Synthesis, Characterization, Fluorescence Properties, and DFT Modeling of Difluoroboron Biindolediketonates.

We report a simple and efficient strategy to enhance the fluorescence of biocompatible biindole diketonates (bdks) in the visible spectrum through difluoroboronation (BF2bdks complexes). Emission spectroscopy testifies an increase in the fluorescence quantum yields from a few percent to as much as >0.7. This massive increment is essentially independent of substitutions at the indole (-H, -Cl, and -OCH3) and corresponds to a significant stabilization of the excited state with respect to non-radiative decay mechanisms: the non-radiative decay rates are reduced by as much as an order of magnitude, from 109 s-1 to 108 s-1, upon difluoroboronation. The stabilization of the excited state is large enough to enable sizeable 1O2 photosensitized production. Different time-dependent (TD) density functional theory (DFT) methods were assessed in their ability to model the electronic properties of the compounds, with TD-B3LYP-D3 providing the most accurate excitation energies. The calculations associate the first active optical transition in both the bdks and BF2bdks electronic spectra to the S0 → S1 transition, corresponding to a shift in the electronic density from the indoles to the oxygens or the O-BF2-O unit, respectively.

Mercury Clathration-Driven Phase Transition in a Luminescent Bipyrazolate Metal-Organic Framework: A Multitechnique Investigation.

Mercury is one of the most toxic heavy metals. By virtue of its triple bond, the novel ligand 1,2-bis(1H-pyrazol-4-yl)ethyne (H2BPE) was expressly designed and synthesized to devise metal-organic frameworks (MOFs) exhibiting high chemical affinity for mercury. Two MOFs, Zn(BPE) and Zn(BPE)·nDMF [interpenetrated i-Zn and noninterpenetrated ni-Zn·S, respectively; DMF = dimethylformamide], were isolated as microcrystalline powders. While i-Zn is stable in water for at least 15 days, its suspension in HgCl2 aqueous solutions prompts its conversion into HgCl2@ni-Zn. A multitechnique approach allowed us to shed light onto the observed HgCl2-triggered i-Zn-to-HgCl2@ni-Zn transformation at the molecular level. Density functional theory calculations on model systems suggested that HgCl2 interacts via the mercury atom with the carbon-carbon triple bond exclusively in ni-Zn. Powder X-ray diffraction enabled us to quantify the extent of the i-Zn-to-HgCl2@ni-Zn transition in 100-5000 ppm HgCl2 (aq) solutions, while X-ray fluorescence and inductively coupled plasma-mass spectrometry allowed us to demonstrate that HgCl2 is quantitatively sequestered from the aqueous phase. Irradiating at 365 nm, an intense fluorescence is observed at 470 nm for ni-Zn·S, which is partially quenched for i-Zn. This spectral benchmark was exploited to monitor in real time the i-Zn-to-HgCl2@ni-Zn conversion kinetics at different HgCl2 (aq) concentrations. A sizeable fluorescence increase was observed, within a 1 h time lapse, even at a concentration of 5 ppb. Overall, this comprehensive investigation unraveled an intriguing molecular mechanism, featuring the disaggregation of a water-stable MOF in the presence of HgCl2 and the self-assembly of a different crystalline phase around the pollutant, which is sequestered and simultaneously quantified by means of a luminescence change. Such a case study might open the way to new-conception strategies to achieve real-time sensing of mercury-containing pollutants in wastewaters and, eventually, pursue their straightforward and cost-effective purification.

Asymmetric Phenyl Substitution: An Effective Strategy to Enhance the Photosensitizing Potential of Curcuminoids.

Curcumin has been demonstrated to exhibit photosensitized bactericidal activity. However, the full exploitation of curcumin as a photo-pharmaceutical active principle is hindered by fast deactivation of the excited state through the transfer of the enol proton to the keto oxygen. Introducing an asymmetry in the molecular structure through acting on the phenyl substituents is expected to be a valuable strategy to impair this undesired de-excitation mechanism competing with the therapeutically relevant ones. In this study, two asymmetric curcumin analogs were synthesized and characterized as to their electronic-state transition spectroscopic properties. Fluorescence decay distributions were also reconstructed. Their analysis confirmed the substantial stabilization of the fluorescent state with respect to the parent compound. Nuclear magnetic resonance experiments were performed with the aim of determining the structural features of the keto-enol ring and the strength of the keto-enol hydrogen bond. Electronic structure calculations were also undertaken to elucidate the effects of substitution on the features of the keto-enol semi-aromatic system and the proneness to proton transfer. Finally, their singlet oxygen-generation efficiency was compared to that of curcumin through the 9,10-dimethylanthracene fluorescent assay.

Double-stranded flanking ends affect the folding kinetics and conformational equilibrium of G-quadruplexes forming sequences within the promoter of KIT oncogene.

G-quadruplexes embedded within promoters play a crucial role in regulating the gene expression. KIT is a widely studied oncogene, whose promoter contains three G-quadruplex forming sequences, c-kit1, c-kit2 and c-kit*. For these sequences available studies cover ensemble and single-molecule analyses, although for kit* the latter were limited to a study on a promoter domain comprising all of them. Recently, c-kit2 has been reported to fold according to a multi-step process involving folding intermediates. Here, by exploiting fluorescence resonance energy transfer, both in ensemble and at the single molecule level, we investigated the folding of expressly designed constructs in which, alike in the physiological context, either c-kit2 or c-kit* are flanked by double stranded DNA segments. To assess whether the presence of flanking ends at the borders of the G-quadruplex affects the folding, we studied under the same protocols oligonucleotides corresponding to the minimal G-quadruplex forming sequences. Data suggest that addition of flanking ends results in biasing both the final equilibrium state and the folding kinetics. A previously unconsidered aspect is thereby unravelled, which ought to be taken into account to achieve a deeper insight of the complex relationships underlying the fine tuning of the gene-regulatory properties of these fascinating DNA structures.

Synthesis, Characterization and DNA-Binding Affinity of a New Zinc(II) Bis(5-methoxy-indol-3-yl)propane-1,3-dione Complex.

The novel zinc(II) µ-oxo-bridged-dimeric complex [Zn2(µ-O)2(BMIP)2] (BMIP = 1,3-bis(5-methoxy-1-methyl-1H-indol-3-yl)propane-1,3-dione), 1, was synthetized and fully characterized. The spectral data indicate a zincoxane molecular structure, with the BMIP ligand coordinating in its neutral form via its oxygen atoms. Structural changes in 1 in dimethylsulfoxide (DMSO) were evidenced by means of spectroscopic techniques including infrared absorption and nuclear magnetic resonance, showing DMSO entrance in the coordination sphere of the metal ion. The resulting complex [Zn2(µ-O)2(BMIP)2(DMSO)], 2, readily reacts in the presence of N-methyl-imidazole (NMI), a liquid-phase nucleoside mimic, to form [Zn2(µ-O)2(BMIP)2(NMI)], 3, through DMSO displacement. The three complexes show high thermal stability, demonstrating that 1 has high affinity for hard nucleophiles. Finally, with the aim of probing the suitability of this system as model scaffold for new potential anticancer metallodrugs, the interactions of 1 with calf thymus DNA were investigated in vitro in pseudo-physiological environment through UV-Vis absorption and fluorescence emission spectroscopy, as well as time-resolved fluorescence studies. The latter analyses revealed that [Zn2(µ-O)2(BMIP)2(DMSO)] binds to DNA with high affinity upon DMSO displacement, opening new perspectives for the development of optimized drug substances.

Nanomechanics of G-quadruplexes within the promoter of the KIT oncogene.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

Demonstration of fibrinogen-FcRn binding at acidic pH by means of Fluorescence Correlation Spectroscopy.

The neonatal Fc receptor (FcRn) interacts with IgG and albumin at acidic pH within endosomes, thus protecting these plasma proteins from degradation. Recently, we proposed fibrinogen as a new binding partner of FcRn. This work was aimed at providing a direct demonstration of FcRn-fibrinogen binding at acidic pH by Fluorescence Correlation Spectroscopy. The increase in diffusion time between free and fibrinogen-bound FITC-labelled FcRn was assumed as the binding indicator. We observed that, at acidic pH (pH = 5.3), FcRn diffusion time shifted from ≈730 µs (FITC-labelled FcRn alone) to >1200 µs (FITC-labelled FcRn added with fibrinogen). A similar trend was exhibited by albumin, a known FcRn interactor, while no significant variations in diffusion time were observed upon incubation with catalase as negative control. Our results demonstrate a binding interaction between fibrinogen, one of the most abundant plasma proteins, and FcRn, a receptor involved in the regulation of the levels of IgG and albumin. This interaction is likely responsible for fibrinogen protection from intracellular degradation and recycling in plasma. Fibrinogen is crucial not only in haemostasis but also in acute inflammatory response and in some pathological conditions. The interaction with FcRn can influence not only the levels of fibrinogen in plasma and other tissues, but also the levels of other FcRn binding partners, among which are some plasma proteins of clinical relevance.

Synthesis and Spectroscopic Characterization of 2-(het)Aryl Perimidine Derivatives with Enhanced Fluorescence Quantum Yields.

Perimidines are a particularly versatile family of heterocyclic compounds, whose properties are exploited in several applications ranging from industrial to medicinal chemistry. The molecular structure of perimidine incorporates a well-known efficient fluorophore, i.e.: 1,8-diaminonaphthalene. The high fluorescence quantum yield shared by most naphthalene derivatives, has enabled their use as stains for bio-imaging and biophysical characterizations. However, fluorescence is dramatically depressed in perimidine as well as in the few of its derivatives analysed so far to this respect. The use of perimidine-like molecules in life sciences might be notably fostered by enhancement of their fluorescence emission. Even more excitingly, the concomitance of both biologically active moieties and a fluorophore in the same molecular structure virtually discloses application of perimidines as drug compounds in state-of-art theranostics protocols. However, somewhat surprisingly, relatively few attempts were made until now in the direction of increasing the performances of perimidines as fluorescent dyes. In this work we present the synthesis and spectroscopic characterization of four perimidine derivatives designed to this aim, two of which result to be endowed with fluorescence quantum yields comparable to 1,8-diaminonaphthalene. A rationalization for such improved behaviour has been attempted employing TD-DFT calculations, which have unravelled the interrelations among bond structure, lone pair conjugation, local electron density changes and fluorescence quantum yield.