Binding Of CB Research Articles

"Gear-driven"-type chirality transfer of tetraphenylethene-based supramolecular organic frameworks for peptides in water.

Chirality transfer for natural chiral biomolecules can reveal the indispensable role of chiral structures in life and can be used to develop the chirality-sensing biomolecular recognition. Here, we report the synthesis and characterization of a series of achiral supramolecular organic frameworks (SOF-1, SOF-2, and SOF-3), constructed from cucurbit[8]uril (CB[8]) and tetraphenylethene (TPE) derivatives (1, 2, and 3), respectively, as chirality-sensing platforms to explore their chirality transfer mechanism for peptides in water. Given the right-handed (P) and left-handed (M) rotational conformation of TPE units and the selective binding of CB[8] to aromatic amino acids, these achiral SOFs can be selectively triggered in water by peptides containing N-terminal tryptophan (W) and phenylalanine (F) residues into their P- or M-rotational conformation, exhibiting significantly different circular dichroism (CD) spectra. Although various peptides have the same l-type chiral configuration, they can induce positive CD signals of SOF-1 and negative CD signals of SOF-2 and SOF-3, respectively. Based on the structural analysis of the linkage units between CB[8] and TPE units in these SOFs, a "gear-driven"-type chirality transfer mechanism has been proposed to visually illustrate the multiple-step chirality transfer process from the recognition site in the CB[8]'s cavity to TPE units. Furthermore, by utilizing the characteristic CD signals generated through the "gear-driven"-type chirality transfer, these SOFs can serve as chiroptical sensor arrays to effectively recognize and distinguish various peptides based on their distinctive CD spectra.

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.

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.

Sequence-dependent nanomolar binding of tripeptides containing N-terminal phenylalanine by Cucurbit[7]uril: A theoretical study

Molecular recognition towards peptides with high affinity and sequence selectivity by synthetic macrocyclic receptors is a great challenge in supramolecular chemistry. In this work, we investigate the molecular recognition of tripeptides containing N-terminal phenylalanine (Phe, F) by synthetic macrocyclic receptor Cucurbit[7]uril (CB[7]) in aqueous solution by molecular dynamics simulations. We found that among twenty amino acids (AAs), the binding of CB[7] to Phe is the strongest. Based on that a series of tripeptides containing N-terminal Phe (with the second residue Gly (G) fixed) are designed to explore the effects of adjacent AA residues on the binding to CB[7]. It is indicated that, in contrast to FGG, only when the 3rd-residue is Glu (E), Lys (K), or Arg (R), the binding affinity with CB[7] can reach the nanomolar level. For the most prominent FGE, the binding free energy with CB[7] is −12.8 kcal/mol, with association constant (Ka) of 2.1 × 109 M−1. It is surprising that once reversing the sequence order of the 2nd-residue and 3rd-residue from FGX to FXG (X = E, K, R), or inserting a second Gly in the middle of FGX, such as FGGX, the binding affinity is significantly changed, with Ka decreased more than 3 orders of magnitude. These results predicted the high binding affinity and sequence selectivity of CB[7] to containing N-terminal Phe peptides, which are beneficial to predict the recognition sites of specific proteins/peptides and to design host-guest complexes with high affinity.

In Vitro and In Vivo Sequestration of Phencyclidine by Me4 Cucurbit[8]uril*.

We report investigations of the use of cucurbit[8]uril (CB[8]) macrocycles as an antidote to counteract the in vivo biological effects of phencyclidine. We investigate the binding of CB[8] and its derivative Me4 CB[8] toward ten drugs of abuse (3-9, 12-14) by a combination of 1 H NMR spectroscopy and isothermal titration calorimetry in phosphate buffered water. We find that the cavity of CB[8] and Me4 CB[8] are able to encapsulate the 1-amino-1-aryl-cyclohexane ring system of phencyclidine (PCP) and ketamine as well as the morphinan skeleton of morphine and hydromorphone with Kd values ≤50 nm. In vitro cytotoxicity (MTS metabolic and adenylate kinase cell death assays in HEK293 and HEPG2 cells) and in vivo maximum tolerated dose studies (Swiss Webster mice) which were performed for Me4 CB[8] indicated good tolerability. The tightest host⋅guest pair (Me4 CB[8]⋅PCP; Kd =2 nm) was advanced to in vivo efficacy studies. The results of open field tests demonstrate that pretreatment of mice with Me4 CB[8] prevents subsequent hyperlocomotion induction by PCP and also that treatment of animals previously dosed with PCP with Me4 CB[8] significantly reduces the locomotion levels.

Capture of Phenylalanine and Phenylalanine-Terminated Peptides Using a Supramolecular Macrocycle for Surface-Enhanced Raman Scattering Detection.

The cucurbit[n]uril (CB[n]) family of macrocycles are known to bind a variety of small molecules with high affinity. These motifs thus have promise in an ever-growing list of trace detection methods. Surface-enhanced Raman scattering (SERS) detection schemes employing CB[n] motifs exhibit increased sensitivity due to selective concentration of the analyte at the nanoparticle surface, coupled with the ability of CB[n] to facilitate the formation of well-defined electromagnetic hot spots. Herein, we report a CB[7] SERS assay for quantification of phenylalanine (Phe) and further demonstrate its utility for detecting peptides with an N-terminal Phe. The CB[7]-guest interaction improves the sensitivity 5-25-fold over direct detection of Phe using citrate-capped silver nanoparticle aggregates, enabling use of a portable Raman system. We further illustrate detection of insulin via binding of CB[7] to the N-terminal Phe residue on its B-chain, suggesting a general strategy for detecting Phe-terminated peptides of clinically relevant biomolecules.

Synthesis of a [6]rotaxane with singly threaded γ-cyclodextrins as a single stereoisomer.

A series of hetero [4]-, [5]- and [6]rotaxanes containing both cucurbit[6]uril (CB[6]) and γ-cyclodextrin (γ-CD) as the macrocyclic components have been synthesized via a threading-followed-by-stoppering approach. Due to the orthogonal binding of CB[6] to ammonium and γ-CD to biphenylene/tetra(ethylene glycol), the [n]rotaxanes display a specific sequence of the interlocked macrocycles. In addition, despite of the asymmetry of γ-CD with respect to the orthogonal plane of the axle, only one stereoisomer of the [6]rotaxane was obtained.

Detection of Analytes from Gas Phase Using Fluorophores and Host–Guest Complexes Incorporated into Lipid Bilayers on Silica Gel Nanoparticles

The aim of this work was to develop chemosensors by analogy with bioreceptors based on lipid membranes stabilized on silica gel nanoparticles. Dansylglycine (DG) and a host–guest complex of DG with cucurbituril CB[8] were used as receptor fluorophores, and naphthalene and trimethylamine (TMA) in a gas phase were used as analytes. Stabilized lipid bilayers (SLBs) were obtained by the sorption of bilayer liposomes on nanoparticles under conditions that ensure the rupture of liposomes and the formation of a flat bilayer. The binding of CB[8] to SLB was performed for the first time and detected by measuring the fluorescence of the thiazole orange complex with CB[8] in a supernatant. The formation of the DG@CB[8] complex in water and a membrane manifested itself in FDG quenching and a shift of the FDG maximum toward short or long waves in water and in the membrane, respectively. On naphthalene excitation at 290 nm, the FDG response was due to energy transfer from naphthalene to DG. The FDG sharply decreased in the presence of TMA–H2O vapors, but it increased above the initial value upon the removal of H2O vapor. It was found that, unlike dyes embedded in a lipid matrix or sorbed on nanoparticles, the FDG response for host–guest complexes was proportional to analyte concentration, and the FDG wavelength did not change with analyte concentration.

Host–Guest Interaction at Molecular Interfaces: Binding of Cucurbit[7]uril on Ferrocenyl Self-Assembled Monolayers on Gold

Ferrocene (Fc)-encapsulated cucurbit[7]uril (CB[7]) supramolecular host–guest complex (Fc@CB[7]) as a synthetic recognition pair has been widely adapted for coupling biomolecules and nanomaterials due to its ultrahigh binding affinity. In this paper, we have explored the binding of CB[7] on binary ferrocenylundecanethiolate/octanethiolate self-assembled monolayer on gold (FcC11S-/C8S-Au), a model system to develop our understanding of host–guest chemistry at molecular interfaces. It has been shown that upon incubation with CB[7] solution the redox behavior of FcC11S-/C8S-Au changes remarkably; i.e., a new pair of peaks appeared at more positive potential with narrowed widths. The ease of quantitation of surface bound-redox species (Fc+/Fc and Fc+@CB[7]/ Fc@CB[7]) enabled us to determine the thermodynamic formation constant of Fc@CB[7] at FcC11S-/C8S-Au (7.3 ± 1.8 × 104 M–1). With time-dependent redox responses, we were able to, for the first time, deduce both the binding and dissociation rate constants, 2...

Influence of hydrophobic residues on the binding of CB[7] toward diammonium ions of common ammonium···ammonium distance.

We report the binding constants of CB[7] toward a series of naphthalene diammonium and 4,4'-dipiperidinium derivatives and compare the results with those obtained previously for CB[7]·3b by (1)H NMR and X-ray crystallography. The nature of binding in the host·guest complexes was investigated using quantum mechanical tools.

Supramolecular Inhibition of Amyloid Fibrillation by Cucurbit[7]uril

AbstractAmyloid fibrils are insoluble protein aggregates comprised of highly ordered β‐sheet structures and they are involved in the pathology of amyloidoses, such as Alzheimer’s disease. A supramolecular strategy is presented for inhibiting amyloid fibrillation by using cucurbit[7]uril (CB[7]). CB[7] prevents the fibrillation of insulin and β‐amyloid by capturing phenylalanine (Phe) residues, which are crucial to the hydrophobic interactions formed during amyloid fibrillation. These results suggest that the Phe‐specific binding of CB[7] can modulate the intermolecular interaction of amyloid proteins and prevent the transition from monomeric to multimeric states. CB[7] thus has potential for the development of a therapeutic strategy for amyloidosis.

Supramolecular inhibition of amyloid fibrillation by cucurbit[7]uril.

Amyloid fibrils are insoluble protein aggregates comprised of highly ordered β-sheet structures and they are involved in the pathology of amyloidoses, such as Alzheimer's disease. A supramolecular strategy is presented for inhibiting amyloid fibrillation by using cucurbit[7]uril (CB[7]). CB[7] prevents the fibrillation of insulin and β-amyloid by capturing phenylalanine (Phe) residues, which are crucial to the hydrophobic interactions formed during amyloid fibrillation. These results suggest that the Phe-specific binding of CB[7] can modulate the intermolecular interaction of amyloid proteins and prevent the transition from monomeric to multimeric states. CB[7] thus has potential for the development of a therapeutic strategy for amyloidosis.

Cucurbit[7]uril complexations of bis(isoquinolinium)alkane dications in aqueous solution

The 1:1 and 2:1 host–guest complexation of a series of 1,n-bis(isoquinolinium)alkane dications (Iq(CH2)nIq2+, n = 2, 4, 5, 6, 8, 9, 10 and 12, and Iq(p-xylene)Iq2+) by cucurbit[7]uril (CB[7]) in aqueous solution has been investigated by 1H NMR spectroscopy and ESI mass spectrometry. The site of binding of the first CB[7] is dependent on the nature of the central linker group, with encapsulation of the p-xylene group or the polymethylene chain when n = 6–10.With shorter (n = 2–5) or longer (n = 12) chains, the first CB[7] binds over an isoquinolinium group. With a second CB[7], the binding of the central group is abandoned in favour of the CB[7] hosts encapsulating the two cationic isoquinolinium termini. The 1:1 and 2:1 host–guest stability constants are related to modes of binding and the nature of the central linkers, and are compared with dicationic guests bearing different terminal groups.

Direct Visualization of Symmetry Breaking During Janus Nanoparticle Formation

The straight-forward synthesis of Janus nanoparticles composed of Ag and AgBr is reported. For their formation, cucurbit[n]uril (CB)-stabilized AgBr nanoparticles are first generated in water by precipitation. Subsequent irradiation with an electron beam transforms a fraction of each AgBr nanoparticle into Ag(0) , leading to well-defined Janus particles, stabilized by the binding of CB to the surface of both AgBr and Ag(0) . With the silver ion reduction being triggered by the electron beam, the progress of the transformation can be directly monitored with a transmission electron microscope.

Coomassie Blue Binding to Amyloid Fibrils and Native Proteins

Coomassie brilliant blue G-250 (CB) is the dye used in the common Bradford assay for protein concentration determination. In this work we investigated the binding of CB to lysozyme and insulin in the native and amyloid fibril states by several optical spectroscopic techniques. We found that Coomassie blue binds both to the native proteins and to amyloid fibrils, but give distinctly different spectral responses. In addition, we investigated how the solvent polarity and viscosity affect the CB absorption and fluorescence spectra, and applied this understanding to the protein observations. The absorption and fluorescence spectra of CB indicated that the binding sites in the fibrils are less polar and holds the CB dye more rigidly than in the native forms. The spectral comparison of CB bound to the two different fibrils showed that the binding sites are different. This was most likely due to differences in secondary structure, which was monitored by circular dichroism. Linear dichroism was used to show that the fibril-bound CB is oriented preferentially parallel to the insulin amyloid fibril axis.

Spectroscopic characterization of Coomassie blue and its binding to amyloid fibrils

Coomassie brilliant blue G-250 (CB) is the dye used frequently in the Bradford assay for protein concentration determination. In this study, we investigated how the solvent polarity and viscosity affect the CB absorption and fluorescence spectra and apply this understanding to investigate the binding of CB to lysozyme and insulin in the native and amyloid fibril states. Coomassie blue binds both to the native protein and to amyloid fibrils but gives distinctly different spectral responses. The absorption and fluorescence spectra of CB indicate that binding sites in the fibrils are less polar and hold the CB dye more rigidly than in the native forms. The spectral comparison of CB bound to the two different fibrils showed that the binding sites are different, and this was most likely due to differences in secondary structure as monitored by circular dichroism. Finally, linear dichroism was used to show that the fibril-bound CB is oriented preferentially parallel to the insulin amyloid fibril axis.

Recognition Properties of Acyclic Glycoluril Oligomers

The fragmentation reaction of bis-nor-seco-CB[10] with 3,5-dimethylphenol (3) delivers methylene bridged glycoluril pentamer 5 in 81% yield. The host-guest recognition properties of the previously known tetramer 4 and those of pentamer 5 and hexamer 6 toward cationic guests in water are used to delineate some important features of the binding of acyclic CB[n]-type receptors.

The cannabinoid type‐1 receptor carboxyl‐terminus, more than just a tail

The cannabinoid type-1 (CB(1)) receptor is a G protein-coupled receptor that binds the main active ingredient of marijuana, Δ(9)-tetrahydrocannabinol, and has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. In the two decades since the discovery of CB(1), studies at the molecular level have centered on the transmembrane core. This interest has now expanded as we discover that other regions of CB(1), including the CB(1) carboxyl-terminus, have critical structures that are important for CB(1) activity and regulation. Following the recent description of the three dimensional structure of the full-length CB(1) carboxyl-terminal tail [Biopolymers (2009) vol. 91, pp. 565-573], several residues and structural motifs including two α-helices (termed H8 and H9) have been postulated to interact with common G protein-coupled receptor accessory proteins, such as G-proteins and β-arrestins. This discourse will focus on the CB(1) carboxyl-terminus; our current understanding of the structural features of this region, evidence for its interaction with proteins, and the impact of structure on the binding and regulatory function of CB(1) accessory proteins. The involvement of the carboxyl-terminus in the receptor life cycle including activation, desensitization, and internalization will be highlighted.

Layer-by-Layer Characterization of a Model Biofuel Cell Anode by (in Situ) Vibrational Spectroscopy

Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) and (in situ) surface-enhanced (resonant) Raman scattering (SE(R)RS) were used as “quality control” tools during the construction of a model biofuel cell anode. The model anode was a layered structure formed by 4-hydroxythiophenol (HTP) attached to a coenzyme analog, cibacron blue F3G-A (CB), which then supported the adsorption of the enzyme formaldehyde dehydrogenase (FalDH). Ex situ SERS and PM-IRRAS showed the HTP attached to a gold surface through its thiol group. The covalent binding of CB to the HTP layer was confirmed by a combination of ex situ and in situ SERS data. In situ SERS spectra after adsorption of FalDH to the CB layer confirmed successful enzyme immobilization.

Molecular mimicry between a monoclonal antibody and one subunit of crotoxin, a heterodimeric phospholipase A2 neurotoxin.

Crotoxin is a heterodimeric phospholipase A2 neurotoxin formed by the non-covalent association of an acidic and non-toxic subunit, CA, and a basic and weakly toxic phospholipase A2, CB. The two subunits behave in a synergistic manner. CA enhances the lethal potency of CB by increasing its selectivity of action. The mAb A-56.36, directed against the non-toxic subunit CA, was previously shown to neutralize crotoxin toxicity by dissociating the crotoxin complex. In the present report, a polypeptide sequence similarity was observed between some CDRs of mAb A-56.36 and two regions of CB (pos. 60-80 and 95-110). Phage displayed peptides corresponding to VH2 and VH3 of mAb A-56.36 and to their homologous sequences in CB bind CA to different extents. This observation shows that mAb A-56.36 interacts with a region of CA involved in its interaction with CB, therefore mimicking the binding of CB to CA. A similar approach was used to determine the regions of ammodytoxin A and of agkistrodotoxin, two phospholipase A2 neurotoxins similar to CB, which are involved in the formation of heterocomplexes with CA. The analysis of these data contributes to the determination of stretches of amino acids which could constitute the paratope of mAb A-56.36, as well as the region of association of CB with CA in crotoxin.