Manipulation of Electrostatic and Saccharide Linker Interactions in the Design of Efficient Glycopolypeptide‐Based Cholera Toxin Inhibitors

Multivalent, glycopolymer inhibitors designed for the treatment of disease and pathogen infection have shown improvements in binding correlated with general changes in glycopolymer architecture and composition. We have previously demonstrated that control of glycopolypeptide backbone extension and ligand spacing significantly impacts the inhibition of the cholera toxin B subunit pentamer (CT B₅) by these polymers. In the studies reported here, we elucidate the role of backbone charge and linker length in modulating the inhibition event. Peptides of the sequence AXPXG (where X is a positive, neutral or negative amino acid), equipped with the alkyne functionality of propargyl glycine, were designed and synthesized via solid‐phase peptide synthetic methods and glycosylated via Cu(I)‐catalyzed alkyne‐azide cycloaddition reactions. The capacity of the glycopeptides to inhibit the binding of the B₅ subunit of cholera toxin was evaluated. These studies indicated that glycopeptides with a negatively charged backbone show improved inhibition of the binding event relative to the other glycopeptides. In addition, variations in the length of the linker between the peptide and the saccharide ligand also affected the inhibition of CT by the glycopeptides. Our findings suggest that, apart from appropriate saccharide spacing and polypeptide chain extension, saccharide linker conformation and the systematic placement of charges on the polypeptide backbone are also significant variables that can be tuned to improve the inhibitory potencies of glycopolypeptide‐based multivalent inhibitors.

     

Synthesis of Highly Selective Submicromolar Microcystin‐Based Inhibitors of Protein Phosphatase (PP)2A over PP1

Research and therapeutic targeting of the phosphoserine/threonine phosphatases PP1 and PP2A is hindered by the lack of selective inhibitors. The microcystin (MC) natural toxins target both phosphatases with equal potency, and their complex synthesis has complicated structure–activity relationship studies in the past. We report herein the synthesis and biochemical evaluation of 11 MC analogues, which was accomplished through an efficient strategy combining solid‐ and solution‐phase approaches. Our approach led to the first MC analogue with submicromolar inhibitory potency that is strongly selective for PP2A over PP1 and does not require the complex lipophilic Adda group. Through mutational and structural analyses, we identified a new key element for binding, as well as reasons for the selectivity. This work gives unprecedented insight into how selectivity between these phosphatases can be achieved with MC analogues.     

A Smart Library of Epoxide Hydrolase Variants and the Top Hits for Synthesis of (S)‐β‐Blocker Precursors

Microtuning of the enzyme active pocket has led to a smart library of epoxide hydrolase variants with an expanded substrate spectrum covering a series of typical β‐blocker precursors. Improved activities of 6‐ to 430‐fold were achieved by redesigning the active site at two predicted hot spots. This study represents a breakthrough in protein engineering of epoxide hydrolases and resulted in enhanced activity toward bulky substrates.     

Design,Synthesis and Biological Evaluation of Potent Azadipeptide Nitrile Inhibitors and Activity‐Based Probes as Promising Anti‐Trypanosoma brucei Agents

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas disease and African sleeping sickness, respectively. There is an urgent need for the development of new drugs against both diseases due to the lack of adequate cures and emerging drug resistance. One promising strategy for the discovery of small‐molecule therapeutics against parasitic diseases has been to target the major cysteine proteases such as cruzain for T. cruzi, and rhodesain/TbCatB for T. brucei. Azadipeptide nitriles belong to a novel class of extremely potent cysteine protease inhibitors against papain‐like proteases. We herein report the design, synthesis, and evaluation of a series of azanitrile‐containing compounds, most of which were shown to potently inhibit both recombinant cruzain and rhodesain at low nanomolar/picomolar ranges. A strong correlation between the potency of rhodesain inhibition (i.e., target‐based screening) and trypanocidal activity (i.e., whole‐organism‐based screening) of the compounds was observed. To facilitate detailed studies of this important class of inhibitors, selected hit compounds from our screenings were chemically converted into activity‐based probes (ABPs), which were subsequently used for in situ proteome profiling and cellular localization studies to further elucidate potential cellular targets (on and off) in both the disease‐relevant bloodstream form (BSF) and the insect‐residing procyclic form (PCF) of Trypanosoma brucei. Overall, the inhibitors presented herein show great promise as a new class of anti‐trypanosome agents, which possess better activities than existing drugs. The activity‐based probes generated from this study could also serve as valuable tools for parasite‐based proteome profiling studies, as well as bioimaging agents for studies of cellular uptake and distribution of these drug candidates. Our studies therefore provide a good starting point for further development of these azanitrile‐containing compounds as potential anti‐parasitic agents.     

Non‐Covalent Polyvalent Ligands by Self‐Assembly of Small Glycodendrimers: A Novel Concept for the Inhibition of Polyvalent Carbohydrate–Protein Interactions In Vitro and In Vivo

Polyvalent carbohydrate–protein interactions occur frequently in biology, particularly in recognition events on cellular membranes. Collectively, they can be much stronger than corresponding monovalent interactions, rendering it difficult to control them with individual small molecules. Artificial macromolecules have been used as polyvalent ligands to inhibit polyvalent processes; however, both reproducible synthesis and appropriate characterization of such complex entities is demanding. Herein, we present an alternative concept avoiding conventional macromolecules. Small glycodendrimers which fulfill single molecule entity criteria self‐assemble to form non‐covalent nanoparticles. These particles—not the individual molecules—function as polyvalent ligands, efficiently inhibiting polyvalent processes both in vitro and in vivo. The synthesis and characterization of these glycodendrimers is described in detail. Furthermore, we report on the characterization of the non‐covalent nanoparticles formed and on their biological evaluation.     

Trimodal Control of Ion‐Transport Activity on Cyclo‐oligo‐(1→6)‐β‐D‐glucosamine‐Based Artificial Ion‐Transport Systems

Cyclo‐oligo‐(1→6)‐β‐D ‐glucosamines functionalized with hydrophobic tails are reported as a new class of transmembrane ion‐transport system. These macrocycles with hydrophilic cavities were introduced as an alternative to cyclodextrins, which are supramolecular systems with hydrophobic cavities. The transport activities of these glycoconjugates were manipulated by altering the oligomericity of the macrocycles, as well as the length and number of attached tails. Hydrophobic tails of 3 different sizes were synthesized and coupled with each glucosamine scaffold through the amide linkage to obtain 18 derivatives. The ion‐transport activity increased from di‐ to tetrameric glucosamine macrocycles, but decreased further when flexible pentameric glucosamine was introduced. The ion‐transport activity also increased with increasing length of attached linkers. For a fixed length of linkers, the transport activity decreased when the number of such tails was reduced. All glycoconjugates displayed a uniform anion‐selectivity sequence: Cl^?>Br^?>I^?. From theoretical studies, hydrogen bonding between the macrocycle backbone and the anion bridged through water molecules was observed.     

In Situ Study of the Function of Bacterioruberin in the Dual‐Chromophore Photoreceptor Archaerhodopsin‐4

While certain archaeal ion pumps have been shown to contain two chromophores, retinal and the carotenoid bacterioruberin, the functions of bacterioruberin have not been well explored. To address this research gap, recombinant archaerhodopsin‐4 (aR4), either with retinal only or with both retinal and bacterioruberin chromophores, was successfully expressed together with endogenous lipids in H. salinarum L33 and MPK409 respectively. In situ solid‐state NMR, supported by molecular spectroscopy and functional assays, revealed for the first time that the retinal thermal equilibrium in the dark‐adapted state is modulated by bacterioruberin binding through a cluster of aromatic residues on helix E. Bacterioruberin not only stabilizes the protein trimeric structure but also affects the photocycle kinetics and the ATP formation rate. These new insights may be generalized to other receptors and proteins in which metastable thermal equilibria and functions are perturbed by ligand binding.     

Design and Synthesis of (13S)‐Methyl‐Substituted Arachidonic Acid Analogues: Templates for Novel Endocannabinoids

Two novel methyl‐substituted arachidonic acid derivatives were prepared in an enantioselective manner from commercially available chiral building blocks, and were found to be excellent templates for the development of (13S)‐methyl‐substituted anandamide analogues. One of the compounds synthesized, namely, (13S,5Z,8Z,11Z,14Z)‐13‐methyl‐eicosa‐5,8,11,14‐tetraenoic acid N‐(2‐hydroxyethyl)amide, is an endocannabinoid analogue with remarkably high affinity for the CB1 cannabinoid receptor.     

Towards Molecular Design Rationalization in Branched Multi‐Thiophene Semiconductors: The 2‐Thienyl‐Persubstituted α‐Oligothiophenes

The introduction of branching in multi‐thiophene semiconductors, although granting the required solubility for processing, results in an increased molecular fluxionality and a higher level of distortion, thus hampering π conjugation. Accordingly, branched oligothiophenes require rationalization of their structure–reactivity relationships for target‐oriented design and optimization of the synthetic effort. Our current research on spiderlike oligothiophenes affords deep insight into the subject, and introduces new, easily accessible molecules with attractive functional properties. In particular, a regular series, T′X _Y , of five new multi‐thiophene systems, T′5₃ , T′8₄ , T′11₅ , T′14₆ , and T′17₇ , constituted by five, eight, 11, 14, and 17 thiophene units, respectively, their longest α‐conjugated chain consisting of tri‐, tetra‐, penta‐, hexa‐, and heptathiophene moieties, respectively, has been synthesized and fully characterized from the structural, spectroscopic, and electrochemical point of view. The electronic properties of the monomers and their electropolymerization ability are discussed and rationalized as a function of their molecular structure, particularly in comparison with the series of 5‐(2,2′‐dithiophene)yl‐persubstituted α‐oligothiophenes ( TX _Y ) previously reported by us. These oligothiophenes are easily accessible materials, with promising properties for applications as active layers in multifunctional organic devices including solar cells.     

Alphabet‐Inspired Design of (Hetero)Aromatic Push–Pull Chromophores

Push–pull molecules represent a unique and fascinating class of organic π‐conjugated materials. Herein, we provide a summary of their recent extraordinary design inspired by letters of the alphabet, especially focusing on H‐, L‐, T‐, V‐, X‐, and Y‐shaped molecules. Representative structures from each class were presented and their fundamental properties and prospective applications were discussed. In particular, emphasis is given to molecules recently prepared in our laboratory with T‐, X‐, and Y‐shaped arrangements based on indan‐1,3‐dione, benzene, pyridine, pyrazine, imidazole, and triphenylamine. These push–pull molecules turned out to be very efficient charge‐transfer chromophores with tunable properties suitable for second‐order nonlinear optics, two‐photon absorption, reversible pH‐induced and photochromic switching, photocatalysis, and intercalation.

     

Aerobic Iron‐Based Cross‐Dehydrogenative Coupling Enables Efficient Diversity‐Oriented Synthesis of Coumestrol‐Based Selective Estrogen Receptor Modulators

An iron‐based cross‐dehydrogenative coupling (CDC) approach was applied for the diversity‐oriented synthesis of coumestrol‐based selective estrogen receptor modulators (SERMs), representing the first application of CDC chemistry in natural product synthesis. The first stage of the two‐step synthesis of coumestrol involved a modified aerobic oxidative cross‐coupling between ethyl 2‐(2,4‐dimethoxybenzoyl)acetate and 3‐methoxyphenol, with FeCl₃ (10 mol %) as the catalyst. The benzofuran coupling product was then subjected to sequential deprotection and lactonization steps, affording the natural product in 59 % overall yield. Based on this new methodology other coumestrol analogues were prepared, and their effects on the proliferation of the estrogen receptor (ER)‐dependent MCF‐7 and of the ER‐independent MDA‐MB‐231 breast cancer cells were tested. As a result, new types of estrogen receptor ligands having an acetamide group instead of the 9‐hydroxyl group of coumestrol were discovered. Both 9‐acetamido‐coumestrol and 8‐acetamidocoumestrol were found more active than the natural product against estrogen‐dependent MCF‐7 breast cancer cells, with IC₅₀ values of 30 and 9 nM , respectively.     

A Strategy for Neuraminidase Inhibitors Using Mechanism‐Based Labeling Information

A potent inhibitor for Vibrio cholerae neuraminidase (VCNA) was developed by using a novel two‐step strategy, a target amino acid validation using mechanism‐based labeling information, and a potent inhibitor search using a focused library. The labeling information suggested the hidden dynamics of a loop structure of VCNA, which can be a potential target of the novel inhibitor. A focused library composed of 187 compounds was prepared from a 9‐azide derivative of 2,3‐dehydro‐N‐acetylneuraminic acid (DANA) to interrupt the function of the loop of the labeled residues. Inhibitor 3c showed potent inhibition properties and was the strongest inhibitor with FANA, a N‐trifluoroacetyl derivative of DANA. Validation studies of the inhibitor with a detergent and a Lineweaver–Burk plot suggested that the 9‐substitution group would interact hydrophobically with the target loop moiety, adding a noncompetitive inhibition property to the DANA skeleton. This information enabled us to design compound 4 having the combined structure of 3c and FANA. Compound 4 showed the most potent inhibition (K_i=73 nM , mixed inhibition) of VCNA with high selectivity among the tested viral, bacterial, and mammal neuraminidases.     

Selective Inhibition of the Immunoproteasome by Structure‐Based Targeting of a Non‐catalytic Cysteine

Clinically applied proteasome inhibitors induce cell death by concomitant blockage of constitutive and immunoproteasomes. In contrast, selective immunoproteasome inhibition is less cytotoxic and has the potential to modulate chronic inflammation and autoimmune diseases. In this study, we rationally designed decarboxylated peptides that covalently target a non‐catalytic cysteine of the immunoproteasome subunit β5i with α‐chloroacetamide‐containing sidechains. The enhanced isoform specificity decreased cytotoxic effects and the compound suppressed the production of inflammatory cytokines. Structure‐based optimization led to over 150‐fold selectivity for subunit β5i over β5c. This new compound class provides a promising starting point for the development of selective immunoproteasome inhibitors as potential anti‐inflammatory agents.     

Chemical Synthesis of Burkholderia Lipid A Modified with Glycosyl Phosphodiester‐Linked 4‐Amino‐4‐deoxy‐β‐L‐arabinose and Its Immunomodulatory Potential

Modification of the Lipid A phosphates by positively charged appendages is a part of the survival strategy of numerous opportunistic Gram‐negative bacteria. The phosphate groups of the cystic fibrosis adapted Burkholderia Lipid A are abundantly esterified by 4‐amino‐4‐deoxy‐β‐L ‐arabinose (β‐L ‐Ara4N), which imposes resistance to antibiotic treatment and contributes to bacterial virulence. To establish structural features accounting for the unique pro‐inflammatory activity of Burkholderia LPS we have synthesised Lipid A substituted by β‐L ‐Ara4N at the anomeric phosphate and its Ara4N‐free counterpart. The double glycosyl phosphodiester was assembled by triazolyl‐tris‐(pyrrolidinyl)phosphonium‐assisted coupling of the β‐L ‐Ara4N H‐phosphonate to α‐lactol of β(1→6) diglucosamine, pentaacylated with (R)‐(3)‐acyloxyacyl‐ and Alloc‐protected (R)‐(3)‐hydroxyacyl residues. The intermediate 1,1′‐glycosyl‐H‐phosphonate diester was oxidised in anhydrous conditions to provide, after total deprotection, β‐L ‐Ara4N‐substituted Burkholderia Lipid A. The β‐L ‐Ara4N modification significantly enhanced the pro‐inflammatory innate immune signaling of otherwise non‐endotoxic Burkholderia Lipid A.     

A Bioorthogonal Click Chemistry Toolbox for Targeted Synthesis of Branched and Well‐Defined Protein–Protein Conjugates

Bioorthogonal chemistry holds great potential to generate difficult‐to‐access protein–protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels–Alder cycloaddition with inverse electron demand (DA_inv). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin‐based targeted cancer therapies.     

Dual Chemical Modification of a Polytheonamide Mimic: Rational Design and Synthesis of Ion‐Channel‐Forming 48‐mer Peptides with Potent Cytotoxicity

Polytheonamide B ( 1 ) is a natural peptide that displays potent cytotoxicity against P388 mouse leukemia cells (IC₅₀=0.098 nm ). Linear 48‐mer 1 is known to form monovalent cation channels on binding to lipid bilayers. We previously developed a fully synthetic route to 1 , and then achieved the design and synthesis of a structurally simplified analogue of 1 , namely, dansylated polytheonamide mimic 2 . Although the synthetically more accessible 2 was found to emulate the channel function of 1 , its cytotoxicity was decreased 120‐fold. Herein, the chemical preparation and biological evaluation of seven analogues 3 – 9 of 2 are reported. Compounds 3 – 9 were modified at their N terminus and/or the side chain of residue 44 of 2 to alter their physicochemical properties. The total synthesis of 3 – 9 was accomplished in a unified fashion by a combination of solid‐phase and solution‐phase chemistry. Systematic evaluation of the hydrophobicities, single‐channel currents, ion‐exchange activities, and cytotoxicities of 3 – 9 revealed that their hydrophobicities are correlated with the total magnitude of ion exchange and determine their cytotoxic potency. Consequently, the most hydrophobic analogue 9 exhibited the lowest IC₅₀ value, which is comparable to that of 1 . Therefore, these results clarified that the bioactivity of the polytheonamide‐based peptides can be rationally controlled by changing their hydrophobicity at the N and C termini of the 48‐amino‐acid sequence.