Characterizing Lomerizine metabolites in camel urine: High-resolution mass spectrometry method development and validation for enhanced doping control.

RATIONALE: Lomerizine (LMZ) is an antimigraine drug that works as a calcium channel blocker and has selective effects on the central nervous system. It is metabolized into trimetazidine (TMZ), which is a prohibited substance owing to its performance-enhancing effects in both human and animal sports. Effective doping control measures are imperative to distinguish the source of TMZ in samples to ensure integrity and fairness of the sport, therefore a comprehensive analysis of LMZ metabolites is essential to identify potential biomarkers in camel urine for effective doping control. METHODS: Camel urine samples were collected from four healthy animals following a single oral administration of LMZ at a dosage of 1 mg/kg body weight. In vitro studies were conducted using homogenized camel liver samples. Lomerizine and its metabolites were extracted using solid-phase extraction and analyzed with a Thermo Fisher Orbitrap Exploris liquid chromatography mass spectrometry system. The acquired data was processed with the Compound Discoverer software. RESULTS: The study conducted a comprehensive analysis of LMZ metabolites in camels and identified 10 phase I and one phase II metabolites. The primary pathway for the formation of phase I metabolites was de-alkylation, while phase II metabolite was formed through alkylation of the parent drug. The study provided valuable insights into the unique metabolic pathways of LMZ in camels under specific experimental conditions. CONCLUSION: The developed method enables the detection and characterization of LMZ and its metabolites in camels. The identified metabolites has the potential to act as marker metabolites for the distinctive detection of LMZ in camel urine to ensure efficient analytical strategies for routine doping control applications.

Fluorescence Sensor for Water in Organic Solvent Using Graphene Oxide- Rhodamine B and Cucurbit[7]uril.

Detection of water in organic solvents gained much importance as these solvents have been used as a medium for conducting organic reactions and water was considered as an inhibitor, when it is present in the reaction medium. There are number of methods available to measure the water content in organic solvents, however, such methods are time consuming and expensive. Here, we developed a facile method for detecting water in organic solvents using an inexpensive fluorescent probe - Rhodamine B decorated Graphene oxide (RBGO). The fluorescent probe, RBGO can be conveniently prepared by mixing the rhodamine B (RB) with graphene oxide (GO) in water. However, the probe will function as a sensor for water in the organic solvents through the release of dye upon interaction with the water present in organic solvents. Surprisingly, combination of cucurbit[7]uril (CB[7]) and RBGO increased the sensitivity of this sensor dramatically for the detection of water. This is the first example of water sensor with best detection limit by the involvement of host molecules such as CB[7]. This sensor displayed the low limit of detection (LOD) for organic solvents (LOD: 0.0015% for DMSO, 0.0025% for DMF), through the two-way process such as decomplexation and encapsulation. We presume that the role of CB [7] can be implemented in other similar sensors to enhance the sensitivity.

Pyrene-Hydrazone-π···Hole Coupled Turn-on Fluorescent and Naked- Eye Colorimetric Sensor for Cyanide: Role of Homogeneous π-Hole Dispersion in Anion Selectivity.

A pyrene based probe associated with π···hole - hydrazone as one of the recognizing elements is synthesized and its turn in to a selective colorimetric and turn-on fluorescent sensor, (L3) for cyanide anion. This chemo sensor show high selectivity towards cyanide anion through photo electron transfer (PET) mechanism. The binding strength and sensitivity of the chemo sensor L3 towards cyanide are found to be 2.0 X 104, and 4.44 x 10-4 respectively. We have compared this high selectivity of the receptor towards cyanide, with our previously reported receptors L1 and L2. The detailed UV-Vis, Emission, 1H-NMR, IR spectroscopic and Molecular Electrostatic Potential (MEP) studies reveals that the homogeneous π···hole dispersion in the aromatic ring governing the selectivity of the receptor towards cyanide anion. Such a positive π···hole homogeneous dispersion is missing in the case of sensor L2, instead we have polarized π···hole dispersion towards 2nd and 4th position of di-nitrophenyl chromophoric unit in L2.

Electrochemiluminescent Chiral Discrimination with a Pillar[5]arene Molecular Universal Joint-Coordinated Ruthenium Complex.

A bicyclic pillar[5]arene derivative fused with a bipyridine side ring, a so-called molecular universal joint (MUJ), was synthesized, and the pair of enantiomers was resolved by high-performance liquid chromatography enantioresolution. The electrochemiluminescent detection based on the ruthenium complex of the enantiopure MUJ showed excellent chiral discrimination toward certain amino acids.

Anion-binding-induced and reduced fluorescence emission (ABIFE & ABRFE): A fluorescent chemo sensor for selective turn-on/off detection of cyanide and fluoride.

A new hydrazine based π-hole assisted, electron deficient turn-on/off fluorescent and colorimetric sensor L with anion-binding induced/reduced fluorescent emission (ABIFE & ABRFE) has been designed and synthesized. The prepared sensor L exhibited excellent turn-on fluorescence emission in the presence of cyanide ion through ABIFE. On the other hand, the receptor L turns-off its fluorescence intensity upon binding with fluoride; however, the reduced emission intensity of this complex of L is recovered by addition of cyanide. The sensor L is almost intact with other tested anions. To the best of our knowledge, this is the first report on sensing of cyanide and fluoride by a neutral hydrazine based receptor via anion-binding-induced fluorescence emission (ABIFE) and anion-binding-reduced fluorescence emission (ABRFE) mechanisms respectively. The observed ABIFE and ABRFE phenomena of L with cyanide is further supported by UV-Vis, Emission, 1H NMR and IR spectroscopic studies.

Supramolecular Assembly of Acriflavine on Graphene Oxide for the Sensing of Adenosine Phosphates.

An acriflavine-graphene oxide (GAF) supramolecular assembly has been prepared from water-soluble graphene oxide (GO) and a fluorescent dye, acriflavine (AF). Upon binding this non-covalently to the GO, the fluorescence of acriflavine has been "turned off" effectively, competitive binding potential of the sensor substrates such as ATP, ADP, AMP and the pyrophosphate weakens the supramolecular assembly of GAF, which allows the release of acriflavine quantitatively, which also "turns-on" the fluorescence of the dye under UV irradiation. Interestingly, GAF displayed the highest sensitivity towards ATP within the family of adenosine phosphates. We have developed a naked eye detection method for the adenosine phosphates biomolecules. For the first time, acriflavine has been utilized for the sensing of adenosine phosphates in combination with GO, which can be useful for the detection of other biomolecules.

C3V symmetric tripodal molecular pocket for linear anions: Selective colorimetric detection of azide through N4-Cu-π···hole interactions.

Tris(3-amino propyl)amine (TRPN) based C3v symmetric tripodal molecular pocket, L has been prepared and mono copper complex of L, (1) having Cu-N4-π··· hole as recognizing elements, becoming a potential and selective colorimetric chemo sensor for perfect linear recognition of N3-, generate a Cu-NNN- π··· hole unit inside the tripodal pocket. Systematic spectrometric and naked-eye colorimetric studies reveal that, this chemo sensor is also colorimetrically recognizing the cyanide ion by its cavity via Cu-N4-π···hole interactions; nevertheless, when azide anion is entering as a guest into the molecular pocket of 1 which is already hosted cyanide anion, then host displaces cyanide ion, subsequently azide is getting inside the cavity. The strength of the copper complex, 1 towards azide and cyanide are found to be 2.36 × 103M-1, and 1.87 × 103 M-1 respectively in 7:3 acetonitrile:water solvent medium. Further, the ability of cyanide displacement by azide in complex 1 is found to be 2.53 × 103 M-1. To the best of our knowledge, this is the first example of naked-eye detection of azide via cyanide displacement assay by a tripodal receptor.

Selective colorimetric sensing and perfect linear recognition of azide: Formation of Cu-azide-Cu cascade complex within the cavity of cryptand.

Bis-trpn [tris(3-aminopropyl)amine], capped dicopper complex of bicyclic cryptand L, 1, became a potential selective colorimetric chemosensor for azide anion. Complex 1 is generating a space inside the cylindrical cavity which will be opt for perfect linear recognition of azide anion through as N4-Cu⋯N3-⋯Cu-N4 axle. Naked eye colorimetric and UV-Vis spectrometric investigations shows the complex 1 has the capability of selective sensing of azide anion. The association constant and limits of detection (LoD) of complex 1 towards azide are found to be 2.754 × 103 M-1 and 1.91 × 10-6 M. To the best of our knowledge, this is the first example of selective colorimetric sensing of azide by a bis­copper cryptate 1 via ideal linear orientation of N4-Cu⋯N3⋯Cu-N4 axle inside the cylindrical shaped cavity.

A molecular phototropic system for cyanide: Detection and sunlight driven harvesting of cyanide with molecular sunflower.

We are reporting a simple, easy to prepare, and conformation switchable first molecular phototropic system L, "(E)-2-(2,4-dinitrophenyl)-1-((pyren-8-yl)methylene)hydrazine, for cyanide harvesting. This molecular phototropic system behaves as a molecular sunflower in which the conformation of this molecular sunflower can be altered in response to the sunlight. This molecular flower can sense and bind the cyanide anion colorimetrically through its transition state. Further, upon exposure of this transition state cyanide complex 1, under sunlight, this system is capable to release the bound cyanide via -C=N- free rotation to reach its lower energy stable conformation. Similar behaviors were observed for acetate and fluoride with L. The strength of the phototropic system L towards cyanide, acetate and fluoride is found to be 4.5 × 105, 1.53 × 102 and 6.09 × 102 M-1.

Most Efficient Tris(3-Aminopropyl) Amine Based Electron Deficient Tripodal Receptor for Azide.

Investigation on strength of the tris(2-amino ethyl) amine and tris (3-amino propyl) amine backboned tripodal receptors, L and L1 (incorporated with tripodal C3ν frame, thio urea-amide linkage and π-hole assisting functionality) which are premeditated to explore the prospect for a particular anion recognition are studied. UV-Vis, 1H- NMR, and IR spectroscopy studies indicates that both the receptors sensing azide anion, colorimetrically and binds azide anion stronger than any other anions such as acetate, and cyanide. In particular the receptor L1 shows the highest binding strength towards azide anion. To the best of our knowledge this is the first receptor showing highest binding ability with azide anion. We used Molecular Electrostatic Potential Surface analyses to support our spectroscopic findings. The association constant and limits of detection for receptor L1 with azide is found to be 8.4X105M-1 and 3.16X10-6 M respectively. The observed highest binding strength of L1 with azide is, could be due to the cooperative effect of extended traditional hydrogen bonding via thiourea-amide functionality, anion-π interaction and C3ν suitable framework.

High Affinity Host-Guest FRET Pair for Single-Vesicle Content-Mixing Assay: Observation of Flickering Fusion Events.

Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged host-guest pair, cucurbit[7]uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.

Reversible morphological transformation between polymer nanocapsules and thin films through dynamic covalent self-assembly.

A facile method has been developed for synthesizing polymer nanocapsules and thin films using multiple in-plane stitching of monomers by the formation of reversible disulfide linkages. Owing to the reversibility of the disulfide linkages, the nanostructured materials readily transform their structures in response to environmental changes at room temperature. For example, in reducing environments, the polymer nanocapsules release loaded cargo molecules. Moreover, reversible morphological transformations between these structures can be achieved by simple solvent exchanges. This work is a novel approach for the formation of robust nano/microstructured materials that dynamically respond to environmental stimuli.

3D tissue engineered supramolecular hydrogels for controlled chondrogenesis of human mesenchymal stem cells.

Despite a wide investigation of hydrogels as an artificial extracellular matrix, there are few scaffold systems for the facile spatiotemporal control of mesenchymal stem cells (MSCs). Here, we report 3D tissue engineered supramolecular hydrogels prepared with highly water-soluble monofunctionalized cucurbit[6]uril-hyaluronic acid (CB[6]-HA), diaminohexane conjugated HA (DAH-HA), and drug conjugated CB[6] (drug-CB[6]) for the controlled chondrogenesis of human mesenchymal stem cells (hMSCs). The mechanical property of supramolecular HA hydrogels was modulated by changing the cross-linking density for the spatial control of hMSCs. In addition, the differentiation of hMSCs was temporally controlled by changing the release profiles of transforming growth factor-ß3 (TGF-ß3) and/or dexamethasone (Dexa) from the hydrolyzable Dexa-CB[6]. The effective chondrogenic differentiation of hMSCs encapsulated in the monoCB[6]/DAH-HA hydrogel with TGF-ß3 and Dexa-CB[6] was confirmed by biochemical glycosaminoglycan content analysis, real-time quantitative PCR, histological, and immunohistochemical analyses. Taken together, we could confirm the feasibility of cytocompatible monoCB[6]/DAH-HA hydrogels as a platform scaffold with controlled drug delivery for cartilage regeneration and other various tissue engineering applications.

Guest binding dynamics with cucurbit[7]uril in the presence of cations.

The binding dynamics of R-(+)-2-naphthyl-1-ethylammonium cation (NpH(+)) with cucurbit[7]uril (CB[7]) was investigated. Competitive binding with Na(+) or H(3)O(+) cations enabled the reaction to be slowed down sufficiently for the kinetics to be studied by fluorescence stopped-flow experiments. The binding of two Na(+) cations to CB[7], i.e., CB[7]·Na(+) (K(01) = 130 ± 10 M(-1)) and Na(+)·CB[7]·Na(+) (K(02) = 21 ± 2 M(-1)), was derived from the analysis of binding isotherms and the kinetic studies. NpH(+) binds only to free CB[7] ((1.06 ± 0.05) × 10(7) M(-1)), and the association rate constant of (6.3 ± 0.3) × 10(8) M(-1) s(-1) is 1 order of magnitude lower than that for a diffusion-controlled process and much higher than the association rate constant previously determined for other CB[n] systems. The high equilibrium constant for the NpH(+)@CB[7] complex is a consequence of the slow dissociation rate constant of 55 s(-1). The kinetics results showed that formation of a complex between a positively charged guest with CB[n] can occur at a rate close to the diffusion-controlled limit with no detection of a stable exclusion complex.

A new photo-switchable "on-off" host-guest system.

A new photo-switchable "on-off" host-guest system comprising cucurbit[7]uril (CB[7]) and a photoresponsive cinnamamide derivative (trans-(3-phenyl-acryloylamino)-acetic acid, E-1) is studied. The cinnamamide derivative and CB[7] forms a stable 1 : 1 host-guest complex (CB[7]·E-1) with a high binding constant (K = 2.1 × 10(4) M(-1)). Irradiation of UV light (300 nm) to an aqueous solution of CB[7]·E-1 induces the E- to Z-conformational change of the cinnamamide derivative, which then leads to the dissociation of the complex as evidenced by UV-visible and (1)H-NMR spectroscopy. The reverse process, photo-induced host-guest complex formation between CB[7] and Z-1 is achieved by irradiation of UV light at 254 nm. The photo-switchable "on-off" host-guest system shows high reversibility and switching efficiency, which makes it potentially useful in designing photoresponsive gating systems.

Supramolecular fishing for plasma membrane proteins using an ultrastable synthetic host-guest binding pair.

Membrane proteomics, the large-scale global analysis of membrane proteins, is often constrained by the efficiency of separating and extracting membrane proteins. Recent approaches involve conjugating membrane proteins with the small molecule biotin and using the receptor streptavidin to extract the labelled proteins. Despite the many advantages of this method, several shortcomings remain, including potential contamination by endogenously biotinylated molecules and interference by streptavidin during analytical stages. Here, we report a supramolecular fishing method for membrane proteins using the synthetic receptor-ligand pair cucurbit[7]uril-1-trimethylammoniomethylferrocene (CB[7]-AFc). CB[7]-conjugated beads selectively capture AFc-labelled proteins from heterogeneous protein mixtures, and AFc-labelling of cells results in the efficient capture of membrane proteins by these beads. The captured proteins can be recovered easily at room temperature by treatment with a strong competitor such as 1,1'-bis(trimethylammoniomethyl)ferrocene. This synthetic but biocompatible host-guest system may be a useful alternative to streptavidin-biotin for membrane proteomics as well as other biological and biotechnological applications.

Complexation of aliphatic ammonium ions with a water-soluble cucurbit[6]uril derivative in pure water: isothermal calorimetric, NMR, and X-ray crystallographic study.

Complexation of a water-soluble cucurbituril (CB) derivative, cyclohexanocucurbit[6]uril (CB*[6]), the cavity dimensions of which are essentially the same as those of CB[6], with various organic mono- and diammonium ions has been studied by isothermal titration calorimetry and 1H NMR spectroscopy. The binding affinity of CB*[6] with the guest molecules in water is 3-5 and 2-3 orders of magnitude higher than those of CB[6] in 50 % formic acid and in 0.05 M NaCl solution, respectively, which is mainly due to the larger enthalpic gains upon complex formation in the absence of interfering ions, such as protons and Na+. In particular, the binding constant (K) of spermine to CB*[6] was measured to be 3.4 x 10(12) M(-1), which is the highest binding constant ever reported for CB[6] or its derivatives. We also obtained the X-ray crystal structures of alpha,omega-alkanediammonium ions (C(n)DA2+, n=4-8) and spermine complexes with CB[6], in which the aliphatic chains of the guest molecules take an extended or partially bent conformation inside the CB[6] cavity, depending on the chain length. The hexamethylene chain of C6DA2+ takes a twisted conformation, which not only allows strong ion-dipole interactions between the ammonium groups and the carbonyl groups at the portals, but also increases hydrophobic interactions between the alkyl part of the guest and the inner wall of the host, which results in the largest enthalpic gain among alpha,omega-alkanediammonium ions. The thermodynamic parameters associated with the complexation are discussed in relation to the binding modes and conformations of the aliphatic chain of the guest molecules inside the host, which were investigated by 1H NMR spectroscopy and X-ray crystallography.