A circular dichroism study of the protective role of polyphosphoesters polymer chains in polyphosphoester-myoglobin conjugates.

Protein-polymer conjugates are a blooming class of hybrid systems with high biomedical potential. Despite a plethora of papers on their biomedical properties, the physical-chemical characterization of many protein-polymer conjugates is missing. Here, we evaluated the thermal stability of a set of fully-degradable polyphosphoester-protein conjugates by variable temperature circular dichroism, a common but powerful technique. We extensively describe their thermodynamic stability in different environments (in physiological buffer or in presence of chemical denaturants, e.g., acid or urea), highlighting the protective role of the polymer in preserving the protein from denaturation. For the first time, we propose a simple but effective protocol to achieve useful information on these systems in vitro, useful to screen new samples in their early stages.

Glucose-coated superparamagnetic iron oxide nanoparticles prepared by metal vapor synthesis can target GLUT1 overexpressing tumors: In vitro tests and in vivo preliminary assessment.

Superparamagnetic iron oxide nanoparticles (SPIONs) coated with glucose (Glc-SPIONs) were prepared by a new approach called Metal Vapor Synthesis (MVS) and their morphological/structural features were investigated by transmission electron microscopy (TEM) and dynamic light scattering. TEM analysis revealed the presence of small roundish crystalline iron oxide nanoparticles in the organic amorphous phase of glucose, The particles were distributed in a narrow range (1.5 nm-3.5 nm) with a mean diameter of 2.7 nm. The hydrodynamic mean diameter of the Glc-SPIONs, was 15.5 nm. From 4 mg/mL onwards, there was a constant level of positive contrast in a T1-weighted sequence. In vitro experiments were performed in three cell lines: pancreatic cancer (PSN-1), human thyroid cancer (BCPAP), and human embryonic kidney non-tumor cells. We evaluated GLUT1 expression in each cell line and demonstrated that the exposure time and concentration of the Glc-SPIONs we used did not affect cell viability. PSN-1 cells were the most effective at internalizing Glc-SPIONs. Although significantly higher than the control cells, a lower Fe content was detected BCPAP cells treated with Glc-SPIONs. To confirm the involvement of GLUT1 in Glc-SPIONs internalization, cellular uptake experiments were also conducted by pre-treating cancer cells with specific GLUT1 inhibitors, All the inhibitors reduced the cancer cell uptake of Glc-SPIONs In vivo tests were performed on mice inoculated with Lewis lung carcinoma. Mice were treated with a single i.v. injection of Glc-SPION and our results showed a great bioavailability to the malignant tissue by the i.v. administration of Glc-SPIONs. Glc-SPIONs were efficiently eliminated by the kidney. To the best of our knowledge, our study demonstrates for the first time that Glc-SPIONs prepared with MVS can be electively internalized by tumor cells both in vitro and in vivo by exploiting one of the most universal metabolic anomalies of cancer.

Inhomogeneous Diastereomeric Composition of Mongersen Antisense Phosphorothioate Oligonucleotide Preparations and Related Pharmacological Activity Impairment.

Mongersen is a 21-mer antisense oligonucleotide designed to downregulate Mothers against decapentaplegic homolog 7 (SMAD7) expression to treat Crohn's disease. Mongersen was manufactured in numerous batches at different scales during several years of clinical development, which all appeared identical, using common physicochemical analytical techniques, while only phosphorous-31 nuclear magnetic resonance (31P-NMR) in solution showed marked differences. Close-up analysis of 27 mongersen batches revealed marked differences in SMAD7 downregulation in a cell-based assay. Principal component analysis of 31P-NMR profiles showed strong correlation with SMAD7 downregulation and, therefore, with pharmacological efficacy in vitro. Mongersen contains 20 phosphorothioate (PS) linkages, whose chirality (Rp/Sp) was not controlled during manufacturing. A different diastereomeric composition throughout batches would lead to superimposable analytical data, but to distinct 31P-NMR profiles, as indeed we found. We tentatively suggest that this may be the origin of different biological activity. As similar manifolds are expected for other PS-based oligonucleotides, the protocol described here provides a general method to identify PS chirality issues and a chemometric tool to score each preparation for this elusive feature.

From Mesocates to Helicates: Structural, Magnetic and Chiro-Optical Studies on Nickel(II) Supramolecular Assemblies Derived from Tetradentate Schiff Bases.

The systematic reactions of a family of tetradentate pyridyl/imine and quinolyl/imine racemic or enantiopure Schiff bases with Ni(NO3 )2 or Ni(ClO4 )2 in the presence of sodium azide yielded, as a function of the starting racemic, chiral or achiral base, a set of chiral, meso or achiral complexes. In all cases, the compounds consist of two NiII cations linked by a double azido bridge in its end-on coordination mode. All the dimers exhibit a mesocate supramolecular structure and one of them, the unprecedented mix of helicate and mesocate in 2:1 ratio. The transition from mesocate to helicate conformation has been reached by tuning the flexibility of the central spacers of the Schiff bases and the size of the substituents. Electronic circular dichroism (ECD) studies have been performed for two pairs of enantiomers and interpreted by means of DFT calculations. Susceptibility measurements show a ferromagnetic coupling between the NiII cations mediated by the end-on azido bridges.

Polyoxygenated cyathane diterpenoids from the mushroom Cyathus africanus, and their neurotrophic and anti-neuroinflammatory activities.

In a previous study, we reported ten new polyoxygenated cyathane diterpenoids, neocyathins A-J, and their anti-neuroinflammatory effects from the liquid culture of the medicinal Basidiomycete Cyathus africanus. In the present study, eight new highly polyoxygenated cyathane diterpenoids, named neocyathins K-R (1-8), were isolated from the solid culture of C. africanus cultivated on cooked rice, together with three known congeners (9-11). The structures and the absolute configurations of the new compounds were elucidated through comprehensive NMR and HRESIMS spectroscopic data, electronic circular dichroism (ECD) data, and chemical conversion. Compounds 1 and 2 represent the first reported naturally occurring compounds with 4,9-seco-cyathane carbon skeleton incorporating an unprecedented medium-sized 9/7 fused ring system, while the 3,4-seco-cyathane derivative (3) was isolated from Cyathus species for the first time. All compounds were evaluated for their neurotrophic and anti-neuroinflammatory activity. All the isolates at 1-25 µM displayed differential nerve growth factor (NGF)-induced neurite outgrowth-promoting activity in PC-12 cells, while one of the compounds, allocyathin B2 (11), inhibited NO production in lipopolysaccharide (LPS)-stimulated microglia BV-2 cells. In addition, molecular docking studies showed that compound 11 generated interactions with the inducible nitric oxide synthase (iNOS) protein.

The folding of a metallopeptide.

We have applied solid-phase synthesis methods for the construction of tris(bipyridyl) peptidic ligands that coordinate Fe(ii) ions with high affinity and fold into stable mononuclear metallopeptides. The main factors influencing the folding pathway and chiral control of the peptidic ligands around the metal ions have been studied both by experimental techniques (CD, UV-vis and NMR) and molecular modeling tools. Amongst the numerous molecular variables that have been studied, this study clearly illustrates how the chirality of a given set of aminoacids (proline in this case) of the peptide dictates the chirality of the metal center of the resulting metallopeptide. Moreover, the relatively hydrophobic peptidic models used in this work show that the most stable structures present reduced solvent contacts and, in counterpart, stabilize the cis configuration of the proline residues.

Glucose-coated superparamagnetic iron oxide nanoparticles prepared by metal vapour synthesis are electively internalized in a pancreatic adenocarcinoma cell line expressing GLUT1 transporter.

Iron oxide nanoparticles (IONP) can have a variety of biomedical applications due to their visualization properties through Magnetic Resonance Imaging (MRI) and heating with radio frequency or alternating magnetic fields. In the oncological field, coating IONP with organic compounds to provide specific features and to achieve the ability of binding specific molecular targets appears to be very promising. To take advantage of the high avidity of tumor cells for glucose, we report the development of very small glucose-coated IONP (glc-IONP) by employing an innovative technique, Metal Vapor Synthesis (MVS). Moreover, we tested the internalization of our gl-IONP on a tumor line, BxPC3, over-expressing GLUT 1 transporter. Both glc-IONP and polyvinylpyrrolidone-IONP (PVP-IONP), as control, were prepared with MVS and were tested on BxPC3 at various concentrations. To evaluate the role of GLUT-1 transporter, we also investigated the effect of adding a polyclonal anti-GLUT1 antibody. After proper treatment, the iron value was assessed by atomic absorption spectrometer, reported in mcg/L and expressed in mg of protein. Our IONP prepared with MVS were very small and homogeneously distributed in a narrow range (1.75-3.75 nm) with an average size of 2.7 nm and were super-paramagnetic. Glc-IONP were internalized by BxPC3 cells in a larger amount than PVP-IONP. After 6h of treatment with 50 mcg/mL of IONPs, the content of Fe was 1.5 times higher in glc-IONP-treated cells compared with PVP-IONP-treated cells. After 1h pre-treatment with anti-GLUT1, a reduction of 41% cellular accumulation of glc-IONP was observed. Conversely, the uptake of PVP-IONPs was reduced only by 14% with antibody pretreatment. In conclusion, MVS allowed us to prepare small, homogeneous, super-paramagnetic glc-IONP, which are electively internalized by a tumor line over-expressing GLUT1. Our glc-IONP appear to have many requisites for in vivo use.

New potent antibacterials against Gram-positive multiresistant pathogens: effects of side chain modification and chirality in linezolid-like 1,2,4-oxadiazoles.

The effects of side chain modification and chirality in linezolid-like 1,2,4-oxadiazoles have been studied to design new potent antibacterials against Gram-positive multidrug-resistant pathogens. The adopted strategy involved a molecular modelling approach, the synthesis and biological evaluation of new designed compounds, enantiomers separation and absolute configuration assignment. Experimental determination of the antibacterial activity of the designed (S)-1-((3-(4-(3-methyl-1,2,4-oxadiazol-5-yl)phenyl)-oxazolidin-2-one-5-yl)methyl)-3-methylthiourea and (S)-1-((3-(3-fluoro-4-(3-methyl-1,2,4-oxadiazol-5-yl)phenyl)-oxazolidin-2-one-5-yl)methyl)-3-methylthiourea against multidrug resistant linezolid bacterial strains was higher than that of linezolid.

Vibronic coupling dominates the electronic circular dichroism of the benzene chromophore ¹L(b) band.

The alkylbenzene derivatives (R)-PhCH(CH3)(t)Bu (1) and (R)-PhCH(CH3)(i)Pr (2) were taken as paradigms of chiral benzene compounds and their vibronic electronic circular dichroism (ECD) spectrum in the (1)L(b) band region analyzed in detail. The (1)L(b) ECD band of chiral benzene compounds is often used to assign absolute configurations on the basis of sector rules. However, (1)L(b) ECD bands of several benzene derivatives are associated with a forbidden character and show marked vibrational progressions strongly modulating their shape. This is also true for compounds 1 and 2, the latter also showing a peculiar thermochromism. The low-temperature ECD spectrum of 2 displays in fact an alternation of positive and negative ECD maxima. Vibronic ECD calculations performed within a TDDFT scheme allowed a full rationalization of the observed ECD spectra of 1 and 2. Especially in the case of 2, the ECD spectrum in the (1)L(b) band region results from a complex balance of Franck-Condon and Herzberg-Teller effects, as well as of conformational factors. Therefore, straightforward sector rules cannot be safely used to assign the absolute configuration of even these simple derivatives.

Presence of a putative steroidal allosteric site on glycoprotein hormone receptors.

In a previous work we found that the insecticide 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), inhibits the accumulation of cAMP as induced by the bovine thyroid stimulating hormone (bTSH) in cells transfected with the TSH receptor. In this work, we demonstrate that the DDT molecular analogues, diethylstilbestrol and quercetine, are more potent inhibitors of the TSH receptor activity than DDT itself. The notion that all these compounds interfere with nuclear estrogen receptors, as either agonists (DDT and diethylstilbestrol) or antagonists (quercetin), prompted us to test the ability of the steroid hormone 17-beta-estradiol to inhibit the TSH receptor activity. We found that estrogen exposure causes a modest but significant inhibition of the bTSH induced cAMP accumulation both in transfected CHO-TSH receptor and Fischer Rat Thyroid Low Serum 5% (FRTL-5) cells. When applied to CHO cells transfected with the luteinizing hormone receptor, 17-beta-estradiol proved capable of inhibiting the hCG induced cAMP accumulation at a concentration as low as 10nM, though the effect was not greater than 35%. The effect of 17-beta-estradiol was not estrogen receptors mediated, as co-transfection of the estrogen receptor alpha and beta subunits with LH receptor caused cAMP to increase above the level attained by the sole hCG stimulation, and not to decrease it as expected. These data suggest the presence of a steroidal-like allosteric binding site on glycoprotein hormone receptors.

The prediction of the circular dichroism of the benzene chromophore: TDDFT calculations and sector rules.

The CD spectra of the series PhCH(Me)R, with R = Et (1), nPr (2), iPr (3), and tBu (4), are reported (1-3 for the first time) at room temperature in the 185-280 nm range and at 183 K. These purely hydrocarbon compounds represent the simplest chiral systems containing the phenyl chromophore and exhibit Cotton effects exclusively allied with the benzene transitions. The bands in 1La and 1Lb regions were checked against the available sector rules, with discordant outcomes. Time-dependent density-functional theory calculations, with various functionals and basis sets tested, correctly reproduced the prominent CD bands observed for 1-4.

Chiroptical and computational studies of a bridled chiroporphyrin and of its nickel(II), copper(II), and zinc(II) complexes.

Circular dichroism (CD) spectra and density functional theory (DFT) calculations are reported for a series of conformationally bistable chiroporphyrins with 8-methylene bridles MBCP-8, which can display either an alphaalphaalphaalpha or an alphabetaalphabeta orientation of their meso substituents. From DFT geometry optimizations, the most stable form of ZnBCP-8 is found to be the alphaalphaalphaalpha conformer. By passing to NiBCP-8, there is a strong stabilization of the alphabetaalphabeta conformation with respect to the alphaalphaalphaalpha conformation, consistent with the X-ray structures of alphaalphaalphaalpha-ZnBCP-8 and alphabetaalphabeta-NiBCP-8. A correlation between the sign of the CD signal in the Soret region and the conformation of the BCP-8 compounds is reported: the alphaalphaalphaalpha conformers H2BCP-8 and ZnBCP-8 show a positive CD signal, whereas the alphabetaalphabeta conformers NiBCP-8 and CuBCP-8 exhibit a negative signal. The possible contributions to the rotational strengths of alphabetaalphabeta-NiBCP-8 and alphaalphaalphaalpha-ZnBCP-8, calculated on the basis of their crystal structures, have been analyzed. The CD signals are found to result from a combination of both the inherent chirality of the porphyrin and of extrinsic contributions due to the chiral bridles. These results may have a broad significance for understanding the chiroptical properties of chiral porphyrins and hemoproteins and for monitoring stimuli-responsive, conformationally bistable chiroporphyrin compounds.