Inverse Electron Demand Diels-Alder Reaction Research Articles

Click'n lock: rapid exchange between unsymmetric tetrazines and thiols for reversible, chemoselective functionalisation of biomolecules with on-demand bioorthogonal locking.

The late-stage functionalisation and diversification of complex structures including biomolecules is often achieved with the help of click chemistry. Besides employing irreversible click-like reactions, many synthetic applications benefit from reversible click reaction strategies, so called de-/trans-click approaches. Yet, the combination of both, reversible and irreversible click chemistry - while still respecting the stringent criteria of click transformations - remains so far elusive for modifications of biomolecular structures. Here, we report click'n lock as a concept that enables reversible click reactions and on-demand locking of chemical entities, thus switching from reversible to irreversible modifications of complex biomolecules. For this purpose, we employ the tetrazine-thiol exchange (TeTEx) reaction as a fully traceless click reaction with second order rate constants k2 higher than 2 M-1 s-1 within aqueous environments. Employing TeTEx as a reversible click reaction for the chemoselective modification of biomolecules is made possible by the use of 3,6-disubstituted 1,2,4,5-tetrazines bearing a single sulfide residue. The inherent reactivity of tetrazines towards inverse electron demand Diels-Alder (IEDDA) reactions allows to stabilize the clicked structure, switching from reversible to irreversible systems (click'n lock).

Toward Precise Antitumoral Photodynamic Therapy Using a Dual Receptor-Mediated Bioorthogonal Activation Approach.

Targeted delivery and specific activation of photosensitizers can greatly improve the treatment outcome of photodynamic therapy. To this end, we report herein a novel dual receptor-mediated bioorthogonal activation approach to enhance the tumor specificity of the photodynamic action. It involves the targeted delivery of a biotinylated boron dipyrromethene (BODIPY)-based photosensitizer, which is quenched in the native form by the attached 1,2,4,5-tetrazine unit, and an epidermal growth factor receptor (EGFR)-targeting cyclic peptide conjugated with a bicycle[6.1.0]non-4-yne moiety. Only for cancer cells that overexpress both the biotin receptor and EGFR, the two components can be internalized preferentially where they undergo an inverse electron-demand Diels-Alder reaction, leading to restoration of the photodynamic activity of the BODIPY core. By using a range of cell lines with different expression levels of these two receptors, we have demonstrated that this stepwise "deliver-and-click" approach can confine the photodynamic action on a specific type of cancer cells.

Making functional connections: A summary of the Nobel Prize in Chemistry 2022

<p indent="0mm">The Nobel Prize in Chemistry 2022 was awarded to three scientists for their work in the development of click chemistry and bioorthogonal chemistry: Carolyn R. Bertozzi from Stanford University, Morten Meldal from the University of Copenhagen, and K. Barry Sharpless from Scripps Research. Notably, this is the second time Sharpless was awarded this prize. In 2001, he received his first Nobel Prize for the development of catalytic asymmetric oxidation reactions. This year’s prize, however, is in a completely different area, as will be summarized below. For both click chemistry and bioothogonal chemistry, their focus is on making easy connections between molecules and snapping them together. Although modern chemistry has experienced substantial development since its birth in the eighteenth century, dealing with complex molecules and systems remains a challenging and laborious process that requires skill and knowledge. Synthesis has become the bottleneck for the discovery of molecular functions. In 2001, Sharpless and co-workers coined the concept of click chemistry, by defining a set of stringent criteria so that few good reactions could be sorted out for making easy and modular connections: Molecules could be linked together simply like buckling up through a “click”. Later in 2002, Sharpless group published the first click-type reaction, namely the copper-catalyzed azide-alkyne cycloaddition (CuAAC). The reaction is silent without catalyst due to the high kinetic barrier, but become as fast as a “click” to generate triazoles in the presence of copper(I) with a strong thermodynamic driving force. Independently, Meldal made a similar discovery during the peptide synthesis. Over the years, CuAAC has proved itself to be a reliable tool for connecting chemical units of various forms together, nearly under any circumstances with high fidelity. In the late 1990s, in Bertozzi’s search for chemical tools for sugar labeling on the surface of living cell, she realized that the key for successful utilization of a chemical handle was the non-interfering nature of the involved reactions with the surrounding biological environments. She termed this kind of non-interfering reactivity as bioothogonal chemistry. By metabolically incorporating unnatural sialic acids to the cell surface, including the azide derivatives, Bertozzi successfully used Staudinger reaction to attach imaging probes on the cell with amide connections. The iconic breakthrough took place in 2004 when click chemistry was merged with bioothogonal chemistry for the first time. Bertozzi developed a stain-promoted azide-alkyne cycloaddition (SPAAC), which gave improved connection capability for biorthogonal cell imaging without the need of a catalyst, nor any compromise on bioorthogonality. The impact of click chemistry and bioorthogonal chemistry is tremendous and ever-growing. They have inspired the innovation of many connection tools, such as the inverse electron-demand Diels-Alder reaction, photoclick chemistry, and the sulfur(VI)-fluoride exchange (SuFEx) reaction. Apart from serving as tools for tracking and manipulating biomolecules and related events <italic>in vivo</italic>, they have also been used for material sciences and medicinal chemistry. For instance, sacituzumab govitecan is a Trop-2-directed antibody and topoisomerase inhibitor drug conjugate approved by the FDA for the treatment of metastatic triple-negative breast cancer and metastatic urothelial cancer. The key linkage is a CuAAC-derived 1,2,3-triazole. In short, there remain a lot of challenges in chemistry and the related fields to be addressed through diverse strategies and chemical approaches nowadays. The mission of chemistry is to bring the greatest benefit to mankind through continuous innovation and integration.

Pyridazine Synthesis from 1,2,4,5-Tetrazines and Alkynes in 1,1,1,3,3,3-Hexafluoro-2-propanol through the Inverse Electron Demand Diels–Alder Reaction

Abstract A facile method to synthesize pyridazines from 1,2,4,5-tetrazines and alkynes is disclosed. The reaction was significantly facilitated by 1,1,1,3,3,3-hexafluoro-2-propanol. The wide scope and the predictable regioselectivity by theoretical calculations will serve to synthesize a wide range of multisubstituted pyridazines.

First Synthesis of 3-Glycopyranosyl-1,2,4-Triazines and Some Cycloadditions Thereof.

C-glycopyranosyl derivatives of six-membered heterocycles are scarcely represented in the chemical literature and the title 3-glycopyranosyl-1,2,4-triazines are completely unknown. In this paper, the first synthesis of this compound class is accomplished by the cyclocondensation of C-glycosyl formamidrazones and 1,2-dicarbonyl derivatives. In addition, the synthesis of C-glycopyranosyl 1,2,4-triazin-5(4H)-ones was also carried out by the transformation of the above formamidrazones with α-keto-carboxylic esters. Inverse electron demand Diels-Alder reactions of 3-glycopyranosyl-1,2,4-triazines with a bicyclononyne derivative yielded the corresponding annulated 2-glycopyranosyl pyridines.

The fullerene awakens

Recently in <i>Angewandte Chemie</i>, Kim and co-workers employed a large Zn-porphyrin paneled cage to activate C₆₀ toward inverse electron-demand Diels-Alder reaction with a tetrazine through precise pre-organization of the reaction partners. Subsequent hydration-induced rearrangement resulted in displacement of the reaction product, enabling catalytic turnover.

Application of "Click" Chemistry in Biomedical Hydrogels.

Since “click” chemistry was first reported in 2001, it has remained a popular research topic in the field of chemistry due to its high yield without byproducts, fast reaction rate, simple reaction, and biocompatibility. It has achieved good applications in various fields, especially for the preparation of hydrogels. The development of biomedicine presents new challenges and opportunities for hydrogels, and “click” chemistry provides a library of chemical tools for the preparation of various innovative hydrogels, including cell culture, 3D bioprinting, and drug release. This article summarizes several common “click” reactions, including copper-catalyzed azide–alkyne cycloaddition reactions, strain-promoted azide–alkyne cycloaddition (SPAAC) reaction, thiol–ene reaction, the Diels–Alder reaction, and the inverse electron demand Diels–Alder (IEDDA) reaction. We introduce the “click” reaction in the nucleic acid field to expand the concept of “click” chemistry. This article focuses on the application of “click” chemistry for preparing various types of biomedical hydrogels and highlights the advantages of “click” reactions for cross-linking to obtain hydrogels. This review also discusses applications of “click” chemistry outside the field of hydrogels, such as drug synthesis, targeted delivery, and surface modification, hydrogels have great application potential in these fields in the future and hopefully inspire other applications of hydrogels.

Dramatically Enhanced Reactivity of Fullerenes and Tetrazine towards the Inverse-Electron-Demand Diels-Alder Reaction inside a Porous Porphyrinic Cage.

Inverse-electron-demand Diels-Alder reaction (IEDDA) between fullerenes and 1,2,4,5-tetrazine generally requires harsh conditions and long reaction times due to their strong electron-accepting nature. Herein, we report a dramatic enhancement in the reactivity of the fullerenes (C60 /C70 )-tetrazine reaction inside a porous Zn-porphyrinic cage (Zn-PB) under sustainable conditions by installing a tetrazine-based axle (LA) via metal-ligand coordination bond, which modulates the cavity size to facilitate the encapsulation of fullerenes. Upon encapsulation, the close proximity of fullerenes and the tetrazine group of LA dramatically increase their reactivity towards the IEDDA reaction to form fullerene-tetrazine adducts. Furthermore, the C60 -tetrazine adduct is rearranged upon hydration to a bent-shaped C60 -pyrazoline adduct that can be released from the Zn-PB cavity in the presence of excess LA, thus catalyzing the formation of C60 -pyrazoline adduct inside Zn-PB without product inhibition.

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.