NMR and Docking Calculations Reveal Novel Atomistic Selectivity of a Synthetic High-Affinity Free Fatty Acid vs. Free Fatty Acids in Sudlow's Drug Binding Sites in Human Serum Albumin.

Saturation transfer difference (STD), inter-ligand NOEs (INPHARMA NMR), and docking calculations are reported for investigating specific binding sites of the high-affinity synthetic 7-nitrobenz-2-oxa-1,3-diazoyl-4-C12 fatty acid (NBD-C12 FA) with non-labeled human serum albumin (HSA) and in competition with the drugs warfarin and ibuprofen. A limited number of negative interligand NOEs between NBD-C12 FA and warfarin were interpreted in terms of a short-range allosteric competitive binding in the wide Sudlow's binding site II (FA7) of NBD-C12 FA with Ser-202, Lys-199, and Trp-214 and warfarin with Arg-218 and Arg-222. In contrast, the significant number of interligand NOEs between NBD-C12 FA and ibuprofen were interpreted in terms of a competitive binding mode in Sudlow's binding site I (FA3 and FA4) with Ser-342, Arg-348, Arg-485, Arg-410, and Tyr-411. NBD-C12 FA has the unique structural properties, compared to short-, medium-, and long-chain saturated and unsaturated natural free fatty acids, of interacting with well-defined structures with amino acids of both the internal and external polar anchor sites in Sudlow's binding site I and with amino acids in both FA3 and FA4 in Sudlow's binding site II. The NBD-C12 FA, therefore, interacts with novel structural characteristics in the drug binding sites I and II and can be regarded as a prototype molecule for drug development.

Ultrasound-Assisted Extraction of Taro Leaf Antioxidants Using Natural Deep Eutectic Solvents: An Eco-Friendly Strategy for the Valorization of Crop Residues.

Colocasia esculenta L. leaves are considered a by-product of taro cultivation and are discarded as environmental waste, despite their valuable phenolic composition. Their valorization to obtain value-added substances for medicinal, food, and cosmetic applications is the aim of the current work. An ultrasound-assisted extraction was developed for the environmentally friendly and sustainable isolation of taro leaf antioxidants using natural deep eutectic solvents (NaDESs). Among the utilized solvents, the NaDES based on betaine and ethylene glycol provided the best extraction efficiencies in terms of polyphenolic content and antioxidant activity. Multi-response optimization suggested a solvent-to-solid ratio of 10 mL g-1, a processing time of 60 min, an extraction temperature of 60 °C, and a water content of 33.8% (w/w) as optimal extraction parameters. Leaf extract obtained under these optimum operational parameters demonstrated a strong radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (65.80 ± 0.87%), a high ferric reducing antioxidant power (126.62 ± 1.92 µmol TE g-1 sample), and significant protection against oxidative stress-induced DNA damage. The chromatographic characterization of the optimum extract revealed its richness in flavonoids (flavones and flavonols). The outcomes of the present study suggest that the proposed method could serve as a highly efficient and green alternative for the recovery of polyphenols from agricultural wastes.

Structural Studies of Monounsaturated and ω-3 Polyunsaturated Free Fatty Acids in Solution with the Combined Use οf NMR and DFT Calculations-Comparison with the Liquid State.

Molecular structures, in chloroform and DMSO solution, of the free fatty acids (FFAs) caproleic acid, oleic acid, α-linolenic acid, eicosapentanoic acid (EPA) and docosahexaenoic acid (DHA) are reported with the combined use of NMR and DFT calculations. Variable temperature and concentration chemical shifts of the COOH protons, transient 1D NOE experiments and DFT calculations demonstrate the major contribution of low molecular weight aggregates of dimerized fatty acids through intermolecular hydrogen bond interactions of the carboxylic groups, with parallel and antiparallel interdigitated structures even at the low concentration of 20 mM in CDCl3. For the dimeric DHA, a structural model of an intermolecular hydrogen bond through carboxylic groups and an intermolecular hydrogen bond between the carboxylic group of one molecule and the ω-3 double bond of a second molecule is shown to play a role. In DMSO-d6 solution, NMR and DFT studies show that the carboxylic groups form strong intermolecular hydrogen bond interactions with a single discrete solvation molecule of DMSO. These solvation species form parallel and antiparallel interdigitated structures of low molecular weight, as in chloroform solution. This structural motif, therefore, is an intrinsic property of the FFAs, which is not strongly affected by the length and degree of unsaturation of the chain and the hydrogen bond ability of the solvent.

Molecular Basis for the Selectivity of DHA and EPA in Sudlow's Drug Binding Sites in Human Serum Albumin with the Combined Use of NMR and Docking Calculations.

Medium- and long-chain saturated and unsaturated free fatty acids (FFAs) are known to bind to human serum albumin (HSA), the main plasma carrier protein. Atomic-level structural data regarding the binding mode in Sudlow's sites I (FA7) and II (FA4, FA3) of the polyunsaturated ω-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), however, are largely unknown. Herein, we report the combined use of saturation transfer difference (STD) and Interligand NOEs for Pharmacophore Mapping (INPHARMA) NMR techniques and molecular docking calculations to investigate the binding mode of DHA and EPA in Sudlow's sites Ι and ΙΙ of HSA. The docking calculations and the significant number of interligand NOEs between DHA and EPA and the drugs warfarin and ibuprofen, which are stereotypical ligands for Sudlow's sites I and II, respectively, were interpreted in terms of competitive binding modes and the presence of two orientations of DHA and EPA at the binding sites FA7 and FA4. The exceptional flexibility of the long-chain DHA and EPA and the formation of strongly folded structural motives are the key properties of HSA-PUFA complexes.

ANTIAGE-DB: A Database and Server for the Prediction of Anti-Aging Compounds Targeting Elastase, Hyaluronidase, and Tyrosinase.

Natural products bear a multivariate biochemical profile with antioxidant, anti-inflammatory, antibacterial, and antitumoral properties. Along with their natural sources, they have been widely used both as anti-aging and anti-melanogenic agents due to their effective contribution in the elimination of reactive oxygen species (ROS) caused by oxidative stress. Their anti-aging activity is mainly related to their capacity of inhibiting enzymes like Human Neutrophil Elastase (HNE), Hyaluronidase (Hyal) and Tyrosinase (Tyr). Herein, we accumulated literature information (covering the period 1965-2020) on the inhibitory activity of natural products and their natural sources towards these enzymes. To navigate this information, we developed a database and server termed ANTIAGE-DB that allows the prediction of the anti-aging potential of target compounds. The server operates in two axes. First a comparison of compounds by shape similarity can be performed against our curated database of natural products whose inhibitory potential has been established in the literature. In addition, inverse virtual screening can be performed for a chosen molecule against the three targeted enzymes. The server is open access, and a detailed report with the prediction results is emailed to the user. ANTIAGE-DB could enable researchers to explore the chemical space of natural based products, but is not limited to, as anti-aging compounds and can predict their anti-aging potential. ANTIAGE-DB is accessed online.

Structural Basis of Artemisinin Binding Sites in Serum Albumin with the Combined Use of NMR and Docking Calculations.

Artemisinin is known to bind to the main plasma protein carrier serum albumin (SA); however, there are no atomic level structural data regarding its binding mode with serum albumin. Herein, we employed a combined strategy of saturation transfer difference (STD), transfer nuclear Overhauser effect spectroscopy (TR-NOESY), STD-total correlation spectroscopy (STD-TOCSY), and Interligand Noes for PHArmacophore Mapping (INPHARMA) NMR methods and molecular docking calculations to investigate the structural basis of the interaction of artemisinin with human and bovine serum albumin (HSA/BSA). A significant number of inter-ligand NOEs between artemisinin and the drugs warfarin and ibuprofen as well as docking calculations were interpreted in terms of competitive binding modes of artemisinin in the warfarin (FA7) and ibuprofen (FA4) binding sites. STD NMR experiments demonstrate that artemisinin is the main analyte for the interaction of the A. annua extract with BSA. The combined strategy of NMR and docking calculations of the present work could be of general interest in the identification of the molecular basis of the interactions of natural products with their receptors even within a complex crude extract.

Density functional theory calculations of δ(13 C) and δ(1 H) chemical shifts and 3 J(13 COO1 H) coupling constants as structural and analytical tools in hydroperoxides: Prospects and limitations of 1 H13 C heteronuclear multiple bond correlation experiments.

Density functional theory (DFT) calculations of δ(13 C) and δ(1 H) chemical shifts and 3 J(13 COO1 H) coupling constants of three model hydroperoxides of the naturally occurring cis-11-OOH and trans-9-OOH isomers of oleate and 9-cis, 11-trans-16-OOH endo hydroperoxide of methyl linolenate are reported. The computational δ(OOH) for various functionals and basis sets were found to be nearly identical for the cis/trans geometric isomers. The chemical shifts of the methine CHOOH protons and carbons, on the contrary, are highly diagnostic for the identification of cis/trans geometric isomerism. The chemical shifts of the olefinic protons and carbons strongly depend on the orientation of the hydroperoxide unit relative to the double bond and, thus, of importance in conformational analysis. The results are in very good agreement with the available experimental data. For the various diastereomeric pairs of the model endo-hydroperoxide, the strongly deshielded OOH resonances, due to the presence of an intramolecular hydrogen bond between the hydroperoxide proton and an oxygen of the endo-peroxide ring, along with the δ(CHOOH), are highly diagnostic for identification and structure elucidation of complex erythro- and threo- diastereomeric pairs of endo-hydroperoxides; the computational results are in very good agreement with the available experimental data. The 3 J(13 COO1 H) coupling constants were found to be < 2  Hz for the cis-trans geometric models and < 0.5  Hz for the endo-hydroperoxide and, thus, unimportant in stereochemical analysis. Sharp resonances of the hydroperoxide protons, with Δν1/2 < 3 Hz, are required for the successful implementation of the 1 H13 C heteronuclear multiple bond correlation (HMBC) technique.

NMR and computational studies reveal novel aspects in molecular recognition of unsaturated fatty acids with non-labelled serum albumin.

An approach based on the combined use of saturation transfer difference (STD), Tr-NOESY and Inter-ligand NOEs for PHArmacophore Mapping (INPHARMA) NMR techniques and docking calculations is reported, for the first time, for mapping interactions and specific binding sites of caproleic acid (10 : 1 cis-9), oleic acid (18 : 1 cis-9), linoleic acid (18 : 2 cis-9,12) and linolenic (18 : 3, cis-9,12,15) free fatty acids (FFAs) with non-labelled serum albumin (BSA/HSA). Significant negative inter-ligand NOEs between the FFAs and the drugs ibuprofen and warfarin, through competition experiments, were observed. The inter-ligand NOEs and docking calculations were interpreted in terms of competitive binding mode, the significant folding of the bis allylic region and the presence of two orientations of the FFAs in the warfarin binding site (FA7), due to two potential distinctive anchoring polar groups of amino acids. This conformational flexibility is the reason that, the location and conformational states of the FFAs in the binding site of warfarin could not be determined accurately, despite numerous available X-ray structural studies. α-Linolenic acid competes favourably with warfarin at the binding site FA7. Isothermal titration calorimetry experiments of the preformed HSA/α-linolenic acid complex upon titration with warfarin show a significant reduction in the binding constant of warfarin, in very good agreement with NMR and computational data. The combined use, therefore, of STD, Tr-NOESY and INPHARMA NMR, ITC and docking calculations may find promising applications in the field of protein-lipid recognition research.

Analytical and Structural Tools of Lipid Hydroperoxides: Present State and Future Perspectives.

Mono- and polyunsaturated lipids are particularly susceptible to peroxidation, which results in the formation of lipid hydroperoxides (LOOHs) as primary nonradical-reaction products. LOOHs may undergo degradation to various products that have been implicated in vital biological reactions, and thus in the pathogenesis of various diseases. The structure elucidation and qualitative and quantitative analysis of lipid hydroperoxides are therefore of great importance. The objectives of the present review are to provide a critical analysis of various methods that have been widely applied, and more specifically on volumetric methods, applications of UV-visible, infrared, Raman/surface-enhanced Raman, fluorescence and chemiluminescence spectroscopies, chromatographic methods, hyphenated MS techniques, NMR and chromatographic methods, NMR spectroscopy in mixture analysis, structural investigations based on quantum chemical calculations of NMR parameters, applications in living cells, and metabolomics. Emphasis will be given to analytical and structural methods that can contribute significantly to the molecular basis of the chemical process involved in the formation of lipid hydroperoxides without the need for the isolation of the individual components. Furthermore, future developments in the field will be discussed.

Serum albumin as a primary non-covalent binding protein for nitro-oleic acid.

This work explores the interaction of 9/10-nitro-oleic acid (NO2-OA) with human serum albumin (HSA). The molecular mechanism of the biological action of NO2-OA is to our knowledge based on a reversible covalent reaction-Michael addition of nucleophilic amino acid residues of proteins. Since HSA is an important fatty acid transporter, a key question is whether NO2-OA can bind covalently or non-covalently to HSA, similarly to oleic acid (OA), which can interact with the FA1-FA7 binding sites of the HSA molecule. 1H NMR studies and competition analysis with OA and the drugs ibuprofen and warfarin were used to investigate a potential non-covalent binding mode. NO2-OA/HSA binding was confirmed to compete with warfarin for FA-7 with significantly higher affinity. NO2-OA competes with ibuprofen for FA-3 and FA-6, however, in contrast to the situation with warfarin, the binding affinities are not significantly different. The described interactions are based exclusively on non-covalent binding. No covalent binding of NO2-OA to HSA was detected by MS/MS. More detailed studies based on MALDI-TOF-MS and Ellman's assay indicated that HSA can be covalently modified in the presence of NO2-OA to a very limited extent. It was also shown that NO2-OA has a higher affinity to HSA than that of OA.

Characterization of Protocatechuate 4,5-Dioxygenase from Pseudarthrobacter phenanthrenivorans Sphe3 and In Situ Reaction Monitoring in the NMR Tube.

The current study aims at the functional and kinetic characterization of protocatechuate (PCA) 4,5-dioxygenase (PcaA) from Pseudarthrobacter phenanthrenivorans Sphe3. This is the first single subunit Type II dioxygenase characterized in Actinobacteria. RT-PCR analysis demonstrated that pcaA and the adjacent putative genes implicated in the PCA meta-cleavage pathway comprise a single transcriptional unit. The recombinant PcaA is highly specific for PCA and exhibits Michaelis-Menten kinetics with Km and Vmax values of 21 ± 1.6 µM and 44.8 ± 4.0 U × mg-1, respectively, in pH 9.5 and at 20 °C. PcaA also converted gallate from a broad range of substrates tested. The enzymatic reaction products were identified and characterized, for the first time, through in situ biotransformation monitoring inside an NMR tube. The PCA reaction product demonstrated a keto-enol tautomerization, whereas the gallate reaction product was present only in the keto form. Moreover, the transcriptional levels of pcaA and pcaR (gene encoding a LysR-type regulator of the pathway) were also determined, showing an induction when cells were grown on PCA and phenanthrene. Studying key enzymes in biodegradation pathways is significant for bioremediation and for efficient biocatalysts development.

DFT Calculations of 1H NMR Chemical Shifts of Geometric Isomers of Conjugated Linolenic Acids, Hexadecatrienyl Pheromones, and Model Triene-Containing Compounds: Structures in Solution and Revision of NMR Assignments.

A DFT study of the 1H NMR chemical shifts, δ(1H), of geometric isomers of 18:3 conjugated linolenic acids (CLnAs), hexadecatrienyl pheromones, and model triene-containing compounds is presented, using standard functionals (B3LYP and PBE0) as well as corrections for dispersion interactions (B3LYP-D3, APFD, M06-2X and ωB97XD). The results are compared with literature experimental δ(1H) data in solution. The closely spaced "inside" olefinic protons are significantly more deshielded due to short-range through-space H…H steric interactions and appear close to or even beyond δ-values of aromatic systems. Several regularities of the computational δ(1H) of the olefinic protons of the conjugated double bonds are reproduced very accurately for the lowest-energy DFT-optimized single conformer for all functionals used and are in very good agreement with experimental δ(1H) in solution. Examples are provided of literature studies in which experimental resonance assignments deviate significantly from DFT predictions and, thus, should be revised. We conclude that DFT calculations of 1H chemical shifts of trienyl compounds are powerful tools (i) for the accurate prediction of δ(1H) even with less demanding functionals and basis sets; (ii) for the unequivocal identification of geometric isomerism of conjugated trienyl systems that occur in nature; (iii) for tackling complex problems of experimental resonance assignments due to extensive signal overlap; and (iv) for structure elucidation in solution.

On the molecular basis of H2O/DMSO eutectic mixtures by using phenol compounds as molecular sensors: a combined NMR and DFT study.

NMR and DFT studies of phenol compounds as molecular sensors were carried out to investigate H2O/DMSO eutectic mixtures at a molecular level. The experimental 1H NMR chemical shifts of the OH groups, δexp(OH), of phenol, paracoumaric acid, and vanillic acid show maximum deshielding and, thus, hydrogen bond interactions in the range of mole fractions 0.20 < χ(DMSO) < 0.33. In the mole fractions χ(DMSO) < 0.2, a progressive decrease in δexp(OH) was observed which demonstrates a decrease in hydrogen bond interactions at infinite dilution in H2O, despite the increase in the number of available hydrogen bond acceptor and donor sites. DFT calculated δcalc(OH) of minimum energy solvation clusters were shown to be in reasonable agreement with the pattern in experimental δexp(OH) data. The chemical shift deshielding and, thus, increased hydrogen bond interactions in the natural product + DMSO + nH2O (n = 2, 3) solvation clusters, relative to complexes in DMSO or H2O solutions, cannot be attributed to a single structural parameter of the cooperative interactions between H2O and DMSO molecules with the phenol OH groups of the natural products. The minimum energy conformers of phenol compounds + 2H2O + DMSO complexes are in excellent agreement with a recent low temperature neutron diffraction experiment of 3D2O + DMSO and demonstrate a general structural motif of solvation complexes. The combined use of 1H NMR and DFT studies with emphasis on δ(OH) of phenol compounds, as molecular sensors, can provide an effective method for the study of solute-solvent interactions at the atomic level.

NMR and Computational Studies as Analytical and High-Resolution Structural Tool for Complex Hydroperoxides and Diastereomeric Endo-Hydroperoxides of Fatty Acids in Solution-Exemplified by Methyl Linolenate.

A combination of selective 1D Total Correlation Spectroscopy (TOCSY) and 1H-13C Heteronuclear Multiple Bond Correlation (HMBC) NMR techniques has been employed for the identification of methyl linolenate primary oxidation products without the need for laborious isolation of the individual compounds. Complex hydroperoxides and diastereomeric endo-hydroperoxides were identified and quantified. Strongly deshielded C-O-O-H 1H-NMR resonances of diastereomeric endo-hydroperoxides in the region of 8.8 to 9.6 ppm were shown to be due to intramolecular hydrogen bonding interactions of the hydroperoxide proton with an oxygen atom of the five-member endo-peroxide ring. These strongly deshielded resonances were utilized as a new method to derive, for the first time, three-dimensional structures with an assignment of pairs of diastereomers in solution with the combined use of 1H-NMR chemical shifts, Density Functional Theory (DFT), and Our N-layered Integrated molecular Orbital and molecular Mechanics (ONIOM) calculations.

DFT Calculations of 1H- and 13C-NMR Chemical Shifts of Geometric Isomers of Conjugated Linoleic Acid (18:2 ω-7) and Model Compounds in Solution.

A density functional theory (DFT) study of the 1H- and 13C-NMR chemical shifts of the geometric isomers of 18:2 ω-7 conjugated linoleic acid (CLA) and nine model compounds is presented, using five functionals and two basis sets. The results are compared with available experimental data from solution high resolution nuclear magnetic resonance (NMR). The experimental 1H chemical shifts exhibit highly diagnostic resonances due to the olefinic protons of the conjugated double bonds. The "inside" olefinic protons of the conjugated double bonds are deshielded than those of the "outside" protons. Furthermore, in the cis/trans isomers, the signals of the cis bonds are more deshielded than those of the trans bonds. These regularities of the experimental 1H chemical shifts of the olefinic protons of the conjugated double bonds are reproduced very accurately for the lowest energy DFT optimized single conformer, for all functionals and basis sets used. The other low energy conformers have negligible effects on the computational 1H-NMR chemical shifts. We conclude that proton NMR chemical shifts are more discriminating than carbon, and DFT calculations can provide a valuable tool for (i) the accurate prediction of 1H-NMR chemical shifts even with less demanding functionals and basis sets; (ii) the unequivocal identification of geometric isomerism of CLAs that occur in nature, and (iii) to derive high resolution structures in solution.

The NMR tube bioreactor.

Enzymes are pliable systems and core cellular components allowing the performance of several processes. They can also be utilized as "green" synthetic factories to generate bioactive therapeutic, diagnostic or theranostic compounds. Methods to enable the mapping of enzyme substrates as well as the understanding of the interactions of the formed products with target proteins could be of importance. This chapter describes the utilization of the "NMR tube bioreactor" method. This method, carried out inside an NMR tube, can allow for the prediction of compounds that are able to serve as potential enzyme substrates, and also exploit the regioselectivity of the enzymatic reactions. Furthermore, it enables the real time monitoring of multiple-biotransformation products in the NMR tube without the need of fractionation or isolation of each individual component. Finally, it allows for the screening of the resulting biotransformation products as ligands for protein targets.

Simultaneous determination of artemisinin and its analogs and flavonoids in Artemisia annua crude extracts with the use of NMR spectroscopy.

Artemisia annua is a promising and potent antimalarial herbal drug. This activity has been ascribed to its component artemisinin, a sesquiterpene lactone. The ability to determine artemisinin and its known analogs in plant extracts is an especially difficult task because the compounds are present in low concentrations, are thermolabile, and lack ultraviolet or fluorescent chromophores. We report herein a facile and rapid 1-D 1 H, 1-D total correlation spectroscopy, 2-D 1 H-13 C heteronuclear single quantum coherence, and 1 H-13 C heteronuclear multiple bond correlation nuclear magnetic resonance techniques for the simultaneous identification and quantification of artemisinin and five of its analogs along with five flavonoids, an aromatic ketone, and camphor (in total, 13 compounds) in crude diethyl ether A. annua extract without the need of laborious isolation of the individual analytes. The above method was validated in terms of precision, linearity, and limit of detection. The analytical results were found to be in excellent agreement with those obtained with the use of the time consuming high-performance liquid chromatography with diode-array detection and liquid chromatography with tandem mass spectrometry for the compounds that standards were available.