Aryl Crown Ethers Based on d-Glucose Scaffold – Highly Selective Receptors of Amino Acid Ester Hydrochlorides

Amino acids are important biomolecules with a broad scope of applications in chemical and biological sciences. Their functions and properties depend on their absolute configuration. Therefore, methods for chiral recognition and separation of amino acids are highly sought after. For the purposes of diagnostic and medicinal applications chiral recognition of amino acids in water is particularly relevant. However synthetic receptors for enantioselective binding of amino acids in aqueous media are rare. Recently we reported a d -glucose-based crown ether for chiral recognition of amino acid esters in water. We achieved enantioselectivities towards amino acids with hydrophobic sidechains which were among the highest ever reported for a small molecule receptor. The binding affinities were however moderate. Herein we disclose analogs of that receptor, containing aryl functionalities in the crown ether fragment. The new receptors show considerably improved binding affinities for amino acid ester hydrochlorides in water, while retaining high enantio- or chemoselectivities.     

Calix[4]azacrowns as ionophores for liquid?Cliquid extraction and facilitated transport of biological supramolecular complexes

The native and methyl ester amino acids have been extracted by calix[4]azacrowns 1 (1,3-[ethylene-bis-aminocarbonylmethoxy)]-p-tert-butylcalix[4]arene) and 2 (1,3-[propylene-bis-aminocarbonylmethoxy)]-p-tert-butyl-calix[4]arene) from an aqueous phase into a chloroform phase and transported through a chloroform liquid membrane as ion pairs in the presence of picrate or tropaeolin 00 as counter ions aiming their separation. The amino acids under study exhibited good extractability by calix[4]azacrowns 1 and 2. Both receptors 1 and 2 showed good extractability towards amino acids under study. The results are discussed in term of correlation of structural properties of amino acids and calix[4]azacrowns involved in experiments. In this respect, the influence of chain length of receptors upon extraction and transport of amino acids, and the nature of anions used as counter ions are evaluated.     

Recognition of Free Tryptophan in Water by Synthetic Pseudopeptides: Fluorescence and Thermodynamic Studies

Pseudopeptidic receptors containing an acridine unit have been prepared and their fluorescence response to a series of amino acids was measured in water. Free amino acids, not protected either at the C or the N terminus, were used for this purpose. The prepared receptors display a selective response to tryptophan (Trp) versus the other assayed amino acids under acidic conditions. The macrocyclic nature of the receptor is important as the fluorescence quenching is higher for the macrocyclic compound than for the related open‐chain receptor. Notably, under the experimental acidic conditions used, both the receptor and guest are fully protonated and positively charged; thus, the experimental results suggest the formation of supramolecular species that contain two positively charged organic molecular components in proximity stabilized through aromatic–aromatic interactions and a complex set of cation‐anion‐cation interactions. The selectivity towards Trp seems to be based on the existence of a strong association between the indole ring of the monocharged amino acid and the acridinium fragment of the triprotonated form of the receptor, which is established to be assisted by the interaction of the cationic moieties with hydrogen sulfate anions.     

双吲哚烯类受体对氨基酸的比色识别作用

设计合成了一系列3,3′-双吲哚烯类受体分子,应用紫外-可见吸收光谱研究了该类受体对二十种天然氨基酸分子的识别作用. 结果表明,在乙腈-水溶液中,双吲哚烯受体分子可以通过其氢键受体位点的质子化作用比色识别天冬氨酸、谷氨酸等酸性氨基酸;而硝基取代的双吲哚烯受体分子则通过其氢键供体位点的去质子化作用比色识别精氨酸、赖氨酸等碱性氨基酸. 在乙腈-水的中性缓冲溶液体系中,硝基取代的双吲哚烯类受体分子通过与巯基发生亲核加成反应高选择性比色识别半胱氨酸和同型半胱氨酸.     

Chiral Recognition of Amino Acid Esters in Organic Solvents Using a Glucose-Based Receptor

Due to the chemical and biological relevance of amino acids, efficient methods for the recognition and separation of their enantiomers are highly sought after. Chiral receptors based on extended molecular scaffolds are typically employed for this purpose. These receptors are often effective only in specific environments and towards a narrow scope of amino acid guests. Recently we reported a simple, glucose-based macrocycle capable of enantioselective binding of a broad range of amino acid methyl esters in water. Herein we demonstrate that the same receptor can be used for chiral recognition of amino acid esters in organic solvents. We show that the binding affinity and selectivity of the receptor are highly dependent on the coordinating strength of the solvent. An in-depth analysis of the receptor’s conformation and its interactions with amino acid methyl esters allowed us to propose a binding mode of amino acids to the receptor in CDCl₃. The binding modes in CDCl₃ and D₂O were then compared, highlighting the main interactions responsible for binding affinity and selectivity in each solvent. We envision that the insight provided by this study will facilitate the development of further amino acid receptors based on monosaccharides with improved binding affinities and both enantio- as well as chemoselectivities.     

Complexation of amino acids derivatives in water by calix[4]arene phosphonic acids

Series of the calix[4]arene phosphonic acids with various substituents at the lower rim was synthesized. Complexing properties of these receptors towards methyl esters of six amino acids strongly depended on the calix[4]arene conformation flexibility. The complex formation processes were monitored using ¹H NMR spectroscopy (deuterated phosphate buffer at pD 7.3, 22 °C) and association constant values were evaluated. Inherently mobile calix[4]arene molecule 3 occurred in cone conformation in aqueous solution turned out to be more effective in complexation of the basic amino acids methyl esters compared to the rigid 2 and flexible 4. Mixed 1:2 and 2:1 (host–guest) complexes were observed for compound 1 with all amino acids methyl esters.     

Electro chromatography on paper

The term 'electrochromatography' is used to denote the specific cases where chromatography and ionophoresis operate simultaneously. The electric field may be applied either in the direction of flow of the solvent or at right angles to it. The filter paper is previously treated with the relevant buffer solution and the developing solvent too is saturated with it. Colloidal graphite painted on the two sides of the paper serves as electrodes.A detailed study has been made of a number of amino acids at different pH's with phenol-buffer as the developing solvent. On ordinary paper acidic amino acids move towards the anode, basic ones towards the cathode, neutral amino acids do not move at all. Using buffer-treated papers, however, the movement depends upon the pH of the buffer and the isoelectric points of the amino acids. By choice of suitable pH and application of electric field in proper direction amino acids having very close RF values (except isomers) can be separated from one another.     

A combinatorial approach to obtain affinity media with binding properties towards the aflatoxins

In our work we performed a combinatorial solid-phase synthesis in aqueous medium to prepare peptide libraries from which to select an amino acid sequence with binding properties towards aflatoxins.We used polystyrene beads, functionalised with carboxylic groups as solid support and eight amino acids as monomers. During the first step 64 different sequences of two amino acids were prepared by exploiting the principles of combinatorial chemistry; then the binding properties of all sequences towards aflatoxin B(1 )were checked. We determined binding constants towards aflatoxin B(1) and towards aflatoxins B(2), G(1) and G(2). Results were promising, so we prepared a new library by using the selected dipeptide as the starting solid phase. After selecting the best tetrapeptide sequences, we determined binding constants towards the quoted aflatoxins. We obtained binding constants ( K>10(4) M(-1)) similar to those shown by human serum albumin for similar compounds. Preliminary studies on an extraction column were promising for the development of an SPE system and for its application in food matrices.     

Synthesis and chiral recognition abilities of new calix[6]arenes bearing amino alcohol moieties

Herein the synthesis and recognition abilities towards amino acids and amino alcohols of new d-/l-phenylalaninol substituted p-tert-butylcalix[6]arenas are reported. These compounds, 6 and 7 have been synthesized via nucleophilic substitution reactions involving 5,11,17,23,29,35-tert-butyl-37,38-dimethoxy-39,40,41,42-(p-tosylethoxy)calix[6]arene 5 with d-/l-phenylalaninol in dry THF. The extraction properties of 6 and 7 towards some selected amino acid methylesters and amino alcohols have been studied by liquid–liquid extraction. These results show that chiral calix[6]arene derivatives exhibit a good affinity towards all amino acids and amino alcohols.     

Amino alcohol based chiral solvating agents: synthesis and applications in the NMR enantiodiscrimination of carboxylic acids

Four optically active amino alcohols were synthesized via the ring opening of (R)-N-(2,3-epoxypropyl)phthalimide with (R)-2-phenyl glycinol, (1R,2S)-cis-1-amino-2-indanol, (R)-2-amino-1-butanol and (S)-phenyl ethylamine in 73-93% yields. The enantioselective recognition of these receptors towards the enantiomers of racemic carboxylic acids was studied by ¹H NMR spectroscopy. The molar ratio and the association constants of the chiral compounds with each of the enantiomers of the guests were determined by using Job plots and a non-linear least-squares fitting method, respectively. Large non-equivalent chemical shifts (up to 30.0 Hz) can be achieved in the presence of chiral amino alcohols 2 and 5. Amongst the chiral receptors used, compound 5 was found to be the best chiral shift reagent, and was effective in the determination of the enantiomeric excess of chiral carboxylic acids.     

Chiral bis(amino amides) as chiral solvating agents for enantiomeric excess determination of α-hydroxy and arylpropionic acids

A family of bis(amino amides) derived from natural amino acids has been synthesized and tested for the NMR enantiodiscrimination, as chiral solvating agents, for enantiomeric excess determination of some carboxylic acids. Those bis(amino amide) receptors contain different structural modifications and the splitting of the signals of the acids, after addition of the corresponding CSAs, depends on those structural variables. The influence of aminoacid side chain and the nature of the aliphatic spacer are important parameters to obtain good chiral discriminations. The results obtained clearly show the chiral recognition abilities of these bis(amino amide) ligands and suggest their advantageous use as chiral solvating agents for carboxylic acids. The binding between bis(amino amides) and carboxylic acids has been studied by ESI-MS, NMR, DSC, and molecular modeling. The data suggest that enantiodiscrimination involves the formation of an ionic pair after proton transfer from the carboxylic substrate to the bis(amino amides).     

Anion influence on extraction and transport of amino acid methyl esters by functionalised calix[4]arene

The liquid–liquid extraction of a series of amino acid methyl esters has been carried out with functionalised calix[4]arene (5,11,17,23-tetrakis(N-methylpiperazino)-25,26,27,28-tetrahydroxycalix[4]arene) from an aqueous phase into a chloroform phase as ion pairs in the presence of picrate ion or tropaeolin 00 as counter ion in order to study the molecular recognition properties of this receptor. The active transport assisted by pH gradient of amino acids as ion pairs through liquid membrane employing the functionalised calix[4]arene as carrier has been investigated. The results showed that the receptor exhibits good extractability towards amino acids and it can also act as carrier through liquid membrane aiming to the separation of amino acids. It was highlighted that the anion nature used as counter ion, the structure of calix[4]arene, and the structure of amino acids are responsible for the experimental results obtained. High yields in both amino acids extraction and transport were obtained for picrate ion used as counter ion.     

Tryptophan receptors containing acridine-based thiourea

Four derivatives of acridine and acridinium compounds (L1, L2, L1H and L2H) comprised thiourea-binding sites were synthesised. The binding abilities of receptors L1, L2, L1H and L2H towards amino acids (l-Trp, l-Phe, l-Leu, l-Ala and l-Gly) were studied by ¹H NMR spectroscopy, UV–vis and fluorescence spectrophotometry. Hydrogen bonding interactions between thiourea-binding site of the ligand and the carboxylate groups in zwitterionic amino acids were found to be the main interactions driving complexation to take place. The stoichiometry of 1:1 ligand to amino acid was observed in all cases. Neutral ligands L1 and L2 showed weak binding towards all studied amino acids. The cyclic ligand L1 showed better binding ability towards tryptophan (Trp) than the acyclic ligand L2 did (K for Trp is 307 and 266 M^? 1 for L1 and L2, respectively). Interestingly, binding abilities of the protonated ligands, L1H and L2H, towards studied amino acids, especially Trp (K for Trp is 3157 and 2873 M^? 1 for L1H and L2H, respectively), were increased due to R–COO^? …H…N⁺–acridinium interactions. Calculated structures of L1H·Trp and L2H·Trp showed that the polyglycol moiety in L1H provided a hydrophobic cavity for binding Trp resulting in a stronger binding affinity of L1H over L2H.     

Dielectric spectroscopy in the GHz region on fully hydrated zwitterionic amino acids

The complex dielectric permittivity of eight different amino acids in water solutions was determined in the frequency range from 0.2 to 20 GHz at room temperature, trying to span the whole range of solubility in each case. Two relaxations were observed at room temperature in this frequency range, which can be mainly assigned to the rotation of amino acids in the aqueous environment, and the reorientational motion of water molecules, respectively. Although the amino acids have a charged (zwitterionic) nature with huge dipole moments, the tendency towards dipolar alignment seems to be very weak, over the investigated concentration ranges. For these small bio-molecules, water screens solute-solute interactions and amino acids remain typically as isolated hydrated monomers. The dielectric results were used to estimate the number of water molecules restrained by each solute molecule. Finally, the comparison between the amino acid relaxation times made it possible to discuss the relationship between rotational dynamics and the structure and hydrodynamic coupling of the amino acid studied.     

Solid-phase-assisted synthesis of targeting peptide-PEG-oligo(ethane amino)amides for receptor-mediated gene delivery

In the forthcoming era of cancer gene therapy, efforts will be devoted to the development of new efficient and non-toxic gene delivery vectors. In this regard, the use of Fmoc/Boc-protected oligo(ethane amino)acids as building blocks for solid-phase-supported assembly represents a novel promising approach towards fully controlled syntheses of effective gene vectors. Here we report on the synthesis of defined polymers containing the following: (i) a plasmid DNA (pDNA) binding domain of eight succinoyl-tetraethylenpentamine (Stp) units and two terminal cysteine residues; (ii) a central polyethylene glycol (PEG) chain (with twenty-four oxyethylene units) for shielding; and (iii) specific peptides for targeting towards cancer cells. Peptides B6 and c(RGDfK), which bind transferrin receptor and α(v)β(3) integrin, respectively, were chosen because of the high expression of these receptors in many tumoral cells. This study shows the feasibility of designing these kinds of fully controlled vectors and their success for targeted pDNA-based gene transfer.     

TAC-scaffolded tripeptides as artificial hydrolytic receptors: a combinatorial approach toward esterase mimics

In this report, we present the first library of tripodal synthetic receptor molecules containing three different, temporarily N-terminal protected peptide arms capable of performing hydrolytic reactions. To construct this library, the orthogonally protected triazacyclophane (TAC)-scaffold was used in the preparation of a split-mix library of 19 683 resin bound tripodal receptor molecules. For the construction of the peptide arms, three different sets of amino acids were used, each focused on one part of the catalytic triad as found in several families of hydrolytic enzymes. Therefore, in the sets of amino acids used to assemble these tripeptides, basic (containing His and Lys), nucleophilic (containing Ser and Cys), or acidic (containing Asp and Glu) amino acid residues were present. In addition, nonfunctional hydrophobic amino acid residues were introduced. Possible unfavorable electrostatic interactions of charged N-termini or their acetylation during screening were circumvented by trifluoroacetylation of the N-terminal amines. Screening was performed with a known esterase substrate, 7-acetoxycoumarin, which upon hydrolysis gave the fluorescent 7-hydroxycoumarin, leading to fluorescence of beads containing a hydrolytically active synthetic receptor. Although many synthetic receptors contain catalytic triad combinations, apparently, only a few showed hydrolytic activity. Sequence analysis of the active receptors showed that carboxylate-containing amino acids are frequently found in the acidic arm and that substrate cleavage is mediated by lysine (noncatalytic) or histidine (catalytic) residues. Kinetic analysis of resynthesized receptors showed that catalysis depended on the number of histidine residues and was not assisted by significant substrate binding.     

A cation-pi binding interaction with a tyrosine in the binding site of the GABAC receptor

GABA(C) (rho) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HT(3), and glycine receptors. As in other members of this family, the agonist binding site of GABA(C) receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-pi interaction to a tryptophan, the GABA(C) binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-pi binding ability of the side chain and EC(50) for receptor activation, thus demonstrating a cation-pi interaction between a tyrosine side chain and a neurotransmitter. Comparisons among four homologous receptors show variations in cation-pi binding energies that reflect the nature of the cationic center of the agonist.     

PEG-supported synthesis of pyrazole oligoamides with peptide beta-sheet affinity

Pyrazole amino acid oligoamides were prepared on polyethylene glycol starting from nitro pyrazole carboxylic acids or protected pyrazole amino acids. The polymer support facilitates product isolation during synthesis and makes the target oligoamides soluble in chloroform and water. This allows the determination of their binding properties towards peptides. Moderate affinity, which increases with the number of pyrazole units, is observed in chloroform and water.     

Amperometric detection of amino acids with a passivated copper electrode

The amperometric behaviour of a copper electrode towards amino acids is studied by means of a rotating disc electrode. A theoretical model describing the anodic background current in a buffer solution and the increase of the current caused by amino acids is checked experimentally. The influences of the amino acid concentration, the rotation speed of the electrode and the composition of the buffer solution are studied. It is proved that chemical dissolution of a passivating film on the electrode surface, which is enhanced by the complexation reaction between the amino acid and copper(II) ions, is the principle of the phenomena observed. The applicability to flow systems is demonstrated.     

Electrochemical determination of L-Tryptophan, L-Tyrosine and L-Cysteine using electrospun carbon nanofibers modified electrode

A novel and simple method for the direct and quantitative determination of L-tryptophan (Trp), L-tyrosine (Tyr) and L-cysteine (Cys) was proposed in this work. Carbon nanofibers (CNFs), made by electrospinning technique, were used to modify carbon paste electrode (CPE) without any treatment to study the electrochemical behaviors of the three amino acids using cyclic voltammetry (CV) and constant potential amperometric method. The results demonstrated that the CNFs modified carbon paste electrode (CNF-CPE) exhibited high electrocatalytic activity and good analytical performance towards the oxidation of the three amino acids. The linear ranges of Trp, Tyr and Cys were 0.1-119, 0.2-107 and 0.15-64 μM with correlation coefficients of 0.9994, 0.9985 and 0.9996, respectively. All the detection limits of the analytes were 0.1 μM (S/N = 3). In addition, the CNF-CPE displayed good reproducibility, high sensitivity and good selectivity towards the determination of the amino acids, making it suitable for the determination of Trp, Tyr and Cys in clinical and medicine.