Advances in the Synthesis of Bioorthogonal Reagents: s-Tetrazines, 1,2,4-Triazines, Cyclooctynes, Heterocycloheptynes, and trans-Cyclooctenes.

Aligned with the increasing importance of bioorthogonal chemistry has been an increasing demand for more potent, affordable, multifunctional, and programmable bioorthogonal reagents. More advanced synthetic chemistry techniques, including transition-metal-catalyzed cross-coupling reactions, C-H activation, photoinduced chemistry, and continuous flow chemistry, have been employed in synthesizing novel bioorthogonal reagents for universal purposes. We discuss herein recent developments regarding the synthesis of popular bioorthogonal reagents, with a focus on s-tetrazines, 1,2,4-triazines, trans-cyclooctenes, cyclooctynes, hetero-cycloheptynes, and -trans-cycloheptenes. This review aims to summarize and discuss the most representative synthetic approaches of these reagents and their derivatives that are useful in bioorthogonal chemistry. The preparation of these molecules and their derivatives utilizes both classical approaches as well as the latest organic chemistry methodologies.

Ratiometric Flapping Force Probe That Works in Polymer Gels.

Polymer gels have recently attracted attention for their application in flexible devices, where mechanically robust gels are required. While there are many strategies to produce tough gels by suppressing nanoscale stress concentration on specific polymer chains, it is still challenging to directly verify the toughening mechanism at the molecular level. To solve this problem, the use of the flapping molecular force probe (FLAP) is promising because it can evaluate the nanoscale forces transmitted in the polymer chain network by ratiometric analysis of a stress-dependent dual fluorescence. A flexible conformational change of FLAP enables real-time and reversible responses to the nanoscale forces at the low force threshold, which is suitable for quantifying the percentage of the stressed polymer chains before structural damage. However, the previously reported FLAP only showed a negligible response in solvated environments because undesirable spontaneous planarization occurs in the excited state, even without mechanical force. Here, we have developed a new ratiometric force probe that functions in common organogels. Replacement of the anthraceneimide units in the flapping wings with pyreneimide units largely suppresses the excited-state planarization, leading to the force probe function under wet conditions. The FLAP-doped polyurethane organogel reversibly shows a dual-fluorescence response under sub-MPa compression. Moreover, the structurally modified FLAP is also advantageous in the wide dynamic range of its fluorescence response in solvent-free elastomers, enabling clearer ratiometric fluorescence imaging of the molecular-level stress concentration during crack growth in a stretched polyurethane film.

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.

Discovery of 2-Sulfinyl-Diazabicyclooctane Derivatives, Potential Oral ß-Lactamase Inhibitors for Infections Caused by Serine ß-Lactamase-Producing Enterobacterales.

Coadministration of ß-lactam and ß-lactamase inhibitor (BLI) is one of the well-established therapeutic measures for bacterial infections caused by ß-lactam-resistant Gram-negative bacteria, whereas we have only two options for orally active BLI, clavulanic acid and sulbactam. Furthermore, these BLIs are losing their clinical usefulness because of the spread of new ß-lactamases, including extended-spectrum ß-lactamases (ESBLs) belonging to class A ß-lactamases, class C and D ß-lactamases, and carbapenemases, which are hardly or not inhibited by these classical BLIs. From the viewpoints of medical cost and burden of healthcare personnel, oral therapy offers many advantages. In our search for novel diazabicyclooctane (DBO) BLIs possessing a thio-functional group at the C2 position, we discovered a 2-sulfinyl-DBO derivative (2), which restores the antibacterial activities of an orally available third-generation cephalosporin, ceftibuten (CTB), against various serine ß-lactamase-producing strains including carbapenem-resistant Enterobacteriaceae (CRE). It can be orally absorbed via the ester prodrug modification and exhibits in vivo efficacy in a combination with CTB.

General, Divergent Platform for Diastereoselective Synthesis of trans-Cyclooctenes with High Reactivity and Favorable Physiochemical Properties*.

trans-Cyclooctenes (TCOs) are essential partners in the fastest known bioorthogonal reactions, but current synthetic methods are limited by poor diastereoselectivity. Especially hard to access are hydrophilic TCOs with favorable physicochemical properties for live cell or in vivo experiments. Described is a new class of TCOs, "a-TCOs", prepared in high yield by stereocontrolled 1,2-additions of nucleophiles to trans-cyclooct-4-enone, which itself was prepared on a large scale in two steps from 1,5-cyclooctadiene. Computational transition-state models rationalize the diastereoselectivity of 1,2-additions to deliver a-TCO products, which were also shown to be more reactive than standard TCOs and less hydrophobic than even a trans-oxocene analogue. Illustrating the favorable physicochemical properties of a-TCOs, a fluorescent TAMRA derivative in live HeLa cells was shown to be cell-permeable through intracellular Diels-Alder chemistry and to wash out more rapidly than other TCOs.

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.

Synthetic approaches towards avibactam and other diazabicyclooctane ß-lactamase inhibitors.

Avibactam is a non ß-lactam ß-lactamase inhibitor that has recently been approved in association with a ß-lactam antibiotic for the treatment of severe infections caused by otherwise resistant bacteria. Its therapeutic success encouraged the development of many congeners based on its particular diazabicyclooctane scaffold. This review presents a detailed overview of the synthetic strategies that have been implemented to acces these complex bicyclic compounds with a particular focus on those that are currently on the market or in clinical trials.

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.

Electronic Donation or Steric Contraction: A Spectroscopic and Structural Analysis of Medium-Sized Constrained Rings for Potential Long-Range Hyperconjugation.

Herein, we report the 1JCH analyses, natural bond orbital analyses, and X-ray crystal structures of a number of C, O, and N constrained tricyclic cycles. These experiments provide access into the nature of the apparent Perlin effect previously reported in constrained tricyclic cycles, as well as evidence suggesting both steric contraction and long-range hyperconjugation account for the observed 1JCH perturbations. We report a true Perlin effect of 10.9 Hz in an azocane and large steric effect resulting in Δ1JC-H = 10.9 Hz in a cyclooctane.

Synthesis and Fabrication of Surface-Active Microparticles Using a Membrane Emulsion Technique and Conjugation of Model Protein via Strain-Promoted Azide-Alkyne Click Chemistry in Physiological Conditions.

The rapid surface immobilization of protein on monodispersed polyester microcarriers is reported. A model protein, functionalized with a dibenzocyclooctyne core, immobilizes on the surface of azide-terminal polycaprolactone microcarriers within 10 min compared to 12 h for other conjugation techniques, and it is conducted in physiological conditions and in the absence of coupling reagents.

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.

Enzymatic Fluorination of Biotin and Tetrazine Conjugates for Pretargeting Approaches to Positron Emission Tomography Imaging.

The use of radiolabelled antibodies and antibody-derived recombinant constructs has shown promise for both imaging and therapeutic use. In this context, the biotin-avidin/streptavidin pairing, along with the inverse-electron-demand Diels-Alder (iEDDA) reaction, have found application in pretargeting approaches for positron emission tomography (PET). This study reports the fluorinase-mediated transhalogenation [5'-chloro-5'-deoxyadenosine (ClDA) substrates to 5'-fluoro-5'-deoxyadenosine (FDA) products] of two antibody pretargeting tools, a FDA-PEG-tetrazine and a [18 F]FDA-PEG-biotin, and each is assessed either for its compatibility towards iEDDA ligation to trans-cyclooctene or for its affinity to avidin. A protocol to avoid radiolytically promoted oxidation of biotin during the synthesis of [18 F]FDA-PEG-biotin was developed. The study adds to the repertoire of conjugates for use in fluorinase-catalysed radiosynthesis for PET and shows that the fluorinase will accept a wide range of ClDA substrates tethered at C-2 of the adenine ring with a PEGylated cargo. The method is exceptional because the nucleophilic reaction with [18 F]fluoride takes place in water at neutral pH and at ambient temperature.

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.

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.

Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes.

Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.

Organocatalyzed Synthesis of [3.2.1] Bicyclooctanes.

Organocatalysis constitutes one of the main research areas in organic chemistry from the last two decades. This chemistry has been applied to the synthesis of many natural products and structures in a manner that reduces the residues and so the ecological impact. In this review, we consider the work that has been done for the synthesis of bicyclo[3.2.1]octane framework. This structure is present in many natural products with very important biological activities.