Synthesis, Biological Evaluation and Docking Studies of Ring-Opened Analogues of Ipomoeassin F.

The plant-derived macrocyclic resin glycoside ipomoeassin F (Ipom-F) binds to Sec61α and significantly disrupts multiple aspects of Sec61-mediated protein biogenesis at the endoplasmic reticulum, ultimately leading to cell death. However, extensive assessment of Ipom-F as a molecular tool and a therapeutic lead is hampered by its limited production scale, largely caused by intramolecular assembly of the macrocyclic ring. Here, using in vitro and/or in cellula biological assays to explore the first series of ring-opened analogues for the ipomoeassins, and indeed all resin glycosides, we provide clear evidence that macrocyclic integrity is not required for the cytotoxic inhibition of Sec61-dependent protein translocation by Ipom-F. Furthermore, our modeling suggests that open-chain analogues of Ipom-F can interact with multiple sites on the Sec61α subunit, most likely located at a previously identified binding site for mycolactone and/or the so-called lateral gate. Subsequent in silico-aided design led to the discovery of the stereochemically simplified analogue 3 as a potent, alternative lead compound that could be synthesized much more efficiently than Ipom-F and will accelerate future ipomoeassin research in chemical biology and drug discovery. Our work may also inspire further exploration of ring-opened analogues of other resin glycosides.

Microcystin-LR Removal from Water via Enzymatic Linearization and Ultrafiltration.

Microcystin-LR (MC-LR) is a toxin produced by cyanobacteria that can bloom in freshwater supplies. This study describes a new strategy for remediation of MC-LR that combines linearization of the toxin using microcystinase A, MlrA, enzyme with rejection of linearized byproducts using membrane filtration. The MlrA enzyme was expressed in Escherichia coli (E. coli) and purified via a His-tag with 95% purity. Additionally, composite membranes made of 95% polysulfone and 5% sulfonated polyether ether ketone (SPEEK) were fabricated and used to filter a solution containing cyclic and linearized MC-LR. Tests were also performed to measure the adsorption and desorption of MC-LR on polysulfone/SPEEK membranes. Liquid chromatography-mass spectrometry (LC-MS) was used to characterize the progress of linearization and removal of MC-LR. Results indicate that the MlrA was successful at linearizing MC-LR. Membrane filtration tests showed rejection of 97% of cyclic MC-LR and virtually all linearized MC-LR, with adsorption to the membranes being the main rejection mechanism. Adsorption/desorption tests indicated that methanol could be used to strip residual MC-LR from membranes to regenerate them. This study demonstrates a novel strategy of remediation of microcystin-tainted water, combining linearization of MC-LR to a low-toxicity byproduct along with removal by membrane filtration.

Cationic Covalent Organic Framework as an Ion Exchange Material for Efficient Adsorptive Separation of Biomolecules.

Although covalent organic frameworks (COFs) have earned significant interest in separation applications, the use of COFs in biomolecule separation remains unexplored. We examined the ionic COF Py-BPy2+-COF as an ion exchange material for biomolecule separation. After characterizing the properties of the synthesized COF with a variety of techniques, binding experiments with both large and small biomolecules were performed. High adsorption capacities of amino acids with different hydrophobicity and charge, as well as proteins of different isoelectric points and molecular weights, were determined in batch equilibrium experiments. Desorption experiments with mixtures of model proteins demonstrated an ability to successfully separate one protein from another with the selectivity hypothesized to be a combination of the isoelectric point, hydrophobicity, and ability to penetrate the crystalline material. Overall, the results demonstrated that Py-BPy2+-COF can be exploited as a robust crystalline anion exchange biomolecule separation material.

Recombinant peptide fusion construction for protein-templated catalytic palladium nanoparticles.

Although peptide-enabled synthesis of nanostructures has garnered considerable interest for use in catalytic applications, it has so far been achieved mostly via Fmoc based solid phase peptide synthesis. Consequently, the potential of longer peptides in nanoparticle synthesis have not been explored largely due to the complexities and economic constraints of this chemical synthesis route. This study examines the potential of a 45-amino acid long peptide expressed as fusion to green fluorescence protein (GFPuv) in Escherichia coli for use in palladium nanoparticle synthesis. Fed-batch fermentation with E. coli harboring an arabinose-inducible plasmid produced a product containing three copies of Pd4 peptide fused to N-terminus of GFPuv ((Pd4)3 -GFPuv). Using the intrinsic fluorescence of GFPuv, expression and enrichment of the fusion product was easily monitored. Crude lysate, desalted lysate, and an ion-exchange enriched fraction containing (Pd4)3 -GFPuv were used to test the hypothesis that high purity of the biologic material used as the nanoparticle synthesis template may not be necessary. Nanoparticles were characterized using a variety of material science techniques and used to catalyze a model Suzuki-Miyaura coupling reaction. Results demonstrated that palladium nanoparticles can be synthesized using the soluble cell extract containing (Pd4)3 -GFPuv without extensive purification or cleavage steps, and as a catalyst the crude mixture is functional.

Revealing the Pharmacophore of Ipomoeassin F through Molecular Editing.

Ipomoeassin F, the flagship congener of a resin glycoside family exhibited single-digit nanomolar IC50 values against several cancer cell lines. To facilitate drug discovery based on this unique yet underexplored natural product, we performed the most sophisticated SAR studies of ipomoeassin F to date, which not only greatly bettered our understanding of its pharmacophore but also led to the discovery of two new derivatives (3 and 27) with similar potency but improved synthetic profile. The work presented here opens new avenues toward harnessing the medicinal potential of the ipomoeassin family of glycolipids in the future.

Total Synthesis and Biological Evaluation of Ipomoeassin F and Its Unnatural 11R-Epimer.

Ipomoeassin F, a macrolide glycoresin containing an embedded disaccharide, possesses potent in vitro antitumor activity with an unknown mechanism of function. It inhibits tumor cell growth with single-digit nanomolar IC50 values, superior to many clinical chemotherapeutic drugs. To facilitate translation of its bioactivity into protein function for drug development, we report here a new synthesis for the gram-scale production of ipomoeassin F (3.8% over 17 linear steps) from commercially available starting materials. The conformation-controlled subtle reactivity differences of the hydroxyl groups in carbohydrates were utilized to quickly construct the disaccharide core, which, along with judicial selection of protecting groups, made the current synthesis very efficient. The same strategy was also applied to the smooth preparation of the 11R-epimer of ipomoeassin F for the first time. Cytotoxicity assays demonstrated the crucial role of the natural 11S configuration. In addition, cell cycle analyses and apoptosis assays on ipomoeassin F and/or its epimer were conducted. This work has laid a solid foundation for understanding the medicinal potential of the ipomoeassin family of glycolipids in the future.