Iodine-Mediated Tryptathionine Formation Facilitates the Synthesis of Amanitins.

Synthetic methods on the macrocyclization of peptides are of high interest since they facilitate the synthesis of various types of potentially bioactive compounds, e.g. addressing targets like protein-protein-interactions. Herein, we report on an efficient method to construct tryptathionine-cross-links in peptides between the amino acids Trp and Cys. This reaction not only is the basis for the total synthesis of the death cap toxin α-amanitin but also provides rapid access to various new amanitin analogues. This study for the first time presents a systematic compilation of structure-activity relations (SAR) of amatoxins with regard to RNA polymerase II inhibition and cytotoxicity with one amanitin derivative of superior RNAP II inhibition. The present approach paves the way for the synthesis of structurally diverse amatoxins as future payloads for antibody-toxin conjugates in cancer therapy.

Nitidumpeptins A and B, Cyclohexapeptides Isolated from Zanthoxylum nitidum var. tomentosum: Structural Elucidation, Total Synthesis, and Antiproliferative Activity in Cancer Cells.

Nitidumpeptins A and B (1 and 2), two novel cyclic hexapeptides, were isolated from the herb Zanthoxylum nitidum var. tomentosum. Their planar structures were elucidated based on NMR and MS spectrometric analysis, and the absolute configurations were determined by the Marfey's method. Structurally, 1 is a unique peptide with a backbone bearing a pyrrolidine-2,5-dione unit, which is the first occurrence moiety specifically in a naturally occurring cyclohexapeptide. The total synthesis of 1 and 2 was achieved by solution-phase in parallel with solid-phase peptide synthesis, and their absolute configurations were further confirmed. The combination of 2 with gefitinib exhibited synergistic antiproliferative activity in acquired gefitinib-resistant non-small cell lung cancer cells (HCC827-gef). The underlying mechanism for the antiproliferative activity of 2 was in part associated with the suppression of YAP expression in HCC827-gef cells.

Total Synthesis of the Death Cap Toxin Phalloidin: Atropoisomer Selectivity Explained by Molecular-Dynamics Simulations.

Phallotoxins and amatoxins are a group of prominent peptide toxins produced by the death cap mushroom Amanita phalloides. Phalloidin is a bicyclic cyclopeptide with an unusual tryptathionin thioether bridge. It is a potent stabilizer of filamentous actin and in a fluorescently labeled form widely used as a probe for actin binding. Herein, we report the enantioselective synthesis of the key amino acid (2S,4R)-4,5-dihydroxy-leucine as a basis for the first total synthesis of phalloidin, which was accomplished by two different synthesis strategies. Molecular-dynamics simulations provided insights into the conformational flexibility of peptide intermediates of different reaction strategies and showed that this flexibility is critical for the formation of atropoisomers. By simulating the intermediates, rather than the final product, molecular-dynamics simulations will become a decisive tool in orchestrating the sequence of ring formation reactions of complex cyclic peptides.

Efficient Synthesis and Stereochemical Revision of Coibamide A.

Coibamide A is a highly potent antiproliferative cyclodepsipeptide originally isolated from a Panamanian marine cyanobacterium. Herein we report an efficient solid-phase strategy for assembly of highly N-methylated cyclodepsipeptides, which is invaluable in generating coibamide A derivatives for structure-activity relationship studies. As a consequence of our synthetic studies, two stereochemical assignments of coibamide A were revised and the total synthesis of this natural compound was achieved for the first time.

Novel Coumarin-Containing Aminophosphonatesas Antitumor Agent: Synthesis, Cytotoxicity, DNA-Binding and Apoptosis Evaluation.

A series of novel coumarin-containing α-aminophosphonates were synthesized and evaluated for their antitumor activities against Human colorectal (HCT-116), human nasopharyngeal carcinoma (human KB) and human lung adenocarcinoma (MGC-803) cell lines in vitro. Compared with 7-hydroxy-4-methylcoumarin (4-MU), most of the derivatives showed an improved antitumor activity. Compound 8j (diethyl 1-(3-(4-methyl-2-oxo-2H-chromen-7-yloxy) propanamido)-1-phenylethyl-Phosphonate), with IC50 value of 8.68 µM against HCT-116 cell lines, was about 12 fold than that of unsubstituted parent compound. The mechanism investigation proved that 8c, 8d, 8f and 8j were achieved through the induction of cell apoptosis by G1 cell-cycle arrest. In addition, the further mechanisms of compound 8j-induced apoptosis in HCT-116 cells demonstrated that compound 8j induced the activations of caspase-9 and caspase-3 for causing cell apoptosis, and altered anti- and pro-apoptotic proteins. DNA-binding experiments suggested that some derivatives bind to DNA through intercalation. The results seem to imply the presence of an important synergistic effect between coumarin and aminophosphonate, which could contribute to the strong chelating properties of aminophosphonate moiety.

Synthesis and antitumor activities of novel rhein α-aminophosphonates conjugates.

Several rhein α-aminophosphonates conjugates (5a-5q) were synthesized and evaluated for in vitro cytotoxicity against HepG-2, CNE, Spca-2, Hela and Hct-116 cell lines. Some compounds showed relatively high cytotoxicity. Especially, compound 5i exhibited the strongest cytotoxicity against Hct-116 cells (IC50 was 5.32 µM). All the synthesized compounds exhibited low cytotoxicity against HUVEC cells. The mechanism of compound 5i was preliminarily investigated by Hoechst 33258 staining, JC-1 mitochondrial membrane potential staining and flow cytometry, which indicated that the compound 5i induced apoptosis in Hct-116 cancer cells. Cell cycle analysis showed that these compound 5i mainly arrested Hct-116 cells in G1 stage. The effects of 5i on the activation of caspases expression indicated that 5i might induce apoptosis via the membrane death receptor pathways. In addition, the binding properties of a model analog 5i to DNA were investigated by methods (UV-vis, fluorescence, CD spectroscopy and FRET-melting) in compare with that of rhein. Results indicated that 5i showed moderate ability to interact ct-DNA.

Computationally Designed Epitope-Mediated Imprinted Polymers versus Conventional Epitope Imprints for the Detection of Human Adenovirus in Water and Human Serum Samples.

Detection of pathogenic viruses for point-of-care applications has attracted great attention since the COVID-19 pandemic. Current virus diagnostic tools are laborious and expensive, while requiring medically trained staff. Although user-friendly and cost-effective biosensors are utilized for virus detection, many of them rely on recognition elements that suffer major drawbacks. Herein, computationally designed epitope-imprinted polymers (eIPs) are conjugated with a portable piezoelectric sensing platform to establish a sensitive and robust biosensor for the human pathogenic adenovirus (HAdV). The template epitope is selected from the knob part of the HAdV capsid, ensuring surface accessibility. Computational simulations are performed to evaluate the conformational stability of the selected epitope. Further, molecular dynamics simulations are executed to investigate the interactions between the epitope and the different functional monomers for the smart design of eIPs. The HAdV epitope is imprinted via the solid-phase synthesis method to produce eIPs using in silico-selected ingredients. The synthetic receptors show a remarkable detection sensitivity (LOD: 102 pfu mL-1) and affinity (dissociation constant (Kd): 6.48 × 10-12 M) for HAdV. Moreover, the computational eIPs lead to around twofold improved binding behavior than the eIPs synthesized with a well-established conventional recipe. The proposed computational strategy holds enormous potential for the intelligent design of ultrasensitive imprinted polymer binders.

Cation Dependence of Enniatin B/Membrane-Interactions Assessed Using Surface-Enhanced Infrared Absorption (SEIRA) Spectroscopy.

Enniatins are mycotoxins with well-known antibacterial, antifungal, antihelmintic and antiviral activity, which have recently come to attention as potential mitochondriotoxic anticancer agents. The cytotoxicity of enniatins is traced back to ionophoric properties, in which the cyclodepsipeptidic structure results in enniatin:cation-complexes of various stoichiometries proposed as membrane-active species. In this work, we employed a combination of surface-enhanced infrared absorption (SEIRA) spectroscopy, tethered bilayer lipid membranes (tBLMs) and density functional theory (DFT)-based computational spectroscopy to monitor the cation-dependence (Mz+=Na+, K+, Cs+, Li+, Mg2+, Ca2+) on the mechanism of enniatin B (EB) incorporation into membranes and identify the functionally relevant EBn : Mz+ complexes formed. We find that Na+ promotes a cooperative incorporation, modelled via an autocatalytic mechanism and mediated by a distorted 2 : 1-EB2 : Na+ complex. K+ (and Cs+) leads to a direct but less efficient insertion into membranes due to the adoption of "ideal" EB2 : K+ sandwich complexes. In contrast, the presence of Li+, Mg2+, and Ca2+ causes a (partial) extraction of EB from the membrane via the formation of "belted" 1 : 1-EB : Mz+ complexes, which screen the cationic charge less efficiently. Our results point to a relevance of the cation dependence for the transport into the malignant cells where the mitochondriotoxic anticancer activity is exerted.

Atom-economical chemoselective synthesis of 1,4-enynes from terminal alkenes and propargylic alcohols catalyzed by Cu(OTf)2.

A novel and efficient Cu(OTf)2-catalyzed sp(3)-sp(2) C-C bond formation reaction through the direct coupling of propargylic alcohols with terminal alkenes has been realized under mild conditions. The reaction is tolerant to air and is atom-economical, in accordance with the concept of modern green chemistry. The present protocol provides an attractive approach to a diverse range of 1,4-enynes in high to excellent yields.

Synthesis and antitumor activity evaluation of maleopimaric acid N-aryl imide atropisomers.

Maleopimaric acid N-aryl imides (2) and methyl maleopimaric acid N-aryl imides (3) were designed and synthesized. Their atropisomers (A and B) were separated into their enantiomeric pure forms and the anti-proliferative activity was tested against NCI, A549, Hep G-2, MGC-803 and Hct-116 cell lines, respectively. A significant difference in the level of cytotoxicity was observed between R and S conformers. Atropisomers A with an R configuration exhibited significant toxicity (the IC50 values ranging from 7.51 to 32.1 µM). Further experiments proved that antitumor activity of 2A was achieved through the induction of cell apoptosis by G1 cell-cycle arrest.

Synthesis, cytotoxicity, DNA binding and apoptosis of rhein-phosphonate derivatives as antitumor agents.

Several rhein-phosphonate derivatives (5a-c) were synthesized and evaluated for in vitro cytotoxicity against HepG-2, CNE, Spca-2, Hela and Hct-116 cell lines. Some compounds showed relatively high cytotoxicity. Especially compounds 5b exhibited the strongest cytotoxicity against HepG-2 and Spca-2 cells (IC50 was 8.82 and 9.01 µM), respectively. All the synthesized compounds exhibited low cytotoxicity against HUVEC cells. Further experiments proved that 5b could disturb the cell cycle in HepG-2 cells and induce apoptosis. In addition, the binding properties of a model conjugate 5b to DNA were investigated by methods (UV-Vis, fluorescence, CD spectroscopy). Results indicated that 5b showed moderate ability to interact ct-DNA.

Potential Distribution across Model Membranes.

Membrane models assembled on electrodes are widely used tools to study potential-dependent molecular processes at or in membranes. However, the relationship between the electrode potential and the potential across the membrane is not known. Here we studied lipid bilayers immobilized on mixed self-assembled monolayers (SAM) on Au electrodes. The mixed SAM was composed of thiol derivatives of different chain lengths such that between the islands of the short one, mercaptobenzonitrile (MBN), and the tethered lipid bilayer an aqueous compartment was formed. The nitrile function of MBN, which served as a reporter group for the vibrational Stark effect (VSE), was probed by surface-enhanced infrared absorption spectroscopy to determine the local electric field as a function of the electrode potential for pure MBN, mixed SAM, and the bilayer system. In parallel, we calculated electric fields at the VSE probe by molecular dynamics (MD) simulations for different charge densities on the metal, thereby mimicking electrode potential changes. The agreement with the experiments was very good for the calculations of the pure MBN SAM and only slightly worse for the mixed SAM. The comparison with the experiments also guided the design of the bilayer system in the MD setups, which were selected to calculate the electrode potential dependence of the transmembrane potential, a quantity that is not directly accessible by the experiments. The results agree very well with estimates in previous studies and thus demonstrate that the present combined experimental-theoretical approach is a promising tool for describing potential-dependent processes at biomimetic interfaces.

The occurrence of ansamers in the synthesis of cyclic peptides.

α-Amanitin is a bicyclic octapeptide composed of a macrolactam with a tryptathionine cross-link forming a handle. Previously, the occurrence of isomers of amanitin, termed atropisomers has been postulated. Although the total synthesis of α-amanitin has been accomplished this aspect still remains unsolved. We perform the synthesis of amanitin analogs, accompanied by in-depth spectroscopic, crystallographic and molecular dynamics studies. The data unambiguously confirms the synthesis of two amatoxin-type isomers, for which we propose the term ansamers. The natural structure of the P-ansamer can be ansa-selectively synthesized using an optimized synthetic strategy. We believe that the here described terminology does also have implications for many other peptide structures, e.g. norbornapeptides, lasso peptides, tryptorubins and others, and helps to unambiguously describe conformational isomerism of cyclic peptides.

Improved Total Synthesis and Biological Evaluation of Coibamide A Analogues.

To enable the large-scale synthesis of coibamide A, we developed an improved synthetic strategy for this class of cyclodepsipeptide. The versatility of the synthetic procedure was demonstrated by the preparation of a series of designed coibamide A analogues, which enabled the preliminary structure-activity relationship (SAR) studies for this compound. Although most modifications of coibamide A resulted in decrease or loss of the antiproliferativity, we found that versatile substitution at position 3 was well tolerated. Remarkably, a simplified analogue, [MeAla3-MeAla6]-coibamide (1f), not only showed nearly the same inhibition as coibamide A against the tested cancer cells but also significantly inhibited tumor growth in vivo. The improved synthetic strategy and the relevant trends of SAR disclosed in this study will be valuable for further optimization of the overall profile of coibamide A.

N-terminal α-amino group modification of antibodies using a site-selective click chemistry method.

Site-specific conjugation of small molecules to antibody molecules is a promising strategy for generation of antibody-drug conjugates. In this report, we describe the successful synthesis of a novel bifunctional molecule, 6-(azidomethyl)-2-pyridinecarboxyaldehyde (6-AM-2-PCA), which was used for conjugation of small molecules to peptides and antibodies. We demonstrated that 6-AM-2-PCA selectively reacted with N-terminal amino groups of peptides and antibodies. In addition, the azide group of 6-AM-2-PCA enabled copper-free click chemistry coupling with dibenzocyclooctyne-containing reagents. Bifunctional 6-AM-2-PCA mediated site-specific conjugation without requiring genetic engineering of peptides or antibodies. A key advantage of 6-AM-2-PCA as a conjugation reagent is its ability to modify proteins in a single step under physiological conditions that are sufficiently moderate to retain protein function. Therefore, this new click chemistry-based method could be a useful complement to other conjugation methods.

Myristic acid-modified thymopentin for enhanced plasma stability and immune-modulating activity.

Natural albumin ligand (fatty acids)-conjugated peptides can rapidly bind to circulating albumin and form complexes to control the release of peptides. The purpose of this study was to prolong the half-life and immune-modulating effects of thymopentin (TP5) by using the albumin binding strategy. We synthesized myristic acid-modified TP5 (TP5-MA) by conjugating a myristic acid-acylated lysine to a permissive site of TP5, which improved the albumin binding affinity of TP5-MA and dramatically enhanced its stability in human plasma. We observed well-preserved bioactivities of TP5-MA in RAW264.7 macrophages using a tumor necrosis factor (TNF)-α stimulation assay. Moreover, the prolonged immune-modulating effect of TP5-MA was confirmed by the normalized CD4+/CD8+ ratio in immune-depressed rat models, which resulted in a reduced administration frequency (twice per week). In general, the enhanced pharmacokinetic and pharmacodynamic properties of TP5-MA make it a promising product for the treatment of immunodeficiency diseases.

Terminal functionalized thiourea-containing dipeptides as multidrug-resistance reversers that target 20S proteasome and cell proliferation.

A series of inhibitors of 20S proteasome based on terminal functionalized dipeptide derivatives containing the thiourea moiety were synthesized and evaluated for inhibition of 20S proteasome and the effects of multidrug-resistance reversers. These compounds exhibited significant selectivity to the ß5-subunit of the human 20S proteasome with IC50 values at submicromolar concentrations. A docking study of the most active compound 6i revealed key interactions between 6i and the active site of the 20S proteasome in which the thiourea moiety and a nitro group were important for improving activity. In particular, compound 6i appeared to be the most potent compound against the NCI-H460 cell line, and displayed similar efficiency in drug-sensitive versus drug-resistant cancer cell lines, at least partly, by inhibition of the activity of 20S proteasome and induce apoptosis. In addition, 6i-induced apoptosis was significantly facilitated in NCI-H460/DOX cells that had been pretreated with inhibitors of P-gp. Mechanistically, compound 6i might trigger apoptotic signalling pathway. Thus, we conclude that dipeptide derivatives containing the thiourea moiety may be the potential inhibitors of proteasome with the ability to reverse multidrug resistance.

Total Synthesis and Stereochemical Assignment of Gymnopeptides A and B.

Gymnopeptides A and B are unprecedented highly N-methylated cyclic ß-hairpin octadecapeptides with striking antiproliferative activities isolated from the mushroom Gymnopus fusipes. Using Fmoc-based solid-phase peptide synthesis, followed by macrolactamization of the resulting linear peptides, the first total synthesis of gymnopeptides A and B was successfully achieved in this study. The coupling methods used for the solid-phase synthesis and the cyclization were optimized, and the configuration of the Ser1/Thr1 residue in gymnopeptide A/B was determined to be l.

Synthesis and Pharmacological Evaluation of Maleopimaric N-arylimides: Identification of Novel Proapoptotic Agents.

Several N-aryl maleopimaric acid diimides (3a-3d, 4a-4g) were synthesized and evaluated their topoisomerase I inhibitory activities along with cytotoxicities against NCI, MGC-803, Bel-7404 and Hct-116 cell lines. The pharmacological dates revealed that most of structure analogs exhibited moderate to high levels of anticancer activities against the tested cancer cell lines. Compound 4g with phenylalanine substituent exhibited significant cytotoxicity against MGC-803 and Hct-116 cells (IC50 was 9.85±1.24 and 8.47±0.95 µM, respectively). All the synthesized compounds exhibited no cytotoxicity against HUVEC cells. In addition, maleopimaric diimides showed stronger cytotoxicity and topoisomerase I inhibitory activity compared to that of maleopimaric acid. Structure-activity relationship study showed that carboxyl and diimide moieties were important to display Topo I inhibitory activities. Further experiments proved that 4g could induce apoptosis of MGC-803 cells. In addition, the further mechanisms of compound 4g-induced apoptosis in MGC-803 cells demonstrated that compound 4g induced the activations of caspase-4, caspase-8 and caspase-3 for causing cell apoptosis, and altered antiand pro-apoptotic proteins. Moreover, cell cycle analysis indicated that the derivative 4g mainly arrested MGC-803 cells in S stage.

Fully automated synthesis of DNA-binding Py-Im polyamides using a triphosgene coupling strategy.

The fully automated solid-phase synthetic strategy of hairpin pyrrole-imidazole polyamides is described. A key advance is the development of methodology for the application of triphosgene as a coupling agent in the automated synthesis of hairpin polyamides without racemization. This automated methodology is compatible with all the typical building blocks, enabling the facile synthesis of polyamide libraries in good yield (9-15%) and crude purity.