Selective extraction and recovery of cytochrome c by liquid-liquid extraction using a calix[6]arene carboxylic acid derivative

Recently, we reported that a calix[6]arene carboxylic acid derivative can selectively extract the lysine-rich protein cytochrome c by interacting with amino groups on the protein surface. In the present article, quantitative extraction and recovery of cytochrome c using this calix[6]arene carboxylic acid derivative are described. Both adjustment of the pH under acidic conditions and addition of an alcohol are necessary to strip the extracted protein from an organic solution to an aqueous solution. Separation of cytochrome c and lysozyme using the calix[6]arene was achieved under the optimal conditions. In the forward extraction stage, 93% of the cytochrome c was extracted, while lysozyme remained in the solution. In the subsequent stripping stage, the extracted cytochrome c was quantitatively recovered in an aqueous solution. Finally, separation of these proteins, which have similar molecular weights and isoelectric points, was accomplished.     

Synthesis and binding properties of carboxylphenyl-modified calix[4]arenes and cytochrome c

Two novel carboxylphenyl-modified calix[4]arenes, tetrakis-carboxylphenylcalix[4]arene (TCPC) and 1,3-bis-carboxylphenylcalix[4]arene (BCPC), as well as a corresponding analogue for comparison, tetrakis-phenylcalix[4]arene (TPC), have been synthesized by palladium-catalyzed Suzuki cross-coupling of arylboronic acid and tetrabromocalix[4]arene as a key step. The binding properties of these calix[4]arene derivatives with bovine heart cytochrome c (cyt c) in dimethylformamide (DMF) was investigated by fluorescence spectroscopy. The binding affinity in the order of TCPC > BCPC ? TPC reflects a clear dependence on the number of carboxyl ligating groups attached onto a receptor and suggests the electrostatic force may be the predominant factor driving the complexing process. The stable 1:1 complexes of TCPC and BCPC with cyt c were evidenced with the binding constants of 3.15 × 10⁶ and 5.85 × 10⁵ L mol⁻¹, respectively. Due to a large overlap between the emission spectrum of TCPC and the absorption spectrum of cyt c, and a short interaction distance (estimated to be 5.6 nm) between them, the fluorescence quenching of TCPC upon complexation with cyt c is attributed to an efficient energy transfer.     

Extraction and separation of a lysine-rich protein by formation of supramolecule between crown ether and protein in aqueous two-phase system

The macrocyclic calixarenes and crown ethers have recently been found to form hydrophobic complexes with the cationic protein cytochrome c (Cyt-c), by recognizing lysine residues on the protein surface. In the present study, it was found that the distribution of cytochrome c in Li₂SO₄/PEG aqueous two-phase system (ATPS) can be controlled by complexation with the crown ether dicyclohexano-18-crown-6 (DCH18C6). The protein was quantitatively extracted into the PEG-rich phase in the presence of DCH18C6 and perchlorate ion. Of various crown ethers and their analogues that were investigated, only DCH18C6 was able to extract cytochrome c into the PEG-rich phase. Extraction of cytochrome c in the ATPS using DCH18C6 is complete within 5 min. Cytochrome c complexed with DCH18C6 in the PEG-rich phase was quantitatively recovered into a salt-rich phase using K₂SO₄ by ion exchange of potassium ion and cationic protein in the cationic protein complex with DCH18C6. Selective extraction of cationic proteins was demonstrated in the ATPS. Under optimum conditions, the lysine-rich protein cytochrome c was selectively extracted over other cationic proteins using DCH18C6.     

Extraction Behavior of Amino Acids by Calix[6]arene Carboxylic Acid Derivatives

A series of calixarene carboxylic acid derivatives were synthesized for the extraction of amino acids. A calix[6]arene carboxylic acid derivative showed the highest extractability to the target tryptophan ester. The main driving forcefor the complexation was the interaction between the ammonium cation of the aminoacid and the oxygen atoms of the host molecule. Stripping of amino acids was alsoaccomplished by contacting the organic solution with a fresh acidic solution. Basedon slope and Job method analyses, it was confirmed that the calix[6]arene formsa 1 : 1 complex with the amino acid ester. The structure of the complex between the calix[6]arene and the amino acid was investigated by ¹H-NMR and CD spectra. The calix[6]arene includes a guest molecule in the cavity, and the inclusion induces the asymmetrization of the host molecule. This host compound functions as a novel recognition tool for amino acids.     

Multiple proton ionizable calixarene derivatives with different ring sizes and complexation ability as efficient ionophores for complexation and solvent extraction of trivalent indium

The solvent extraction behavior of multiple proton ionizable p-tert-butylcalix[4]arene and [6]arene carboxylic acid derivatives towards indium has been investigated along with an acyclic monomeric analogue from weakly acidic media into chloroform. The extraction mechanism is ion exchange and carboxylic acid groups are adequate ligating sites for extraction. The cyclic structure of calixarene ligands to accommodate the potential guest species and the cooperativity effect of multifunctional groups significantly affect the complexation behavior and calixarene derivatives are found to be excellent extractants over the monomeric analogue. The composition of the extracted complex depends on the solution pH and attempts to determine the composition of the extracted complex for the extraction of indium have been stymied by complications arising from the formation of polynuclear species of indium and bridged polymeric species of calixarene carboxylic acid derivatives. One mole of calix[4]arene derivative extracts 2.5 moles of indium whereas the calix[6]arene derivative tends to extract 4.0 moles of indium. The loaded indium is back extracted with 1 mol dm(-3) hydrochloric acid solution. Though quantitative back extraction of indium was achieved from the fully loaded calix[6]arene derivative, it was only achieved up to 85% in the case of the calix[4]arene derivative.     

Chiral calix[4]arenes bearing α-hydroxy amide units as membrane carriers for amino acid methyl esters

Novel chiral calix[4]arene derivatives functionalized at the lower rim have been prepared from the reaction of p-tert-butylcalix[4]arene diamine or acylhydrazine derivative with mandelic acid or hydroxyisovaleric acid. The structures of these receptors were characterized by FTIR, ¹H, ¹³C and 2D COSY NMR spectroscopy and elemental analysis. The transport of amino acid derivatives (phenylalanine, phenylglycine and tryptophan methyl ester hydrochlorides) was studied through bulk liquid membrane in the presence of chiral calix[4]arene derivatives. The receptors have been found to act as carriers for transport of aromatic amino acid methylesters from the aqueous source phase to the aqueous receiving phase. The transport rate and L/D selectivity of amino acid esters studied depend strongly upon the structure of the chiral receptors and guests. The best enantioselectivity was obtained in the case of phenylglycine methyl ester for all chiral carriers.     

Selective functionalization at the small rim of calix[6]arene. Synthesis of novel non-symmetrical N3, N4 and N3ArO biomimetic ligands

Eight novel calix[6]arene-based biomimetic ligands for transition metal ions have been synthesized. They display a non-symmetrical N₃, N₄ or N₃ArO binding core that mimics enzyme active sites presenting histidine and tyrosine residues. The key step for their synthesis is the mono-alkylation at the small rim of the C_3v symmetrical trimethyl ether derivative of tBu-calix[6]arene with N-Boc-2-chloroethylamine to yield a novel calix[6]arene synthon. Its combined O-alkylation with a chloromethyl aromatic amine and N-deprotection or alkylation or reductive alkylation with a salicylaldehyde derivative yielded the calix[6]arene-based ligands with mixed N/O donors.     

CMPO-substituted calix[6]- and calix[8]arene extractants for the separation of An/Ln from radioactive waste

Three calix[6]arene derivatives (1a-c) and two calix[8]arene derivatives (2a,b), with six and eight CMPO residues, respectively, attached to the narrow/lower rim via ether links, were synthesised. Preliminary liquid-liquid extraction studies for Eu(III) and Am(III) from aqueous nitric acid to o-nitrophenylhexyl ether reveal remarkable properties with respect to efficiency and selectivity, especially for the tert-butylcalix[6]arene derivative with a -(CH₂)₃- spacer.     

Specific extraction behavior of amide derivative of calix[4]arene for silver (I) and gold (III) ions from highly acidic chloride media

25,26,27,28-tetrakis(N,N-diethylaminocarbonylmethoxy)-5,11,17,23-tetrakis(1,1,3,3-tetramethylbutyl)calix[4]arene, a macrocyclic extraction reagent, and p-(1,1,3,3-tetra-methylbutyl)phenoxymethyl-N,N-diethylamide, an acyclic extraction reagent corresponding to the former one, were synthesized to investigate their extraction behavior for silver(I), gold(III), palladium(II), and platinum(IV) from highly acidic solution into chloroform. In the extraction of silver and gold from hydrochloric acid solution, a completely different extraction behavior was observed between these two types of the reagents. The extraction behavior was examined in detail for silver and was found to be dependent on whether silver ion was extracted as a cationic species or a anionic species complexed with chloride ion. This was supported by proton nuclear magnetic resonance study of the calix[4]arene derivative. As a result, the extraction of silver ion with calix[4]arene derivative was very peculiar which was attributable to the fitting between cyclic size of calix[4]arene and ionic radius of silver.     

Inherently chiral biscalixarene cone–cone conformers consisting of calix[4]arene and calix[5]arene subunits

Inherently chiral biscalixarenes with hetero-cavities were synthesized by a covalent assembly of p-tert-butylcalix[5]arene with a 1,3-substituted calix[4]arene via 1,3-alkylation reaction and subsequent desymmetrization. The racemates were resolved by chiral HPLC method. ¹H NMR spectra, VT-NMR spectra, and theoretical calculations support that the calix[5]arene subunit of the inherently chiral calix[4][5]arene ester adopts a cone-in conformation, with the aromatic ring bearing the CH₂CO₂Et group tilting inward the calix[5]arene cavity. By contrast, such a cone-in structural feature of the calix[5]arene subunit disappears for the corresponding inherently chiral calix[4][5]arene carboxylic acid, due to the intramolecular hydrogen bonding between the carboxyl group and an ethereal oxygen of the glycolic chain.     

Calix[n]arene Carboxylic Acid Derivatives as Regulators of Enzymatic Reactions: Enhanced Enantioselectivity in Lipase-Catalyzed Hydrolysis of (R/S)-Naproxen Methyl Ester

Candida rugosa lipase was immobilized with a sol–gel encapsulation procedure in the presence and absence of a calix[n]arene carboxylic acid derivative grafted onto magnetic nanoparticles or in the presence of the calix[n]arene carboxylic acid derivative with Fe₃O₄ magnetic nanoparticles as an additive. Through the enantioselective hydrolysis of racemic naproxen methyl ester and the hydrolysis of p-nitrophenylpalmitate, the relative enzyme activity was evaluated and tested. These results show that the encapsulated lipase without supports has lower conversion and enantioselectivity compared to the Calix[n]COOH-based encapsulated lipase. It has also been observed that the Calix[4]COOH-based encapsulated lipase has excellent enantioselectivity (enantiomeric ratio (E)?>?400) as compared to encapsulated-free lipase enantioselectivity (E?=?137), and it also has an enantiomeric excess value of ~98 % for S-naproxen.     

Transport of Na+, K+ ions in a bubbling pseudo-emulsion liquid membrane system with p-tert-butylcalixarenes as carriers

The liquid membrane transport of Na⁺ ions by p-tert-butylcalix[6]arene and that of K⁺ ions by p-tert-butylcalix[8]arene were investigated by means of a bubbling pseudo-emulsion liquid membrane system. This system represents a proton-coupled transport with a flow of protons in the opposite direction. The driving force for the transport is the pH gradient between the source and receiving phases. When the pH difference between the two phases is sufficient, the carriers calix[6]arene(or calix[8]arene) can successfully transport Na⁺ (or K⁺) ions from the source phase with a lower Na⁺ (or K⁺) concentration into the receiving phase with a higher Na⁺ (or K⁺) concentration, like a Na⁺ (or K⁺) ion pump.     

Multivalent calixarene-based C-fucosyl derivative: a new Pseudomonas aeruginosa biofilm inhibitor

The first example of multivalent conjugate in which four α-l-C-fucosyl units are clustered by means of a calix[4]arene platform was designed as a new potential Pseudomonas aeruginosa biofilm inhibitor. The anti-biofilm activity of the synthesized compound (6) against PAO1 strain was assayed and it was found to be dose-dependent. The presence of the fucose cluster improves the biofilm inhibitor efficiency as proven by the lower inhibitor activity of the analogous glycyl-calix[4]arene derivative (3) lacking in the fucose moieties.     

Surfactant Ion Selective Membrane Electrodes

Abstract

Calix[8]arene ethers, carboxylic esters having various p-substituents and acid are synthesized and studied. In a bulk membrane transport of dodecylpyridinium the better carrier is the simplest host, calix[8]arene octamethyl ether. Inversely, it is the worst carrier for dodecyltrimethylammonium; the better here is carboxy ester with adamantyl p-substituents. Various calixarenes are successfully used in plasticized membrane ion-selective electrodes (ISE) for both above ammonium and pyridinium surfactants. Operational characteristics, potentiometric selectivity and optimal membrane compositions are reported. Typical limit of detection is at the level of nx10^?6 - 1x10^?5 M, response time 5-10 s, and lifetime is at least 6 months. ISE's selectivity is significantly better than that of ion-association and crown-ether based electrodes.     

Characterization of 1,3-alternate calix[4]arene-silica bonded stationary phases and their comparison to selected commercial columns by using principal component analysis

Twelve calix[4]arene stationary phases in 1,3-alternate conformation, synthesized in the authors’ laboratory, were characterized in terms of their surface coverage, hydrophobic selectivity, aromatic selectivity, shape selectivity, hydrogen bonding capacity and ion-exchange capacity. The set of tests commonly used for evaluation of commercially available stationary phases was applied to assess fundamental chromatographic properties of the calixarene phases. The new calixarene phases were compared to each other, to Caltrex and LiChrosorb C-18 columns. Principal component analysis has been used to provide comparison between 1,3-alternate calix[4]arene phases and commercially available phenyl, fluorophenyl and fluoroalkyl columns.     

High yield preparation of a novel tetrakis[ruthenium tris(bipyridine)]calix[6]arene derivative with good diastereomeric purity

In order to develop methodology for the selective preparation of complex molecular structures with potential photochemical or electron transfer functions, the diastereoselective synthesis of multiple ruthenium tris(bipyridine) complexes tethered to a central calix[6]arene core was investigated. Applying recently developed methodology, the resolved precursor cis-Δ-[Ru(bpy)₂(DMSO)Cl]PF₆ (98.6% ee) was efficiently reacted with a novel calix[6]arene derivative, to give the tetrakis[Ru(bpy)₂(bpy′)]calix[6]arene derivative (9) with almost complete retention of absolute stereochemistry at each of the four metal centres, as seen by the unusually strong molar circular dichroism (CD) spectrum. The synthesis of racemic 9 was also carried out, and demonstrated to have an inactive CD spectrum.     

Complexation and extraction behavior of trivalent indium with multiple proton ionizable p�Ct-butylcalix[5]arene pentacaarboxylic acid derivative: a new efficient solvent extraction reagent for indium

Complexation behavior of plural ion-exchangeable p?Ct-butylcalix[5]arene pentacarboxylic acid derivative towards trivalent indium has been investigated along with its monomeric analog from weakly acidic media into chloroform. The cyclic structure of calixarene ligand providing certain cavity and cooperativity of functional groups significantly affect the complexation behavior and calixarene derivative is an excellent extractant over monomeric analog. The extraction mechanism is ion exchange and carboxylic acid groups are adequate functional sites for extraction. Mononuclear and/or polynuclear species of indium and monomeric or bridged dimeric species of calixarene are involved in complexation and the composition of extracted complex varied with solution pH. One mole of calix[5]arene derivative tend to extract 3.5 mol of indium. The loaded indium was quantitatively back extracted with 1 mol dm^?3 hydrochloric acid solution.     

tert-Butylthiacalix[4]arene monolayers as a biomimetic model for the oxidation of antioxidants with cytochrome c

The possibility of designing a model of sensor to antioxidants based on thiacalix[4]arene monolayers with immobilized cytochrome c was shown. The molecular surface area S ₀ of thiacalix[4]arene in the monolayers and the surface pressure coefficient ?dπ/dS (elasticity) reflect changes in the redox state of cytochrome c in the presence of dihydroquercetin and ascorbic acid in the aqueous subphase. The absorption spectra in the visible and UV ranges of solutions of the subphase and transferred thiacalix[4]arene monolayers with immobilized cytochrome c confirm the oxidation of the antioxidants to quinones and formation of the reduced form of cytochrome c.     

Synthesis of calix[4]arene(amido)monocrowns and their photoresponsive derivatives

A series of new calix[4]arene(amido)mono-crown compounds have been synthesized through aminolysis of calix[4]arene esters and intramolecular cyclization of the intermediates. The title compounds were converted into their nitro and azo substituted derivatives to provide novel photoresponsive molecular receptors for transition metal ions. Single crystal X-ray analysis of calix[4]arene(ethyleneamido)mono-crown (2a) revealed that the compound is present in a cone conformation with an amido loop that caps the lower rim of calix[4]arene cavity to result in stacking along axis a and axis c to provide supramolecular aggregates in the solid state. Evaluation of synthesized macrocycles in the solution phase for recognition of transition metal cations (Cr³⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Ag⁺, Cd²⁺, Pb²⁺, Hg⁺, Hg²⁺, Pd²⁺, and Pt²⁺) by UV-visible spectroscopy revealed that p-tert-butyl-calix[4]arene mono-(amidocrown) 1c selectively shows a blue shift at 38 nm on interaction with Hg⁺ ions.     

A novel hybrid macrocyclic receptor based on bile acid and calix[4]arene frameworks for metal ion recognition

A novel hybrid macrocyclic receptor based on bile acid and calix[4]arene has been synthesized using the α-face of the bile acid and cone-conformation of calix[4]arene. Metal recognition properties of receptor were investigated. The receptor showed the strongest affinity for Hg²⁺ in acetonitrile. The stoichiometry of the Receptor:Hg²⁺ was 1:1.