1H NMR Titration Research Articles

Bromine atom introduction improves the F- sensing ability of an indolo[3,2-b]carbazole-salicylaldehyde-based fluorescence turn-on sensor.

The heavy-atom effect usually quenches fluorescence, but scarcely enhances it. Herein, fluorescence turn-on sensors without or with a bromine atom for F- detection are presented, achieving fast response time within 1 min, and the LODs of 1.9 × 10-7 and 8.5 × 10-8 M, reflecting that halogen atom introduction is beneficial for F- detection ability improvement. The sensing mechanism of -OH unit deprotonation is confirmed based on the results of a 1 : 2 stoichiometric ratio, 1H NMR titration and TD-DFT calculation. The water environment F- detection and spiked recovery experiments demonstrate their potential for real sample detection.

Enhanced binding of methyl alkylammonium cations through preorganization of a water-soluble calix[4]pyrrole.

We describe the synthesis of two tetra-α aryl-extended calix[4]pyrroles (C[4]Ps) 4a-b bearing four terminal carboxylic groups in their meso-propyl chains defining the lower rims. The synthesized C[4]Ps became soluble (1-3 mM) in water at pD = 10. We probed the interaction of 4a towards tetra-methylammonium (G1) chloride in water using 1H NMR spectroscopy. The C[4]P 4a includes G1 in the shallow aromatic cavity defined by the pyrrole rings in cone conformation forming a 1 : 1 complex G1⊂4a. Pyridine-N-oxide (PNO) binding in the larger polar aromatic cavity of 4a results in the quantitative self-assembly of the supramolecular receptor PNO@4a featuring the pyrrole rings preorganized in cone conformation. The PNO@4a receptor displays improved binding properties towards G1 than the parent C[4]P 4a. We thermodynamically characterized (1H NMR titrations and ITC experiments) the 1 : 1 complexes of PNO@4a with a series of tetra-alkylammonium salts, including biologically relevant examples. The PNO@4a supramolecular receptor displays significant affinity (log K = 3-4) but lacks selectivity in water binding of methyl trialkyl ammonium cations. Cation-π and coulombic interactions are the main intermolecular forces stabilizing the complexes. We also performed DFT calculations to gain some insights into the complexes' structures.

Probing and evaluating transmembrane chloride ion transport in double walled trifluorophenyl/phthalimide extended calix[4]pyrrole-based supramolecular receptors.

Therapeutic applications have sparked increased interest in the use of synthetic anion receptors for ion transport across lipid membranes. In this context, the construction of synthetic transmembrane transporters for the physiologically important chloride ion is currently of enormous interest. As a result, considerable effort is being devoted to the design and synthesis of artificial transmembrane chloride ion transporters. However, only inadequate progress has been made in developing macrocyclic chloride ion transporters using the fundamental principles of supramolecular chemistry, and hence this field entails fostering investigations. In this investigation, the synthesis of two new double walled trifluorophenyl/phthalimide extended calix[4]pyrrole (C4P) receptors (3 and 7) has been successfully reported. 1H-NMR titration and HRMS studies confirmed the 1 : 1 binding stoichiometry of the chloride ion with these receptors in the solution phase (only receptor 3b was studied by 1H-NMR). Regarding ion transport of 3b and 7, when studied in the HPTS-based vesicular system, 3b showed better activity with an EC50 value of 0.39 μM. The detailed ion transport studies on 3b have revealed that ion transport occurs through the Cl-/NO3- antiport mode.

Solution and gaseous phase sensing of formaldehyde with economical triphenylmethane based sensors: a tool to estimate formaldehyde content in stored fish samples.

The use of formalin to preserve raw food items such as fish, meat, vegetables etc. is very commonly practiced in the present day. Also, formaldehyde (FA), which is the main constituent of formalin solution, is known to cause serious health issues on exposure. Considering the ill effects of formaldehyde, herein we report synthesis of highly sensitive triphenylmethane based formaldehyde (FA) sensors from a single step reaction of inexpensive reagents namely 4-hydroxy benzaldehyde and 2,6-dimethyl phenol. The synthetic method also provides highly pure product in bulk quantity. The analytical activity of the triphenylmethane sensor 1 with a limit of detection (LOD) value of 2.31 × 10-6 M for FA was significantly enhanced through induced deprotonation and thereafter a LOD value of 1.82 × 10-8 M could be achieved. To the best of our knowledge, the LOD value of the deprotonated form (sensor 2) for FA was superior to those of all the FA optical sensors reported so far. The mechanism of sensing was demonstrated by 1H-NMR titration and recording mass spectra before and after addition of FA to a solution of sensor 2. Both sensor 1 and sensor 2 exhibit quenching in emission upon addition of FA. A fluorescence study also demonstrates enhancement in analytical activity of the sensor upon induced deprotonation. Then the sensor was effectively immobilized into a hydrophilic and biocompatible starch-PVA polymer matrix which enabled detection of FA in a 100% aqueous system reversibly. Again, quick and effective sensing of FA in real food samples (stored fish) with the help of a computational application was demonstrated. The sensors have significant practical applicability as they effectively detect FA in real food samples qualitatively and quantitatively.

Duplex-forming oligocarbamates with tunable nonbonding sites.

In biopolymers such as proteins and nucleic acids, monomer sequence encodes for highly specific intra- and intermolecular interactions that direct self-assembly into complex architectures with high fidelity. This remarkable structural control translates into precise control over the properties of the biopolymer. Polymer scientists have sought to achieve similarly precise control over the structure and function of synthetic assemblies. A common strategy for achieving this goal has been to exploit existing biopolymers, known to associate with specific geometries and stoichiometries, for the assembly of synthetic building blocks. However, such systems are neither scalable nor amenable to the relatively harsh conditions required by various materials science applications, particularly those involving non-aqueous environments. To overcome these limitations, we have synthesized sequence-defined oligocarbamates (SeDOCs) that assemble into duplexes through complementary hydrogen bonds between thymine (T) and diaminotriazine (D) pendant groups. The SeDOC platform makes it simple to incorporate non-hydrogen-bonding sites into an oligomer's array of recognition motifs, thereby enabling an investigation into this unexplored handle for controlling the hybridization of complementary ligands. We successfully synthesized monovalent, divalent, and trivalent SeDOCs and characterized their self-assembly via diffusion ordered spectroscopy, 1H-NMR titration, and isothermal titration calorimetry. Our findings reveal that the binding strength of monovalent oligomers with complementary pendant groups is entropically driven and independent of monomer sequence. The results further show that the hybridization of multivalent oligomers is cooperative, that their binding enthalpy (ΔH) and entropy (TΔS) depend on monomer sequence, and that sequence-dependent changes in ΔH and TΔS occur in tandem to minimize the overall change in binding free energy.

A corannulene-based metallobox for the encapsulation of fullerenes.

A corannulene-bis-N-imidazolium salt was used for the synthesis of two corannulene-bis-N-heterocyclic carbenes of dirhodium(i) complexes of formula (corannulene-di-NHC)[RhCl(COD)]2 and (corannulene-di-NHC)[RhCl(CO)2]2. Both complexes were characterized by spectroscopic techniques, and the electron-donating properties of the corannulene-di-NHC ligand were studied by means of infrared spectroscopy and cyclic voltammetry. The complex (corannulene-di-NHC)[RhCl(COD)]2 was used for the encapsulation of fullerenes C60 and C70, generating host-guest complexes with 2 : 1 stoichiometry, as evidenced by 1H NMR and ITC titrations. Then, a tetra-rhodium(i) metallo-rectangle supported by two corannulene-bis-imidazolylidene ligands and two cofacial 4,4'-bipyridine ligands was prepared and characterized. This metallobox is capable of quantitatively encapsulating fullerenes C60 and C70, forming complexes that are highly stable even at high temperatures. The molecular structure of the metallobox with encapsulated C60 reveals a perfect size and shape complementarity that benefits from the concave-convex π-π interaction between the polyaromatic surfaces of the host and the guest.

A ferrocene-based chemo-dosimeter for colorimetric and electrochemical detection of cyanide and its estimation in cassava flour.

A simple chemo-dosimeter VDP2 bearing a ferrocene moiety was designed, synthesized, and characterized, and exhibited both chromogenic and electrochemical responses selectively for CN- in H2O-DMSO (9 : 1, v/v) medium. The probe VDP2 showed an instantaneous color change from colorless to yellow with CN- that can readily be observed visually. The deprotonation of the benzimidazole -NH, followed by nucleophilic addition of CN- to the olefinic C-atom, as evidenced by 1H and 13C NMR titration experiments, caused the colorimetric and electrochemical responses. The mass spectral study, CV, FTIR and Mulliken charges computed well supported the proposed mechanism. The electrochemical limit of detection was calculated to be 72 nM. The results of DFT and TD-DFT calculations suggested that the colorless nature of the probe VDP2 is due to weak intramolecular charge transfer (ICT) transition and the yellow color of the VDP2+CN adduct is due to through-space ICT transition. Above all, the probe could be an ideal candidate for monitoring cyanide in water samples and cassava flour with practical significance. A simple and convenient colorimetric method was developed to determine cyanide content in cassava flour.

Ammonium halide selective ion pair recognition and extraction with a chalcogen bonding heteroditopic receptor.

The first example of a heteroditopic receptor capable of cooperative recognition and extraction of ammonium salt (NH4X) ion-pairs is described. Consisting of a bidentate 3,5-bis-tellurotriazole chalcogen bond donor binding cleft, the appendage of benzo-15-crown-5 (B15C5) substituents to the tellurium centres facilitates binding of the ammonium cation via a co-facial bis-B15C5 sandwich complex, which serves to switch on chalcogen bonding-mediated anion binding potency. Extensive quantitative ion-pair recognition 1H NMR titration studies in CD3CN/CDCl3 (1 : 1, v/v) solvent media reveal impressive ion-pair binding affinities towards a variety of ammonium halide, nitrate and thiocyanate salts, with the heteroditopic receptor displaying notable ammonium halide salt selectivity. The prodigious solution phase NH4X recognition also translates to efficient solid-liquid and liquid-liquid extraction capabilities.

A pyridine-N-oxide catenane for cation recognition.

The rapid preparation of a pyridine-N-oxide containing [2]catenane is described. The [2]catenane was characterized by NMR spectroscopy, mass spectrometry and X-ray single crystal structure determination. 1H NMR titration experiments reveal the [2]catenane may be reversibly protonated, as well as an ability to bind lithium cations more strongly than sodium cations.

Lithium chloride selective ion-pair recognition by heteroditopic [2]rotaxanes.

The first heteroditopic [2]rotaxane host systems capable of strong and selective binding of lithium chloride ion-pair species are described. Importantly, a cooperative 'switch on' mechanism was found to operate, in which complexation of a lithium metal cation enhances the halide anion affinity of the rotaxanes via a combination of favourable proximal electrostatic and preorganised allosteric effects. The mechanically bonded rotaxane host design features a macrocycle component possessing a 2,6-dialkoxy pyridyl cation binding motif and an isophthalamide anion binding group, as well as an axle component functionalised with either a halogen bonding (XB) iodotriazole or hydrogen bonding (HB) prototriazole moiety. Extensive quantitative 1H NMR titration studies in CD3CN/CDCl3 solvent mixtures determined enhanced ion-pair binding affinities for lithium halides over the corresponding sodium or potassium halide salts, with the axle prototriazole-containing HB rotaxane in particular demonstrating a marked selectivity for lithium chloride. Solid-state X-ray crystallographic studies and computational DFT investigations provide evidence for a [2]rotaxane host axle-separated ion-pair binding mode, in which complementary cation and anion binding motifs from both the macrocycle and axle components act convergently to recognise each of the charged guest species.

Highly sensitive AIEE active fluorescent probe for detection of deferasirox: extensive experimental and theoretical studies.

High concentrations of deferasirox (DFX) in living organisms cause hepatic, gastric and renal malfunctions. Therefore, it is significant to establish an accurate and efficient approach for the detection of deferasirox (DFX) to protect public health. Herein, we synthesized a thiourea-based diphenylacetamide probe MPT for the effective sensing of deferasirox through the fluorescence quenching phenomenon. The designed probe MPT shows a fluorescence quenching response toward deferasirox (DFX) through photo-induced electron transfer (PET). Furthermore, DFT studies were performed to support the experimental results. 1H-NMR titration experiment was used to explore the interaction type between probe MPT and DFX. The existence of non-covalent interactions was verified with spectroscopic studies that were assisted by NCI studies, QTAIM and SAPT0 analysis. Dynamic light scattering (DLS) analysis and scanning electron microscopy (SEM) were used to investigate the complexation of probe MPT with DFX. Moreover, the on-site solution phase and solid-state detection of DFX by probe MPT are executed. Additionally, the practical applications of probe MPT to sense DFX were also revealed in human plasma as well as in artificial urine samples.

Selective Separation of Americium(III), Curium(III), and Lanthanide(III) by Aqueous and Organic Competitive Extraction.

Adding hydrophilic ligands into aqueous solutions for the selective binding of actinides(III) is acknowledged as an advanced strategy in Ln(III)/An(III) separation. In view of the recycling and radioactive waste disposal of the minor actinide, there remains an urgent need to design and develop the appropriate ligand for selective separation of An(III) from Ln(III). Herein, four novel hydrophilic ligands with hard-soft hybrid donors, derived from the pyridine and phenanthroline skeletons, were designed and synthesized as masking agents for selective complexation of An(III) in the aqueous phase. The known N,N,N',N'-tetraoctyl diglycolamide (TODGA) was used as lipophilic extractant in the organic phase for extraction of Ln(III), and a new strategy for the competitive extraction of An(III) and Ln(III) was developed based on TODGA and the above hydrophilic ligands. The optimal hydrophilic ligand of N,N'-bis(2-hydroxyethyl)-2,9-dicarboxamide-1,10-phenanthroline (2OH-DAPhen) displayed exceptional selectivity toward Am(III) over Ln(III), with the concentrations of HNO3 ranging from 0.05 to 3.0 M. The maximum separation factors were up to 1365 for Eu/Am, 417.66 for Eu/Cm, and 42.38 for La/Am. The coordination mode and bonding property of 2OH-DAPhen with Ln(III) were investigated by 1H NMR titration, UV-vis spectrophotometric titration, luminescence titration, FT-IR, ESI-HRMS analysis, and DFT calculations. The results revealed that the predominant species formed in the aqueous phase was a 1:1 ligand/metal complex. DFT calculations also confirmed that the affinity of 2OH-DAPhen for Am(III) was better than that for Eu(III). The present work using a competitive extraction strategy developed a feasible alternative method for the selective separation of trivalent actinides from lanthanides.

Fluorescent turn on detection of zinc(II) ion using a hydrazone derived from 4-tert-butyl-2,6-diformylphenol and 3-hydrazineylquinoxalin-2(1H)-one

A polydentate chelating ligand (LH) was synthesized from 4-tertbutyl-2,6-diformylphenol and 3-hydrazineylquinoxalin-2(1H)-one and characterized by mass spectrometry, IR and NMR spectroscopy. LH is weakly fluorescent in EtOH – HEPES buffer (5 mM, pH = 7.4, 8:2, v/v) but exhibited a turn on response with Zn2+ ion having a workable pH range of 6–12 and thus is compatible with biological pH. This sensor is selective for Zn2+ ion, over various metal ions and anions as evident from competition experiments. The probable mechanism of sensing by LH is restriction of ESIPT, PET and C = N isomerization processes and on-set of CHEF process upon coordination to Zn(II) ion. Reversibility was established using EDTA test and applicability in various real samples were evaluated. The calculated limit of detection for Zn2+ ion was 95 nM and Job plot as well as 1H NMR titration suggested a 2:1 binding ratio between Zn2+ ion and LH. From the reaction (in ethanol) of ZnCl2 with LH, in 2:1 ratio, crystalline solids of composition [Zn2(L)Cl3]⋅2H2O 2.5DMF was isolated and its molecular structure was established by single crystal XRD studies. DFT/TDDFT calculation supported the experimental electronic absorption spectrum.

Visible to near-infrared broadband electrochromic switching of π-extended viologen exhibiting highly efficient photothermal conversion

This study describes a new π-extended viologen molecule (TFBV) characterized by an electron-deficient structure inserted between two pyridinium groups. The single-layered electrochromic devices (ECDs) using TFBV all-in-one ion gel exhibit remarkable transparency under neutral conditions and display strong absorption bands in the visible and near-infrared (NIR) regions. The TFBV ECDs undergo a black coloration at a low operating voltage (−1.4 V), and at higher voltages (>−1.5 V), they display notable absorption in the NIR-II region. The two-electron reduction to neutral TFBV was confirmed through the chemical reduction of TFBV using Na2S, monitored through UV–vis spectra and 1H NMR titrations. The TFBV ECDs demonstrate an impressive color contrast of up to 96 % and a coloration efficiency of 150.18 cm2/C. The ECDs show outstanding kinetic stability through the 2000th cycle and moderate switching stability up to the 10000th cycle, accompanied by fast coloration times. Flexible TFBV ECDs, built on ITO PET, sustain superior performance even during flat and semicircle bending maneuvers, ensuring stability across repeated bending cycles. A uniform color transition is achieved in large-area ECDs of 10 cm × 10 cm. The strong NIR absorption of TFBV ECDs enables the photothermal heater to harvest the heat from the light energy, inducing a temperature rise of 45 °C. Furthermore, these devices perform a photothermal conversion efficiency of 52.5 % during multiple on–off cycles. TFBV ECDs, offering high transparency and intense darkness while blocking UV–Vis-NIR spectra, prove promising candidates for broadband ECDs with integrated photothermal conversion capabilities.

A Photoswitchable Macrocycle Controls Anion‐Templated Pseudorotaxane Formation and Axle Relocalization

AbstractImportant biological processes, such as signaling and transport, are regulated by dynamic binding events. The development of artificial supramolecular systems in which binding between different components is controlled could help emulate such processes. Herein, we describe stiff‐stilbene‐containing macrocycles that can be switched between (Z)‐ and (E)‐isomers by light, as demonstrated by UV/Vis and 1H NMR spectroscopy. The (Z)‐isomers can be effectively threaded by pyridinium halide axles to give pseudorotaxane complexes, as confirmed by 1H NMR titration studies and single‐crystal X‐ray crystallography. The overall stability of these complexes can be tuned by varying the templating counteranion. However, upon light‐induced isomerization to the (E)‐isomer, the threading capability is drastically reduced. The axle component, in addition, can form a heterodimeric complex with a secondary isophthalamide host. Therefore, when all components are combined, light irradiation triggers axle exchange between the macrocycle and this secondary host, which has been monitored by 1H NMR spectroscopy and simulated computationally.

An AIE active anthracene-based Schiff base probe for “turn-on” detection of Cu2+ ions: Demonstrations with nanostructural investigations, DFT, cellular imaging, and real water analysis

An aggregation induced emission (AIE) active anthracene-based Schiff base probe “(2-((anthracen-9-ylmethylene) amino) phenol); AP” was synthesized via one-pot reaction and utilized in “turn-on” detection of Cu2+ ions. Ultraviolet–visible and photoluminescence (UV–vis/PL) interrogations confirm the AIE between 0 and 99 % water fraction (fw) and high selectivity of AP to Cu2+. PL studies establish the linear regression of Cu2+ between 0 and 24 µM with a detection limit (LOD) of 53.2 nM. The Job plot and high-resolution mass (HR-mass) data affirm 2:1 stoichiometry and the association constant (Ka) value appraised as 1.88 × 10−4 M−2. Time-resolved photoluminescence (TRPL) studies reveal decay kinetics of AIE, Cu2+ sensors and complex's reversibility. Moreover, 1H NMR titration and density functional theory (DFT) investigations identify the binding mode, π-π stacking in AIE, AP − Cu2+ − AP* excimer formation, band gap variations and involvement of photoinduced electron transfer (PET) and chelation enhanced fluorescence (CHEF) mechanism. Crystallinity and nanostructural variations of AIE and Cu2+ sensors are investigated by using powder-X-ray diffraction (PXRD), scanning electron microscope (SEM), transmission electron microscope (TEM), and dynamic light scattering (DLS). AIE and Cu2+ sensory method are validated by MDA-MB-231 cellular imaging studies. Finally, real water-based titrations show nanomolar LODs and recoveries > 97 % with RSDs < 3.5 %.

Sequence-Specific Relay Recognition of Multiple Anions: An Interplay between Proton Donors and Acceptors.

Ratiometric detection of analyte is highly deserving since the technique is free from background correction. This work reports the design and synthesis of a pyridine-end oligo p-phenylenevinylene (OPV) derivative, 1 and its application in ratiometric dual-mode (both colorimetric and fluorogenic) recognition of dual anions, bisulfate (LOD=12.5 ppb) followed by fluoride (LOD=18.2 ppb) by sequence-specific relay (SPR) technique. The colorless probe turns brown with addition of bisulfate and again becomes colorless with the sequential addition of fluoride ion. In addition to such naked-eye color change, interestingly the ratiometric spectroscopic signals are reversible and evidently, the probe is reusable for several cycles. Besides, in presence of bisulfate, the protonated probe molecules, owing to their larger amphiphilic characteristics, formed self-assembled nanostructures. In addition to colorimetric and fluorescent changes, 1 H NMR titration and systematic DFT study evidently establish the underneath proton transfer mechanisms. Such reusable OPV-based chemosensor particularly with the capability of naked-eye recognition of dual anions using the SPR technique is seminal and possibly the first report in the literature.

Precisely designed NIR based conjugated framework as a solid state emitter for selective recognition of cyanide ion in solid state and cancer cells

Cyanide is undoubtedly dangerous and fast acting chemical employed in various industrial applications with toxic side effects, which raises urgent need for its monitoring. In this context, oxazolonaphthoimidazo[1,2-a]pyridine-malononitrile (ONIM) conjugate linked by vinyl linkage was designed, synthesized and characterised by FT-IR, NMR and mass spectrometry. The developed probe has been evaluated for the colorimetric and fluorometric detection of cyanide ion. An obvious color change with unique rapid “turn-off” fluorometric response was observed within 5 s upon addition of CN– thus reflecting its better acuteness and sensitivity over other competing anions. The developed probe exhibits a detection limit of 9.54 nM which is very low in comparison to maximum permitable amount of cyanide in drinking water (1.9 µM). The probe is stable within the pH range of 2–9 and suitable for the recognition of CN– in the pH range of 7 to 10, specially at neutral pH conditions. The chemodosimetric approach based detection mechanism was authenticated by 1H NMR titration and DFT calculations. The solid state applications were established by utilising ONIM impregnated TLC plates and filter paper in cyanide recognition. The solid state emissive behaviour of ONIM offers on-spot detection of the cyanide anion. Additionally, the practicability of ONIM has been extended in live cell imaging of CN– in human breast cancer cell (MDA-MB-231), which revealed a “turn-off” fluorescence response for the probe in the presence of CN– ion.

A novel coumarin-derived fluorescent probe for real-time detection of pH in living zebrafish and actual food samples

A coumarin-based fluorescent probe DIC (3-(2-(5-(4-(diphenylamino)phenyl)isoxazol-3-yl)-1-hydroxyvinyl)-7-hydroxy-2H-chromen-2-one) with high performance was exploited for detecting pH changes in biological system and food samples. The probe DIC could sensitively sense alkaline pH fluctuations in the range of 6.50‒9.98 with the pKa value of 8.12 through a remarkable fluorescence turn-on process. The probe DIC exhibited excellent photostability and reversibility in monitoring the pH fluctuations. The detection mechanism of DIC for pH was confirmed by 1H NMR titration experiment and theoretical calculation. This probe owned a low cytotoxicity and was successfully employed for in-suit tracking of pH fluctuations in living zebrafish. In addition, the probe DIC could be applied in the visualization of meat and prawn freshness.

A New Azomethine Chemosensor for Naked-Eye Detection of CN− and AcOˉ: Application to Determination of Cyanide in Some Agro-Products and Poisoned Human Blood Samples

A new symmetrical azomethine probe, H2L, has been synthesized via a condensation reaction of N, N´-Ethylene bis (3-formyl-5-methyl salicylaldimine), with Isonicotinic hydrazide in EtOH. The anion recognition studies exhibited that H2L acts as a chemosensor for the detection of CN− and AcO− in pure DMSO and DMSO/H2O 90:10(V/V) media over other basic anions such as F−, Cl−, Br−, I−, N3 −, SO4 2−, SO3 2−, PO4 3−, H2PO4 −, HSO4 −, NO2 −, and NO3 −. The ability of the H2L for rapid colorimetric sensing of CN− and AcOˉ was studied using UV–Vis, and 1H NMR titration methods. The detection limits of H2L toward CN− and AcO− in the semi-aqueous medium were found to be 2.12 and 2.63 μM, respectively, which are lower than the LOD of some existing azomethine probes. Effectively, H2L was used to qualitatively and quantitatively detect cyanide ions in the human-poisoned blood and some of the agro-products containing cyanogenic samples without relying on expensive instruments.