Inverse Electron Demand Diels-Alder Reaction Research Articles

Divergent Reactivity of Phosphorylated and Related Allenes: [4 + 2] Cycloaddition with 3,6-Diphenyltetrazine, Self-Addition Leading to Dimers and [Pd]-Complex Formation.

Phosphorus-based naphthalenes are formed by self-dimerization-cum-cyclization of α-aryl allenylphosphonates or allenylphosphine oxides using catalytic Pd(OAc)2in the presence of PPh3 and Et3N . This reaction involves [4 + 2]-cycloaddition with the (β,γ) double bond of one allene as the dienophile; the double bonds at the α-aryl-(β',γ') group and (α,β)-carbons of the second allene act as the diene part. A subsequent proton shift also takes place. Upon treating allenylphosphine oxides with Pd(OAc)2 [stoichiometry 2:1] in the presence of PPh3/Ag2CO3, a [Pd]-complex is isolated and structurally characterized. This complex can be used as a catalyst for C-C bond-forming reactions of phosphorus-based allenes with 2-iodophenol. Densely substituted 3,6-diphenylpyridazines are conveniently obtained in excellent yields by a thermally induced regioselective Inverse Electron Demand Diels-Alder (IEDDA) reaction of allenes with 3,6-diphenyltetrazine, followed by a [1,3]-H shift.

Investigation of the Impact of Hydrolytically Cleavable Groups on the Stability of Poly(ethylene glycol) Based Hydrogels Cross-Linked via the Inverse Electron Demand Diels-Alder (iEDDA) Reaction.

Eight-armed poly(ethylene glycol) (PEG) hydrogels cross-linked via inverse electron demand Diels-Alder reaction between norbornene and tetrazine groups are promising materials for long-term protein delivery. While a controlled release over 265 days is achieved for 15% w/v hydrogels in the previous study, the material shows high stability over 500 days despite having cleavable ester linkages between the PEG macromonomers and their functionalities. In this study, the hydrolyzable ester linkers in the PEG-norbornene precursor structure are exchanged to reduce the degradation time. To this end, 3,6-epoxy-1,2,3,6-tetrahydrophthalimide, phenyl carbamate, carbonate ester, and phenyl carbonate ester are introduced as degradable functional groups. Oscillatory shear experiments reveal that they are not affected the in situ gelation. All hydrogel types have gel points of less than 20 s even at a low polymer concentration of 5% w/v. Hydrogels with varying polymer concentrations have similar mesh sizes, all of which fell in the range of 4-12nm. The inclusion of phenyl carbonate ester accelerates degradation considerably, with complete dissolution of 15% w/v hydrogels after 302 days of incubation in phosphate buffer (pH 7.4). Controlled release of 150kDa fluorescein isothiocyanate-dextran over a period of at least 150 days is achieved with 15% w/v hydrogels.

Preclinical Evaluation of hnRNPA2B1 Antibody in Human Triple-Negative Breast Cancer MDA-MB-231 Cells via PET Imaging.

Triple-negative breast cancer (TNBC) does not express estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Because TNBC lacks the expression of commonly targeted receptors, it is challenging to develop a new imaging agent for this cancer subtype. Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA–protein complexes that have been linked to tumor development and progression. Considering the high expression of hnRNPA2B1, an hnRNP subtype, in TNBC MDA-MB-231 cells, this study aimed to develop a novel hnRNPA2B1 antibody-based nuclear imaging agent. The hnRNPA2B1-specific antibody was radiolabeled with 64Cu and evaluated in vitro and in vivo. The trans-cyclooctene (TCO) was functionalized on the antibody to obtain hnRNP-PEG4-TCO and reactive tetrazine (Tz) on the ultrastable bifunctional chelator PCB-TE2A-alkyne to yield PCB-TE2A-Tz for the inverse electron demand Diels–Alder reaction. The 64Cu-radiolabeled antibody was administered and imaged at 1–18 h time points for conventional imaging. Alternatively, the unlabeled antibody conjugate was administered, and 48 h later radiolabeled 64Cu-PCB-TE2A-Tz was administered to the same mice for the pretargeting strategy and imaged at the same time intervals for direct comparison. The tumor was successfully visualized in both strategies, and comparatively, pretargeting showed superior results. The 64Cu-PCB-TE2A-Tz was successfully clicked at the tumor site with hnRNP-PEG4-TCO and the non-clicked were concurrently eliminated. This led to increase the tumor uptake with extremely high tumor-to-background ratio manifested by positron emission tomography (PET) imaging and biodistribution studies.

Live-Cell Imaging of Sterculic Acid-a Naturally Occurring 1,2-Cyclopropene Fatty Acid-by Bioorthogonal Reaction with Turn-On Tetrazine-Fluorophore Conjugates.

In the field of lipid research, bioorthogonal chemistry has made the study of lipid uptake and processing in living systems possible, whilst minimising biological properties arising from detectable pendant groups. To allow the study of unsaturated free fatty acids in live cells, we here report the use of sterculic acid, a 1,2‐cyclopropene‐containing oleic acid analogue, as a bioorthogonal probe. We show that this lipid can be readily taken up by dendritic cells without toxic side effects, and that it can subsequently be visualised using an inverse electron‐demand Diels–Alder reaction with quenched tetrazine‐fluorophore conjugates. In addition, the lipid can be used to identify changes in protein oleoylation after immune cell activation. Finally, this reaction can be integrated into a multiplexed bioorthogonal reaction workflow by combining it with two sequential copper‐catalysed Huisgen ligation reactions. This allows for the study of multiple biomolecules in the cell simultaneously by multimodal confocal imaging.

Reduction-responsive and bioorthogonal carboxymethyl cellulose based soft hydrogels cross-linked via IEDDA click chemistry for cancer therapy application

In this work, novel biocompatible and reduction-responsive soft hydrogels were formulated from norbornene (Nb)-functionalized carboxymethyl cellulose (CMCNb). To cross-link the CMC-Nb via a highly bioorthogonal inverse electron demand Diels–Alder (IEDDA) reaction, we employed a water-soluble and reduction-responsive diselenide-based cross-linker possessing two terminal tetrazine (Tz) groups with varying molar concentrations (Nb/Tz molar ratios of 10/10, 10/05, and 10/2.5). The N2 microbubbles liberated as a by-product during the IEDDA reaction generated in-situ pores in hydrogel networks. The resulting hydrogels had highly porous structures and relatively soft mechanical properties (storage moduli in the range 74 ⁓160 Pa). The hydrogels showed high swelling ratios (>35 times), tunable gelation times (1–5 min), and excellent doxorubicin (DOX) loading efficiencies (>85 %). The hydrogels exhibited stimuli-responsive and fast release of DOX (99 %, after 12 h) in the presence of 10 mmol of glutathione as compared to the normal PBS solution (38 %). The cytotoxic effects of blank hydrogels were not observed against HEK-239 cells, while the DOX-encapsulated hydrogels exhibited anti-tumor activity in BT-20 cancer cells. The results indicate potential applications of the CMC-based soft hydrogels in injectable drug delivery systems.

The inverse-electron demand Diels–Alder reaction of tetrazines with cyclic enol ethers

Inverse electron-demand Diels–Alder additions (iEDDA) between 1,2,4,5-tetrazines and suitable unsaturated dienophiles such as olefins, alkynes, or enol ethers provide facile access to pyridazines. Herein the use of cyclic enol ether derivatives for preparing pyridazines bearing 2-hydroxyethyl, 3-hyproxypropyl, and 3-oxopropyl substituents at the 4-position is disclosed and second order rate constants for the reactions with 2,3-dihydrofuran, 3,4-dihydro-2H-pyran, and 2-methoxy-3,4-dihydro-2H-pyran are presented.Graphical abstract

Bridgehead Alkene-Enabled Strain-Driven Bioorthogonal Reaction.

Herein, we report a novel bioorthogonal reaction that hinges on a bridgehead alkene (BHA)-enabled inverse-electron-demand Diels-Alder (IEDDA) cycloaddition. Readily accessible from natural product β-caryophyllene, the strained BHA displays high reactivity toward the IEDDA reaction while maintaining excellent biocompatibility. The developed IEDDA reaction has been applied to in vitro protein labeling and pretargeted live cell imaging.

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.

Inverse-electron demand Diels Alder Reactions between glycals and tetrazines

The inverse-electron demand Diels Alder reaction (IEDDA) of substituted tetrazines with 2,3-unsaturated sugars (glycals) has been investigated to prepare novel carbohydrate-based heterocycles. The cycloaddition reactions occurred in moderate to good, isolated yields and gave acyclic, C-linked pyranose diazines as the major products (33–90%). The effects of variations in sugars, sugar protecting groups, and reaction solvents on the yields and products obtained in these reactions were studied. Lower yields of adducts were isolated for TBDMS-protected glucals and for 4,6-O-benzylidene protected glucals. When unprotected sugars were used, the reactions failed to give the desired cycloadducts. A range of substituted tetrazines were also evaluated in these reactions. For comparison, HOMO-[LUMO + 1] gaps for glycal-tetrazine pairs were calculated using Density Functional (DFT) calculations at the B3LYP/631G+ level.

A Dehydrogenative Inverse Electron Demand Diels-Alder Reaction for the Synthesis of Functionalized Pyranones.

An efficient dehydrogenative inverse electron demand Diels-Alder reaction of isopropyl and prenyl benzene derivatives with electron-deficient dienes followed by decarboxylation has been reported for the first time. The much broader substrate scope of dienophiles and electron-deficient dienes led to biologically valuable pyranones in good to excellent yields.

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.

Copper Catalyzed Inverse Electron Demand [4+2] Cycloaddition for the Synthesis of Oxazines

A copper catalyzed tandem CuAAC/ring cleavage/[4+2] annulation reaction of terminal ynones, sulfonyl azides, and imines has been developed to synthesize the functionalized oxazines under mild conditions. Particularly, the intermediate N-sulfonyl acylketenimines undergo cycloaddition of an inverse electron demand Diels–Alder reaction with imines and a series of 1,3-oxazine derivatives were obtained successfully in good yields.

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.

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.

Synthesis of Multiple Bispecific Antibody Formats with Only One Single Enzyme Based on Enhanced Trypsiligase.

Bispecific antibodies (bsAbs) were first developed in the 1960s and are now emerging as a leading class of immunotherapies for cancer treatment with the potential to further improve clinical efficacy and safety. Many different formats of bsAbs have been established in the last few years, mainly generated genetically. Here we report on a novel, flexible, and fast chemo–enzymatic, as well as purely enzymatic strategies, for generating bispecific antibody fragments by covalent fusion of two functional antibody Fab fragments (Fabs). For the chemo–enzymatic approach, we first modified the single Fabs site-specifically with click anchors using an enhanced Trypsiligase variant (eTl) and afterward converted the modified Fabs into the final heterodimers via click chemistry. Regarding the latter, we used the strain-promoted alkyne-azide cycloaddition (SPAAC) and inverse electron-demand Diels–Alder reaction (IEDDA) click approaches well known for their fast reaction kinetics and fewer side reactions. For applications where the non-natural linkages or hydrophobic click chemistry products might interfere, we developed two purely enzymatic alternatives enabling C- to C- and C- to N-terminal coupling of the two Fabs via a native peptide bond. This simple system could be expanded into a modular system, eliminating the need for extensive genetic engineering. The bispecific Fab fragments (bsFabs) produced here to bind the growth factors ErbB2 and ErbB3 with similar KD values, such as the sole Fabs. Tested in breast cancer cell lines, we obtained biologically active bsFabs with improved properties compared to its single Fab counterparts.

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.