Tetrazine-trans-cyclooctene ligation: Unveiling the chemistry and applications within the human body.

Bioorthogonal reactions have revolutionized chemical biology by enabling selective chemical transformations within living organisms and cells. This review comprehensively explores bioorthogonal chemistry, emphasizing inverse-electron-demand Diels-Alder (IEDDA) reactions between tetrazines and strained dienophiles and their crucial role in chemical biology and various applications within the human body. This highly reactive and selective reaction finds diverse applications, including cleaving antibody-drug conjugates, prodrugs, proteins, peptide antigens, and enzyme substrates. The versatility extends to hydrogel chemistry, which is crucial for biomedical applications, yet it faces challenges in achieving precise cellularization. In situ activation of cytotoxic compounds from injectable biopolymer belongs to the click-activated protodrugs against cancer (CAPAC) platform, an innovative approach to tumor-targeted prodrug delivery and activation. The CAPAC platform, relying on click chemistry between trans-cyclooctene (TCO) and tetrazine-modified biopolymers, exhibits modularity across diverse tumor characteristics, presenting a promising approach in anticancer therapeutics. The review highlights the importance of bioorthogonal reactions in developing radiopharmaceuticals for positron emission tomography (PET) imaging and theranostics, offering a promising avenue for diverse therapeutic applications.

Synthesis and biological evaluation of novel schisanhenol derivatives as potential hepatoprotective agents.

Twenty-one new schisanhenol derivatives were synthesized, and their hepatoprotective effects against liver injury induced by concanavalin A (Con A) were evaluated in vitro using an MTT assay. The data indicated that most derivatives exhibited equivalent or better protective activity than the positive control (dimethyl dicarboxylate biphenyl, DDB) under the same conditions. Among them, compound 1b showed the most potent hepatoprotective activity against Con A-induced immunological injury. Mechanistic studies in vitro revealed that 1b inhibited cell apoptosis and inflammatory responses caused by Con A treatment via IL-6/JAK2/STAT3 signaling pathway. Consistently, it also exhibited significant hepatoprotective activity in mice with Con A-induced immunological liver injury. These results clearly indicated that 1b might be a highly potent hepatoprotective agent targeting IL-6/STAT3 signaling pathway.

Inhibition of amyloid formation of amyloid ß (1-42), amylin and insulin by 1,5-diazacyclooctanes, a spermine-acrolein conjugate.

Amyloid aggregates of proteins are known to be involved in various diseases such as Alzheimer's disease (AD). It is therefore speculated that the inhibition of amyloid formation can play an important role in the prevention of various diseases involving amyloids. Recently, we have found that acrolein reacts with polyamines, such as spermine, and produces 1,5-diazacyclooctane, such as cyclic spermine (cSPM). cSPM could suppress the aggregation of amyloid ß 1-40 (Aß40), one of the causative proteins of AD. This result suggests the potential inhibitory effect of cSPM against Aß 1-42 (Aß42) and other amyloid protein aggregation which are the main pathological features of AD and other diseases. However, the effect on the aggregation of such proteins remains unclear. In this study, the effect of cSPM on the amyloid formation of Aß42, amylin, and insulin was investigated. These three amyloidogenic proteins forming amyloids under physiological conditions (pH 7.4 and 37℃) served as model and are thought to be the causative proteins of AD, type 2 diabetes, and insulin-derived amyloidosis, respectively. Our results indicate that cSPM can suppress the amyloid aggregation of these proteins and reduce cytotoxicity. This study contributes to a better understanding of means to potentially counteract diseases by the means of polyamine and acrolein.

Dibenzocyclooctadiene lignans from Kadsura coccinea alleviate APAP-induced hepatotoxicity via oxidative stress inhibition and activating the Nrf2 pathway in vitro.

Phytochemical investigation on the roots of Kadsura coccinea led to the isolation five previously unknown dibenzocyclooctadiene lignans, named heilaohusuins A-E (1-5). Their structures determined by NMR spectroscopy, HR-ESI-MS, and ECD spectra. Hepatoprotection effects of a series of dibenzocyclooctadiene derivatives (1-68) were investigated against acetaminophen (APAP) induced HepG2 cells. Compounds 2, 10, 13, 21, 32, 41, 46, and 49 showed remarkable protective effects, increasing the viabilities to > 52.2% (bicyclol, 52.1 ± 1.3%) at 10 µM. The structure-activity relationships (SAR) for hepatoprotective activity were summarized, according to the activity results of dibenzocyclooctadiene derivatives. Furthermore, we found that one new dibenzocyclooctadiene lignan heilaohusuin B attenuates hepatotoxicity, the mechanism might be closely correlated with oxidative stress inhibition via activating the Nrf2 pathway.

Design, Synthesis, Conjugation, and Reactivity of Novel trans,trans-1,5-Cyclooctadiene-Derived Bioorthogonal Linkers.

The tetrazine/trans-cyclooctene (TCO) inverse electron-demand Diels-Alder (IEDDA) reaction is the fastest bioorthogonal "click" ligation process reported to date. In this context, TCO reagents have found widespread applications; however, their availability and structural diversity is still somewhat limited due to challenges connected with their synthesis and structural modification. To address this issue, we developed a novel strategy for the conjugation of TCO derivatives to a biomolecule, which allows for the creation of greater structural diversity from a single precursor molecule, i.e., trans,trans-1,5-cyclooctadiene [(E,E)-COD] 1, whose preparation requires standard laboratory equipment and readily available reagents. This two-step strategy relies on the use of new bifunctional TCO linkers (5a-11a) for IEDDA reactions, which can be synthesized via 1,3-dipolar cycloaddition of (E,E)-COD 1 with different azido spacers (5-11) carrying an electrophilic function (NHS-ester, N-succinimidyl carbonate, p-nitrophenyl-carbonate, maleimide) in the ω-position. Following bioconjugation of these electrophilic linkers to the nucleophilic residue (cysteine or lysine) of a protein (step 1), the resulting TCO-decorated constructs can be subjected to a IEDDA reaction with tetrazines functionalized with fluorescent or near-infrared (NIR) tags (step 2). We successfully used this strategy to label bovine serum albumin with the TCO linker 8a and subsequently reacted it in a cell lysate with the fluorescein-isothiocyanate (FITC)-derived tetrazine 12. The same strategy was then used to label the bacterial wall of Gram-positive Staphylococcus aureus, showing the potential of these linkers for live-cell imaging. Finally, we determined the impact of structural differences of the linkers upon the stability of the bioorthogonal constructs. The compounds for stability studies were prepared by conjugation of TCO linkers 6a, 8a, and 10a to mAbs, such as Rituximab and Obinutuzumab, and subsequent labeling with a reactive Cy3-functionalized tetrazine.

Synthesis and biological evaluation of Schizandrin derivatives as tubulin polymerization inhibitors.

A series of oxime ester-derivatives were prepared by utilizing the schizandrin (1), a major compound isolated from Schisandra grandiflora, which is deployed in different traditional system of medicine. The in vitro antiproliferative activities of the synthesized compounds were assessed against a selected panel of human cancer cell lines (A549, RKO P3, DU145 and Hela) and normal cell (HEK293). Several of these derivatives were found more potent in comparison to parent compound, schizandrin (1). Particularly, 4a and 4b demonstrated potent activity against DU-145 and RKOP3 cell lines with IC50 values of 3.42 µM and 3.35 µM respectively. To characterize the molecular mechanisms involved in antitumoral activity, these two compounds, 4a and 4b were selected for further studies. Cell cycle analysis revealed that both the compounds were able to induce apoptosis and cell cycle arrest at G0/G1 phase. To know the extent of apoptosis in DU145 and RKOP3 cell lines, Annexin V-FITC were performed. Moreover, the tubulin polymerization assay indicated that 4a and 4b exhibits potent inhibitory effect on the tubulin assembly. Molecular docking studies and competitive binding assay also indicated that 4a and 4b effectively bind at the colchicine binding site of the tubulin.

A bioorthogonally activatable photosensitiser for site-specific photodynamic therapy.

A boron dipyrromethene based photosensitiser substituted with a 1,2,4,5-tetrazine moiety has been prepared of which the photoactivity can be activated upon an inverse-electron-demand Diels-Alder reaction with trans-cyclooctene derivatives. By using a biotin-conjugated trans-cyclooctene to tag the biotin-receptor-positive HeLa cells, this photosensitiser exhibits site-specific activation through cycloaddition, leading to high photocytotoxicity.

Cell surface clicking of antibody-recruiting polymers to metabolically azide-labeled cancer cells.

Triggering antibody-mediated innate immune mechanisms to kill cancer cells is an attractive therapeutic avenue. In this context, recruitment of endogenous antibodies to the cancer cell surface could be a viable alternative to the use of monoclonal antibodies. We report on antibody-recruiting polymers containing multiple antibody-binding hapten motifs and cyclooctynes that can covalently conjugate to azides introduced onto the glycocalyx of cancer cells by metabolic labeling with azido sugars.

Macrophage cell tracking PET imaging using mesoporous silica nanoparticles via in vivo bioorthogonal F-18 labeling.

We introduce an efficient cell tracking imaging protocol using positron emission tomography (PET). Since macrophages are known to home and accumulate in tumor tissues and atherosclerotic plaque, we design a PET imaging protocol for macrophage cell tracking using aza-dibenzocyclooctyne-tethered PEGylated mesoporous silica nanoparticles (DBCO-MSNs) with the short half-life F-18-labeled azide-radiotracer via an in vivo strain-promoted alkyne azide cycloaddition (SPAAC) covalent labeling reaction inside macrophage cells in vivo. This PET imaging protocol for in vivo cell tracking successfully visualizes the migration of macrophage cells into the tumor site by the bioorthogonal SPAAC reaction of DBCO-MSNs with [18F]fluoropentaethylene glycolic azide ([18F]2) to form 18F-labeled aza-dibenzocycloocta-triazolic MSNs (18F-DBCOT-MSNs) inside RAW 264.7 cells. The tissue radioactivity distribution results were consistent with PET imaging findings. In addition, PET images of atherosclerosis in ApoE-/- mice fed a western diet for 30 weeks were obtained using the devised macrophage cell-tracking protocol.

The efficiency of 18F labelling of a prostate specific membrane antigen ligand via strain-promoted azide-alkyne reaction: reaction speed versus hydrophilicity.

Here we report the 18F labeling of a prostate specific membrane antigen (PSMA) ligand via a strain promoted oxa-dibenzocyclooctyne (ODIBO)- or bicyclo[6.1.0]nonyne (BCN)-azide reaction. Although ODIBO reacts with azide 20 fold faster than BCN, in vivo PET imaging suggests that 18F-BCN-azide-PSMA demonstrated much higher tumor uptake and a much higher tumor to background contrast.

A Compact and Synthetically Accessible Fluorine-18 Labelled Cyclooctyne Prosthetic Group for Labelling of Biomolecules by Copper-Free Click Chemistry.

A new fluorine-containing azadibenzocyclooctyne (ADIBO-F) was designed using a synthetically accessible pathway. The fluorine-18 prosthetic group was prepared from its toluenesulfonate precursor and isolated in 21-35 % radiochemical yield in 30 minutes of synthetic time. ADIBO-F has been incorporated into azide-functionalized, cancer-targeting peptides through a strain-promoted alkyne-azide cycloaddition with high radiochemical yields and purities. The final products are novel peptide-based positron emission tomography (PET) imaging agents that possess high affinities for their targets, growth hormone secretagogue receptor 1a (GHSR-1a) and gastrin-releasing peptide receptor (GRPR), with IC50 values of 9.7 and 0.50 nm, respectively. This is a new and rapid labelling option for the incorporation of fluorine-18 into biomolecules for PET imaging.

Biomarker-Based Metabolic Labeling for Redirected and Enhanced Immune Response.

Installation of an antibody-recruiting moiety on the surface of disease-relevant cells can lead to the selective destruction of targets by the immune system. Such an approach can be an alternative strategy to traditional chemotherapeutics in cancer therapy and possibly other diseases. Herein we describe the development of a new strategy to selectively label targets with an antibody-recruiting moiety through its covalent and stable installation, complementing existing methods of employing reversible binding. This is achieved through selective delivery of 1,3,4- O-acetyl- N-azidoacetylmannosamine (Ac3ManNAz) to folate receptor-overexpressing cells using an Ac3ManNAz-folate conjugate via a cleavable linker. As such, Ac3ManNAz is converted to cell surface glycan bearing an azido group, which serves as an anchor to introduce l-rhamnose (Rha), a hapten, via a click reaction with aza-dibenzocyclooctyne (DBCO)-Rha. We tested this method in several cell lines including KB, HEK-293, and MCF7 and were able to demonstrate the following: 1) Rha can be selectively installed to the folate receptor overexpressing cell surface and 2) the Rha installed on the target surface can recruit anti-rhamnose (anti-Rha) antibodies, leading to the destruction of target cells via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).

Synthesis and biological evaluation of Schizandrin derivatives as potential anti-cancer agents.

A new series of Schizandrin (1) derivatives were synthesized utilizing the C-9 position of the Schizandrin core and evaluated for their cytotoxic activities against HeLa (cervical cancer), A549 (lung cancer), MCF-7 (breast cancer) and DU-145 (prostate cancer) cell lines. Among the synthesized series, 4e, 4f, 4g and 5 showed potent activities against tested cell lines. More significantly, compound 5 exhibited most potent cytotoxic activity against DU-145 with an IC50 value of 1.38 µM which is comparable to the standard agent, doxorubicin. Further, flow cytometry analysis indicated that 5 arrested cells in G2/M phase and consequently leading to apoptosis. Molecular docking analysis showed that 5 occupied the colchicine binding pocket of tubulin. Overall, the present study demonstrates that 5, as a mitotic-agent.

Annonalide and derivatives: Semisynthesis, cytotoxic activities and studies on interaction of annonalide with DNA.

The cytotoxic activity of the pimarane diterpene annonalide (1) and nine of its semisynthetic derivatives (2-10) was investigated against the human tumor cell lines HL-60 (leukemia), PC-3 (prostate adenocarcinoma), HepG2 (hepatocellular carcinoma), SF-295 (glioblastoma) and HCT-116 (colon cancer), and normal mouse fibroblast (L929) cells. The preparation of 2-10 involved derivatization of the side chain of 1 at C-13. Except for 2, all derivatives are being reported for the first time. Most of the tested compounds presented IC50s below 4.0 µM, being considered potential antitumor agents. The structures of all new compounds were elucidated by spectroscopic analyses including 2D NMR and HRMS. Additionally, the interaction of annonalide (1) with ctDNA was evaluated using spectroscopic techniques, and the formation of a supramolecular complex with the macromolecule was confirmed. Competition assays with fluorescent probes (Hoechst and ethidium bromide) and theoretical studies confirmed that 1 interacts preferentially via DNA intercalation with stoichiometric ratio of 1:1 (1:ctDNA). The ΔG value was calculated as -28.24 kJ mol-1, and indicated that the interaction process occurs spontaneously. Docking studies revealed that van der Walls is the most important interaction in 1-DNA and EB-DNA complexes, and that both ligands (1 and EB) interact with the same DNA residues (DA6, DA17 and DT19).

Novel Gomisin B analogues as potential cytotoxic agents: Design, synthesis, biological evaluation and docking studies.

As part of pharmacological-phytochemical integrated studies on medicinal flora, Gomisin B (1) was isolated as major phytochemical lead from schisandra grandiflora, a plant traditionally used in different Asian systems of medicine. A series of 1,2,3-triazoles derivatives were synthesized at the C-7' position of the gomisin B core through diastereoselective Michael addition followed by regioselective Huisgen 1,3-dipolar cycloaddition reactions. All these triazolyl derivatives (5a-5q) were well characterized using modern spectroscopic techniques and evaluated for their anti-cancer activity against a panel of five human cancerous cell-lines. Among them, compound 5b exhibited the best cytotoxicity against SIHA cell (IC50 0.24 µM) which was more than the standard drug doxorubicin, while the other derivatives were exhibited moderate to low activities in tested cell lines. The cell cycle analysis indicated that compound 5b stalled HeLa cells at G2/M phase. 5b promoted tubulin polymerization and this was supported by the docking studies, wherein 5b showed significant binding affinity at the colchicine binding pocket of tubulin. Overall, we identified a novel small molecule that demonstrated anticancer activity by promoting in vitro tubulin assembly.

Rhodium(I) N-Heterocyclic Carbene Bioorganometallics as in Vitro Antiproliferative Agents with Distinct Effects on Cellular Signaling.

Organometallics with N-heterocyclic carbene (NHC) ligands have triggered major interest in inorganic medicinal chemistry. Complexes of the type Rh(I)(NHC)(COD)X (where X is Cl or I, COD is cyclooctadiene, and NHC is a dimethylbenzimidazolylidene) represent a promising type of new metallodrugs that have been explored by advanced biomedical methods only recently. In this work, we have synthesized and characterized several complexes of this type. As observed by mass spectrometry, these complexes remained stable over at least 3 h in aqueous solution, after which hydrolysis of the halido ligands occurred and release of the NHC ligand was evident. Effects against mitochondria and general cell tumor metabolism were noted at higher concentrations, whereas phosphorylation of HSP27, p38, ERK1/2, FAK, and p70S6K was induced substantially already at lower exposure levels. Regarding the antiproliferative activity in tumor cells, a clear preference for iodido over chlorido secondary ligands was noted, as well as effects of the substituents of the NHC ligand.

Constrained bithiazoles: small molecule correctors of defective ΔF508-CFTR protein trafficking.

Conformationally constrained bithiazoles were previously found to have improved efficacy over nonconstrained bithiazoles for correction of defective cellular processing of the ΔF508 mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. In this study, two sets of constrained bithiazoles were designed, synthesized, and tested in vitro using ΔF508-CFTR expressing epithelial cells. The SAR data demonstrated that modulating the constraining ring size between 7- versus 8-membered in these constrained bithiazole correctors did not significantly enhance their potency (IC50), but strongly affected maximum efficacy (Vmax), with constrained bithiazoles 9e and 10c increasing Vmax by 1.5-fold compared to benchmark bithiazole corr4a. The data suggest that the 7- and 8-membered constrained ring bithiazoles are similar in their ability to accommodate the requisite geometric constraints during protein binding.

A concise total synthesis of deoxyschizandrin and exploration of its antiproliferative effects and those of structurally related derivatives.

The natural product deoxyschizandrin has been shown to have a wide range of biological activities. In recent years the therapeutic potential of this compound against cancers has attracted significant interest. Herein we describe a concise de novo total synthesis of deoxyschizandrin based around a double organocuprate oxidation strategy. In addition, we present the results of biological studies exploring the ability of deoxyschizandrin and synthetic precursors lacking the medium ring biaryl unit to inhibit the proliferation of a human cancer cell line. These studies led to the identification of a structurally novel agent with in vitro anticancer activity.

Nickel-catalyzed tetramerization of alkynes: synthesis and structure of octatetraenes.

In the presence of a catalytic system comprised of NiBr(2)(dppe) and Zn, arylacetylenes undergo tetramerization to form linear octatetraenes, generally in good to excellent yields. The structure of the coupling products was verified by X-ray crystallography.