Cucurbit[8]uril-based supramolecular theranostics.

Different from most of the conventional platforms with dissatisfactory theranostic capabilities, supramolecular nanotheranostic systems have unparalleled advantages via the artful combination of supramolecular chemistry and nanotechnology. Benefiting from the tunable stimuli-responsiveness and compatible hierarchical organization, host-guest interactions have developed into the most popular mainstay for constructing supramolecular nanoplatforms. Characterized by the strong and diverse complexation property, cucurbit[8]uril (CB[8]) shows great potential as important building blocks for supramolecular theranostic systems. In this review, we summarize the recent progress of CB[8]-based supramolecular theranostics regarding the design, manufacture and theranostic mechanism. Meanwhile, the current limitations and corresponding reasonable solutions as well as the potential future development are also discussed.

Adamantylglycine as a high-affinity peptide label for membrane transport monitoring and regulation.

The non-canonical amino acid adamantylglycine (Ada) is introduced into peptides to allow high-affinity binding to cucurbit[7]uril (CB7). Introduction of Ada into a cell-penetrating peptide (CPP) sequence had minimal influence on the membrane transport, yet enabled up- and down-regulation of the membrane transport activity.

Using a biocatalyzed reaction cycle for transient and pH-dependent host-guest supramolecular hydrogels.

The formation of transient structures plays important roles in biological processes, capturing temporary states of matter through influx of energy or biological reaction networks catalyzed by enzymes. These natural transient structures inspire efforts to mimic this elegant mechanism of structural control in synthetic analogues. Specifically, though traditional supramolecular materials are designed on the basis of equilibrium formation, recent efforts have explored out-of-equilibrium control of these materials using both direct and indirect mechanisms; the preponderance of such works has been in the area of low molecular weight gelators. Here, a transient supramolecular hydrogel is realized through cucurbit[7]uril host-guest physical crosslinking under indirect control from a biocatalyzed network that regulates and oscillates pH. The duration of transient hydrogel formation, and resulting mechanical properties, are tunable according to the dose of enzyme, substrate, or pH stimulus. This tunability enables control over emergent functions, such as the programmable burst release of encapsulated model macromolecular payloads.

Supermolecular Confined Silicon Phosphorescence Nanoprobes for Time-Resolved Hypoxic Imaging Analysis.

Room temperature phosphorescence (RTP) nanoprobes play crucial roles in hypoxia imaging due to their high signal-to-background ratio (SBR) in the time domain. However, synthesizing RTP probes in aqueous media with a small size and high quantum yield remains challenging for intracellular hypoxic imaging up to present. Herein, aqueous RTP nanoprobes consisting of naphthalene anhydride derivatives, cucurbit[7]uril (CB[7]), and organosilicon are reported via supermolecular confined methods. Benefiting from the noncovalent confinement of CB[7] and hydrolysis reactions of organosilicon, such small-sized RTP nanoprobes (5-10 nm) exhibit inherent tunable phosphorescence (from 400 to 680 nm) with microsecond second lifetimes (up to ∼158.7 µs) and high quantum yield (up to ∼30%). The as-prepared RTP nanoprobes illustrate excellent intracellular hypoxia responsibility in a broad range from ∼0.1 to 21% oxygen concentrations. Compared to traditional fluorescence mode, the SBR value (∼108.69) of microsecond-range time-resolved in vitro imaging is up to 2.26 times greater in severe hypoxia (<0.1% O2), offering opportunities for precision imaging analysis in a hypoxic environment.

Metabolic Investigation and Auxiliary Enzyme Modelization of the Pyrrocidine Pathway Allow Rationalization of Paracyclophane-Decahydrofluorene Formation.

Fungal paracyclophane-decahydrofluorene-containing natural products are complex polycyclic metabolites derived from similar hybrid PKS-NRPS pathways. Herein we studied the biosynthesis of pyrrocidines, one representative of this family, by gene inactivation in the producer Sarocladium zeae coupled to thorough metabolic analysis and molecular modeling of key enzymes. We characterized nine pyrrocidines and analogues as well as in mutants a variety of accumulating metabolites with new structures including rare cis-decalin, cytochalasan, and fused 6/15/5 macrocycles. This diversity highlights the extraordinary plasticity of the pyrrocidine biosynthetic gene cluster. From accumulating metabolites, we delineated the scenario of pyrrocidine biosynthesis. The ring A of the decahydrofluorene is installed by PrcB, a membrane-bound cyclizing isomerase, on a PKS-NRPS-derived pyrrolidone precursor. Docking experiments in PrcB allowed us to characterize the active site suggesting a mechanism triggered by arginine-mediated deprotonation at the terminal methyl of the substrate. Next, two integral membrane proteins, PrcD and PrcE, each predicted as a four-helix bundle, perform hydroxylation of the pyrrolidone ring and paracyclophane formation, respectively. Modelization of PrcE highlights a topological homology with vitamin K oxido-reductase and the presence of a disulfide bond. Our results suggest a previously unsuspected coupling mechanism via a transient loss of aromaticity of tyrosine residue to form the strained paracyclophane motif. Finally, the lipocalin-like protein PrcX drives the exo-cycloaddition yielding ring B and C of the decahydrofluorene to afford pyrrocidine A, which is transformed by a reductase PrcI to form pyrrocidine B. These insights will greatly facilitate the microbial production of pyrrocidine analogues by synthetic biology.

An antibiotic preorganized for ribosomal binding overcomes antimicrobial resistance.

We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes evolutionarily diverse forms of antimicrobial resistance that render modern antibiotics ineffective. CRM exhibits in vitro and in vivo efficacy against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We show that CRM is highly preorganized for ribosomal binding by determining its density functional theory-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Lastly, we report two additional x-ray crystal structures of CRM in complex with bacterial ribosomes separately modified by the ribosomal RNA methylases, chloramphenicol-florfenicol resistance (Cfr) and erythromycin-resistance ribosomal RNA methylase (Erm), revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.

Imaging GPCR Dimerization in Living Cells with Cucurbit[7]uril and Hemicyanine as a "Turn-On" Fluorescence Probe.

Although multiple forms of dimers have been described for GPCR, their dynamics and function are still controversially discussed field. Fluorescence microscopy allows GPCR to be imaged within their native context; however, a key challenge is to site-specifically incorporate reporter moieties that can produce high-quality signals upon formation of GPCR dimers. To this end, we propose a supramolecular sensor approach to detect agonist-induced dimer formation of µ-opioid receptors (µORs) at the surface of intact cells. With the macrocyclic host cucurbit[7]uril and its guest hemicyanine dye tethered to aptamer strands directed against the histidine residues, the sensing module is assembled by host-guest complexation once the histidine-tagged µORs dimerize and bring the discrete supramolecular units into close proximity. With the enhanced sensitivity attributed by the "turn-on" fluorescence emission and high specificity afforded by the intermolecular recognition, in situ visualization of dynamic GPCR dimerization was realized with high precision, thereby validating the supramolecular sensing entity as a sophisticated and versatile strategy to investigate GPCR dimers, which represent an obvious therapeutic target.

Characterization and heterologous reconstitution of Taxus biosynthetic enzymes leading to baccatin III.

Paclitaxel is a well known anticancer compound. Its biosynthesis involves the formation of a highly functionalized diterpenoid core skeleton (baccatin III) and the subsequent assembly of a phenylisoserinoyl side chain. Despite intensive investigation for half a century, the complete biosynthetic pathway of baccatin III remains unknown. In this work, we identified a bifunctional cytochrome P450 enzyme [taxane oxetanase 1 (TOT1)] in Taxus mairei that catalyzes an oxidative rearrangement in paclitaxel oxetane formation, which represents a previously unknown enzyme mechanism for oxetane ring formation. We created a screening strategy based on the taxusin biosynthesis pathway and uncovered the enzyme responsible for the taxane oxidation of the C9 position (T9αH1). Finally, we artificially reconstituted a biosynthetic pathway for the production of baccatin III in tobacco.

Cucurbit[7]uril-mediated Histidine Dimerization: Exploring the Structure and Binding Mechanism.

Cucurbit[7,8]urils are known to form inclusion complexes with hydrophobic amino acids such as Trp, Tyr, Phe, and Met, as well as peptides containing these residues at the N-terminus. Despite their widespread use in protein purification, the affinity of histidine (His) for cucurbit[7,8]urils has not been extensively explored. In this study, X-ray diffraction experiments were conducted to investigate the binding of two histidine moieties to the cucurbit[7]uril (CB7) cavity, resulting in a network of π-π and hydrogen bonds. This assembly was found to induce a His pKa shift of ΔpKa=-4. Histidine weakly bound to CB7 or CB8; however, isothermal titration calorimetry revealed micromolar equilibrium dissociation constant values for CB7 and CB8 when bound to dipeptides containing His at the C-terminus. Conversely, dipeptides with His at the N-terminus exhibited millimolar values. Additionally, the His-Gly-Gly tripeptide formed a 2 : 1 complex with CB7. These findings suggest the potential use of histidine and histidine-containing tags in conjunction with CB7 for various biological applications.

Complexation of Fluorofenidone by Cucurbit[7]uril and ß-Cyclodextrin: Keto-Enol Tautomerization to Enhance the Solubility.

This study is designed to compare drug encapsulation by cucurbit[7]uril and ß-cyclodextrin, using fluorofenidone as a model drug. Single-crystal X-ray diffraction analysis was employed to successfully determine the crystal structures of fluorofenidone·H+@cucurbit[7]uril Form, fluorofenidone@cucurbit[7]uril Form, and fluorofenidone@ß-cyclodextrin Form. Keto-enol tautomerization of fluorofenidone mediated by cucurbit[7]uril in acid solution is confirmed by crystal structures, pH titration, and nuclear magnetic resonance experiments. However, ß-cyclodextrin cannot cause the keto-enol tautomerization of fluorofenidone under similar conditions. The phase solubility study demonstrates that cucurbit[7]uril has a much higher solubilization capacity for fluorofenidone than ß-cyclodextrin in 0.1 M HCl since the Kc values of fluorofenidone with cucurbit[7]uril and ß-cyclodextrin were 1223.97 ± 452.68 and 78.49 ± 10.56 M-1, respectively. Excellent solubility can be attributed to the keto-enol tautomerization of fluorofenidone under the conditions of cucurbit[7]uril in acid solution. The enol form of fluorofenidone is encapsulated by cucurbit[7]uril by hydrogen bonding interaction and hydrophobic interaction to increase binding affinity. Rat pharmacokinetic studies demonstrate that the area under the plasma concentration-time curve from time 0 to 7 h value of fluorofenidone@cucurbit[7]uril complex is 1.70-fold greater than that of free fluorofenidone, and the mean residence time from time 0 to 7 h is slightly prolonged from 1.29 to 1.76 h (P < 0.01) after oral administration. However, no significant difference is found between fluorofenidone and fluorofenidone@ß-cyclodextrin complex. This work indicates that the induction of keto-enol tautomerization of drugs using macrocyclic molecules has the potential to be an effective method to improve their solubility and bioavailability, providing valuable insights for the application of macrocyclic molecules in the biomedical field.

Theoretical study of the rearrangement reaction in bisorbicillinoid biosynthesis: insights into the molecular mechanisms involved.

Bisorbibutenolide and bisorbicillinolide are polyketide compounds with complex skeletons that are formed by the dimerization of sorbicillin. These compounds have long been of interest, with several reports of their biosynthesis, biological activity, and total synthesis. In this study, we theoretically investigated the detailed biosynthetic mechanism of the rearrangement reaction to form bisorbicillinolide. We showed that the presence of water molecules facilitates the intramolecular aldol reaction, determined the rate-limiting steps, and revealed that a cyclopropane intermediate is formed during the rearrangement process. Although computational chemistry has been widely applied to the carbocation chemistry present in terpene biosynthesis, it has seldom been used to investigate the carbonyl chemistry responsible for polyketide biosynthesis. This study shows that computational chemistry is a useful tool for studying anionic skeletal rearrangement reactions.

Modulation of the Fibrillation Kinetics and Morphology of a Therapeutic Peptide by Cucurbit[7]uril.

Fibrillation is a challenge commonly encountered in the formulation and development of therapeutic peptides. Cucurbit[7]urils (CB[7]), a group of water soluble macrocycles, have been reported to suppress fibrillation in insulin and human calcitonin through association with Phe and Tyr residues which drive fibril formation. Here, we report the effect of CB[7] on the fibrillation behavior of the HIV fusion inhibitor enfuvirtide (ENF) that contains N-terminal Tyr and C-terminal Phe residues. Thioflavin T fluorescence, CD spectroscopy, and transmission electron microscopy were used to monitor fibrillation behavior. Fibrillation onset showed a strong pH dependency, with pH 6.5 identified as the condition most suitable to monitor the effects of CB[7]. Binding of CB[7] to wild-type ENF was measured by isothermal titration calorimetry and was consistent with a single site (Ka = 2.4 × 105 M-1). A weaker interaction (Ka = 2.8 × 103 M-1) was observed for an ENF mutant with the C-terminal Phe substituted for Ala (ENFm), suggesting that Phe was the specific site for CB[7] recognition. The onset of ENF fibrillation onset was delayed, rather than fully suppressed, in the presence of CB[7]. The ENFm mutant showed a greater delay in fibrillation onset but with no observable effect on fibrillation kinetics in the presence of CB[7]. Interestingly, ENF/CB[7] and ENFm fibrils exhibited comparable morphologies, differing from those observed for ENF alone. The results indicate that CB[7] is capable of modulating fibrillation onset and the resulting ENF fibrils by specifically binding to the C-terminal Phe residue. The work reinforces the potential of CB[7] as an inhibitor of fibrillation and highlights its role in determining fibril morphologies.

Electrochemical Detection of Drugs via a Supramolecular Cucurbit[7]uril-Based Indicator Displacement Assay.

Electrochemical detection methods are attractive for developing miniaturized, disposable, and portable sensors for molecular diagnostics. In this article, we present a cucurbit[7]uril-based chemosensor with an electrochemical signal readout for the micromolar detection of the muscle relaxant pancuronium bromide in buffer and human urine. This is possible through a competitive binding assay using a chemosensor ensemble consisting of cucurbit[7]uril as the host and an electrochemically active platinum(II) compound as the guest indicator. The electrochemical properties of the indicator are strongly modulated depending on the complexation state, a feature that is exploited to establish a functional chemosensor. Our design avoids cumbersome immobilization approaches on electrode surfaces, which are associated with practical and conceptual drawbacks. Moreover, it can be used with commercially available screen-printed electrodes that require minimal sample volume. The design principle presented here can be applied to other cucurbit[n]uril-based chemosensors, providing an alternative to fluorescence-based assays.

Supramolecular Complexation of Azobenzene Dyes by Cucurbit[7]uril.

This report describes cucurbit[7]uril (CB7) complexation of azobenzene dyes that have a 4-(N,N'-dimethylamino) or 4-amino substituent. Absorption and NMR data show that CB7 encapsulates the protonated form of the azobenzene and that the complexed dye exists as its azonium tautomer with a trans azo conformation and substantial quinoid resonance character. Because CB7 complexation stabilizes the dye conjugate acid, there is an upward shift in its pKa, and in one specific case, the pKa of the protonated azobenzene is increased from 3.09 to 4.47. Molecular modeling indicates that the CB7/azobenzene complex is stabilized by three major noncovalent factors: (i) ion-dipole interactions between the partially cationic 4-(N,N'-dimethylamino) or 4-amino group on the encapsulated protonated azobenzene and the electronegative carbonyl oxygens on CB7, (ii) inclusion of the upper aryl ring of the azobenzene within the hydrophobic CB7 cavity, and (iii) a hydrogen bond between the proton on the azo nitrogen and CB7 carbonyls. CB7 complexation enhances azobenzene stability and increases azobenzene hydrophilicity; thus, it is a promising way to improve azobenzene performance as a pigment or prodrug. In addition, the striking yellow/pink color change that accompanies CB7 complexation can be exploited to create azobenzene dye displacement assays with naked eye detection.

A pH- and Metal-Actuated Molecular Shuttle in Water.

The structure of the Viologen-Phenylene-Imidazole (VPI) guest, previously shown to be bound by cucurbit[7]uril (CB[7]) with binding modes depending on pH and silver ions, has been extended by adding hydrophobic groups on the two extremities of VPI before investigations of CB[7] binding by NMR, ITC, X-ray diffraction, UV-vis and fluorescence spectroscopies. With an imidazole station extended by a naphthalene group (VPI-N), binding modes of CB[7] are similar to those previously observed. However, with the viologen extended by a tolyl group (T-VPI), CB[7] preferentially sits on station T, shuttling between the T and P stations at acid pH or after Ag+ addition. The CB[7] ⋅ T-VPI complex thus behaves as a metal-actuated thermodynamic stop-and-go molecular shuttle featured by fast and autonomous ring translocation between two stations and a continuum for fractional station occupancy solely and easily controlled by Ag+ concentration.

Theoretical prediction of nanomolar and sequence-selective binding of synthetic supramolecular cucurbit[7]uril to N-terminal Leu-containing tripeptides.

Molecular recognition towards peptides and proteins with high affinity by synthetic supramolecular hosts is important but challenging. In this work, we investigate the molecular recognition of the synthetic cucurbit[7]uril (CB[7]) to 17 designed N-terminal Leu-containing tripeptides in aqueous medium by molecular dynamics (MD) simulation and screen out tripeptides with high binding affinity. It is found that, compared to LGG, only the third residue is Arg (R), the binding affinity of CB[7] to LGR reaches nanomolar level with binding equilibrium constant (Ka) of 1.1 × 109 M-1. The CB[7] recognition to the N-terminal Leu-containing tripeptides is highly sequence dependent; whether changing the sequence order (from LGR to LRG) or increasing the sequence length (from LGR to LGGR), Ka decreases by about three orders of magnitude. Interestingly, substituting N-terminal Leu for its isomer Ile, the binding of CB[7] to tripeptides weakens significantly with Ka decreasing by 3-8 orders of magnitude. Thus CB[7] can effectively distinguish N-terminal Leu-containing tripeptides from N-terminal Ile-containing tripeptides. Importantly, we predict that when R is as C-terminus, regardless of N-terminal residue being of aromatic type or Leu, the binding strength is always close to the nanomolar level. Therefore, R can be introduced to rationally design novel peptides with high binding affinity to CB[7] in practical applications.

Metal-Assisted Complexation of Fluorogenic Dyes by Cucurbit[7]uril and Cucurbit[8]uril: A DFT Evaluation of the Key Factors Governing the Host-Guest Recognition.

With the emergence of host-guest systems, a novel branch of complexation chemistry has found wide application in industries such as food, pharmacy, medicine, environmental protection and cosmetics. Along with the extensively studied cyclodextrins and calixarenes, the innovative cucurbiturils (CB) have enjoyed increased popularity among the scientific community as they possess even better qualities as cavitands as compared to the former molecules. Moreover, their complexation abilities could further be enhanced with the assistance of metal cations, which can interestingly exert a dual effect on the complexation process: either by competitively binding to the host entity or cooperatively associating with the CB@guest structures. In our previous work, two metal species (Mg2+ and Ga3+) have been found to bind to CB molecules in the strongest fashion upon the formation of host-guest complexes. The current study focuses on their role in the complex formation with three dye molecules: thiazole orange, neutral red, and thioflavin T. Various key factors influencing the process have been recognized, such as pH and the dielectric constant of the medium, the cavity size of the host, Mn+ charge, and the presence/absence of hydration shell around the metal cation. A well-calibrated DFT methodology, solidly based and validated and presented in the literature experimental data, is applied. The obtained results shed new light on several aspects of the cucurbituril complexation chemistry.

Stability and pKa Modulation of Aminophenoxazinones and Their Disulfide Mimics by Host-Guest Interaction with Cucurbit[7]uril. Direct Applications in Agrochemical Wheat Models.

Aqueous solubility and stability often limit the application of aminophenoxazinones and their sulfur mimics as promising agrochemicals in a sustainable agriculture inspired by allelopathy. This paper presents a solution to the problem using host-guest complexation with cucurbiturils (CBn). Computational studies show that CB7 is the most suitably sized homologue due to its strong affinity for guest molecules and its high water solubility. Complex formation has been studied by direct titrations monitored using UV-vis spectroscopy, finding a preferential interaction with protonated aminophenoxazinone species with high binding affinities (CB7·APOH+, Ka = (1.85 ± 0.37) × 106 M-1; CB7·DiS-NH3+, Ka = (3.91 ± 0.53) × 104 M-1; and DiS-(NH3+)2, Ka= (1.27 ± 0.42) × 105 M-1). NMR characterization and stability analysis were also performed and revealed an interesting pKa modulation and stabilization by cucurbiturils (2-amino-3H-phenoxazin-3-one (APO), pKa = 2.94 ± 0.30, and CB7·APO, pKa = 4.12 ± 0.15; 2,2'-disulfanediyldianiline (DiS-NH2), pKa = 2.14 ± 0.09, and CB7·DiS-NH2, pKa = 3.26 ± 0.09), thus favoring applications in different kinds of crop soils. Kinetic studies have demonstrated the stability of the CB7·APO complex at different pH media for more than 90 min. An in vitro bioassay with etiolated wheat coleoptiles showed that the bioactivity of APO and DiS-NH2 is enhanced upon complexation.

DNA-based assemblies with bischromophoric styryl dye-chromene conjugates and cucurbit[7]uril.

Novel conjugates consist of 4-styrylpyridinium dye and 2,2-diphenyl-2H-chromene moiety were obtained, and their affinity to double stranded DNA and cucurbit[7]uril was investigated. With a combination of absorption, fluorescence and circular dichroism spectroscopies as well as MALDI-TOF mass spectrometry, we demonstrate that these compounds can interact with macromolecules to form of the supramolecular assemblies due to two suitable binding sites. The ternary complex is formed as a result of the intercalation of a positively charged styryl part between DNA base pairs, while cucurbit[7]uril is located on the alkyl chain between two moieties of conjugate. All these findings provide valuable information into controlling the interaction between organic molecules, DNA and cucurbit[7]uril.

Supramolecular Protein Stabilization with Zwitterionic Polypeptide-Cucurbit[7]uril Conjugates.

Protein aggregation is an obstacle for the development of new biopharmaceuticals, presenting challenges in shipping and storage of vital therapies. Though a variety of materials and methods have been explored, the need remains for a simple material that is biodegradable, nontoxic, and highly efficient at stabilizing protein therapeutics. In this work, we investigated zwitterionic polypeptides prepared using a rapid and scalable polymerization technique and conjugated to a supramolecular macrocycle host, cucurbit[7]uril, for the ability to inhibit aggregation of model protein therapeutics insulin and calcitonin. The polypeptides are based on the natural amino acid methionine, and zwitterion sulfonium modifications were compared to analogous cationic and neutral structures. Each polymer was end-modified with a single cucurbit[7]uril macrocycle to afford supramolecular recognition and binding to terminal aromatic amino acids on proteins. Only conjugates prepared from zwitterionic structures of sufficient chain lengths were efficient inhibitors of insulin aggregation and could also inhibit aggregation of calcitonin. This polypeptide exhibited no cytotoxicity in human cells even at concentrations that were five-fold of the intended therapeutic regime. We explored treatment of the zwitterionic polypeptides with a panel of natural proteases and found steady biodegradation as expected, supporting eventual clearance when used as a protein formulation additive.