Reactivity of amino acids and short peptide sequences: identifying bioactive compounds via DFT calculations.

Bioactive peptides are short amino acid sequences that play important roles in various physiological processes, including antioxidant and protective effects. These compounds can be obtained through protein hydrolysis and have a wide range of potential applications in a variety of areas. However, despite the potential of these compounds, more in-depth knowledge is still necessary to better understand details regarding their chemical reactivity and electronic properties. In this study, we used molecular modeling techniques to investigate the electronic structure of isolated amino acids (AA) and short peptide sequences. Details on the relative alignments between the frontier electronic levels, local chemical reactivity and donor-acceptor properties of the 20 primary amino acids and some di- and tripeptides were evaluated in the framework of the density functional theory (DFT). Our results suggest that the electronic properties of isolated amino acids can be used to interpret the reactivity of short sequences. We found that aromatic and charged amino acids, as well as Methionine, play a key role in determining the local reactivity of peptides, in agreement with experimental data. Our analyses also allowed us to identify the influence of the relative position of AA and terminations on the local reactivity of the sequences, which can guide experimental studies and help to propose/evaluate possible mechanisms of action. In summary, our data indicate that the position of active sites of polypeptides can be predicted from short sequences, providing a promising strategy for the synthesis and bioprospection of new optimized compounds.

The Antioxidant Peptide Salamandrin-I: First Bioactive Peptide Identified from Skin Secretion of Salamandra Genus (Salamandra salamandra).

Amphibian skin is a multifunctional organ that plays key roles in defense, breathing, and water balance. In this study, skin secretion samples of the fire salamander (Salamandra salamandra) were separated using RP-HPLC and de novo sequenced using MALDI-TOF MS/MS. Next, we used an in silico platform to screen antioxidant molecules in the framework of density functional theory. One of the identified peptides, salamandrin-I, [M + H]+ = 1406.6 Da, was selected for solid-phase synthesis; it showed free radical scavenging activity against DPPH and ABTS radicals. Salamandrin-I did not show antimicrobial activity against Gram-positive and -negative bacteria. In vitro assays using human microglia and red blood cells showed that salamandrin-I has no cytotoxicity up to the concentration of 100 µM. In addition, in vivo toxicity tests on Galleria mellonella larvae resulted in no mortality at 20 and 40 mg/kg. Antioxidant peptides derived from natural sources are increasingly attracting interest. Among several applications, these peptides, such as salamandrin-I, can be used as templates in the design of novel antioxidant molecules that may contribute to devising strategies for more effective control of neurological disease.

A combined Far-FTIR, FTIR Spectromicroscopy, and DFT Study of the Effect of DNA Binding on the [4Fe4S] Cluster Site in EndoIII.

Endonuclease III (EndoIII) is a DNA glycosylase that contains the [4Fe4S] cluster, which is essential for the protein to bind to damaged DNA in a process called base excision repair (BER). Here we propose that the change in the covalency of Fe-S bonds of the [4Fe4S] cluster caused by double-stranded (ds)-DNA binding is accompanied by a change in their strength, which is due to alterations of the electronic structure of the cluster. Micro-FTIR spectroscopy in the mid-IR region and FTIR spectroscopy in the far IR (450 and 300 cm-1) were used independently to study the structural changes in EndoIII and the behavior of the [4Fe4S] cluster it contains, in the native form and upon its binding to ds-DNA. Structural changes in the DNA itself were also examined. The characteristics vibrational modes, corresponding to Fe-S (sulfide) and Fe-S (thiolate) bonds were identified in the cluster through far IR spectroscopy as well through quantum chemistry calculations. Based on the experimental results, these vibrational modes shift in their spectral positions caused by negatively charged DNA in the vicinity of the cluster. Modifications of the Fe-S bond lengths upon DNA binding, both of the Fe-S (sulfide) and Fe-S (thiolate) bonds in the [4Fe4S] cluster of EndoIII are responsible for the stabilization of the cluster towards higher oxidation state (3+), and hence its redox communication along the ds-DNA helix.

Interplay of non-uniform charge distribution on the electrochemical modification of graphene.

Graphene is considered a model material for surfaces because it is stable despite being composed of a single layer of carbon atoms. Although the thermal and electronic properties of graphene are well reported, the behavior of graphene sheets with the addition of charges to the structure is not well understood. Combining infrared spectroscopy, electrochemical analysis, and computational simulations, we report the effect of an electrochemically induced covalent anchoring of 4-carboxyphenyl (4-CP) units on the optical and electronic properties of graphene. Charges in graphene become concentrated at specific sites of the sheet when electrochemically perturbed and the functionalization occurs inhomogeneously along the graphene sheet. We observed that, when graphene is covalently functionalized, the resistance to heterogeneous electron transfer is increased by a factor of 1.4. Furthermore, scattering-type scanning near-field optical microscopy and atomic force microscopy show that the covalent functionalization affects drastically the optical and physical properties of the graphene/SiO2 system, especially the plasmon-phonon coupling after the functionalization. In addition, from these we infer that a comparatively higher degree of functionalization occurs near the electrode edges. These results are supported by computational simulations, which show that the covalent anchoring of 4-CP units weakens electron transfer because the charges are retained on the sp3-hybridized carbon atoms generated upon functionalization, suggesting that graphene properties are deeply influenced by the way the molecules are immobilized on its structure.

Lycopene-rich extract from red guava (Psidium guajava L.) displays cytotoxic effect against human breast adenocarcinoma cell line MCF-7 via an apoptotic-like pathway.

This study investigated a lycopene-rich extract from red guava (LEG) for its chemical composition using spectrophotometry, mass spectrometry, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), and computational studies. The cytotoxic activity of LEG and the underlying mechanism was studied in human breast adenocarcinoma cells (MCF-7), murine fibroblast cells (NIH-3T3), BALB/c murine peritoneal macrophages, and sheep blood erythrocytes by evaluating the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and flow cytometry. Spectrophotometry analysis showed that LEG contained 20% of lycopene per extract dry weight. Experimental and theoretical ATR-FTIR suggests the presence of lycopene, whereas MS/MS spectra obtained after fragmentation of the molecular ion [M]+• of 536.4364 show fragment ions at m/z 269.2259, 375.3034, 444.3788, and 467.3658, corroborating the presence of lycopene mostly related to all-trans configuration. Treatment with LEG (1600 to 6.25µg/mL) for 24 and 72h significantly affected the viability of MCF-7 cells (mean half maximal inhibitory concentration [IC50]=29.85 and 5.964µg/mL, respectively) but not NIH-3T3 cells (IC50=1579 and 911.5µg/mL, respectively). Furthermore LEG at concentrations from 800 to 6.25µg/mL presented low cytotoxicity against BALB/c peritoneal macrophages (IC50≥800µg/mL) and no hemolytic activity. LEG (400 and 800µg/mL) caused reduction in the cell proliferation and induced cell cycle arrest, DNA fragmentation, modifications in the mitochondrial membrane potential, and morphologic changes related to granularity and size in MCF-7 cells; however, it failed to cause any significant damage to the cell membrane or display necrosis or traditional apoptosis. In conclusion, LEG was able to induce cytostatic and cytotoxic effects on breast cancer cells probably via induction of an apoptotic-like pathway.

Metal coordination study at Ag and Cd sites in crown thioether complexes through DFT calculations and hyperfine parameters.

Structural and electronic properties of [C12H24S6X], [C13H26S6OX], and [C14H28S6OX] (X: Ag(+), Cd(2+)) crown thioether complexes were investigated within the framework of the density functional theory (DFT) using the projector augmented wave (PAW) method. The theoretical results were compared with time-differential perturbed γ-γ angular correlations (TDPAC) experiments reported in the literature using the (111)Ag→(111)Cd probe. In the case of X=Ag(+), a refinement of the structure was performed and the predicted equilibrium structures compared with available X-ray diffraction experimental data. Structural distortions induced by replacing Ag(+) with Cd(2+) were investigated as well as the electric-field gradient (EFG) tensor at the Cd(2+) sites. Our results suggest that the EFG at Cd(2+) sites corresponds to the Ag(+) coordination sphere structure, i.e., before the structural relaxations of the molecule with X=Cd(2+) are completed. The results are discussed in terms of the characteristics of the TDPAC (111)Ag→(111)Cd probe and the time window of the measurement, and provide an interesting tool with which to probe molecular relaxations.

Industrial Scale Isolation, Structural and Spectroscopic Characterization of Epiisopiloturine from Pilocarpus microphyllus Stapf Leaves: A Promising Alkaloid against Schistosomiasis.

This paper presents an industrial scale process for extraction, purification, and isolation of epiisopiloturine (EPI) (2(3H)-Furanone,dihydro-3-(hydroxyphenylmethyl)-4-[(1-methyl-1H-imidazol-4-yl)methyl]-, [3S-[3a(R*),4b]]), which is an alkaloid from jaborandi leaves (Pilocarpus microphyllus Stapf). Additionally for the first time a set of structural and spectroscopic techniques were used to characterize this alkaloid. EPI has shown schistomicidal activity against adults and young forms, as well as the reduction of the egg laying adult worms and low toxicity to mammalian cells (in vitro). At first, the extraction of EPI was done with toluene and methylene chloride to obtain a solution that was alkalinized with ammonium carbonate. The remaining solution was treated in sequence by acidification, filtration and alkalinization. These industrial procedures are necessary in order to remove impurities and subsequent application of the high performance liquid chromatography (HPLC). The HPLC was employed also to remove other alkaloids, to obtain EPI purity higher than 98%. The viability of the method was confirmed through HPLC and electrospray mass spectrometry, that yielded a pseudo molecular ion of m/z equal to 287.1 Da. EPI structure was characterized by single crystal X-ray diffraction (XRD), (1)H and (13)C nuclear magnetic resonance (NMR) in deuterated methanol/chloroform solution, vibrational spectroscopy and mass coupled thermal analyses. EPI molecule presents a parallel alignment of the benzene and the methyl imidazol ring separated by an interplanar spacing of 3.758 Å indicating a π-π bond interaction. The imidazole alkaloid melts at 225°C and decomposes above 230°C under air. EPI structure was used in theoretical Density Functional Theory calculations, considering the single crystal XRD data in order to simulate the NMR, infrared and Raman spectra of the molecule, and performs the signals attribution.