-Cyclooctene Linkers.

An increase in the click-to-release reaction rate between cleavable trans-cyclooctenes (TCO) and tetrazines would be beneficial for drug delivery applications. In this work, we have developed a short and stereoselective synthesis route towards highly reactive sTCOs that serve as cleavable linkers, affording quantitative tetrazine-triggered payload release. In addition, the fivefold more reactive sTCO exhibited the same in vivo stability as current TCO linkers when used as antibody linkers in circulation in mice.

Bioorthogonal PROTAC Prodrugs Enabled by On-Target Activation.

Although proteolysis targeting chimeras (PROTACs) have become promising therapeutic modalities, important concerns exist about the potential toxicity of the approach owing to uncontrolled degradation of proteins and undesirable ligase-mediated off-target effects. Precision manipulation of degradation activity of PROTACs could minimize potential toxicity and side effects. As a result, extensive efforts have been devoted to developing cancer biomarker activating prodrugs of PROTACs. In this investigation, we developed a bioorthogonal on-demand prodrug strategy (termed click-release "crPROTACs") that enables on-target activation of PROTAC prodrugs and release of PROTACs in cancer cells selectively. Inactive PROTAC prodrugs TCO-ARV-771 and TCO-DT2216 are rationally designed by conjugating a bioorthogonal trans-cyclooctenes (TCO) group into the ligand of the VHL E3 ubiquitin ligase. The tetrazine (Tz)-modified RGD peptide, c(RGDyK)-Tz, which targets integrin αvβ3 biomarker in cancer cells, serves as the activation component for click-release of the PROTAC prodrugs to achieve targeted degradation of proteins of interest (POIs) in cancer cells versus noncancerous normal cells. The results of studies accessing the viability of this strategy show that the PROTAC prodrugs are selectively activated in an integrin αvβ3-dependent manner to produce PROTACs, which degrade POIs in cancer cells. The crPROTAC strategy might be a general, abiotic approach to induce selective cancer cell death through the ubiquitin-proteasome pathway.

The inverse electron demand Diels-Alder cycloaddition with carbon-11 and fluorine-18: A gateway to pretargeted imaging across the blood-brain barrier.

There is increased focus on developing tools to image large biomolecules, such as antibodies, within the brain using positron emission tomography (PET). The inverse electron demand Diels-Alder cycloaddition (IEDDA) reaction has offered the greatest prospect of achieving such a feat and has gained much interest over the past decade. The fast reaction kinetics of the IEDDA reaction opens up the possibility of utilising a pretargeted approach, whereby the subject is pretreated with a biomolecule that has high specificity for its target. A radiolabelled second component is then administered to the subject, enabling the biomolecule to be visualised by PET. However, for this to become common practice, there is a need for the development of either radiolabelled trans-cyclooctenes (TCOs) or tetrazines that can cross the blood-brain barrier (BBB). This review highlights the advancements in the development of both radiolabelled TCOs and tetrazines, which have been radiolabelled with either carbon-11 or fluorine-18 and show promise or have been evaluated for use in pretargeted PET imaging across the BBB.

Radiolabelling of peptides with tetrazine ligation based on the inverse electron-demand Diels-Alder reaction: rapid, catalyst-free and mild conversion of 1,4-dihydropyridazines to pyridazines.

Click chemistry reactions, such as the tetrazine ligation, based on the inverse-electron demand Diels-Alder (IEDDA), are chemoselective cycloaddition reactions widely used for chemical modifications and synthesis of biomolecule-based radiopharmaceuticals for positron emission tomography (PET). The reactions have potential also for pretargeted PET imaging. When used as a bioconjugation method in production of biomolecule-based radiopharmaceuticals, IEDDA-based tetrazine ligation has one significant drawback, namely the formation of a mixture comprising reduced metastable dihydropyridazines (DHPs) and oxidized cycloadducts. Conversion of the reduced DHPs to stable pyridazines requires oxidation, which is typically achieved by using oxidants or by photo-irradiated air-oxidation, both methods requiring added reagents or reaction times of several hours, not compatible with short-lived radionuclides. Here we report a mild, rapid, and catalyst-free conversion of the DHPs to pyridazines. In this study, a model peptide Tyr3-octreotide (TOC) was modified with polyethylene glycol (PEG) linkers and with trans-cyclooctenes (TCOs) for rapid IEDDA-mediated radiolabeling. Fluorine-18-labelled alkylammoniomethyltrifluoroborate ([18F]AmBF3) tetrazines were conjugated to the TCO-TOC analogs at room temperature for rapid synthesis of PET imaging agent candidates. The formed DHPs were successfully converted to the oxidized form, after heating the radiolabelled bioconjugates in aqueous solution (≥95% water) at 60 °C for a minimum of 10 minutes in the presence of air, resulting in one-pot back-to-back IEDDA reaction and DHP conversion. The water content of the reaction mixture was to be found critical for the coversion. Our finding offers a straightforward method for conversion of the metastable DHPs from the IEDDA-based tetrazine ligation to stable, oxidized pyridazines. The method is especially suitable for applications requiring rapid conversion.

Oxidative Desymmetrization Enables the Concise Synthesis of a trans-Cyclooctene Linker for Bioorthogonal Bond Cleavage.

Modified trans-cyclooctenes (TCO) are capable of highly efficient molecular manipulations in biological environments, driven by the bioorthogonal reaction with tetrazines (Tz). The development of click-cleavable TCO has fueled the field of in vivo chemistry and enabled the design of therapeutic strategies that have already started to enter the clinic. A key element for most of these approaches is the implementation of a cleavable TCO linker. So far, only one member of this class has been developed, a compound that requires a high synthetic effort, mainly to fulfill the multilayered demands on its chemical structure. To tackle this limitation, we developed a dioxolane-fused cleavable TCO linker (dcTCO) that can be prepared in only five steps by applying an oxidative desymmetrization to achieve diastereoselective introduction of the required functionalities. Based on investigation of the structure, reaction kinetics, stability, and hydrophilicity of dcTCO, we demonstrate its bioorthogonal application in the design of a caged prodrug that can be activated by in-situ Tz-triggered cleavage to achieve a remarkable >1000-fold increase in cytotoxicity.

Development and evaluation of an 18F-labeled nanobody to target SARS-CoV-2's spike protein.

COVID-19, caused by the SARS-CoV-2 virus, has become a global pandemic that is still present after more than two years. COVID-19 is mainly known as a respiratory disease that can cause long-term consequences referred to as long COVID. Molecular imaging of SARS-CoV-2 in COVID-19 patients would be a powerful tool for studying the pathological mechanisms and viral load in different organs, providing insights into the disease and the origin of long-term consequences and assessing the effectiveness of potential COVID-19 treatments. Current diagnostic methods used in the clinic do not allow direct imaging of SARS-CoV-2. In this work, a nanobody (NB) - a small, engineered protein derived from alpacas - and an Fc-fused NB which selectively target the SARS-CoV-2 Spike protein were developed as imaging agents for positron emission tomography (PET). We used the tetrazine ligation to 18F-label the NB under mild conditions once the NBs were successfully modified with trans-cyclooctenes (TCOs). We confirmed binding to the Spike protein by SDS-PAGE. Dynamic PET scans in rats showed excretion through the liver for both constructs. Future work will evaluate in vivo binding to the Spike protein with our radioligands.

Pretargeted Imaging beyond the Blood-Brain Barrier-Utopia or Feasible?

Pretargeting is a promising nuclear imaging technique that allows for the usage of antibodies (Abs) with enhanced imaging contrast and reduced patient radiation burden. It is based on bioorthogonal chemistry with the tetrazine ligation—a reaction between trans-cyclooctenes (TCOs) and tetrazines (Tzs)—currently being the most popular reaction due to its high selectivity and reactivity. As Abs can be designed to bind specifically to currently ‘undruggable’ targets such as protein isoforms or oligomers, which play a crucial role in neurodegenerative diseases, pretargeted imaging beyond the BBB is highly sought after, but has not been achieved yet. A challenge in this respect is that large molecules such as Abs show poor brain uptake. Uptake can be increased by receptor mediated transcytosis; however, it is largely unknown if the achieved brain concentrations are sufficient for pretargeted imaging. In this study, we investigated whether the required concentrations are feasible to reach. As a model Ab, we used the bispecific anti-amyloid beta (Aβ) anti-transferrin receptor (TfR) Ab 3D6scFv8D3 and conjugated it to a different amount of TCOs per Ab and tested different concentrations in vitro. With this model in hand, we estimated the minimum required TCO concentration to achieve a suitable contrast between the high and low binding regions. The estimation was carried out using pretargeted autoradiography on brain sections of an Alzheimer’s disease mouse model. Biodistribution studies in wild-type (WT) mice were used to correlate how different TCO/Ab ratios alter the brain uptake. Pretargeted autoradiography showed that increasing the number of TCOs as well as increasing the TCO-Ab concentration increased the imaging contrast. A minimum brain concentration of TCOs for pretargeting purposes was determined to be 10.7 pmol/g in vitro. Biodistribution studies in WT mice showed a brain uptake of 1.1% ID/g using TCO-3D6scFv8D3 with 6.8 TCO/Ab. According to our estimations using the optimal parameters, pretargeted imaging beyond the BBB is not a utopia. Necessary brain TCO concentrations can be reached and are in the same order of magnitude as required to achieve sufficient contrast. This work gives a first estimate that pretargeted imaging is indeed possible with antibodies. This could allow the imaging of currently ‘undruggable’ targets and therefore be crucial to monitor (e.g., therapies for intractable neurodegenerative diseases).

Tetrazine-Triggered Bioorthogonal Cleavage of trans-Cyclooctene-Caged Phenols Using a Minimal Self-Immolative Linker Strategy.

Bond-cleavage reactions triggered by bioorthogonal tetrazine ligation have emerged as strategies to chemically control the function of (bio)molecules and achieve activation of prodrugs in living systems. While most of these approaches make use of caged amines, current methods for the release of phenols are limited by unfavorable reaction kinetics or insufficient stability of the Tz-responsive reactants. To address this issue, we have implemented a self-immolative linker that enables the connection of cleavable trans-cyclooctenes (TCO) and phenols via carbamate linkages. Based on detailed investigation of the reaction mechanism with several Tz, revealing up to 96 % elimination after 2 hours, we have developed a TCO-caged prodrug with 750-fold reduced cytotoxicity compared to the parent drug and achieved in situ activation upon Tz/TCO click-to-release.

EGFR-targeted prodrug activation using bioorthogonal alkene-azide click-and-release chemistry

Epidermal growth factor receptor (EGFR) is overexpressed in many cancers and therefore serves as an excellent target for prodrug activation. Functionalised trans-cyclooctenes (TCO) were conjugated to an EGFR antibody (cetuximab), providing a reagent for pre-targeting and localisation of the bioorthogonal reagent. The TCOs react with a 4-azidobenzyl carbamate doxorubicin prodrug via a [3 + 2]-cycloaddition and subsequent self-immolation leads to release of doxorubicin (click-and-release). In vitro cell-based assays demonstrated proof-of-concept, that cetuximab conjugated to highly strained TCO (AB-d-TCO) could bind to the EGFR in a melanoma cell line, and selectively activate the doxorubicin prodrug. In a non-EGFR expressing melanoma cell line, no significant prodrug activation was observed. In vivo experiments using this combination of AB-d-TCO and the azido-doxorubicin prodrug in a murine melanoma model revealed no significant anti-tumour activity or increased survival, suggesting there was insufficient prodrug activation and drug release at the tumour site.

Clickable Radiocomplexes With Trivalent Radiometals for Cancer Theranostics: In vitro and in vivo Studies.

Pre-targeting approaches based on the inverse-electron-demand Diels-Alder (iEDDA) reaction between strained trans-cyclooctenes (TCO) and electron-deficient tetrazines (Tz) have emerged in recent years as valid alternatives to classic targeted strategies to improve the diagnostic and therapeutic properties of radioactive probes. To explore these pre-targeting strategies based on in vivo click chemistry, a small family of clickable chelators was synthesized and radiolabelled with medically relevant trivalent radiometals. The structure of the clickable chelators was diversified to modulate the pharmacokinetics of the resulting [111In]In-radiocomplexes, as assessed upon injection in healthy mice. The derivative DOTA-Tz was chosen to pursue the studies upon radiolabelling with 90Y, yielding a radiocomplex with high specific activity, high radiochemical yields and suitable in vitro stability. The [90Y]Y-DOTA-Tz complex was evaluated in a prostate cancer PC3 xenograft by ex-vivo biodistribution studies and Cerenkov luminescence imaging (CLI). The results highlighted a quick elimination through the renal system and no relevant accumulation in non-target organs or non-specific tumor uptake. Furthermore, a clickable bombesin antagonist was injected in PC3 tumor-bearing mice followed by the radiocomplex [90Y]Y-DOTA-Tz, and the mice imaged by CLI at different post-injection times (p.i.). Analysis of the images 15 min and 1 h p.i. pointed out an encouraging quick tumor uptake with a fast washout, providing a preliminary proof of concept of the usefulness of the designed clickable complexes for pre-targeting strategies. To the best of our knowledge, the use of peptide antagonists for this purpose was not explored before. Further investigations are needed to optimize the pre-targeting approach based on this type of biomolecules and evaluate its eventual advantages.

Synthesis and formulation of self‐immolative PEG‐aryl azide block copolymers and click‐to‐release reactivity with trans‐cyclooctene

AbstractSelf‐immolative aryl azides can react with trans‐cyclooctenes (TCO), triphenylphosphines or hydrogen sulfide (H2S) to activate prodrugs, imaging probes and drug delivery systems. To date, the synthesis of polymers containing these aryl azide self‐immolative linkers and their reactivity with a strained alkene (i.e., in a bioorthogonal reaction) has not been explored. Also, due to the instability of aryl azides towards light and high temperatures, the polymerization methods compatible with aryl azides are limited. Through systematic investigation of the reversible addition‐fragmentation chain transfer (RAFT) and atom transfer radical polymerization (ATRP) methods, a self‐immolative PEG‐aryl azide block copolymer (PEG45‐b‐ABOC28 2) and a non‐responsive 4‐fluoroaryl block copolymer (PEG45‐b‐FBOC24 3) was prepared. ATRP provided the desired polymers in a highly controlled manner, whereas the RAFT conditions led to higher levels of aryl azide polymer degradation. The ATRP derived polymers 2 and 3 were formulated into nanoparticles of approximately 200 nm diameter, and particle triggering was demonstrated by the [3+2]‐cycloaddition reaction of TCO with PEG45‐b‐ABOC28 2 in solution (pure polymer) and as a formulated nanoparticle. Preliminary in vitro cell viability studies suggested that the stimuli‐responsive aryl azide polymers/nanoparticles are not cytotoxic up to 200 μg/ml concentrations.

Bioorthogonal Reactions Utilizing Nitrones as Versatile Dipoles in Cycloaddition Reactions.

Bioorthogonal chemical reactions have emerged as convenient and rapid methods for incorporating unnatural functionality into living systems. Different prototype reactions have been optimized for use in biological settings. Optimization of 3 + 2 dipolar cycloadditions involving nitrones has resulted in highly efficient reaction conditions for bioorthogonal chemistry. Through substitution at the nitrone carbon or nitrogen atom, stereoelectronic tuning of the reactivity of the dipole has assisted in optimizing reactivity. Nitrones have been shown to react rapidly with cyclooctynes with bimolecular rate constants approaching k2 = 102 M-1 s-1, which are among the fastest bioorthogonal reactions reported (McKay et al. Org. Biomol. Chem. 2012, 10, 3066-3070). Nitrones have also been shown to react with trans-cyclooctenes (TCO) in strain-promoted TCO-nitrone cycloadditions reactions. Copper catalyzed reactions involving alkynes and nitrones have also been optimized for applications in biology. This review provides a comprehensive accounting of the different bioorthogonal reactions that have been developed using nitrones as versatile reactants, and provides some recent examples of applications for probing biological systems.

Core-Shell Microfibers via Bioorthogonal Layer-by-Layer Assembly.

A new technique is described for the construction of core-shell microfibers for biomedical applications. Fibrous scaffolds were fabricated by electrospinning, followed by covalent layer-by-layer deposition based on the rapid bioorthogonal reaction between s-tetrazines (Tz) and trans-cyclooctenes (TCOs). Electrospun poly(ε-caprolactone) (PCL) scaffolds were subjected to surface modifications to install tetrazine groups. The scaffolds were iteratively submerged in aqueous solutions of TCO-modified hyaluronic acid (HA-TCO) and tetrazine-modified hyaluronic acid (HA-Tz), resulting in the controlled growth of a cross-linked HA gel around individual microfibers. Integrin-binding motifs were covalently attached to the surface of the microfibers using TCO-conjugated RGD peptide. The scaffolds fostered the attachment and growth of primary porcine vocal fold fibroblasts without a significant induction of the myofibroblast phenotype. Stimulation with transforming growth factor beta (TGF-β) moderately enhanced fibroblast activation, and inhibition of the Rho/ROCK signaling pathway using Y27632 further decreased the expression of myofibroblastic markers. The bioorthogonally assembled scaffolds with a stiff PCL core and a soft HA shell may find application as therapeutic implants for the treatment of vocal fold scarring.

Fluorogenic Bifunctional trans-Cyclooctenes as Efficient Tools for Investigating Click-to-Release Kinetics.

The inverse electron demand Diels–Alder pyridazine elimination reaction between tetrazines and allylic substituted trans‐cyclooctenes (TCOs) is a key player in bioorthogonal bond cleavage reactions. Determining the rate of elimination of alkylamine substrates has so far proven difficult. Here, we report a fluorogenic tool consisting of a TCO‐linked EDANS fluorophore and a DABCYL quencher for accurate determination of both the click and release rate constants for any tetrazine at physiologically relevant concentrations.

Preclinical Evaluation of a Novel 18F-Labeled dTCO-Amide Derivative for Bioorthogonal Pretargeted Positron Emission Tomography Imaging.

Pretargeted positron emission tomography (PET) imaging based on the bioorthogonal inverse-electron-demand Diels–Alder reaction between tetrazines (Tz) and trans-cyclooctenes (TCO) has emerged as a promising tool for solid tumor imaging, allowing the use of short-lived radionuclides in immune-PET applications. With this strategy, it became possible to achieve desirable target-to-background ratios and at the same time to decrease the radiation burden to nontargeted tissues because of the fast clearance of small PET probes. Here, we show the synthesis of novel 18F-labeled dTCO-amide probes for pretargeted immuno-PET imaging. The PET probes were evaluated regarding their stability, reactivity toward tetrazine, and pharmacokinetic profile. [18F]MICA-213 showed an extremely fast kinetic rate (10,553 M–1 s–1 in 50:50 MeOH/water), good stability in saline and plasma up to 4 h at 37 °C with no isomerization observed, and the biodistribution in healthy mice revealed a mixed hepatobiliary and renal clearance with no defluorination and low background in other tissues. [18F]MICA-213 was further used for in vivo pretargeted immune-PET imaging carried out in nude mice bearing LS174T colorectal tumors that were previously treated with a tetrazine-modified anti-TAG-72 monoclonal antibody (CC49). Pretargeted μPET imaging results showed clear visualization of the tumor tissue with a significantly higher uptake when compared to the control.

Antibody Pretargeting Based on Bioorthogonal Click Chemistry for Cancer Imaging and Targeted Radionuclide Therapy.

Bioorthogonal click chemistry-employing antibody-conjugated trans-cyclooctenes (TCO) and tetrazine (Tz)-based radioligands able to covalently bind in vivo-appeared recently as a potential alternative to circumvent the hematotoxicity induced by radioimmunotherapy of solid tumors. This Review focuses on the recent advances concerning TCO/Tz pretargeting in both cancer imaging and targeted-radionuclide therapy for prospective clinical transfer. We exhaustively identified 25 PubMed publications reporting preclinical imaging and 5 therapy studies with full mAbs as targeting vectors, since its first application in 2010. The fast, safe, modulable, and specific TCO/Tz pretargeting showed high potential as a theranostic tool to get more personalized and precise cancer care. The recent optimizations reported here highlighted a possible first clinical evaluation of IEDDA pretargeting in the coming years.

Cycloadditions of Trans-Cyclooctenes and Nitrones as Tools for Bioorthogonal Labelling.

Trans-cyclooctenes (TCOs) represent interesting and highly reactive dipolarophiles for organic transformations including bioorthogonal chemistry. Herein we show that TCOs react rapidly with nitrones and that these reactions are bioorthogonal. Kinetic analysis of acyclic and cyclic nitrones with strained-trans-cyclooctene (s-TCO) shows fast reactivity and demonstrates the utility of this cycloaddition reaction for bioorthogonal labelling. Labelling of the bacterial peptidoglycan layer with unnatural d-amino acids tagged with nitrones and s-TCO-Alexa488 is demonstrated. These new findings expand the bioorthogonal toolbox, and allow TCO reagents to be used in bioorthogonal applications beyond tetrazine ligations for the first time and open up new avenues for bioorthogonal ligations with diverse nitrone reactants.