Inverse Electron Demand Diels-Alder Research Articles

Stoichiometric Post-Modification of Hydrogel Microparticles Dictates Neural Stem Cell Fate in Microporous Annealed Particle Scaffolds.

Microporous annealed particle (MAP) scaffolds are generated from assembled hydrogel microparticles (microgels). It has been previously demonstrated that MAP scaffoldare porous, biocompatible, and recruit neural progenitor cells (NPCs) to the stroke cavity after injection into the stroke core. Here, the goal is to study NPC fate inside MAPscaffolds in vitro. To create plain microgels that can later be converted to contain different types of bioactivities, the inverse electron-demand Diels-Alder reaction between tetrazine and norbornene is utilized, which allows the post-modification of plain microgels stoichiometrically. As a result of adhesive peptide attachment, NPC spreading leads to contractile force generation which can be recorded by tracking microgel displacement. Alternatively, non-adhesive peptide integration results in neurosphere formation that grows within the void spaceof MAP scaffolds. Although the formed neurospheres do not impose a contractile force on the scaffolds, they are seen to continuously transverse the scaffolds. It is concluded that MAP scaffoldscan be engineered to either promote neurogenesis or enhance stemness depending on the chosen post-modifications of the microgels, which can be key in modulating their phenotypes in various applications in vivo.

Development of a multi faceted platform containing a tetrazine, fluorophore and chelator: synthesis, characterization, radiolabeling, and immuno-SPECT imaging

BackgroundCombining optical (fluorescence) imaging with nuclear imaging has the potential to offer a powerful tool in personal health care, where nuclear imaging offers in vivo functional whole-body visualization, and the fluorescence modality may be used for image-guided tumor resection. Varying chemical strategies have been exploited to fuse both modalities into one molecular entity. When radiometals are employed in nuclear imaging, a chelator is typically inserted into the molecule to facilitate radiolabeling; the availability of the chelator further expands the potential use of these platforms for targeted radionuclide therapy if a therapeutic radiometal is employed. Herein, a novel mixed modality scaffold which contains a tetrazine (Tz)––for biomolecule conjugation, fluorophore—for optical imaging, and chelator—for radiometal incorporation, in one construct is presented. The novel platform was characterized for its fluorescence properties, radiolabeled with single-photon emission computed tomography (SPECT) isotope indium-111 (111In3+) and therapeutic alpha emitter actinium-225 (225Ac3+). Both radiolabels were conjugated in vitro to trans-cyclooctene (TCO)-modified trastuzumab; biodistribution and immuno-SPECT imaging of the former conjugate was assessed.ResultsKey to the success of the platform synthesis was incorporation of a 4,4′-dicyano-BODIPY fluorophore. The route gives access to an advanced intermediate where final chelator-incorporated compounds can be easily accessed in one step prior to radiolabeling or biomolecule conjugation. The DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) conjugate was prepared, displayed good fluorescence properties, and was successfully radiolabeled with 111In & 225Ac in high radiochemical yield. Both complexes were then separately conjugated in vitro to TCO modified trastuzumab through an inverse electron demand Diels–Alder (IEDDA) reaction with the Tz. Pilot small animal in vivo immuno-SPECT imaging with [111In]In-DO3A-BODIPY-Tz-TCO-trastuzumab was also conducted and exhibited high tumor uptake (21.2 ± 5.6%ID/g 6 days post-injection) with low uptake in non-target tissues.ConclusionsThe novel platform shows promise as a multi-modal probe for theranostic applications. In particular, access to an advanced synthetic intermediate where tailored chelators can be incorporated in the last step of synthesis expands the potential use of the scaffold to other radiometals. Future studies including validation of ex vivo fluorescence imaging and exploiting the pre-targeting approach available through the IEDDA reaction are warranted.

Pretargeted PET of Osteodestructive Lesions in Dogs.

The last decade has witnessed the creation of a highly effective approach to in vivo pretargeting based on the inverse electron demand Diels-Alder (IEDDA) click ligation between tetrazine (Tz) and trans-cyclooctene (TCO). Despite the steady progression of this technology toward the clinic, concerns have persisted regarding whether this in vivo chemistry will work in humans given their larger size and blood volume. In this work, we describe the use of a 64Cu-labeled Tz radioligand ([64Cu]Cu-SarAr-Tz) and a TCO-bearing bisphosphonate (TCO-BP) for the pretargeted positron emission tomography (PET) imaging of osteodestructive lesions in a large animal model: companion dogs. First, in a small animal pilot study, healthy mice were injected with TCO-BP followed after 1 or 6 h by [64Cu]Cu-SarAr-Tz. PET images were collected 1, 6, and 24 h after the administration of [64Cu]Cu-SarAr-Tz, revealing that this approach produced high activity concentrations in the bone (>20 and >15%ID/g in the femur and humerus, respectively, at 24 h post injection) as well as high target-to-background contrast. Subsequently, companion dogs (n = 5) presenting with osteodestructive lesions were administered TCO-BP (5 or 10 mg/kg) followed 1 h later by [64Cu]Cu-SarAr-Tz (2.2-7.3 mCi; 81.4-270.1 MBq). PET scans were collected for each dog 4 h after the administration of the radioligand, and SUV values for the osteodestructive lesions, healthy bones, and kidneys were determined. In these animals, pretargeted PET clearly delineated healthy bone and produced very high activity concentrations in osteodestructive lesions. Low levels of uptake were observed in all healthy organs except for the kidneys and bladder due to the renal excretion of excess radioligand. Ultimately, this work not only illustrates that pretargeted PET with TCO-BP and [64Cu]Cu-SarAr-Tz is an effective tool for the visualization of osteodestructive lesions but also demonstrates for the first time that in vivo pretargeting based on IEDDA click chemistry is feasible in large animals.

Synthesis and ex vivo biodistribution of two 68Ga-labeled tetrazine tracers: Comparison of pharmacokinetics

Pretargeted PET imaging allows the use of radiotracers labeled with short-living PET radionuclides for tracing drugs with slow pharmacokinetics. Recently, especially methods based on bioorthogonal chemistry have been under intensive investigation for pretargeted PET imaging. The pharmacokinetics of the radiotracer is one of the factors that determine the success of the pretargeted strategy. Here, we report synthesis and biological evaluation of two 68Ga-labeled tetrazine (Tz)-based radiotracers, [68Ga]Ga-HBED-CC-PEG4-Tz ([68Ga]4) and [68Ga]Ga-DOTA-PEG4-Tz ([68Ga]6), aiming for development of new tracer candidates for pretargeted PET imaging based on the inverse electron demand Diels-Alder (IEDDA) ligation between a tetrazine and a strained alkene, such as trans-cyclooctene (TCO). Excellent radiochemical yield (RCY) was obtained for [68Ga]4 (RCY > 96%) and slightly lower for [68Ga]6 (RCY > 88%). Radiolabeling of HBED-CC-Tz proved to be faster and more efficient under milder conditions compared to the DOTA analogue. The two tracers exhibited excellent radiolabel stability both in vitro and in vivo. Moreover, [68Ga]4 was successfully used for radiolabeling two different TCO-functionalized nanoparticles in vitro: Hepatitis E virus nanoparticles (HEVNPs) and porous silicon nanoparticles (PSiNPs).

Quantitative Analysis of Protein Bioconjugation in Cells with an Expanded Genetic Code

Protein bioconjugation – specific covalent bond formation between a protein of interest (POI) and a label – has a major impact on protein imaging, protein function studies, and drug discovery. Ideal protein bioconjugation should be fast, quantitative, one step with no off‐target binding, and have minimal impact on protein function, no matter the complexity of the biological environment. The impressive selectivity and kinetics of bioorthogonal reactions, specifically the catalyst‐free inverse‐electron demand Diels‐Alder (IEDDA) cycloaddition of tetrazines and strained alkene dienophiles, make IEDDA‐reactive unnatural amino acids an exceptional tool for protein bioconjugation via genetic code expansion. Analyses of these bioconjugation reactions in live cells has been mostly limited to fluorophore labeling, and thus is rarely quantitative. Self‐labeling enzymes, like HaloTag, have been used for protein isolation and purification, studies of protein‐protein interactions, and in vivo and in vitro imaging. The excellent permeability and low reactivity of the chloroalkane substrate of HaloTag yields bioconjugation detection independent of ligand permeability. The objective of this research is to utilize the HaloTag technology in combination with genetic code expansion to develop a generalized method for quantification of intracellular bioconjugation to any POI at defined sites. We hypothesize that bioconjugation efficiency could be assessed through site‐specific installation of a reactive handle on a POI, followed by ligand‐mediated protein labeling via ternary complex formation after cell lysis and exposure to HaloTag. Here we describe the optimization of concentrations, reaction times, and reaction partners for bioconjugation and pulldown for POIs expressing different IEDDA reactive surface residues. Additionally, we describe the attempted application of this method for labeling protein targets, such as kinases and nuclear receptors. With this new methodology, we have demonstrated quantitative assessment of protein bioconjugation reactions in live cells.

Clip to Click: Controlling Inverse Electron-Demand Diels-Alder Reactions with Macrocyclic Tetrazines.

A universal strategy to control tetrazine reactivity in the inverse electron-demand Diels-Alder (IEDDA) reaction was developed as "Clip to Click". Incorporating a chemical bridge into 3,6-diphenyl-1,2,4,5-tetrazine (macrocyclic tetrazine) rendered it unreactive toward trans-cyclooctene. A computational study revealed that the unreactive property of macrocyclic tetrazines is mainly due to the high distortion energy of tetrazine. We demonstrated that the cleavage ("Clip") of the macrocyclic linker can activate the tetrazine moiety for the IEDDA reaction ("Click").

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [89Zr]Zr-DFO-PEG5-Tz.

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-of-concept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new 89Zr-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels–Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t1/2 = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [89Zr]Zr-DFO-PEG5-Tz ([89Zr]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 ± 0.2 vs 17.1 ± 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for 89Zr-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.

Nanoparticular Carriers As Objects to Study Intentional and Unintentional Bioconjugation.

Synthetic nanoparticles are interesting to use in the study of ligation with natural biorelevant structures. That is because they present an intermediate situation between reactions onto soluble polymers or onto solid surfaces. In addition, differently functionalized nanoparticles can be separated and studied independently thereafter. So what would be a "patchy functionalization" on a macroscopic surface results in differently functionalized nanoparticles, which can be separated after the interaction with body fluids. This paper will review bioconjugation of such nanoparticles with a special focus on recent results concerning the formation of a protein corona by unspecific adsorption (lower lines of TOC), which presents an unintentional bioconjugation, and on new aspects of intentionally performed bioconjugation by covalent chemistry (upper line). For this purpose, it is important that polymeric nanoparticles without a protein corona can be prepared. This opens, e.g., the possibility to look for special proteins adsorbed as a result of the natural compound ligated to the nanoparticle by covalent chemistry, like the Fc part of antibodies. At the same time, the use of highly reactive, bioorthogonal functional groups (inverse electron demand Diels-Alder cycloaddition) on the nanoparticles allows an efficient ligation after administration inside the body, i.e., in vivo.

Enhanced Therapeutic Effect of Liposomal Doxorubicin via Bio-Orthogonal Chemical Reactions in Tumors.

Liposomes are highly biocompatible drug carriers in drug delivery systems (DDSs). Preferential accumulation of liposomes and acceleration of drug release at target tumor sites are essential for effective cancer therapy using liposomal formulations; however, conventional liposomes are unsuitable for on-demand drug release. We have previously reported that drug release can be accelerated via a bio-orthogonal inverse electron demand Diels-Alder (IEDDA) reaction between amphiphilic tetrazine (Tz)-containing liposomes and norbornene (NB) derivatives in vitro. In this study, we prepared HSTz-liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC) and Tz compound (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)octadecanamide) with particle sizes of 60-80 nm and ζ-potentials of -5 to 0 mV. Similar to our previous report, the addition of 5-norbornene-2-carboxylic acid (NBCOOH) to HSTz-liposomes accelerated drug release from the liposomes in vitro. In the biodistribution study using colon26 tumor-bearing mice, the radiolabeled HSTz-liposomes were accumulated and retained in the tumor at 6-48 h post-injection, whereas the radioactivity in the blood almost disappeared at 48 h. Therefore, the timing of the injection of NBCOOH was selected to be 48 h after the injection of the HSTz-liposome to avoid the IEDDA reaction in the bloodstream. We investigated the in vivo drug release by evaluating the intratumoral localization of doxorubicin (DOX) encapsulated in HSTz-liposomes labeled with fluorescent lipids. In the tumors treated with HSTz-liposomes and NBCOOH, DOX was more widely dispersed in the tumor compared with fluorescent lipid, suggesting that the release of encapsulated drugs (DOX) from HSTz-liposomes was enhanced in the tumor tissue via the bio-orthogonal IEDDA reaction. Furthermore, the combination of DOX-encapsulated HSTz-liposomes with NBCOOH significantly suppressed tumor growth compared to conventional DOX-encapsulated liposomes. In conclusion, the bio-orthogonal IEDDA reactions in the liposomal membrane enabled the acceleration of drug release from HSTz-liposomes in vivo, suggesting a promising strategy for effective cancer therapy.

Beyond the Bioorthogonal Inverse-Electron-Demand Diels–Alder Reactions of Tetrazines: 2-Pyrone-Functionalized Fluorogenic Probes

AbstractThe applicability of pyrones as a bioorthogonal platform was explored in inverse-electron-demand Diels–Alder (IEDDA) reactions with a strained cyclooctyne. Studies showed that the pyrones are indeed suitable for IEDDA reactions under physiological conditions. Furthermore, the stable pyrone moiety could be utilized to construct easily accessible fluorogenic probes. Mutual orthogonality of the IEDDA reaction of 2-pyrones with SPAAC reactions of azides was also explored.

A Kinetic and Fluorogenic Enhancement Strategy for Labeling of Nucleic Acids.

Chemical modification of nucleic acids in living cells can be sterically hindered by tight packing of bioorthogonal functional groups in chromatin. To address this limitation, we report here a dual enhancement strategy for nucleic acid-templated reactions utilizing a fluorogenic intercalating agent capable of undergoing inverse electron-demand Diels-Alder (IEDDA) reactions with DNA containing 5-vinyl-2'-deoxyuridine (VdU) or RNA containing 5-vinyl-uridine (VU). Reversible high-affinity intercalation of a novel acridine-tetrazine conjugate "PINK" (KD =5±1 μM) increases the reaction rate of tetrazine-alkene IEDDA on duplex DNA by 60 000-fold (590 M-1 s-1 ) as compared to the non-templated reaction. At the same time, loss of tetrazine-acridine fluorescence quenching renders the reaction highly fluorogenic and detectable under no-wash conditions. This strategy enables live-cell dynamic imaging of acridine-modified nucleic acids in dividing cells.

A Kinetic and Fluorogenic Enhancement Strategy for Labeling of Nucleic Acids

AbstractChemical modification of nucleic acids in living cells can be sterically hindered by tight packing of bioorthogonal functional groups in chromatin. To address this limitation, we report here a dual enhancement strategy for nucleic acid‐templated reactions utilizing a fluorogenic intercalating agent capable of undergoing inverse electron‐demand Diels–Alder (IEDDA) reactions with DNA containing 5‐vinyl‐2′‐deoxyuridine (VdU) or RNA containing 5‐vinyl‐uridine (VU). Reversible high‐affinity intercalation of a novel acridine–tetrazine conjugate “PINK” (KD=5±1 μM) increases the reaction rate of tetrazine–alkene IEDDA on duplex DNA by 60 000‐fold (590 M−1 s−1) as compared to the non‐templated reaction. At the same time, loss of tetrazine–acridine fluorescence quenching renders the reaction highly fluorogenic and detectable under no‐wash conditions. This strategy enables live‐cell dynamic imaging of acridine‐modified nucleic acids in dividing cells.

Phosphorogenic Iridium(III) bis-Tetrazine Complexes for Bioorthogonal Peptide Stapling, Bioimaging, Photocytotoxic Applications, and the Construction of Nanosized Hydrogels.

The dual functionality of 1,2,4,5-tetrazine as a bioorthogonal reactive unit and a luminescence quencher has shaped tetrazine-based probes as attractive candidates for luminogenic labeling of biomolecules in living systems. In this work, three cyclometalated iridium(III) complexes featuring two tetrazine units were synthesized and characterized. Upon photoexcitation, the complexes were non-emissive but displayed up to 3900-fold emission enhancement upon the inverse electron-demand Diels-Alder (IEDDA) [4+2] cycloaddition with (1R,8S,9s)-bicyclo[6.1.0]non-4-yne (BCN) substrates. The rapid reaction kinetics (k2 up to 1.47×104 M-1 s-1 ) of the complexes toward BCN substrates allowed effective peptide labeling. The complexes were also applied as live cell bioimaging reagents and photocytotoxic agents. One of the complexes was utilized in the preparation of luminescent nanosized hydrogels that exhibited interesting cargo delivery properties.

Phosphorogenic Iridium(III) bis‐Tetrazine Complexes for Bioorthogonal Peptide Stapling, Bioimaging, Photocytotoxic Applications, and the Construction of Nanosized Hydrogels

AbstractThe dual functionality of 1,2,4,5‐tetrazine as a bioorthogonal reactive unit and a luminescence quencher has shaped tetrazine‐based probes as attractive candidates for luminogenic labeling of biomolecules in living systems. In this work, three cyclometalated iridium(III) complexes featuring two tetrazine units were synthesized and characterized. Upon photoexcitation, the complexes were non‐emissive but displayed up to 3900‐fold emission enhancement upon the inverse electron‐demand Diels–Alder (IEDDA) [4+2] cycloaddition with (1R,8S,9s)‐bicyclo[6.1.0]non‐4‐yne (BCN) substrates. The rapid reaction kinetics (k2 up to 1.47×104 M−1 s−1) of the complexes toward BCN substrates allowed effective peptide labeling. The complexes were also applied as live cell bioimaging reagents and photocytotoxic agents. One of the complexes was utilized in the preparation of luminescent nanosized hydrogels that exhibited interesting cargo delivery properties.

Remotely controllable supramolecular rotor mounted inside a porphyrinic cage

Remotely controllable supramolecular rotor mounted inside a porphyrinic cage

Inverse electron-demand diels-alder reactions of tetrazine and norbornene at the air-water interface

The surface chemistry of the inverse electron-demand Diels-Alder (IEDDA) reaction at the air-water interface is elucidated. Tetrazine (C18-Tz) and norbornene derivatives (C16-NCA) were used as the reactants. Langmuir monolayers of C18-Tz, C16-NCA, and their binary mixtures were prepared on aqueous substrates. The surface properties were analyzed using the surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms, as well as fluorescence microscopy to monitor the progress of the reaction. First, to provide comparison data to evaluate the reaction on the surface, the two components were mixed in stock solutions of organic solvents for the IEDDA reaction. The Langmuir monolayer spread from the reaction solution was characterized as a function of the reaction time. In the subsequent experiments, the Langmuir monolayers were deposited onto the surface of the substrate solutions by spreading from separate stock solutions of C18-Tz and C16-NCA. The variation of the surface behavior of the monolayers with the molecular area, surface composition of the two components, compression speed of the monolayers, and the temperature was studied. We discuss the effects of the air phase in the reaction field on the reaction efficiency by comparing the results obtained from the two methods.

Computational insights into the inverse electron-demand Diels-Alder reaction of norbornenes with 1,2,4,5-tetrazines: norbornene substituents' effects on the reaction rate.

The study of the reaction rates and mechanism of click chemistry reactions still remains an interesting challenge in organic chemistry. In this regard, the inverse electron demand Diels-Alder (IEDDA) reaction represents a promising metal-free alternative with enhanced reaction rates compared to other reactions of the click chemistry toolbox. Among the different types of dienophiles used in the IEDDA reactions, norbornenes have been widely used given their high stability and fast reaction rates. The inverse electron-demand Diels Alder reaction of 3,6-dipyridin-2-yl-1,2,4,5-tetrazine with a series of norbornene derivatives was studied with quantum mechanical calculations at the M06-2X/6-311+G(d,p) level of theory. The theoretical predictions were confirmed with the experimental data and analyzed with the use of the distortion/interaction model. The obtained results will help in obtaining a better understanding of the factors that affect the relative cycloaddition rates of norbornenes with tetrazines, which are crucial for selectively tuning their efficacy.

Intracellular fluorogenic supramolecular assemblies for self-reporting bioorthogonal prodrug activation.

A visual drug delivery system (DDS) is urgently needed for precision medicine. DDS-mediated bioorthogonal prodrug activation strategies have demonstrated remarkable advantages in enlarging a therapeutic index via the alleviation of adverse drug reactions. However, the events of bioorthogonal prodrug activation remain inaccessible. Here, we construct a self-reporting bioorthogonal prodrug activation system using fluorescence emission to interpret prodrug activation events. In designed reactive oxygen species (ROS)-instructed supramolecular assemblies, the bioorthogonal reaction handle of tetrazine carries a dual role as fluorescence quencher and prodrug activator. The subsequent inverse-electron-demand Diels-Alder (IEDDA) reaction simultaneously liberates fluorescence and active drugs, which form a linear relationship. Differentiated by their cellular redox status, ROS-instructed supramolecular assemblies form selectively in both tumor cells and cell spheroids. Upon prodrug treatment, the brightness of fluorescence reflects the liberation of active drugs, which further correlates with the cell survival rate. Therefore, a fluorescence-based visualizable DDS (VDDS) for bioorthogonal prodrug activation is demonstrated, which should be useful to elucidate the multi-step processes in drug delivery and determine prodrug activation efficacy.

Enantioselective construction of cis-hydroindole scaffolds via an asymmetric inverse-electron-demand Diels-Alder reaction: application to the formal total synthesis of (+)-minovincine.

cis-Hydroindole scaffolds widely exist in a large number of natural products, pharmaceuticals, and organocatalysts. Therefore, the development of efficient and enantioselective methods for the construction of cis-hydroindoles is of great interest and importance. Herein, a novel approach for the enantioselective synthesis of cis-hydroindole scaffolds has been realized through a chiral N,N′-dioxide/Mg(OTf)2 complex catalyzed asymmetric inverse-electron-demand Diels–Alder (IEDDA) reaction of 2-pyrones and cyclic enamines. A series of substituted cis-hydroindole derivatives bearing multiple contiguous stereocenters and functional groups were obtained in good to excellent yields and enantioselectivities (up to 99% yield, and 95% ee) under mild reaction conditions. Moreover, the enantioselective formal total synthesis of (+)-minovincine was concisely furnished with high efficiency and stereoselectivity to demonstrate the synthetic potential of this method.

Side-by-Side Comparison of Five Chelators for 89Zr-Labeling of Biomolecules: Investigation of Chemical/Radiochemical Properties and Complex Stability.

Simple SummaryThe positron emitter 89Zr4+ is an important radionuclide for the preparation of radiolabeled antibodies, being applied in highly specific and sensitive positron emission tomography (PET) imaging of malignancies. The introduction of 89Zr4+ into biomolecules is performed using chelating agents, wrapping up the radiometal and preventing its release from the antibody by forming so-called complexes. Desferrioxamine B (DFO) is the clinical gold standard chelator for the preparation of 89Zr antibodies despite its known inability to stably encapsulate the radiometal, resulting in 89Zr release and associated challenges such as decreased image quality and radiation dose to healthy tissues. Therefore, several research groups have been working to develop new chelating agents able to stably encapsulate the 89Zr4+ ion. However, there are no data available directly comparing the stability of the formed 89Zr complexes of the most promising chelating agents developed so far. Here, we report on the comparison of five different chelators with high potential for stable complexation of 89Zr and determined two of them—DFO* and 3,4,3-(LI-1,2-HOPO)—to be highly interesting for the preparation of 89Zr-based radiolabeled agents and routine clinical application.In this work, five different chelating agents, namely DFO, CTH-36, DFO*, 3,4,3-(LI-1,2-HOPO) and DOTA-GA, were compared with regard to the relative kinetic inertness of their corresponding 89Zr complexes to evaluate their potential for in vivo application and stable 89Zr complexation. The chelators were identically functionalized with tetrazines, enabling a fully comparable, efficient, chemoselective and biorthogonal conjugation chemistry for the modification of any complementarily derivatized biomolecules of interest. A small model peptide of clinical relevance (TCO-c(RGDfK)) was derivatized via iEDDA click reaction with the developed chelating agents (TCO = trans-cyclooctene and iEDDA = inverse electron demand Diels-Alder). The bioconjugates were labeled with 89Zr4+, and their radiochemical properties (labeling conditions and efficiency), logD(7.4), as well as the relative kinetic inertness of the formed complexes, were compared. Furthermore, density functional theory (DFT) calculations were conducted to identify potential influences of chelator modification on complex formation and geometry. The results of the DFT studies showed—apart from the DOTA-GA derivative—no significant influence of chelator backbone functionalization or the conjugation of the chelator tetrazines by iEDDA. All tetrazines could be efficiently introduced into c(RGDfK), demonstrating the high suitability of the agents for efficient and chemoselective bioconjugation. The DFO-, CTH-36- and DFO*-modified c(RGDfK) peptides showed a high radiolabeling efficiency under mild reaction conditions and complete 89Zr incorporation within 1 h, yielding the 89Zr-labeled analogs as homogenous products. In contrast, 3,4,3-(LI-1,2-HOPO)-c(RGDfK) required considerably prolonged reaction times of 5 h for complete radiometal incorporation and yielded several different 89Zr-labeled species. The labeling of the DOTA-GA-modified peptide was not successful at all. Compared to [89Zr]Zr-DFO-, [89Zr]Zr-CTH-36- and [89Zr]Zr-DFO*-c(RGDfK), the corresponding [89Zr]Zr-3,4,3-(LI-1,2-HOPO) peptide showed a strongly increased lipophilicity. Finally, the relative stability of the 89Zr complexes against the EDTA challenge was investigated. The [89Zr]Zr-DFO complex showed—as expected—a low kinetic inertness. Unexpectedly, also, the [89Zr]Zr-CTH-36 complex demonstrated a high susceptibility against the challenge, limiting the usefulness of CTH-36 for stable 89Zr complexation. Only the [89Zr]Zr-DFO* and the [89Zr]Zr-3,4,3-(LI-1,2-HOPO) complexes demonstrated a high inertness, qualifying them for further comparative in vivo investigation to determine the most appropriate alternative to DFO for clinical application.