A perylene diimide-based nanoring architecture for exogenous and endogenous ATP detection: biochemical assay for monitoring phosphorylation of glucose.

Positively charged amphiphiles hold great significance in supramolecular chemistry due to their good solubility, and physiochemical and molecular recognition properties. Herein, we report the synthesis, characterization and molecular recognition properties of the dicationic amphiphile based on perylene diimide-tyrosine alkyl amide amine (PDI 3). PDI 3 showed the formation of a nanoring architecture in the self-assembled aggregated state (90% H2O-DMSO mixture) as observed by SEM and TEM studies. The diameter of the nanoring is around 30-50 nm with a height varying from 1 to 2 nm. The self-assembled aggregates of PDI 3 are very sensitive towards nucleoside triphosphates. Upon addition of ATP, PDI 3 showed a decrease in the absorbance and emission intensity at 535 and 580 nm (due to the monomer state), respectively. The lowest detection limit for ATP is 10.8 nM (UV) and 3.06 nM (FI). Upon interaction of ATP with PDI 3, the nanoring morphology transformed into a spherical structure. These changes could be attributed to the formation of ionic self-assembled aggregates between dicationic PDI 3 and negatively charged ATP via electrostatic and H-bonding interactions. The complexation mechanism of PDI 3 and ATP was confirmed by optical, NMR, Job's plot, DLS, SEM and AFM studies. PDI 3 displays low cytotoxicity toward MG-63 cells and can be successfully used for the detection of exogenous and endogenous ATP. The resulting PDI 3 + ATP complex is successfully used as a 'turn-on' biochemical assay for monitoring phosphorylation of glucose.

Influence of core extension and side chain nature in targeting G-quadruplex structures with perylene monoimide derivatives.

A structure-activity relationship (SAR) study in terms of G-quadruplex binding ability and antiproliferative activity of six fluorescent perylenemonoimide (PMIs) derivatives is reported. A positive charge seems to be the key to target G4. This study also reveals the importance of the element substitution in the potential biological activity of PMIs, being the polyethylene glycol (PEG) chains in the peri position responsible for their antiproliferative activity. Among them, the cationic PMI6 with two PEG chains is the most promising compound since its fluorescence is enhanced in the presence of G-quadruplex structures. Moreover, PMI6 binds to the human telomeric G-quadruplex hTelo with high affinity and displays a high antiproliferative potential towards HeLa (cervical adenocarcinoma), A549 (lung adenocarcinoma) and A2780 (ovarian adenocarcinoma) cells. Its fate can be followed inside cells thanks to its fluorescent properties: the compound is found to accumulate in the mitochondria.

On the interaction of an anticancer trisubstituted naphthalene diimide with G-quadruplexes of different topologies: a structural insight.

Naphthalene diimides showed significant anticancer activity in animal models, with therapeutic potential related to their ability to strongly interact with G-quadruplexes. Recently, a trifunctionalized naphthalene diimide, named NDI-5, was identified as the best analogue of a mini-library of novel naphthalene diimides for its high G-quadruplex binding affinity along with marked, selective anticancer activity, emerging as promising candidate drug for in vivo studies. Here we used NMR, dynamic light scattering, circular dichroism and fluorescence analyses to investigate the interactions of NDI-5 with G-quadruplexes featuring either parallel or hybrid topology. Interplay of different binding modes of NDI-5 to G-quadruplexes was observed for both parallel and hybrid topologies, with end-stacking always operative as the predominant binding event. While NDI-5 primarily targets the 5'-end quartet of the hybrid G-quadruplex model (m-tel24), the binding to a parallel G-quadruplex model (M2) occurs seemingly simultaneously at the 5'- and 3'-end quartets. With parallel G-quadruplex M2, NDI-5 formed stable complexes with 1:3 DNA:ligand binding stoichiometry. Conversely, when interacting with hybrid G-quadruplex m-tel24, NDI-5 showed multiple binding poses on a single G-quadruplex unit and/or formed different complexes comprising two or more G-quadruplex units. NDI-5 produced stabilizing effects on both G-quadruplexes, forming complexes with dissociation constants in the nM range.

Regioisomer-manipulating thio-perylenediimide nanoagents for photothermal/photodynamic theranostics.

Thionated perylenediimides (PDIs) can potentially generate thermal and reactive oxygen species and thus can be used as theranostic agents for photothermal/photodynamic therapy. Herein, thionated cis-/trans-isomer PDI-CS and PDI-TS were designed and prepared to investigate thionation engineering on therapeutic performance. The results revealed that the photodynamic performance is less associated with the positon of sulfur atoms. By contrast, trans-isomer PDI-TS showed a photothermal conversion efficiency of up to 58.4%, which was 40% higher than that of PDI-CS (∼41.6%). An in vitro half-maximal inhibitory concentration of ∼7.78 µg mL-1 was achieved for PDI-TS, which was 1.7-fold smaller than that of PDI-CS, strongly reasserting the regioisomer-modulated phototheranostic performance. Notably, the strong π-π and CS interactions in PDI-TS nanoagents are essential factors attributed to their excellent photothermal performance, indicating that the optimization of non-bonding interactions is an ingenious way to improve phototheranostic performance. This work provides a facile means of creating thio-perylenediimides that possess excellent antitumor properties and a novel proof of concept to improve therapeutic performance through the optimization of non-bonding interactions.

Peptide Stapling with Anion-π Catalysts.

We report design, synthesis and evaluation of a series of naphthalenediimides (NDIs) that are bridged with short peptides. Reminiscent of peptide stapling technologies, the macrocycles are conveniently accessible by a chromogenic nucleophilic aromatic substitution of two bromides in the NDI core with two thiols from cysteine sidechains. The dimension of core-bridged NDIs matches that of one turn of an α helix. NDI-stapled peptides exist as two, often separable atropisomers. Introduction of tertiary amine bases in amino-acid sidechains above the π-acidic NDI surface affords operational anion-π catalysts. According to an enolate chemistry benchmark reaction, anion-π catalysis next to peptides occurs with record chemoselectivity but weak enantioselectivity. Catalytic activity drops with increasing distance of the amine base to the NDI surface, looser homocysteine bridges, mismatched, shortened and elongated α-helix turns, and acyclic peptide controls. Elongation of isolated turns into short α helices significantly increases activity. This increase is consistent with remote control of anion-π catalysis from the α-helix macrodipole.

Real-time monitoring of intracellular pH in live cells with fluorescent ionic liquid.

Real-time monitoring of intracellular pH is of great significance due to its essential role in physiological and pathological processes. In present work, the ionic liquid (IL) N-methyl-6-hydroxyquinolinium bis(trifluoromethylsulfonyl) imide ([6MQc][NTf2]) is proposed as a fluorescence probe for the quantitative imaging of intracellular pH in response to external stimuli. The fluorescence of the IL [6MQc][NTf2] exhibits a sensitive response to pH variations, as the deprotonation of [6MQc][NTf2] generates the highly fluorescent zwitterionic product [6MQz]. pH fluctuations in the range of 6.0-7.5 can be accurately sensed by monitoring the fluorescence change at 555 nm. Moreover, this IL probe exhibits favorable biocompatibility, excellent anti-photobleaching properties, and high tolerance to ionic strength. Using the IL probe, real-time sensing of hypoxia- and drug-induced intracellular pH changes in MCF-7 cells is achieved.

Near-IR oxime-based solvatochromic perylene diimide probe as a chemosensor for Pd species and Cu2+ ions in water and live cells.

A near-IR perylene diimide probe (OPR-PDI) containing an oxime-propargyl hybrid moiety at the bay position, was designed and synthesized for detection of Pd species and Cu2+ ions in 90% water, the solid state and MG-63 live cells. The aggregation tendency of OPR-PDI in different polarity solvents transmits solvatochromic and fluorochromic properties to differentiate certain organic solvents. Supramolecular aggregates of OPR-PDI in 90% water act as a dual chemosensor for palladium (Pd) species via de-propargylation or hydrolysis of the Schiff-base and Cu2+ ions via complexation with the O/N binding site with a low limit of detection (LOD) of the order of 7.9 × 10-8 M and 3.4 × 10-7 M respectively. TLC strips coated with OPR-PDI can be applied for sensing of Pd0 and Cu2+ ions in the solid state at levels as low as 34.6 ng cm-2 and 10.5 ng cm-2. OPR-PDI imprinted TLC strips could be used as paper sheets for writing coloured alphabets using Pd0 and Cu2+ ions as ink. Moreover, MTT assay showed that OPR-PDI has very low cytotoxicity (IC50 = 230 µM), good permeability, biocompatibility and can be applied for bio-imaging of Pd species and Cu2+ ions in MG-63 cells. DFT calculations, and cyclic voltammetric (CV) and NMR titration studies have also been discussed.

The Interaction of Cyclic Naphthalene Diimide with G-Quadruplex under Molecular Crowding Condition.

G-quadruplex specific targeting molecules, also termed as G4 ligands, are attracting increasing attention for their ability to recognize and stabilize G-quadruplex and high potentiality for biological regulation. However, G4 ligands recognizing G-quadruplex were generally investigated within a dilute condition, which might be interfered with under a cellular crowding environment. Here, we designed and synthesized several new cyclic naphthalene diimide (cNDI) derivatives, and investigated their interaction with G-quadruplex under molecular crowding condition (40% v/v polyethylene glycol (PEG)200) to mimic the cellular condition. The results indicated that, under molecular crowding conditions, cNDI derivatives were still able to recognize and stabilize G-quadruplex structures based on circular dichroism measurement. The binding affinities were slightly decreased but still comparatively high upon determination by isothermal titration calorimetry and UV-vis absorbance spectroscopy. More interestingly, cNDI derivatives were observed with preference to induce a telomere sequence to form a hybrid G-quadruplex under cation-deficient molecular crowding conditions.

Dyads of G-Quadruplex Ligands Triggering DNA Damage Response and Tumour Cell Growth Inhibition at Subnanomolar Concentration.

Naphthalene diimide (NDI) dyads exhibiting a different substitution pattern and linker length have been synthesised and evaluated as G-quadruplex (G4) ligands, by investigating their cytotoxicity in selected cell lines. The dyads with the long C7 linker exhibit extremely low IC50 values, below 10 nm, on different cancer cell lines. Contrary, the dyads with the shorter C4 linker were much less effective, with IC values increasing up to 1 µm. Among the three dyads with the longest linker, small differences in the IC50 values emerge, suggesting that the linker length plays a more important role than the substitution pattern. We have further shown that the dyads are able to induce cellular DNA damage response, which is not limited to the telomeric regions and is likely the origin of their cytotoxicity. Both absorption titration and dynamic light scattering of the most cytotoxic dyads in the presence of hTel22 highlight their ability to induce effective G4 aggregation, acting as non-covalent cross-linking agents.

4-Substituted benzenesulfonamides featuring cyclic imides moieties exhibit potent and isoform-selective carbonic anhydrase II/IX inhibition.

The synthesis, characterization and biological evaluation of series of cyclic imides incorporating the 4-sulfamoylbenzamide scaffold (16-29) is disclosed. The compounds were designed by application of the "tail approach" to the aromatic sulfonamide scaffold and prepared by reacting the proper acid anhydride with 4-(hydrazinecarbonyl)benzenesulfonamide (15). Phtalimides and cyclic imides are biologically privileged scaffolds, endowed with versatile biological activity, such as an anti-proliferative action. The compounds were investigated for the inhibition of four human (h) isoforms of zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), and more specifically against the cytosolic hCA I and II and the transmembrane hCA IV and IX. Most screened sulfonamides exhibited great potency in inhibiting CA isoforms II, widely involved in glaucoma and other pathologies (KIs in the range of 0.7-62.3 nM), and IX, that is a validated anti-tumor target (KIs in the range of 3.0-50.9 nM), whereas interesting hydrophilicity-dependent inhibitory profiles were measured against isoform CA IV (KIs in the range of 3.9-428.6 nM). In silico studies were carried out to assess the binding mode of selected derivatives to hCA II, IV and IX.

[Asymmetric Synthesis of Unnatural Amino Acid-containing Peptides via Direct Asymmetric Reaction of Peptidyl Compounds].

Unnatural amino acids and the peptides bearing such units have gathered much attention due to their interesting biological activities and synthetic utility as chiral building blocks for the synthesis of complex molecules. This paper descibes an asymmetric synthesis of unnatural amino acid derivatives and their subsequent application to the preparation of unnatural amino acid-containing peptides. The α-imino carboxylic acid derivatives, which are common substrates for the synthesis of unnatural amino acids, could be readily prepared by MnO2-mediated oxidation of N-PMP-protected glycine derivatives. This reaction allowed us to synthesize novel derivatives such as α-imino perfluoroalkylesters, imides or thioesters. These compounds are useful for the synthesis of unnatural amino acid derivatives. MnO2-mediated oxidation was further applied to the synthesis of peptidyl imine. Moreover, a new methodology for the synthesis of unnatural amino acid-containing peptides was developed using the thus obtained imino peptides. This novel approach should be useful for the divergent synthesis of peptides possessing varieties of unnatural amino acid moieties.

l-Dopa and dopamine conjugated naphthalenediimides modulate amyloid ß toxicity.

The process of protein misfolding and aggregation to form neurotoxic species is strongly implicated in most of the neurodegenerative disorders. In particular, amyloid beta (Aß) misfolding and aggregation is central to pathophysiological processes of Alzheimer's disease. The development of aggregation modulators has enormous implications in the discovery of effective therapeutic agents for Alzheimer's disease. Herein, we report the design and synthesis of a series of natural amino acid, l-dopa and dopamine appended derivatives of naphthalenediimide (NDI) to identify efficient aggregation modulators. Furthermore, the molecular docking studies revealed the possible binding sites and binding mode of NDI-conjugates to Aß aggregates. Among the designed NDI-conjugates, l-dopa and dopamine derivatives (NLD and NDP, respectively) showed excellent aggregation modulation efficiency (inhibition and dissolution), as shown by the thioflavin T (ThT) binding assays, dot blot analysis and in cellulo studies. The docking results from in silico studies are in good agreement with the experimental data. In addition to their significant modulation efficiency towards Aß aggregation, NLD and NDP possess antioxidant activity conducive to the development of disease-modifying therapeutic agents for the treatment of Alzheimer's disease.

Synthesis of chalcone-imide derivatives and investigation of their anticancer and antimicrobial activities, carbonic anhydrase and acetylcholinesterase enzymes inhibition profiles.

The new 1-(4-(3-(aryl)acryloyl)phenyl)-1H-pyrrole-2,5-diones (5a-g) were prepared from 4'-aminchalcones (3a-g) and screened for biological activities. All compounds (3a-g and 5a-g), except 3d and 3e displayed good cytotoxic activities with IC50 values in the range of 7.06-67.46 µM. IC50 value of 5-fluorouracil (5-FU) was 90.36 µM. Moreover, most of compounds 5a-g showed high antibacterial activity with 8-20 mm of inhibition zone (19-25 mm of Sulbactam-Cefoperazone (SCF)). In addition, they showed good inhibitory action against acetylcholinesterase (AChE), and human carbonic anhydrase I, and II (hCA I and hCA II) isoforms. Also, these compounds demonstrated effective inhibition profiles with Ki values of 426.47-699.58 nM against hCA I, 214.92-532.21 nM against hCA II, and 70.470-229.42 nM against AChE. On the other hand, acetazolamide, clinically used drug, showed a Ki value of 977.77 ± 227.4 nM against CA I, and 904.47 ± 106.3 nM against CA II, respectively. Also, tacrine inhibited AChE showed a Ki value of 446.56 ± 58.33 nM.

Cyclic perylene diimide: Selective ligand for tetraplex DNA binding over double stranded DNA.

Synthesized cyclic perylene diimide, cPDI, showed the binding constant of 6.3 × 106 M-1 with binding number of n = 2 with TA-core as a tetraplex DNA in 50 mM Tris-HCl buffer (pH = 7.4) containing 100 mM KCl using Schatchard analysis and showed a higher preference for tetraplex DNA than for double stranded DNA with over 103 times. CD spectra showed that TA-core induced its antiparallel conformation upon addition of cPDI in the absence or presence of K+ or Na+ ions. The cPDI inhibits the telomerase activity with IC50 of 0.3 µM using TRAP assay which is potential anti-cancer drug with low side effect.

High-Spin Iron Imido Complexes Competent for C-H Bond Amination.

Reduction of previously reported (ArL)FeCl with potassium graphite furnished a low-spin (S = 1/2) iron complex (ArL)Fe which features an intramolecular η6-arene interaction and can be utilized as an FeI synthon (ArL = 5-mesityl-1,9-(2,4,6-Ph3C6H2)dipyrrin). Treatment of (ArL)Fe with adamantyl azide or mesityl azide led to the formation of the high-spin (S = 5/2), three-coordinate imidos (ArL)Fe(NAd) and (ArL)Fe(NMes), respectively, as determined by EPR, zero-field 57Fe Mössbauer, magnetometry, and single crystal X-ray diffraction. The high-spin iron imidos are reactive with a variety of substrates: (ArL)Fe(NAd) reacts with azide yielding a ferrous tetrazido (ArL)Fe(κ2-N4Ad2), undergoes intermolecular nitrene transfer to phosphine, abstracts H atoms from weak C-H bonds (1,4-cyclohexadiene, 2,4,6-tBu3C6H2OH) to afford ferrous amido product (ArL)Fe(NHAd), and can mediate intermolecular C-H amination of toluene [PhCH3/PhCD3 kH/kD: 15.5(3); PhCH2D kH/kD: 11(1)]. The C-H bond functionalization reactivity is rationalized from a two-step mechanism wherein each step occurs via maximal energy and orbital overlap between the imido fragment and the C-H bond containing substrate.

Stabilization of peptides against proteolysis through disulfide-bridged conjugation with synthetic aromatics.

Peptides have been promising molecular scaffolds for the development of potential therapeutics with high affinity and specificity to biomacromolecules. However, their inherent proteolytic instability significantly hampers their biological applications. Strategies that can stabilize peptides against proteolytic digestion on the basis of noncovalent interactions-without extensive manipulation of the sequence or use of unnatural residues-are greatly desired. In this work, we developed a general, convenient, and efficient strategy for the stabilization of peptides against proteolysis, which involves noncovalent π-π interactions between aromatic amino acid residues in peptides and synthetic electron-deficient aromatics (NDI), together with the implication of steric hindrance (from the bulky NDI moiety), and the enhancement of peptide α-helicity. This strategy is complementary in concept to the conventional well-established covalent approaches for peptide stabilization, and is thus promising for being utilized, in combination with the latter ones, to circumvent the problem of proteolytic instability of peptides. We envisioned that this study should provide invaluable guidelines to the design and synthesis of organic molecule-peptide hybrids with significantly improved proteolytic resistance, and benefit the development of peptide therapeutics and probes.

Synthesis and biological evaluation of cyclic imides incorporating benzenesulfonamide moieties as carbonic anhydrase I, II, IV and IX inhibitors.

A group of cyclic imides was synthesized by reaction of amino-substituted benzenesulfonamides with a series of acid anhydrides such as succinic, maleic, tetrahydrophthalic, pyrazine-2,3-dicarboxylic acid anhydride, and substituted phthalic anhydrides. The synthesized sulfonamides were evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors against the human (h) isoforms hCA I, II, IV and IX, involved in a variety of diseases among which glaucoma, retinitis pigmentosa, etc. Some of these sulfonamides showed effective inhibitory action (in the nanomolar range) against the cytosolic isoform hCA II and the transmembrane, tumor-associated one hCA IX, making them interesting candidates for preclinical evaluation in glaucoma or various tumors in which the two enzymes are involved. hCA I and IV were on the other hand less inhibited by these sulfonamides, with inhibition constants in the micromolar range.

Synthesis and X-ray Structural Analysis of the Ruthenium(III) Complex Na[trans-RuCl4(DMSO) (PyrDiaz)], the Diazene Derivative of Antitumor NAMI-Pyr.

Novel tetrachloridoruthenium(III) complex Na[trans-RuCl4(DMSO)(PyrDiaz)] (3) with pyridine-tethered diazenedicarboxamide PyrDiaz ligand (PyrDiaz = N1-(4-isopropylphenyl)-N2-(pyridin-2-ylmethyl)diazene-1,2-dicarboxamide) was synthesized by direct coupling of PyrDiaz with sodium trans-bis(dimethyl sulfoxide)tetrachloridoruthenate(III) (Na-[trans-Ru(DMSO)2Cl4]). Compound 3 is the analogue of the antimetastatic Ru(III) complex NAMI-A and NAMI-Pyr. Single crystal X.

Solvent free, catalyst free, microwave or grinding assisted synthesis of bis-cyclic imide derivatives and their evaluation for anticancer activity.

Cyclic imides are well known to be very important antitumor agents such as mitonafide and amonafide etc. Based on this fact, we have synthesized two series of cyclic imide derivatives containing two cyclic imide moiety in their structures (bis-cyclic imides) and screened them for in vitro anticancer activity against five human cancer cell lines i.e. breast (T47D), lung (NCl H-522), colon (HCT-15), ovary (PA-1) and liver (Hep G2). One series of bis-cyclic imide derivatives (3a-h) have been synthesized by condensation of acid anhydrides (1a-b) with diamines (2a-d) and another series (9a-f, 10a-f, 11a-f and 12a-f) by condensation of various diamines (4a-f) with diacids (5-8) in good yields. Structures assigned to 3a-h, 9a-f, 10a-f, 11a-f and 12a-f were fully characterized by spectroscopic means and elemental analysis. On screening for in vitro anticancer activity, compounds 3a (breast T47D), 3d (breast T47D, liver Hep G2), 3e (breast T47D, liver Hep G2), 3h (colon HCT-15), 10f (liver Hep G2) and 11a (colon HCT-15, ovary PA-1) exhibited good anticancer activities with IC50 values range from 12.41±3.2 to 17.9±2.5µM.

EMICORON: A multi-targeting G4 ligand with a promising preclinical profile.

BACKGROUND: During the last decade, guanine G-rich sequences folding into G-quadruplex (G4) structures have received a lot of attention and their biological role is now a matter of large debate. Rising amounts of experimental evidence have validated several G-rich motifs as molecular targets in cancer treatment. Despite that an increasing number of small molecules has been reported to possess excellent G4 stabilizing properties, none of them has progressed through the drug-development pipeline due to their poor drug-like properties. In this context, the identification of G4 ligands with more favorable pharmacological properties and with a well-defined target activity could be fruitful for anticancer therapy application. SCOPE OF REVIEW: This manuscript outlines the current state of knowledge regarding EMICORON, a G4-interactive molecule structurally and biologically similar, on the one side, to coronene and, on the other side, to a bay-monosubstituted perylene. MAJOR CONCLUSIONS: Overall this work evidences that EMICORON, a new promising G4 ligand, possesses a marked antitumoral activity both standing alone and in combination with chemotherapeutics. Moreover, EMICORON represents a good example of multimodal class of antitumoral drug, able to simultaneously affect multiple targets participating in several distinct signaling pathways, thus simplifying the treatment modalities and improving the selectivity against cancer cells. GENERAL SIGNIFICANCE: Due to the importance of G4 forming sequences in crucial biological processes participating in tumor progression, their successful targeting with small molecules could represent a very important innovation in the development of effective therapeutic strategies against cancer. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.