Supermolecule-assisted imaging of low-molecular-weight quaternary-ammonium compounds by MALDI-MS of their non-covalent complexes with cucurbit[7]uril

Cucurbit[7]uril was used to form non-covalent complexes with low-molecular-weight quaternary-ammonium compounds for their indirect analysis by MALDI-MS. By shifting the ion signals to a higher and interference-free mass region, the distributions of neurine, choline, and phosphocholine in rat brain were visualized by MALDI imaging with high selectivity and good sensitivity.

Cucurbit[7]uril was used to form non-covalent complexes with low-molecular-weight quaternary-ammonium compounds for their indirect analysis by MALDI-MS.

Matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a powerful tool for the sensitive and multiplexed analysis of molecules in a tissue sample. This technique provides the localization and abundances of a molecule in situ, making it an attractive molecular histology tool in medical, pharmaceutical, and biological research.¹ Despite its many advantages, some practical problems exist in the direct analysis of some low-molecular-weight (LMW) compounds because of matrix-related ion interferences in the low-mass region of MALDI-MS spectra which interfere with the selectivity of the assay.^2,3LMW quaternary-ammonium compounds (QACs) include neurine, choline, phosphocholine, as well as a few other molecules. These compounds are essential biomolecules involved in energy production, lipid metabolism, and neurotransmission.^3–6 Evaluation of the distributions of these compounds in a tissue is of biological significance. Due to their low molecular weights, however, the direct detection of some of these QACs by MALDI-MS is often hampered by interferences from numerous matrix-related signals. Furthermore, the successful determination by MALDI-MS of the spatial distributions of the QAC compounds in a tissue sample is often inaccurate because the observed ion signals of these compounds can partially result from in-source fragmentation of, for example, phospholipids. Therefore, selective MALDI-MSI of such LMW QACs is challenging. In recent years, on-tissue chemical derivatization has emerged as an effective technique to enhance the in situ detection and imaging performance of some low-abundance or hard-to-ionize endogenous compounds by MALDI-MS.^7–10 On-tissue chemical derivatization, however, is not applicable to most LMW QACs because these compounds do not have the functional groups in their molecular structures that are required for chemical derivatization. Therefore, new methods are needed for the molecular imaging of these compounds by MALDI-MS. To ameliorate this situation, we have developed a new approach to enhance the selectivity of MALDI-MSI for in situ imaging of the three LMW QACs using on-tissue inclusion of the analytes within a supermolecule, i.e., cucurbit[7]uril (CB[7]), to form non-covalent complexes (Fig. 1).Open in a separate windowFig. 1The formation of CB[n]–QACs host–guest complexes.Cucurbit[n]urils (CB[n]) consist of n glycouril units that are bound in a ring-like arrangement via methylene bridges, and which can form stable non-covalent host–guest complexes with cations with high binding constants.¹¹ As a soft ionization technique, MALDI has been shown to ionize non-covalent complexes under mild laser energy conditions, without dissociation during desorption or ionization.^12–14 CB[6] and CB[7] have been used as mass-shifting reagents to analyze polyamines (e.g., spermidine, and spermine) in plant tissues by forming the non-covalent CB[n]–analyte complexes.^15,16 By shifting the detected or expected ion signal of an analyte to a higher mass region, the interference signals originating from common MALDI matrices and other coexisting LMW compounds in a sample can be greatly reduced or eliminated, thus increasing the selectivity of the assay. In addition, non-covalent interactions between CB[n] and the LMW analytes often need no activation energy for the formation of the complex.¹¹ In other words, the formation of non-covalent bonds is relatively easy and rapid. Because of these features, we examined the use of CB[n] as host molecules for the on-tissue formation of non-covalent supramolecular complexes with LMW QACs, for MALDI-MSI.CB[n] supermolecules that are composed of n = 5, 6, 7, 8, or 10 repeating units of glycouril are commercially available. Due to the extremely small cavity size, CB[5] is only suitable for the encapsulation of gases, forming portal complexes with alkali, alkaline earth, and ammonium cations.¹¹ At the other extreme, due to their large portal size and large cavity volume, CB[8] and CB[10] can interact with two identical or two different small molecules to form 1 : 2 or 1 : 1 : 1 complexes. This feature would increase the complexity of MALDI MS spectra and would make interpreting these spectra more difficult. In contrast, CB[6] and CB[7] have moderate cavity sizes, with cavity volumes of 164 Å and 279 Å, respectively,¹¹ and thus may have the potential for use in the MALDI-MSI of LMW QACs. CB[6], however, is essentially insoluble in either water or organic solvents, and performed poorly in our preliminary experiments. CB[7], on the other hand, possesses modest solubility in water (∼30 mM),¹¹ and has been shown to be compatible with commonly used organic solvents such as methanol and acetonitrile,^17,18 which would be important for MALDI matrix-deposition procedures using wet-chemistry techniques such as spray coating. Therefore, CB[7] was chosen for the MALDI-MSI presented in this study. To evaluate the proposed strategy for the indirect analysis of LMW QACs, 3 compounds (neurine, choline, and phosphocholine) were tested by MALDI-MS, with or without CB[7] as the non-covalent complex-forming supermolecule. For the inclusion of CB[7], dried-droplet spotting was used by, in turn, depositing 0.5 μL of a solution of one of the 3 QACs (50 μM each in 50% MeOH), 0.5 μL of a CB[7] solution (4 mM in 50% MeOH), and 0.5 μL of an α-cyano-4-hydroxycinnamic acid (CHCA) (7 mg mL⁻¹ in 50% MeOH) MALDI matrix solution on a polished stainless-steel plate. For the CB[7]-free procedure, no CB[7] was deposited. When CB[7] was included, all 3 QACs displayed unique signals corresponding to positive ions of the formed CB[7]–QAC complex, without sodiated or potassiated adduct ions or fragment ions of the complexes being observed (Fig. S1A–C^†). In Fig. S1D to F,^† these 3 compounds were also able to be detected by direct MALDI-MS without the CB[7] inclusion, but the mass spectrum acquired from the spotted choline sample (Fig. S1E^†) showed a weak ion signal corresponding to the m/z of neurine when the power of the laser impacting the sample for desorption/ionization was ≥21 μJ (25% of the full power), indicating in-source fragmentation of choline. Similarly, in the mass spectrum of phosphocholine, two ion signals corresponding to neurine and choline, respectively, were observed in direct MALDI-MS of phosphocholine when the same level of laser power was applied. The comparison of the MALDI-MS spectra of these 3 compounds, with and without the CB[7] inclusion, showed the high selectivity of the CB[7]-assisted strategy for the indirect MALDI-MSI of these QACs.The selectivity of the CB[7]-assisted MALDI detection of the LMW QACs was further evaluated by comparing the interference background signals obtained by direct and indirect analysis of a mixture of the 3 QACs. Fig. 2 shows a comparison of the MALDI mass spectra of the matrix CHCA, 3 QACs, CB[7], and the 3 QACs–CB[7] complexes, respectively, acquired in the reflectron mode of MALDI-TOF operations. In Fig. 2A, a signal at m/z 86.0928, which may be derived from the fragmentation of CHCA, was observed, which interferes with neurine detection. In Fig. 2B, although signals for neurine, choline, and phosphocholine were observed, severe interferences from the matrix and other background ions were also seen in the lower mass region (i.e., below m/z 400), and phosphocholine was only weakly detected, compared with the signals for neurine and choline, demonstrating the inadequate selectivity of direct MALDI-MS for detection of these LMW compounds using the conventional CHCA organic matrix. In Fig. 2C, signals from the CB[7]-related ions ([CB[7] + H]⁺, [CB[7] + Na]⁺, and [CB[7] + K]⁺) can clearly be seen in the mass region from m/z 1160 to m/z 1210 – i.e., in the higher-mass clean-background region of the spectrum (Fig. 2C). For the sample deposited with a mixture of the 3 QACs, CB[7], and CHCA, MALDI signals corresponding to the ions of CB[7]–neurine, CB[7]–choline and CB[7]–phosphocholine were observed as ion clusters around m/z 1148, 1266, and 1346, respectively (see the measured and calculated m/z values in Table S1^† for details), without matrix-related interferences (Fig. 2D). This experiment demonstrated the high selectivity of the CB[7]-assisted MALDI-MS method for the detection of these 3 LMW QACs.Open in a separate windowFig. 2MALDI mass spectra of (A) CHCA; (B) mixture of the three QAC standards; (C) CB[7]; and (D) on-target spotting of CB[7] and the three QAC standards. The inset in A shows a zoom-in of the low mass region of the spectrum. 50 pmol of each QAC and 2 nmol of CB[7] was spotted on the target MALDI plate spot (CB[7] : QACs molar ratio as 40 : 1). CHCA (7 mg mL⁻¹ in 50% MeOH) was used as the MALDI matrix.The analytical sensitivity of the proposed CB[7]-assisted approach was evaluated by performing on-target CB[7] inclusion on serially diluted standard solutions containing the analytes. As a result, the limits of detection (LOD), defined as the lowest amounts of the analytes which produced MALDI signals of the CB[7]–QAC complexes with a signal-to-noise ratio of ≥3, were determined to be 0.2, 0.2, and 2 pmol for neurine, choline, and phosphocholine, respectively, indicating acceptable sensitivities for the on-spot detection using the proposed method. As a comparison, the LODs of neurine, choline, and phosphocholine by MALDI-MS without CB[7] assisting were determined to be 0.1, 0.3, and 2 pmol, respectively. The MALDI-MS analysis of QACs with and without CB[7] assistance showed similar sensitivities of detection in terms of LODs.To demonstrate the capability of the proposed strategy for relative quantitation of the QACs by MALDI-MS as well as for the subsequent sensitivity study, 1 μL of standard solutions of the 3 QACs with varying concentrations were spotted on the MALDI target, followed by deposition of 1 μL of 2 mM of CB[7] solution and 2 μL of the same CHCA matrix solution using the dried-droplet approach. A comparison study without the addition of CB[7] was also performed. MALDI-MS spectra of CB[7]–QAC complexes were acquired under a set of optimized instrument operating conditions and compared, as shown in Fig. 3. The observed signal intensities of the individual QACs or CB[7]–QAC complexes showed linear responses as a function of the concentrations of the applied standard solutions, with correlation coefficients (R²) being equal to or greater than 0.977 over a concentration range of 5 pmol to 100 pmol, for each of the 3 compounds (Fig. S2^†). The slopes of the calibration curves with or without CB[7] were similar, which further showed that the CB[7]-assisted approach increased the selectivity of detection of the 3 LMW QACs while maintaining the sensitivity.Open in a separate windowFig. 3MS Spectra of CB[7]–QACs (neurine, choline and phosphocholine) complexes obtained with (A) 100 pmol (B) 50 pmol (C) 10 pmol and (D) 5 pmol of analyte each. 2 nmol of CB[7] and 2 μL of CHCA (7 mg mL⁻¹ in 50% MeOH) were spotted on the target MALDI-plate spot.Next, the performance of this method for on-tissue detection of LMW QACs was evaluated using 14 μm-thick rat brain tissue sections as representative tissue samples. Two rat brain tissue sections were deposited with the matrix of CHCA (14 mg mL⁻¹ in 50% MeOH), with and without inclusion of CB[7] (2 mM in 50% MeOH) prior to the addition of the matrix coating, using a Bruker Daltonics ImagePrep sprayer. When no pre-coating of CB[7] on the tissue section was used, only very few weak signals were detected in the mass region of m/z 1220–1400 (Fig. 4A). In the upper part of Fig. 4B, signals corresponding to the formed complexes of CB[7]-neurine, CB[7]-choline, and CB[7]-phosphocholine can be clearly detected with the signal-to-noise ratios of ≥300 for the 3 endogenous QACs. These results indicated that the CB[7]-assisted approach delivered signals free from interferences and gave accurate results, demonstrating the high selectivity of the proposed strategy for on-tissue detection of these three compounds.Open in a separate windowFig. 4Average mass spectra obtained by conducting on-tissue inclusion. (A) A rat brain tissue section (14 μm) coated with CB[7] and CHCA, (B) rat brain section coated with CHCA only.To optimize the procedure for the MALDI imaging of these compounds in rat brain, the effects of the amount of CB[7] deposited and the percentage of organic solvent (MeOH) in the CB[7] solutions on the MALDI signal intensities were investigated. The amounts of deposited CB[7] on the tissue were controlled by varying the number of the CB[7] spray cycles. Sufficient CB[7] benefited the formation of CB[7]–QACs. However, excessive amounts of CB[7] induced suppression of the observed ion signals of the CB[7]–QACs. The most intense signals were observed with the application of 15–19 spray cycles of the supermolecule solution at a concentration of 2 mM CB[7] (Fig. S3A^†). The use of 25% and 50% methanol in both CB[7] solutions resulted in about 30% higher signal intensities than with 75% methanol in the aqueous solutions (Fig. S3B^†). However, the lower percentages of methanol in the solutions induced delocalization of the analytes on the tissue sections. Therefore, 75% aqueous methanol was chosen as the solvent for both the CB[7] solution and the matrix solution, and 15 cycles of spray were used for all the subsequent imaging experiments in order to avoid possible delocalization during the CB[7] inclusion and CHCA coating steps.To simplify the procedure for tissue imaging, we explored the feasibility of performing the on-tissue CB[7] inclusion and the matrix coating in a single step, and compared the single-step procedure with the two-step procedure. In the two-step procedure, the CB[7] solution (2 mM in 75% MeOH) and the CHCA solution (14 mg mL⁻¹ in 75% MeOH) were coated, in turn, with 15 spray cycles for each round of the coating. For the single-step operation, a mixed solution of 2 mM of CB[7] and 14 mg mL⁻¹ of CHCA in 75% MeOH was sprayed onto the tissue sections with 15 spray cycles. The mass spectra obtained using the two different deposition procedures are shown in Fig. S4,^† and both showed similar mass spectral quality for the detection of the 3 QACs, without any apparent differences in the observed signal intensities. Thus, the single-step procedure was utilized to simplify the experiment.Under the optimized on-tissue inclusion conditions, the MALDI-MSI of neurine, choline, and phosphocholine in rat brain tissue was performed to characterize the localization and distribution patterns of the three QACs (neurine, choline, and phosphocholine) on transversely sliced tissue sections of a rat brain with a laser irradiation diameter of 200 μm. As shown in Fig. 5, the 3 QACs showed slightly different distributions in the different anatomical regions of the rat brain. Neurine and phosphocholine showed similar distribution patterns to each other, and both of these compounds were detected with higher abundances in the regions of cortex and thalamus, while choline showed the reverse distribution pattern. These observations were consistent with the observations in an earlier ammonium sulfate-assisted MALDI-MSI study.³ It should be noted that the current study could be defined as a feasibility study for the proposed CB[7]-assisted strategy. Better imaging results are expected to be obtained by using the CB[7]-assisted strategy on newer-generation instruments (including both matrix coating devices and MALDI-MS instruments).Open in a separate windowFig. 5MALDI-MS ion images of three QACs obtained by using the CB[7]-assisted approach on a rat brain tissue section and the corresponding H & E staining.For the MALDI-MSI of LMW QACs, the use of ammonium sulfate-assisted MALDI-MSI (i.e., spraying ammonium sulfate together with matrix onto the tissue sections), as reported by Mitsutoshi, et al., improved the detection performance and the distributions of 5 hydrophilic QACs (carnitine, acetylcarnitine, glycerophosphocholine, choline, and phosphocholine) were visualized in rat brain tissues.³ There are also some reports on multiplexed and chemical derivatization-free MALDI-MS imaging of endogenous compounds, including a few QACs in tissue.^19,20 Compared to these earlier studies on the MALDI-MSI of LMW QACs, a significant advantage of our proposed strategy is its high selectivity, resulting from shifting the LMW-QACs to an interference-free higher mass region, with no interferences derived from in-source fragmentation. To our knowledge, neurine has not been previously imaged in tissue specimens by MALDI-MS. In this work, neurine in rat brain was able to be successfully imaged, as a result of the formation of a non-covalent complex with CB[7] which improved its MALDI-MS detection.     

A LSER-based model to predict the solubilizing effect of drugs by inclusion with cucurbit[7]uril

A large number of traditional drugs and the development of new drugs often encounter the problem of poor water solubility. Cucurbit[7]uril, a novel macrocyclic host, has attracted great interest in this field. Investigating the solubilizing effect of drugs by inclusion with cucurbit[7]uril could provide guidance for drug solubilization. In this work, the interactions of drugs with cucurbit[7]uril, drugs with water and the inclusion complexes with water, and the properties of drugs and inclusion complexes, are considered to establish a linear solvation energy relationships (LSER)-based model. This model could be applied to predicting the solubility of drugs with cucurbit[7]uril in water. Density functional theory (DFT) is employed to obtain the properties and interaction parameters. The multi-parameter solubility model obtained by stepwise regression shows good fitting and predicting results. And the surface area of inclusion complexes (A₃), the LUMO energy of inclusion complexes (E_3LUMO), the polarity index of inclusion complexes (I₃), the electronegativity of drugs (χ₁), and the oil–water partition coefficient of drugs (log p_1w) are effective parameters related to the solubilization of drugs with cucurbit[7]uril. Futhermore, the model could be extended to calculate the solubilizing effect of other macrocycles.

A LSER model was built for predicting the solubility of inclusion complexes.     

Light driven molecular lock comprises a Ru(bpy)2(hpip) complex and cucurbit[8]uril

Here, complex 1 ([Ru(bpy)₂(hpip)]²⁺–MV²⁺) and CB[8] can form a stable 1 : 1 inclusion complex in aqueous solution, resembling a U-shaped conformation. Upon light irradiation, two complex 1 were reversibly locked through the formation of a MV˙⁺ radical dimer that is stabilized in the cavity of CB[8] with Ru complexes as blockers, in which complex 1 was transformed from a U-shaped conformation to a interlocked complex. This study provided a feasible strategy for the fabrication of a photo-driven supramolecular machine resembling a “lock”.

Upon light irradiation, complex 1 was transformed from a U-shaped conformation to an interlocked complex.     

HyperCEST detection of cucurbit[6]uril in whole blood using an ultrashort saturation Pre‐pulse train

Xenon based biosensors have the potential to detect and localize biomarkers associated with a wide variety of diseases. The development and nuclear magnetic resonance (NMR) characterization of cage molecules which encapsulate hyperpolarized xenon is imperative for the development of these xenon biosensors. We acquired ¹²⁹Xe NMR spectra, and magnetic resonance images and a HyperCEST saturation map of cucurbit[6]uril (CB6) in whole bovine blood. We observed a mean HyperCEST depletion of 84% (n = 5) at a concentration of 5 mM and 74% at 2.5 mM. Additionally, we collected these data using a pulsed HyperCEST saturation pre‐pulse train with a SAR of 0.025 W/kg which will minimize any potential RF heating in animal or human tissue. Copyright © 2016 John Wiley & Sons, Ltd.     

Preparation,characterization, and biological activity study of thymoquinone-cucurbit[7]uril inclusion complex

In this study, the formation of a host–guest inclusion complex between cucurbit[7]uril (CB[7]) and thymoquinone (TQ) was investigated in aqueous solution. The formation of a stable inclusion complex, CB[7]–TQ, was confirmed by using different techniques, such as ¹H NMR and UV-visible spectroscopy. The aqueous solubility of TQ was clearly enhanced upon the addition of CB[7], which provided an initial indication for supramolecular complexation. The complexation stoichiometry and the binding constant of the inclusion complex were determined through a combination of two sets of titration methods, including UV-visible and fluorescence displacement titrations. Both methods suggested the formation of a 1 : 1 stoichiometry between CB[7] and TQ with moderate binding affinity of 3 × 10³ M⁻¹. Density functional theory (DFT) calculations were also performed to verify the structure of the resulted host–guest complex and to support the complexation stoichiometry. The theoretical calculations were in agreement with experimental results obtained by ¹H NMR spectroscopy. Most importantly, the cytotoxic effect of the CB[7]–TQ complex was investigated against cancer and normal cell lines. The results showed that the anticancer activity of TQ against MDA-MB-231 cells was enhanced by the complexation with CB[7], while no significant effect was observed in MCF-7 cells. The results also confirmed the low toxicity of the CB[7] host molecule that supports the use of CB[7] as a drug carrier.

The host–guest inclusion complexation of thymoquinone by cucurbit[7]uril in aqueous solution is established, which results in an enhanced biological activity.     

Host–guest modes and supramolecular frameworks of complexes of tetramethyl cucurbit[6]uril with 4-chloroaniline and 4,4′-diaminostilbene

Since the first reportal on decamethylcucurbit[5]uril (Me₁₀Q[5]) in 1992, substituted cucurbit[n]urils have attracted considerable research interest. In this study, the host–guest modes between the tetramethyl cucurbit[6]uril (TMeQ[6]) as a host and 4-chloroaniline and 4,4′-diaminostilbene (G1 and G2) as guests were investigated by single-crystal X-ray diffraction, NMR, ITC, UV-Vis spectrum, and MALDI-TOF mass spectrometry analyses. The experimental results showed that TMeQ[6] formed a 1 : 1 inclusion compound with G1, and the carbonyl portal of TMeQ[6] formed a 1 : 1 self-assembly with G2. Further, multi-dimensional supramolecular frameworks were formed driven by weak interaction forces in the system (hydrogen bonding, C–H⋯π interactions, ion–dipole interactions, and dipole–dipole interactions).

Two TMeQ[6]-based multi-dimensional supramolecular frameworks were formed driven by weak interaction forces in the system (hydrogen bonding, C–H... π interactions, ion–dipole interactions, and dipole–dipole interactions).     

Polymerization of a biscalix[5]arene derivative

Recent decades have seen an increased interest in the preparation of polymers possessing host or guest moieties as the end group, which has enabled new polymeric materials such as self-healable, shape-memory, and stimuli-responsive materials. Such polymers are commonly synthesized by tethering the host or guest moieties to polymers. On the other hand, there are limited reports demonstrating the preparation of host- or guest-appended polymers by directly polymerizing the corresponding host- or guest-appended monomers, which is valuable for easy access to diverse polymers from single molecular species. However, reactive host and/or guest moieties of the monomer interfere with the polymerization reaction. Here, we report that a biscalix[5]arene host-appended molecule can be polymerized with various monomers to form the corresponding host-appended polymers. The host–guest complexation behavior of calix[5]arene-appended polymers with fullerene derivatives was studied by ¹H NMR and UV/Vis spectroscopic techniques, which revealed that the long polymer chains did not prevent host–guest complexation even when the fullerene derivative was equipped with a polymer chain. Thus, the present study shows the potential for developing polymers that have various combinations of polymer chains.

A calix[5]arene appended monomer molecule was subjected to polymerization reaction to yield corresponding methacrylate polymers. The calix[5]arene appended polymers showed excellent encapsulation capability for fullerene molecules.     

Multiple cellular electrophysiological effects of a novel antiarrhythmic furoquinoline derivative HA-7 [N-benzyl-7-methoxy-2,3,4,9-tetrahydrofuro[2,3-b]quinoline-3,4-dione] in guinea pig cardiac preparations

We studied the electrophysiological and antiarrhythmic actions of HA-7 [N-benzyl-7-methoxy-2,3,4,9-tetrahydrofuro[2,3-b]quinoline-3,4-dione], a furoquinoline alkaloid derivative, in guinea pig heart preparations. In the perfused whole heart model, HA-7 caused a prolongation in the basic cycle length, ventricular repolarization time, and the atrioventricular (AV) nodal Wenckebach cycle length and prolonged the refractory period of the atrium, AV node, and His-Purkinje system. The atrioventricular conduction interval was also prolonged in a frequency-dependent manner. In isolated hearts, HA-7 significantly raised the threshold for experimental atrial fibrillation and reduced the occurrence of reperfusion-induced ventricular fibrillation. Conventional microelectrode-recording study shows that HA-7, but not d-sotalol, prolonged the action potential duration (APD) and decreased the maximum rate of depolarization in isolated atrial strips. In ventricular papillary muscles, higher concentrations of HA-7 caused a prolongation of APD(90) in a frequency-independent manner, whereas d-sotalol exerted a reverse frequency-dependent action on this parameter. Whole-cell patch clamp results on ventricular myocytes indicate that HA-7 decreased both the slow (I(Ks)) (IC(50) = 4.8 muM) and fast component (I(Kr)) (IC(50) = 1.1 muM) of the delayed rectifier K(+) currents. Similar results could also be observed in atrial myocytes. The inward rectifier K(+) current (I(K1)) was also reduced somewhat by HA-7. HA-7 also suppressed the Na(+) inward current (I(Na)) (IC(50) = 2.9 muM) and inhibited the L-type Ca(2+) current (I(Ca)) (IC(50) = 4.0 muM, maximal inhibition = 69%) to a lesser extent. We conclude that HA-7 blocks multiple ionic currents and that these changes affect the electrophysiological properties of the conduction system as well as the myocardial tissues and may contribute to its antiarrhythmic efficacy.     

Synthesis of a coumarin derivative of resorcin[4]arene with solvent-controlled chirality

This paper presents the synthesis of a coumarin derivative of resorcin[4]arene (1) using a cascade thermolysis/Michael reaction. The influence of the hydrogen bonding system on the conformational rigidity and cyclochirality of the coumarin derivative of resorcin[4]arene was discussed; these properties depended on the proton-donor–acceptor properties of the solvent. Significant differences, which depended on the environment, in the coumarin derivative of resorcin[4]arene fluorescence were observed and discussed.

This paper presents the synthesis of a coumarin derivative of resorcin[4]arene (1) using a cascade thermolysis/Michael reaction.     

Study on the coordination of cyclopentanocucurbit[5,6]uril with Fe3+, Co2+ and Ni2+ ions

This paper reports the coordination of cyclopentanocucurbit[5]uril (CyP₅Q[5]) and cyclopentanocucurbit[6]uril (CyP₆Q[6]) with Fe(ClO₄)₃, Co(ClO₄)₂ and Ni(ClO₄)₂. Single crystal X-ray diffraction analysis shows the metal ions are directly coordinated with the portal of the cucurbit[n]uril to form a one-dimensional supramolecular chain or independent systems in the CyP₅Q[5]@Fe(ClO₄)₃, CyP₅Q[5]@Co(ClO₄)₂, CyP₆Q[6]@Co(ClO₄)₂ and CyP₅Q[5]@Ni(ClO₄)₂ complexes. In CyP₆Q[6]@Fe(ClO₄)₃, the metal ion is not directly coordinated with the cucurbit[n]uril portal, but after forming Fe(H₂O)₆, it interacts with the cucurbit[n]uril portal via a hydrogen bond. The CyP₆Q[6]@Ni(ClO₄)₂ complex is quite special; in this system, there are both metal ions directly coordinated with the cucurbit[n]uril portal and free on the outer surface of the cucurbit[n]uril.

This paper reports the coordination of cyclopentanocucurbit[5]uril (CyP₅Q[5]) and cyclopentanocucurbit[6]uril (CyP₆Q[6]) with Fe(ClO₄)₃, Co(ClO₄)₂ and Ni(ClO₄)₂.     

Application of pH-responsive polymers to oral dosage forms for insulin]

The potential of graft copolymer networks with poly(methacrylic acid-g-ethylene glycol; P(MAA-g-EG)) for oral dosage forms to enhance insulin absorption is reviewed. The polymer exhibited unique pH-responsive characteristics in which interpolymer complexes were formed and dissociated, respectively, in acidic and neutral/basic environments. Correspondingly, the polymer was capable of highly incorporating and rapidly releasing insulin in vitro. This insulin loaded polymer successfully enhanced oral insulin absorption in rats with significant hypoglycemic effects. The polymer was also shown to possess mucoadhesive properties. Furthermore, the polymer demonstrated high calcium binding which may affect the proteolytic activity of calcium-dependent enzymes and/or reduce transepithelial resistances. Thus, the polymer has the potential to be used as a carrier for oral dosage forms of insulin to enhance its mucosal absorption.     

WAY-163909 [(7bR,10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole]: A novel 5-hydroxytryptamine 2C receptor-selective agonist with preclinical antipsychotic-like activity.

Serotonin-2C (5-HT2C) receptor antagonists and agonists have been shown to affect dopamine (DA) neurotransmission, with agonists selectively decreasing mesolimbic DA. As antipsychotic efficacy is proposed to be associated with decreased mesolimbic DA neurotransmission by virtue of DA D2 receptor antagonism, the 5-HT2C-selective receptor agonist, WAY-163909 [(7bR,10aR)-1,2, 3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7, 1hi]indole], was evaluated in animal models of schizophrenia and in vivo microdialysis and electrophysiology to determine the effects on mesolimbic and nigrostriatal DA neurotransmission. Similar to clozapine, WAY-163909 (1.7-30 mg/kg i.p.) decreased apomorphine-induced climbing with little effect on stereotypy and no significant induction of catalepsy. WAY-163909 (0.3-3 mg/kg s.c.) more potently reduced phencyclidine-induced locomotor activity compared with d-amphetamine with no effect on spontaneous activity. WAY-163909 (1.7-17 mg/kg i.p.) reversed MK-801 (5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate)- and DOI [1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane]-disrupted prepulse inhibition of startle (PPI) and improved PPI in DBA/2N mice. In conditioned avoidance responding, WAY-163909 (0.3-3 mg/kg i.p.; 1-17 mg/kg p.o.) reduced avoidance responding, an effect blocked by the 5-HT(2B/2C) receptor antagonist SB 206553 [5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole]. WAY-163909 (10 mg/kg s.c.) selectively decreased extracellular levels of DA in the nucleus accumbens without affecting the striatum. Likewise, in vivo electrophysiological recordings showed a decrease in the number of spontaneously firing DA neurons in the ventral tegmental area but not in the substantia nigra with both acute and chronic (21-day) administration of WAY-163909 (1-10 mg/kg i.p.). Thus, the profile of the 5-HT2C selective receptor agonist WAY-163909 is similar to that of an atypical antipsychotic and additionally may have rapid onset properties.     

Bicyclo[6.1.0]nonyne carboxylic acid for the production of stable molecular probes

Bicyclo[6.1.0]non-4-yn-9-ylmethanol (BCN alcohol) is the most prominent strained-alkyne scaffold in chemical biology. Described herein is the synthesis of an oxidized analogue – BCN acid – whose facile functionalization via amide bond formation yields more stable derivatives than the classically encountered carbamates.

We report a novel strained alkyne, coined BCN acid. This compound participates efficiently in diverse bioorthogonal reactions and allows the facile production of amide probes, which showed superior in vitro stability compared to carbamate analogues.     

A study on the self-assembly mode and supramolecular framework of complexes of cucurbit[6]urils and 1-(4-methoxyphenyl)piperazine

Self-assembly between symmetrical dicyclohexyl-substituted cucurbit[6]uril (abbreviated as (CyH)₂Q[6]) and cyclopentanocucurbit[6]uril (CyP₆Q[6]) as hosts and 1-(4-methoxyphenyl)piperazine (MeOPP) as a guest molecule has been studied by means of single-crystal X-ray diffraction analysis, NMR, MALDI-TOF mass spectrometry, and other characterization methods. The experimental results showed that the self-assembly was driven by the formation of exclusion complexes by the cucurbit[n]uril and the guest, that is, supramolecular interaction between the negative charge of the cucurbit[n]uril portals and a coordination polymer guest. Complexes were formed between the positive charge of the cucurbit[n]uril outer wall and inorganic anions, thus generating self-assemblies with multi-dimensional and multi-level supramolecular frameworks.

Complexes were formed between the positive charge of the cucurbit[n]uril outer wall and inorganic anions, thus generating self-assemblies with multi-dimensional and multi-level supramolecular frameworks.     

Retro Diels Alder protocol for regioselective synthesis of novel [1,2,4]triazolo[4,3-a]pyrimidin-7(1H)-ones

Reactions of diastereochemically varied norbornene-condensed 2-thioxopyrimidin-4-ones 6 and 10 with variously functionalized hydrazonoyl chlorides 2a–h gave regioselectively angular norbornene-based [1,2,4]triazolo[4,3-a]pyrimidin-7(1H)-ones 7a–h and 11a,c–e, respectively. Thermal retro Diels–Alder (RDA) reaction of 7a–h and 11a,c–e resulted in the target compounds 4a–h as single products. On the other hand, reactions of thiouracil 1 and hydrozonoyl chlorides 2a–e gave regioselectively [1,2,4]triazolo[4,3-a]pyrimidinone-5(1H)-ones 3a–e. The opposite regioselectivity of thiouracil 1 and norbornene-condensed 2-thioxopyrimidin-4-ones 6 and 10 was attributed to electronic factors according to DFT calculations. The angular structure of norbornene based [1,2,4]triazolo[4,3-a]pyrimidin-7(1H)-ones was confirmed by single crystal X-ray crystallography.

Norbornene is an efficient motif for blocking the electronic effect of the C C bond to invert the stereochemistry outcome. Then, using the RDA strategy the C C bond was recovered to obtain the target compound with a definite stereochemistry.     

Glucuronidation of [6]-shogaol, [8]-shogaol and [10]-shogaol by human tissues and expressed UGT enzymes: identification of UGT2B7 as the major contributor

Shogaols, mainly [6]-shogaol (6S), [8]-shogaol (8S) and [10]-shogaol (10S), the predominant and characteristic pungent phytochemicals in ginger, are responsible for most of its beneficial effects. However, poor oral bioavailability owing to extensive glucuronidation limits their application. The present study aimed to characterize the glucuronidation pathways of 6S, 8S and 10S by using pooled human liver microsomes (HLM), human intestine microsomes (HIM) and recombinant human UDP-glucosyltransferases (UGTs). The rates of glucuronidation were determined by incubating shogaols with uridine diphosphate glucuronic acid-supplemented microsomes. Kinetic parameters were derived by appropriate model fitting. Reaction phenotyping assays, activity correlation analyses and relative activity factors were performed to identify the main UGT isoforms. As a result, one mono-4′-O-glucuronide was detected after incubating each shogaol with HLM and HIM. Enzymes kinetic analysis demonstrated that glucuronidation of shogaols consistently displayed the substrate inhibition profile, and the liver showed higher metabolic activity for shogaols (CL_int = 1.37–2.87 mL min⁻¹ mg⁻¹) than the intestine (CL_int = 0.67–0.85 mL min⁻¹ mg⁻¹). Besides, reaction phenotyping assays revealed that UGT2B7 displayed the highest catalytic ability (CL_int = 0.47–1.17 mL min⁻¹ mg⁻¹) among all tested UGTs. In addition, glucuronidation of shogaols was strongly correlated with AZT glucuronidation (r = 0.886, 0.803 and 0.871 for glucuronidation of 6S, 8S and 10S, respectively; p < 0.01) in a bank of individual HLMs (n = 9). Furthermore, UGT2B7 contributed to 40.8%, 34.2% and 36.0% for the glucuronidation of 6S, 8S and 10S in HLM, respectively. Taken altogether, shogaols were efficiently metabolized through the glucuronidation pathway, and UGT2B7 was the main contributor to their glucuronidation.

The glucuronidation pathways of shogaols ([6]-shogaol, [8]-shogaol and [10]-shogaol) were characterized in human tissues and recombinant human UDP-glucosyltransferases, and UGT2B7 was identified as the main contributor to their glucuronidation.     

WAY-163909 [(7bR, 10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole], a novel 5-hydroxytryptamine 2C receptor-selective agonist with anorectic activity

The pharmacological profile of WAY-163909 [(7bR, 10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole], a novel 5-hydroxytryptamine (HT)(2C) (serotonin) receptor-selective agonist is presented. WAY-163909 displaced [(125)I]2,5-dimethoxy-4-iodoamphetamine binding from human 5-HT(2C) receptor sites, in Chinese hamster ovary (CHO) cell membranes, with a K(i) value of 10.5 +/- 1.1 nM. Binding affinities determined for the human 5-HT(2A) and 5-HT(2B) receptor subtypes were 212 and 485 nM, respectively. In functional studies, WAY-163909 stimulated the mobilization of intracellular calcium in CHO cells stably expressing the human 5-HT(2C) receptor with an EC(50) value of 8 nM, and E(max) relative to 5-HT of 90%. WAY-163909 failed to stimulate calcium mobilization in cells expressing the human 5-HT(2A) receptor subtype (EC(50) > 10muM) and was a 5-HT(2B) receptor partial agonist (EC(50) 185 nM, E(max) 40%). WAY-163909 exhibited negligible affinity (<50% inhibition at 1 muM) for other receptor sites examined, including human 5-HT(1A), D2, and D3 receptors, and the 5-HT transporter binding site in rat cortical membranes. WAY-163909 exhibited weak affinity for the human D4 (245 nM) and 5-HT(7) (343 nM) receptor subtypes and the alpha1 binding site in rat cortical membranes (665 nM). WAY-163909 produced a dose-dependent reduction in food intake in normal Sprague-Dawley rats (ED(50) = 2.93 mg/kg), an effect blocked by a 5-HT(2C) receptor antagonist but not by a 5-HT(2A) or 5-HT(2B) receptor antagonist. In addition, WAY-163909 decreased food intake in obese Zucker rats and diet-induced obese mice with ED(50) values of 1.4 and 5.19 mg/kg i.p., respectively, consistent with the potential utility of 5-HT(2C) receptor agonists as anti-obesity agents.     

Evaluation of thiazolidinedione derivative drugs for safety]

The first thiazolidinedione derivative drug for diabetes, troglitazone, was found to cause fatal hepatotoxicity, although it was judged as safe during the clinical trial. Subsequently, pioglitazone has been clinically used both in Japan and U.S. and has had no fatal cases but caused heart failure. Therefore, careful follow up observation is necessary in these drugs by checking liver function tests and cardiac function.