Arginine clustering on calix[4]arene macrocycles for improved cell penetration and DNA delivery.

Cell-penetrating peptides are widely used as molecular transporters for the internalization inside cells of various cargo, including proteins and nucleic acids. A special role is played by arginine-rich peptides and oligoarginines covalently linked or simply mixed with the cargo. Here we report cell-penetrating agents in which arginine units are clustered on a macrocyclic scaffold. Instead of using long peptides, four single arginine units were covalently attached to either the upper or lower rim of a calix[4]arene, kept in the cone conformation building a 'parallel' cyclic array. These new macrocyclic carriers show high efficiency in DNA delivery and transfection in a variety of cell lines.

ATP cleavage by cone tetraguanidinocalix[4]arene.

The upper rim cone tetraguanidinocalix[4]arene 1 is a highly effective catalyst of ATP hydrolysis. The catalytically most active species is the triprotonated form of the catalyst. The three protonated guanidinium groups provide the electrostatic driving force for substrate binding and activation, while the neutral guanidine most likely acts as a nucleophilic catalyst.

Lower rim guanidinocalix[4]arenes: macrocyclic nonviral vectors for cell transfection.

Guanidinium groups were introduced through a spacer at the lower rim of calix[4]arenes in the cone conformation to give new potential nonviral vectors for gene delivery. Several structural modifications were explored, such as the presence or absence of a macrocyclic scaffold, lipophilicity of the backbone, length of the spacer, and nature of the charged groups, in order to better understand the factors which affect the DNA condensation ability and transfection efficiency of these derivatives. The most interesting compound was a calix[4]arene unsubstituted at the upper rim and having four guanidinium groups linked at the lower rim through a three carbon atom spacer. This compound, when formulated with DOPE, showed low toxicity and transfection efficiency higher than the commercially available lipofectamine LTX in the treatment of human Rhabdomiosarcoma and Vero cells. Most of the investigated compounds showed a tendency to self-aggregate in pure water or in the presence of salts, as evidenced by NMR and AFM studies, and it was found that the ability to condense DNA plasmids in nanometric globules is a necessary but not sufficient condition for transfection. The superiority of macrocyclic vectors over linear Gemini-type analogues and of guanidinium compared to other ammonium head groups in determining the biological activity of the vectors was also ascertained.

Upper rim guanidinocalix[4]arenes as artificial phosphodiesterases.

Calix[4]arene derivatives, blocked in the cone conformation and functionalized with two to four guanidinium units at the upper rim were synthesized and investigated as catalysts in the cleavage of the RNA model compound 2-hydroxypropyl p-nitrophenyl phosphate. When compared with the behavior of a monofunctional model compound, the catalytic superiority of the calix[4]arene derivatives points to a high level of cooperation between catalytic groups. Combination of acidity measurements with the pH dependence of catalytic rates unequivocally shows that a necessary requisite for effective catalysis is the simultaneous presence, on the same molecular framework, of a neutral guanidine acting as a general base and a protonated guanidine acting as an electrophilic activator. The additional guanidinium (guanidine) group in the diprotonated (monoprotonated) trifunctional calix[4]arene acts as a more or less innocent spectator. This is not the case with the tetrasubstituted calix[4]arene, whose mono-, di-, and triprotonated forms are slightly less effective than the corresponding di- and triguanidinocalix[4]arene derivatives, most likely on account of a steric interference with HPNP caused by overcrowding.

Combining carbohydrate substitutions at bioinspired positions with multivalent presentation towards optimising lectin inhibitors: case study with calixarenes.

Carbohydrate derivatisation and glycocluster formation are both known to enhance avidity for lectin binding. Using a plant toxin and human adhesion/growth-regulatory lectins (inter- and intrafamily comparisons) the effect of their combination is examined. In detail, aromatic substituents were introduced at the 2-N or 3'-positions of N-acetyllactosamine and the products conjugated to two types of calix[n]arenes (n = 4, 6) via thiourea-linker chemistry.

CO2 capture by multivalent amino-functionalized calix[4]arenes: self-assembly, absorption, and QCM detection studies.

The reactivity of CO(2) with polyamino substrates based on calix[4]arenes and on a difunctional, noncyclic model has been studied. All the compounds react with CO(2) in chloroform to form ammonium carbamate salts. However, the number, topology, and conformational features of the amino-functionalized arms present on the multivalent scaffold have a remarkable influence on the reaction efficiency and on the product composition. Tetraaminocalix[4]arenes 1-3 rapidly and efficiently react with 2 equiv of CO(2), yielding highly stable hydrogen-bonded dimers formed by the self-assembly of two bis-ammonium bis-carbamate intramolecular salts. 1,3-Diaminocalix[4]arene 4 absorbs 1 mol of CO(2), affording less stable zwitterionic ammonium carbamates. Gemini compound 5 reacts with CO(2) in a 1:1 stoichiometry, forming hydrogen-bonded dimers of ammonium carbamate derivatives of moderate stability. For upper rim 1,3-diaminocalix[4]arene 6, in addition to the labile intramolecular salt, the presence of a self-assembled polymer was also detected. These systems were fully characterized in solution by (1)H and (13)C NMR spectroscopy, whereas the corresponding gas-solid reactions were further investigated by QCM measurements. Interestingly, the high affinity and reversibility of CO(2) uptake shown by 1,3-diamino calix[4]arene 4 enabled us to attain a promising QCM device for carbon dioxide sensing.

Glucosylthioureidocalix[4]arenes: Synthesis, conformations and gas phase recognition of amino acids.

The gas-phase recognition of native amino acids and the conformational properties of three glucosylthioureidocalix[4]arenes () were studied theoretically and experimentally using ab initio calculations, ESI-FTICR,(1)H and (13)C NMR MS. The conformational and complexation properties of the glucocalixarenes were dependent on the number of glucose units at the upper rim and the length of the alkyl chains at the lower rim of the calixarene skeleton. ESI-MS experiments showed the compounds to form 1 : 1 complexes with the amino acids, with a marked preference for amino acids containing an aromatic nucleus and an additional H-bonding group in their side chain (Trp, Tyr, Phe >> Ser, Leu and Asp). The experimental data were rationalized by the results of ab initio calculations. ESI-MS competitions carried out with enantiomeric-labelled (EL) amino acids showed enantiomeric selectivities ranging from 0.61 (Phe(D)/Phe(L) with ligand ) to 2.58 (Tyr(D)/Tyr(L) with ligand ). In gas-phase hydrogen-deuterium (H/D) exchange reactions, diglucosylcalix[4]arene exhibited extremely slow exchange rates, which were attributed to the close proximity and strong hydrogen bonding between the facing glucosylthioureido groups. H/D exchange rates were much higher for the tetraglucosylcalix[4]arenes and and their amino acid complexes, and the more rigid tetrapropoxy derivative showed more selective H/D exchange reactions than the calixarene . Bi- or trimodal H/D exchange distribution was observed for the tetraglucosyl derivatives indicating that these ligands exist in multiple isomeric forms in gas phase.

Calix[6]arene-picolinamide extractants for radioactive waste: effect of modification of the basicity of the pyridine N atom on the extraction efficiency and An/Ln separation.

The effect of a systematic variation of the basicity of the pyridine nitrogen atom in calix[6]arene-based picolinamide ligands on the actinide(III)/lanthanide(III) separation by solvent extraction has been investigated. The distribution coefficients for trivalent metal ions (D(M)) decrease by increasing the nitric acid concentration, but for ligands (2 and 4) possessing a much less basic aromatic nitrogen atom, D(M) values are considerably higher than those of ligands (1 and 5) having more basic pyridine nuclei. Also in terms of selectivity ligands 2 and 4 behave better than ligand 1 especially at nitric acid concentrations very close to those of the nuclear waste. At [H(+)] = 1 mol L(-1), SF(Am/Eu) are still 3.23 and 1.92 for 2 and 4, respectively. A simple quantitative relationship between the efficiency of these extractants and the gas-phase basicity of suitably chosen model compounds is proposed, in order to explain the experimental extraction data, on one hand, and to orient future syntheses of ligands for An/Ln separation, on the other hand.

The role of building-block metrics in the halogen-bonding-driven self-assembly of calixarenes, inorganic salts and diiodoperfluoroalkanes.

Halogen bonding effectively co-operates with ion-ion interactions in determining the supramolecular structure of three-component heteromeric crystals comprising of calix[4]arenes, inorganic salts and diiodoperfluoroalkanes. The subtle interplay between these two types of interactions, as well as the influence of the valence of the metal ion, the "size" of the calixarene platform and the length of the perfluorocarbon module on the stoichiometry and overall supramolecular organisation of the ternary supramolecular architectures is discussed. The relevance to the overall packing of the size matching between the pitches along the cationic and anionic sub-lattices is also discussed.

Macrocyclic nonviral vectors: high cell transfection efficiency and low toxicity in a lower rim guanidinium calix[4]arene.

New multivalent cationic lipids, one of them showing high efficiency and low toxicity in cell transfection, have been obtained by attaching guanidinium groups at the lower rim of calix[4]arenes.

Calix[n]arene-based glycoclusters: bioactivity of thiourea-linked galactose/lactose moieties as inhibitors of binding of medically relevant lectins to a glycoprotein and cell-surface glycoconjugates and selectivity among human adhesion/growth-regulatory galectins.

Growing insights into the functionality of lectin-carbohydrate interactions are identifying attractive new targets for drug design. As glycan recognition is regulated by the structure of the sugar epitope and also by topological aspects of its presentation, a suitable arrangement of ligands in synthetic glycoclusters has the potential to enhance their avidity and selectivity. If adequately realized, such compounds might find medical applications. This is why we focused on lectins of clinical interest, acting either as a potent biohazard (a toxin from Viscum album L. akin to ricin) or as a factor in tumor progression (human galectins-1, -3, and -4). Using a set of 14 calix[n]arenes (n=4, 6, and 8) with thiourea-linked galactose or lactose moieties, we first ascertained the lectin-binding properties of the derivatized sugar head groups conjugated to the synthetic macrocycles. Despite their high degree of flexibility, the calix[6,8]arenes proved especially effective for the plant AB-toxin, in the solid-phase model system with a single glycoprotein (asialofetuin) and with human tumor cells in vitro. The bioactivity of the calix[n]arenes was also proven for human galectins. Notably, selectivity for the tested tandem-repeat-type galectin-4 among the three subgroups was determined at the level of solid-phase and cell assays, the large flexible macrocycles again figuring prominently as inhibitors. Alternate and cone versions of calix[4]arene with lactose units distinguished between galectins-1 and -4 versus galectin-3 in cell assays. The results thus revealed bioactivity of galactose-/lactose-presenting calix[n]arenes for medically relevant lectins and selectivity within the family of adhesion/growth-regulatory human galectins.

Efficient and selective cleavage of RNA oligonucleotides by calix[4]arene-based synthetic metallonucleases.

Di- and trinuclear copper(II) complexes of [12]aneN3 macrocycles anchored at the upper rim of cone calix[4]arenes in 1,2-, 1,3-, and 1,2,3-positions were investigated as cleaving agents of 6-, 7-, and 17-meric oligoribonucleotides. A kinetic investigation of the cleavage reactions was carried out using gel electrophoresis to separate and analyze reactants and products having a radioactive phosphate label in the terminal 5'-position. The degree of cooperation was assessed on the basis of a comparison with rates of cleavage by mononuclear controls. A remarkable selectivity of cleavage of the CpA phosphodiester bond was observed for all metal complexes, in sharp contrast with the UpU and UpG selectivity previously observed in the cleavage of diribonucleoside monophosphates by the same metal complexes. The highest rate acceleration, brought about in the cleavage of the 5'-pCpA bond in hexanucleotide 9 by 50 muM trinuclear complex 5-Cu3 (water solution, pH 7.4, 50 degrees C), amounts to 5 x 105-fold, as based on the estimated background reactivity of the CpA dimer. Selectivity in the cleavage of oligoribonucleotides by copper(II) complexes closely resembles that experienced by ribonuclease A and by a number of metal-independent RNase A mimicks. The possible role of the dianionic phosphate at the 5'-terminal positions as a primary anchoring site for the metal catalyst is discussed.

N-linked peptidocalix[4]arene bisureas as enantioselective receptors for amino acid derivatives.

Bisurea calix[4]arenes 1 and 2 possessing L-amino acid moieties at the lower rim were synthesized by reaction of the methyl esters of glycine, L-alanine, or L-isoleucine with the appropriate isocyanate (12 or 13), obtained with a safe and efficient Curtius rearrangement from the corresponding carboxylic acid derivatives. The conformational properties of the ligands 1 and 2 were investigated by means of a combined NMR and molecular modeling study which evidences that they are deeply influenced by strong intramolecular H-bonds between the urea NH groups and the vicinal phenolic oxygen atoms or the opposite urea C=O group. Complexation studies performed by ESI-MS and NMR spectroscopy in acetone solution show that the binding ability of these bisurea hosts decreases by increasing the side chain size of the amino acid. Host 2b has a remarkable binding ability for the N-acetyl-D-phenylalaninate anion with an interesting enantioselectivity (KDass/KLass=4.14), which is explained on the basis of a three-point interaction mode of binding.

DNA condensation and cell transfection properties of guanidinium calixarenes: dependence on macrocycle lipophilicity, size, and conformation.

Calix[n]arenes functionalized with guanidinium groups at the upper rim and alkyl chains at the lower rim bind to DNA, condense it, and in some cases, promote cell transfection depending on their structure and lipophilicity. Atomic force microscopy (AFM) studies indicate that upon DNA binding the hydrophobic association of the lipophilic chains of cone guanidinium calix[4]arenes drives the formation of intramolecular DNA condensates, characterized by DNA loops emerging from a dense core. Furthermore, hexyl and octyl chains confer to these calixarenes cell transfection capabilities. Conversely, larger and conformationally mobile calix[6]- and calix[8]arene methoxy derivatives form intermolecular aggregates characterized by "gorgonlike" structures composed of multiple plectomenes. These adducts, in which interstrand connections are dominated by electrostatic interactions, fail to promote cell transfection. Finally, calix[4]arenes in a 1,3-alternate conformation show an intermediate behavior because they condense DNA, but the process is driven by charge-charge interactions.

Catalysis of diribonucleoside monophosphate cleavage by water soluble copper(II) complexes of calix[4]arene based nitrogen ligands.

Calix[4]arenes functionalized at the 1,2-, 1,3-, and 1,2,3-positions of the upper rim with [12]ane-N(3) ligating units were synthesized, and their bi- and trimetallic zinc(II) and copper(II) complexes were investigated as catalysts in the cleavage of phosphodiesters as RNA models. The results of comparative kinetic studies using monometallic controls indicate that the subunits of all of the zinc(II) complexes and of the 1,3-distal bimetallic copper(II) complex 7-Cu(2) act as essentially independent monometallic catalysts. The lack of cooperation between metal ions in the above complexes is in marked contrast with the behavior of the 1,2-vicinal bimetallic copper(II) complex 6-Cu(2), which exhibits high catalytic efficiency and high levels of cooperation between metal ions in the cleavage of HPNP and of diribonucleoside monophosphates NpN'. A third ligated metal ion at the upper rim does not enhance the catalytic efficiency, which excludes the simultaneous cooperation in the catalysis of the three metal ions in 8-Cu(3). Rate accelerations relative to the background brought about by 6-Cu(2) and 8-Cu(3) (1.0 mM catalyst, water solution, pH 7.0, 50 degrees C) are on the order of 10(4)-fold, largely independent of the nucleobase structure, with the exception of the cleavage of diribonucleoside monophosphates in which the nucleobase N is uracil, namely UpU and UpG, for which rate enhancements rise to 10(5)-fold. The rationale for the observed selectivity is discussed in terms of deprotonation of the uracil moiety under the reaction conditions and complexation of the resulting anion with one of the copper(II) centers.

Inclusion of naturally occurring amino acids in water soluble calix[4]arenes: a microcalorimetric and 1H NMR investigation supported by molecular modeling.

The thermodynamic parameters for the inclusion of some naturally occurring amino acids into a series of p-sulfonated calix[4]arenes, were determined via both 1H NMR and calorimetric titrations in buffered aqueous solution at 25 degrees C. The calorimetric data show that inclusion is enthalpically driven in all cases, regardless of flexibility of the host and the nature of the guest. The most efficient receptor is the calix[4]arene tetrasulfonate 1, which exists in solution at pH 7 in a cone conformation, stiffened by H-bonding at the lower rim. Molecular mechanics data help in the understanding of why some hosts do not form inclusion complexes at all. The comparison of our data with literature reports shows that there are dramatic buffer-dependent changes in the binding affinities.

Scanning force microscopy of upright-standing, isolated calixarene-porphyrin heterodimers.

A water soluble calixarene[4]arene 1 with four guanidinium substituents on the upper rim and propyl groups below was anchored in the propylamino coating of smooth silica particles, and a tricarboxylate-tripod porphyrin 2 of 2 nm height was attached to these cationic islands. The molecular complex with a height of 3 nm was unequivocally detected on the particles' surface by atomic force microscopy in the tapping mode. Although deposits of 1 (height: 1 nm) and 2 (height: 2 nm) were also evident on the smooth silica particles, 3 nm seems to be the minimal height to identify single objects. The soft surface of the particles not only allowed tight attachment of molecular edge amphiphiles by the hydrophobic effect but also immobilized the particles on the mica surface by amine-silicate interactions.

Calix[4]arene-based Zn2+ complexes as shape- and size-selective catalysts of ester cleavage.

[structures: see text] The kinetics of methanolysis of a number of esters endowed with a carboxylate anchoring group have been investigated in the presence of di- and trinuclear Zn2+ complexes of calix[4]arenes functionalized at the upper rim with nitrogen ligands. The results (i) emphasize the importance of a good match between ester size and intermetal distance, (ii) reveal a substrate independent superiority of the 1,2-vicinal dinuclear catalyst 1-Zn2 to its 1,3-distal regioisomer 2-Zn2, and (iii) provide further evidence for the concurrence of the three metal ions of 3-Zn3 in the catalytic mechanism.

A synthetic divalent cholera toxin glycocalix[4]arene ligand having higher affinity than natural GM1 oligosaccharide.

A high-affinity ligand of cholera toxin, the divalent glycocalix[4]arene 1, was obtained by exploiting a combination of structure-based design of glycomimetic monovalent ligands and affinity enhancements by multivalent presentation through a calix[4]arene scaffold. It exhibits a slightly higher affinity for the toxin than its natural ligand, the GM1 oligosaccharide.