Cyclic Backbone Research Articles

Monofluoromethyl‐Substituted Sulfonium Ylides: Preparation, Structure‐Reactivity Study and Substrate Scope†

Summary of main observation and conclusionStructure‐reactivity study of a family of electrophilic monofluoromethylating reagents based on sulfonium ylide skeleton with different steric hindrance and electron‐withdrawing properties was described. These studies led us to discover two highly reactive reagents 3 with a cyclic malonate backbone and 6 with an electron‐poor 1,1,1,5,5,5‐hexafluoropentane‐2,4‐dione backbone. The high reactivity of reagent 6 allowed to highly selectively access either C‐monofluoromethylated or O‐monofluoromethylated β‐ketoesters in high yields by the use of different bases. In addition, reactions of reagent 3 with a variety of nucleophiles including phenols, carboxylic acids, thiophenols or heteroaryl nucleophiles occurred in full conversion within 10 min at room temperature and the scopes for these reactions were reported in detail.

Enhancing Robustness of Sortase A by Loop Engineering and Backbone Cyclization.

Staphylococcus aureus sortase A (SaSrtA) is widely used for site‐specific protein modifications, but it lacks the robustness for performing bioconjugation reactions at elevated temperatures or in presence of denaturing agents. Loop engineering and subsequent head‐to‐tail backbone cyclization of SaSrtA yielded the cyclized variant CyM6 that has a 7.5 °C increased melting temperature and up to 4.6‐fold increased resistance towards denaturants when compared to the parent rM4. CyM6 gained up to 2.6‐fold (vs. parent rM4) yield of conjugate in ligation of peptide and primary amine under denaturing conditions.

Lateral cyclic testing and backbone curve development of high-strength steel built-up box columns under axial compression

The AISC 341 (2016) has a more stringent width-to-thickness (b/t) limit for highly ductile hollow box columns (HBCs) than the AIJ (2010) or Taiwan Code (2010), resulting in significant thickness difference in design. Moreover, the cyclic backbone curves based on ASCE 41 (2013) and NIST (2017) underestimate the post-buckling flexural strength of HBCs, particularly in high axial compression force. This paper presents test results of six full-scale, built-up HBCs using SM 570 M steel with the actual yield strength of 460–530 MPa. The lateral cyclic behavior of built-up columns was studied in terms of different b/t ratios, magnitudes of axial compression forces, and lateral drift histories (i.e., cyclically symmetric versus near-fault displacement histories). The built-up box columns were 290–400 mm wide with b/t ratios from 11 to 21, 4000 mm high, and tested laterally with both ends fixed after under a constant axial compression force, 2591–7935 kN. The HBC specimens which were designed based on the highly ductile member requirement in AISC 341 (2016), even under an axial compression force (=40%Py), performed satisfactorily at 4% drift and experienced flange and web fracture at 5% drift. However, the HBC specimens that satisfied the most compact b/t requirement in AIJ (2010) or Taiwan code (2010) did not perform well at 4% drift, losing the axial capacity after significant column local buckling and shortening. The gathered test data, supported by more test data in this work, were analyzed by a multiple regression method to obtain empirical formulations for predicting the maximum column moment, plastic rotation and post-yield hardening parameters. The proposed formulation reasonably predicts the first-cycle envelope curves of built-up HBCs, improving prediction results based on ASCE 41 (2013) and NIST (2017).

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus

Cyclotides are plant-derived peptides characterized by an ∼30-amino acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

Resorcinol-Based Epoxy Resins Hardened with Limonene and Eugenol Derivatives: From the Synthesis of Renewable Diamines to the Mechanical Properties of Biobased Thermosets

Synthesis of fully eco-friendly thermosets based on renewable resorcinol diglycidyl ether (RE) hardenered with different biobased diamines having aliphatic, cyclic, or aromatic backbones were prepa...

1-Hydroxyanthraquinones Containing Aryl Substituents as Potent and Selective Anticancer Agents.

A series of 1,2-, 1,4-disubstituted or 1,2,4-trisubstituted anthraquinone-based compounds was designed, synthesized, characterized and biologically evaluated for anticancer efficacy. 2- or 4-arylated 1-hydroxy-9,10-antraquinones (anthracene-9,10-diones) were prepared by Suzuki–Miyaura cross-coupling reaction of 1-hydroxy-2-bromoanthraquinone, 1-hydroxy-4-iodoanthraquinone or 1-hydroxy-2,4-dibromoanthraquinone with arylboronic acids. The cross-coupling reaction of 2,4-dibromo-9,10-anthraquinone with arylboronic acids provide a convenient approach to 2,4-bis arylated 1-hydroxyanthraquinones with a variety of aryl substituent in the 2 and 4 position. The cytotoxicity of new anthraquinone derivatives was evaluated using the conventional MTT assays. The data revealed that six of the aryl substituted compounds among the entire series 3, 15, 16, 25, 27, 28 were comparable potent with the commercially available reference drug doxorubicin on the human glioblastoma cells SNB-19, prostate cancer DU-145 or breast cancer cells MDA-MB-231 and were relatively safe towards human telomerase (h-TERT)immortalized lung fibroblasts cells. The results suggested that the in vitro antitumor activity of synthesized 2-aryl, 4-aryl- and 2,4-diaryl substituted 1-hydroxyanthraquinones depends on the nature of the substituent within the cyclic backbone. Docking interaction of 2-, 4-substituted and 2,4-disubstituted 1-hydroxyanthraquinones indicates intercalative mode of binding of compounds with DNA topoisomerase. The interaction with the DNA of 4-aryl-13, 15, 16 and 4-(furan-3-yl)-23 1-hydroxyanthraquinones was experimentally confirmed through a change in electroforetic mobility. Further experiments with 1-hydroxy-4-phenyl-anthraquinone 13 demonstrated that the compound induced cell cycle arrest at sub-G1 phase in DU-145 cells in the concentration 1.1 μM, which is probably achieved by inducing apoptosis. 4-Arylsubstituted 1-hydroxyanthraquinones 13 and 16 induced the enhancement of DNA synthesis on SNB19 cell lines.

Application and Structural Analysis of Triazole‐Bridged Disulfide Mimetics in Cyclic Peptides

AbstractRuthenium‐catalysed azide–alkyne cycloaddition (RuAAC) provides access to 1,5‐disubstituted 1,2,3‐triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross‐linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor‐1. NMR and X‐ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole‐bridged peptides also displayed superior half‐lives in liver S9 stability assays compared to disulfide‐bridged peptides. This work establishes a foundation for the application of 1,5‐disubstituted 1,2,3‐triazoles as disulfide mimetics.

Application and Structural Analysis of Triazole-Bridged Disulfide Mimetics in Cyclic Peptides.

Ruthenium-catalysed azide-alkyne cycloaddition (RuAAC) provides access to 1,5-disubstituted 1,2,3-triazole motifs in peptide engineering applications. However, investigation of this motif as a disulfide mimetic in cyclic peptides has been limited, and the structural consequences remain to be studied. We report synthetic strategies to install various triazole linkages into cyclic peptides through backbone cyclisation and RuAAC cross-linking reactions. These linkages were evaluated in four serine protease inhibitors based on sunflower trypsin inhibitor-1. NMR and X-ray crystallography revealed exceptional consensus of bridging distance and backbone conformations (RMSD<0.5 Å) of the triazole linkages compared to the parent disulfide molecules. The triazole-bridged peptides also displayed superior half-lives in liver S9 stability assays compared to disulfide-bridged peptides. This work establishes a foundation for the application of 1,5-disubstituted 1,2,3-triazoles as disulfide mimetics.

Discussion of “A Cyclic Backbone Curve for Shear-Critical Reinforced Concrete Walls” by Siamak Epackachi, Nikhil Sharma, Andrew Whittaker, Ronald O. Hamburger, and Ayse Hortacsu

Discussion of “A Cyclic Backbone Curve for Shear-Critical Reinforced Concrete Walls” by Siamak Epackachi, Nikhil Sharma, Andrew Whittaker, Ronald O. Hamburger, and Ayse Hortacsu

Effects of Cyclization on Activity and Stability of α-Conotoxin TxIB.

α-Conotoxin TxIB specifically blocked α6/α3β2β3 acetylcholine receptors (nAChRs), and it could be a potential probe for studying addiction and other diseases related to α6/α3β2β3 nAChRs. However, as a peptide, TxIB may suffer from low stability, short half-life, and poor bioavailability. In this study, cyclization of TxIB was used to improve its stability. Four cyclic mutants of TxIB (cTxIB) were synthesized, and the inhibition of these analogues on α6/α3β2β3 nAChRs as well as their stability in human serum were measured. All cyclized analogues had similar activity compared to wild-type TxIB, which indicated that backbone cyclization of TxIB had no significant effect on its activity. Cyclization of TxIB with a seven-residue linker improved its stability significantly in human serum. Besides this, the results showed that cyclization maintained the activity of α-conotoxin TxIB, which is conducive to its future application.

A bifunctional asparaginyl endopeptidase efficiently catalyzes both cleavage and cyclization of cyclic trypsin inhibitors

Asparaginyl endopeptidases (AEPs) catalyze the key backbone cyclization step during the biosynthesis of plant-derived cyclic peptides. Here, we report the identification of two AEPs from Momordica cochinchinensis and biochemically characterize MCoAEP2 that catalyzes the maturation of trypsin inhibitor cyclotides. Recombinantly produced MCoAEP2 catalyzes the backbone cyclization of a linear cyclotide precursor (MCoTI-II-NAL) with a kcat/Km of 620 mM−1 s−1, making it one of the fastest cyclases reported to date. We show that MCoAEP2 can mediate both the N-terminal excision and C-terminal cyclization of cyclotide precursors in vitro. The rate of cyclization/hydrolysis is primarily influenced by varying pH, which could potentially control the succession of AEP-mediated processing events in vivo. Furthermore, MCoAEP2 efficiently catalyzes the backbone cyclization of an engineered MCoTI-II analog with anti-angiogenic activity. MCoAEP2 provides enhanced synthetic access to structures previously inaccessible by direct chemistry approaches and enables the wider application of trypsin inhibitor cyclotides in biotechnology applications.

Strain criteria for alkenes: Two different manifestations

Criteria that have been used to describe alkene strain can be divided into energetic and geometrical criteria. An overview of strain criteria from both groups is given, and altogether, 16 different criteria have been calculated with the DFT M062X/TZVP method for several strained alkene series. Factor analysis was used to judge whether these strain criteria measure the same underlying phenomena. The factor analysis showed that for systematically substituted ethene derivatives (series A) all the studied criteria were describing the same thing. The factor analysis showed that for series B, where the double bonds geometry is fixed with a cyclic backbone, at least two different criteria (e.g. gas-phase basicity and hydrogenation reaction enthalpy) are needed to describe the overall strain. We propose that these two different expressions of strain describe strain related to reactivity and thermodynamics. Those two strains were well-correlated in the first series.

Backbone Cyclization and Dimerization of LL-37-Derived Peptides Enhance Antimicrobial Activity and Proteolytic Stability.

Can antimicrobial activity and peptide stability of alpha-helical peptides be increased by making them into dimers and macrocycles? Here, we explore that concept by using KR-12 as the starting point for peptide engineering. KR-12 has previously been determined as the minimalized antimicrobial fragment of the human host defense peptide LL-37. Backbone-cyclized KR-12 dimers, tethered by linkers of two to four amino acid residues, were synthesized and their antimicrobial activity, proteolytic stability and structures characterized. A modified KR-12 sequence, with substitutions at previously identified key residues, were also included in the screening panel. The backbone cyclized KR-12 dimers showed improved antimicrobial activity and increased stability compared to monomeric KR-12. The most active cyclic dimer displayed 16-fold higher antibacterial activity compared to KR-12 against Pseudomonas aeruginosa and Staphylococcus aureus, and 8-fold increased fungicidal activity against Candida albicans. It also showed increased hemolytic and cytotoxic activity. Enhanced antimicrobial activity coincided with increased membrane permeabilization of liposomes with one distinct discrepancy: monomeric KR-12 was much less disruptive of liposomes with bacterial lipid composition compared to liposomes from fungal lipid extract. Circular dichroism showed that the four-residue linked most active cyclic dimer had 65% helical content when bound to lyso-phosphatidylglycerol micelles, indicating that the helical propensity of the parent peptide is maintained in the new macrocyclic form. In conclusion, the current work on KR-12 suggests that dimerization together with backbone cyclization is an effective strategy for improving both potency and stability of linear antimicrobial peptides.

Synthesis and properties of pH-cleavable toothbrush-like copolymers comprising multi-reactive Y junctions and a linear or cyclic backbone

Y-junction-bearing toothbrush-like copolymers can exhibit unique physical properties and hierarchical (co)assembly behaviors dependent on topology, external stimuli and hydrolysis.

Recombinant Expression of Cyclotides Using Expressed Protein Ligation.

Cyclotides are naturally occurring microproteins (≈30 residues long) present in several families of plants. All cyclotides share a unique head-to-tail circular knotted topology containing three disulfide bridges forming a cystine knot topology. Cyclotides possess high stability to chemical, physical, and biological degradation and have been reported to cross cellular membranes. In addition, naturally occurring and engineered cyclotides have shown to possess various pharmacologically relevant activities. These unique features make the cyclotide scaffold an excellent tool for the design of novel peptide-based therapeutics by using molecular evolution and/or peptide epitope grafting techniques. In this chapter, we provide protocols to recombinantly produce a natively folded cyclotide making use of a standard bacterial expression system in combination with an intein-mediated backbone cyclization with concomitant oxidative folding.

Cyclotides: From Structure to Function

This Review explores the class of plant-derived macrocyclic peptides called cyclotides. We include an account of their discovery, characterization, and distribution in the plant kingdom as well as a detailed analysis of their sequences and structures, biosynthesis and chemical synthesis, biological functions, and applications. These macrocyclic peptides are around 30 amino acids in size and are characterized by their head-to-tail cyclic backbone and cystine knot motif, which render them to be exceptionally stable, with resistance to thermal or enzymatic degradation. Routes to their chemical synthesis have been developed over the past two decades, and this capability has facilitated a wide range of mutagenesis and structure-activity relationship studies. In turn, these studies have both led to an increased understanding of their mechanisms of action as well as facilitated a range of applications in agriculture and medicine, as ecofriendly crop protection agents, and as drug leads or scaffolds for pharmaceutical design. Our overall objective in this Review is to provide readers with a comprehensive overview of cyclotides that we hope will stimulate further work on this fascinating family of peptides.

Porous films from cyclic block copolymers

Porous films are attractive materials used in numerous fields, such as catalysis, templating, and separation. In previous examples of porous films prepared from block polymers, the pore size was usually determined by various factors, including mutual interaction between polymer chains, and was hard to precisely manipulate by modification of the block polymers. Herein, we report a novel method of preparing porous films from cyclic block copolymers with the pore size determined by the size of individual components. The incompatibility of the dangling polystyrene arms and cyclic polysiloxane backbones drove the assembly, which then led to the vertical alignment of the cyclic polysiloxane backbone to form porous films. Coarse-grained molecular dynamics simulations were also implemented to gain insight into the assembly process and the film microstructure at the molecular scale. A cyclic block copolymer allows feasible fabrication of porous films through designed interaction between its macromolecular arms. The morphology of pores is directly determined by the structural features of the block copolymer and can be easily tuned by modification of the length of its ring and arms.

Abstract A134: Therapeutic targeting of Hdm2/HdmX E3 ligase with bioactive cyclotides

Abstract Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Hdm2-p53 interface or HdmX-p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Hdm2–HdmX which is mediated by RING–RING interaction has recently appeared as a promising novel anticancer approach. Targeting the E3 ligase of Hdm2-HdmX should overcome HdmX-driven resistance, which is found in many cancers overexpressing HdmX, to p53-Hdm2 antagonists such a nutlins. Cyclotides are a novel class of micro-proteins (≈30-40 residues long) with a unique topology containing a head-to-tail cyclized backbone structure further stabilized by three disulfide bonds that form a cystine knot. This unique molecular framework makes them exceptionally stable to physical, chemical, and biological degradation compared to linear peptides of similar size. The cyclotides are also highly tolerant to sequence variability, aside from the conserved residues forming the cystine knot, orally bioavailable and able to cross cellular membranes to modulate intracellular protein-protein interactions (PPIs) both in vitro and in vivo. These unique properties make them ideal scaffolds for many biotechnological applications, including drug discovery. We report the discovery of a novel cyclotide, MCo-52-2, able to antagonize efficiently the interaction between the RING domains of Hdm2 and HdmX and show p53-specific anticancer activity in several cancer cell lines both in vitro and in vivo. We will also present different strategies to improve the cellular uptake and pharmacokinetic profiles of bioactive cyclotides. Citation Format: Julio A Camarero. Therapeutic targeting of Hdm2/HdmX E3 ligase with bioactive cyclotides [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A134. doi:10.1158/1535-7163.TARG-19-A134

Evaluation of Anti-inflammatory Efficacy of RA-V: a Natural Cyclopeptide.

Inflammation occurs in a cascade reaction to cause damage in the tissues. It involves increased expression of oxidants and cytokines in the injured tissues. Peptides are chosen for the development of alternate and more selective anti-inflammatory therapies. They are highly specific and significant due to their low molecular weight, simple structure, low antigenicity, and low toxicity. They are capable of easy absorption through different routes of administration and rapid clearance from the system. Plant-derived cyclic peptides have great therapeutic values. Peptides from Rubia cordifolia exhibit potent anti-inflammatory, anti-diabetic, anti-arthritic, and antioxidant properties. Cyclotides are small peptides isolated from plants with a cyclic backbone and disulfide bonds. The cyclic cystine knot structure of the cyclic peptide makes them resistant to chemical, enzymatic, and thermal conditions. They are isolated from Rubiaceae, Violaceae, and Cucurbitaceae plant families. The overall aim of this study is to evaluate the biological efficacy of the cyclopeptide, RA-V (deoxybouvardin) isolated from Rubia cordifolia.

Characterization of Tachyplesin Peptides and Their Cyclized Analogues to Improve Antimicrobial and Anticancer Properties.

Tachyplesin I, II and III are host defense peptides from horseshoe crab species with antimicrobial and anticancer activities. They have an amphipathic β-hairpin structure, are highly positively-charged and differ by only one or two amino acid residues. In this study, we compared the structure and activity of the three tachyplesin peptides alongside their backbone cyclized analogues. We assessed the peptide structures using nuclear magnetic resonance (NMR) spectroscopy, then compared the activity against bacteria (both in the planktonic and biofilm forms) and a panel of cancerous cells. The importance of peptide-lipid interactions was examined using surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies showed that tachyplesin peptides and their cyclic analogues were most potent against Gram-negative bacteria and melanoma cell lines, and showed a preference for binding to negatively-charged lipid membranes. Backbone cyclization did not improve potency, but improved peptide stability in human serum and reduced toxicity toward human red blood cells. Peptide-lipid binding affinity, orientation within the membrane, and ability to disrupt lipid bilayers differed between the cyclized peptide and the parent counterpart. We show that tachyplesin peptides and cyclized analogues have similarly potent antimicrobial and anticancer properties, but that backbone cyclization improves their stability and therapeutic potential.