NMR Chemical Shifts Research Articles

Synthesis, crystal growth, structural and physicochemical properties of N-methylurea benzoic acid single crystal for non-linear optical applications

The organic single crystal of N-methylurea benzoic acid (NMUB) was synthesized and successfully grown from a slow evaporation solution growth technique for the first time in literature. The structure of the NMUB single crystal was analyzed by Single crystal X-ray diffraction (SCXRD); the crystallization of the NMUB crystal was a monoclinic crystal system with the space group P21/c. The crystal structure was combined by the carboxylic groups of H- atoms in benzoic acid with protonated N-methylurea anions. The FTIR and 1H, 13C NMR chemical shifts were analyzed by distinct vibrational groups and molecular structures. The PXRD ascertains the grown crystal crystallographic planes and crystalline nature. The Hirshfeld surfaces analysis determines the theoretical inter and intra-molecular interaction with the aid of color coding. The UV-Vis NIR spectra revealed the low absorption edge, and the optical band gap was anticipated. The crystal thermal and mechanical stability was analyzed by TG/DTA, Vickers microhardness, and void volume. The laser utility limitation for the crystal was determined through laser beam irradiated LDT analysis. The third-order optical non-linear parameters were determined from the z-scan technique.

Backbone and side chain chemical shift assignment of diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris, an organophosphorus-degrading enzyme.

NMR chemical shift assignments are reported for backbone (15N, 1H) and partial side chain (13Cα and β, side chain 1H) atoms of diisopropyl fluorophosphatase (DFPase), a calcium-dependent phosphotriesterase capable of hydrolyzing phosphorus - fluorine bonds in a variety of toxic organophosphorus compounds. Analysis of residues lining the active site of DFPase highlight a number of residues whose chemical shifts can be used as a diagnostic of binding and detection of organophosphorus compounds.

Exploring the influence of free radicals on photolytic removal of nadolol from water: Mechanism of degradation and toxicity of intermediates

β-blockers are known to have negative effects on fish and other aquatic animal species, so their removal is key for preserving aquatic ecosystems. To reduce the risks related to β-blockers, it is necessary to assess their effects and develop more effective treatments such as advanced oxidation processes. Improving sewage treatments is a critical approach to reducing β-blockers in aquatic environments. In this work, for the first time, the direct and indirect photolysis of nadolol (NAD) was investigated under different light sources (simulated solar (SS), UV-LED, and UV radiations) in ultrapure water. Indirect photolysis by H2O2 showed 1.5, 2.1, and 5.6 times higher NAD degradation efficiency than direct photolysis under mentioned irradiations. This effect was particularly pronounced in the presence of UV radiation, in which the degradation efficiency of NAD was the highest (80.2%). Computational analysis based on density functional theory calculations, together with the results of NAD photodegradation efficiency in the presence of radical scavengers (isopropanol and benzoquinone), was used to propose the NAD degradation mechanism. Sixteen degradation intermediates were proposed, along with their NMR chemical shifts. Also, this study analyzed the degree of catalase activity, lipid peroxidation, and hydroxyl radicals neutralization of NAD and its photodegradation mixtures obtained after indirect photolysis. The degree of mineralization and in vitro toxicity of NAD and its degradation intermediates obtained in the presence of UV/H2O2 were assessed.

Dynamic Sorting of Mobile and Rigid Molecules in Biomembranes by Magic-Angle Spinning 13C NMR

Understanding the membrane dynamics of complex systems is essential to follow their function. As molecules in membranes can be in a rigid or mobile state depending on external (temperature, pressure) or internal (pH, domains, etc.) conditions, we propose an in-depth examination of NMR methods to filter highly mobile molecular parts from others that are in more restricted environments. We have thus developed a quantitative magic-angle spinning (MAS) 13C NMR approach coupled with cross-polarization (CP) and/or Insensitive Nuclei Enhanced by Polarization Transfer (INEPT) on rigid and fluid unlabeled model membranes. We demonstrate that INEPT can detect only very mobile lipid headgroups in gel (solid-ordered) phases; the remaining rigid parts are only detected with CP. A direct correlation is established between the normalized line intensity as obtained by CP and the C-H (C-D) order parameters measured by wide-line 2H NMR or extracted from molecular dynamics: ICP/IDPeq ≈ 5|SCH|, indicating that when the order is greater than 0.2-0.3 (maximum value of 0.5 for chain CH2), only rigid parts can be filtered and detected using CP techniques. In very fluid (liquid-disordered) membranes, where there are many more active motions, both INEPT and CP detect resonances, with, however, a clear propensity of each technique to detect mobile and restricted molecular parts, respectively. Interestingly, the 13C NMR chemical shift of lipid hydrocarbon chains can be used to monitor order-disorder phase transitions and calculate the fraction of chain defects (rotamers) and the part of the transition enthalpy due to bond rotations (6-7 kJ·mol-1 for dimyristolphosphatidylcholine, DMPC). Cholesterol-containing membranes (liquid-ordered phases) can be dynamically contrasted as the rigid-body sterol is mainly detected by the CP technique, with a contact time of 1 ms, and the phospholipid by INEPT. Our work opens up a straightforward, robust, and cost-effective route for the determination of membrane dynamics by taking advantage of well-resolved conventional 13C NMR experiments without the need of isotopic labeling.

Green synthesis, Single‐Crystal X‐RD, Hirshfeld Analysis and Anti‐Covid‐19 Molecular Docking Investigation of Symmetrical Azines

AbstractThe 1,2‐bis(3,4‐dimethoxybenzylidene)hydrazine (VAHD) and 1,2‐bis(3‐methoxy‐4‐hydroxybenzylidene)hydrazine (VNHD) are synthesised in a solvent free and catalyst free by greener method (MW). Both the compounds are characterized by FT‐IR,1H NMR and 13C NMR spectral studies. Single crystal XRD analysis provides more information on the structure of the compounds VAHD and VNHD. The energy gap (Eg), frontier orbital energies (EHOMO, ELUMO) and reactivity parameters like chemical hardness and global hardness andMulliken charges are calculated using density functional theory with B3LYP/6‐311++G(d,p) basis set. The experimental and theoretical calculated IR frequencies and NMR chemical shifts values are compared by DFT method. Hirshfeld surface analysis was conducted to study structure and molecular properties. Molecular docking of symmetrical azine at the active sites of SARS‐COVID receptors was investigated. Furthermore, the swissADME online application was used to analyse the physicochemical and pharmacokinetic features of the compounds (VAHD and VNHD).

Chemical shift assignments of calmodulin bound to the GluN1 C0 domain (residues 841–865) of the NMDA receptor

Neuroplasticity and synaptic transmission in the brain are regulated by N-methyl-D-aspartate receptors (NMDARs) that consist of hetero-tetrameric combinations of the glycine-binding GluN1 and glutamate-binding GluN2 subunits. Calmodulin (CaM) binds to the cytosolic C0 domain of GluN1 (residues 841–865) that may play a role in the Ca2+-dependent inactivation (CDI) of NMDAR channel activity. Dysregulation of NMDARs are linked to various neurological disorders, including Alzheimer’s disease, depression, stroke, epilepsy, and schizophrenia. Here, we report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the GluN1 C0 domain of the human NMDAR (BMRB no. 51715).

Evaluation of the CHARMM36m force field combined with the OPC water model for protein simulations.

The accuracy of atomistic molecular dynamics (MD) simulations depends on the accuracy of the used force field. For proteins many force fields have been developed so far, each in combination with a particular water model. Typically, combining a protein force field with a water model for which is has not been developed is not expected to yield reliable results. However, recently the combination of the CHARMM36m force field with the OPC water model was shown to accurately estimate the compactness and secondary structure content of intrinsically disordered proteins. Whether CHARMM36m+OPC yields similar accuracy for globular proteins as well has, however, not yet been evaluated systematically. Here, we benchmark this combination on a set of six different globular proteins. To this end, we performed 50 x 1 μs MD simulations per protein using CHARMM36m+OPC and, for comparison, the same simulations using the well-established Amber99SB-ILDN+TIP4P force field. We compared the generated ensembles by means of RMSD, radius of gyration and secondary structure content and also compared the conformational dynamics. We found that CHARMM36m+OPC generates less compact ensembles and shows higher barriers for conformational transitions. Furthermore, we compare ensembles of both force fields to experimental crystal structures, B-factors, and NMR chemical shifts. Here, we found that both force fields compare equally well to experimental data, with CHARMM36m+OPC ensembles agreeing with chemical shifts slightly better, whereas Amber99SB-ILDN+TIP4P ensembles better agree with crystal structures. Our results show that CHARMM36m+OPC and Amber99SB-ILDN+TIP4P yield similar accuracy for globular proteins. Combined with earlier results for disordered proteins, these findings suggest that CHARMM36m+OPC should provide good accuracy for a broad range of disordered and folded proteins as well.

Encapsulation of xenon by bridged resorcinarene cages with high 129Xe NMR chemical shift and efficient exchange dynamics

Encapsulation of xenon by bridged resorcinarene cages with high 129Xe NMR chemical shift and efficient exchange dynamics

Phenylpropanoids and a diarylpentanol from Stellera chamaejasme with acetylcholinesterase inhibitory activity

The chemical investigation on the roots of Stellera chamaejasme led to the identification of five undescribed compounds, including three lignans (1, 2, and 3b), one coumarin (4), one diarylpentanoid (5), and eight known analogues (3a, 6–10, and 11a/11b). Among them, two pairs of enantiomers were separated by chiral resolution. The structures of all compounds were elucidated by NMR spectroscopy and HRESIMS data analyses, quantum chemical calculations of NMR chemical shifts and ECD, as well as single-crystal X-ray diffraction analyses. All isolates were tested for their inhibitory activities against AChE, and compound 2 showed the most potent inhibitory activity, with an inhibition rate of 88.0% at 100 μM. In addition, the possible interaction mechanism of 2 against AChE was predicted by molecular docking.

Structures and solvent effects on the 1H and 13C NMR chemical shifts of the family of three pro-lithospermate molecules: DFT study

The solvent effects on the structures and nuclear magnetic resonance spectroscopy of a series of three pro-lithospermate molecules are studied. These molecules include methyl pro-lithospermate, ethyl pro-lithospermate, and n-butyl pro-lithospermate, which exhibit enantiomers and diastereoisomers. The trans-oriented enantiomers were observed by Yu et al. [Nat. Prod. Res. 31(21), 2505–2512 (2017)] and are the subject of the present study. The density functional theory was validated using the gauge-including atomic orbital/B3LYP-D3BJ/6–31++G(d,p) and continuous set of gauge transformation (CSGT)/the long-range corrected Tao, Perdew, Staroverov, and Scuseria’s τ-dependent gradient-corrected correlation-exchange functional (LC-TPSS)TPSS/cc-pVTZ methods. The chemical shifts of 1H and 13C were calculated in dimethylsulfoxide (DMSO), methanol, and water. It turns out that solvent effects are weak on the geometrical parameters (bond lengths and bond angles). The harmonic oscillator model of the aromaticity index was used to calculate the aromaticity of the subunit rings. With regard to the available experimental results, the chemical shifts of 1H and 13C are reproduced better by B3LYP-D3BJ/6–31++G(d,p) than by LC-TPSSTPSS/cc-pVTZ in the DMSO medium. The solvent effects are significant on the 13C chemical shift, mostly for protic solvents, and insignificant on 1H. The vicinal spin–spin coupling constants of protons were calculated. The coupling constant of trans-olefinic and ortho-aromatic protons fits well with the experiment.

Low information NMR data guided protein-peptide complex structure determination with MELDXMD.

About 40% of the protein-protein interactions are mediated through small peptide epitope. Knowledge of how a peptide or an intrinsically disordered protein fragment interact with a protein would widen of the understanding of gene regulation, transcription and finally drug discovery. However, the folding upon binding nature of majority of the peptide interacting partner, limits the ability of modeling tools to predict structures of such complexes. To address this problem, we show an integrative approach combining cost-effective NMR chemical shift data and physics based molecular simulations to determine structures of protein-peptide complexes. Here in this presentation, we demonstrate our approach for polypeptide complexes formed with the extraterminal (ET) domain of bromo and extraterminal domain (BET) proteins, which exhibit a high degree of binding plasticity. This system is particularly challenging as the binding process includes allosteric changes across the ET receptor upon binding, and the polypeptide binding partners can form different conformations (e.g., helices and hairpins) in the complex. In a blind study, the new approach successfully predicts bound-state structure, using only backbone chemical shift data, in excellent agreement with experimental structure. We show, this approach is successful on not only strongly interacting complexes, but also in the case of weak binder, this method has been successful. MELD+NMR also predicts relative binding affinities of different peptides, consistent with the experimental values. The hybrid MELD+NMR approach provides a powerful new tool for structural analysis of protein-polypeptide complexes involving disorder to order transitions upon complex formation which are not successfully modeled with most other complex predication methods including state of the art docking methods, providing 3D atomistic structures of complexes as well as their relative binding affinities.

Molecular fingerprint in hyaluronan-peptide interactions: Cooperation of electrostatics and side-chain specificity in enhanced arginine effect.

Hyaluronan is a sugar polymer found on the extracellular side, close to the plasma membrane. It is the scaffold of the extracellular matrix due to its high solubility, despite its low negative charge density and unusual length. Hyaluronan is also a known ligand of several glycan-binding proteins. We can improve our understanding of these interactions by characterizing hyaluronan affinity to model amino acid sequences of reduced complexity. Extracted features and fingerprints allow for identifying new potential binding partners and expand the current understanding of hyaluronan's biological role. Here, we use a combination of NMR and molecular dynamics simulations (MD) to study hyaluronan binding to short oligopeptides. We focus on arginine as a known critical amino acid for this interaction and compare it to lysine and glycine to highlight its distinctive features. We can test the binding using 1H NMR chemical shift perturbation (CSP) by titrating hyaluronan with a peptide. CSP not only estimates binding strength but also pinpoints the binding motif involved. Our findings are further explained by MD, which provide an atomistic fingerprint of the hyaluronan-peptide complex. Interestingly, we could not find any NOE signal confirming the CSP observed interactions. The combination of positive CSP with no visible NOEs indicates the presence of a dynamic binding mode in agreement with our MD simulations. This agreement with simulations was only achieved using the prosECCo force field for accounting for electronic polarizability. Overall, we find that arginine, a positively charged amino acid, binds the strongest to the negative hyaluronan, followed by the positive lysine. The hyaluronan's carboxyl and amide groups are the driving force for interacting with the peptides. We prove the importance of the electrostatics interaction but also a specific sidechain affinity that favors arginine.

New nor-chamigrane and bisabolane sesquiterpenoids from the sea hare Aplysia dactylomela

Two new nor-chamigranes and a new bisabolane sesquiterpene, given the trivial names dactylomelanins CE (1–3), were isolated from the sea hare Aplysia dactylomela along with a known compound (4). Their structures and absolute configurations were elucidated by analysis of their HRESIMS, NMR and ECD spectroscopic data. The position of the bromine atom in the structure of 1 was established by quantum chemical calculations of the NMR chemical shifts and by analysis of DP4+ probability. Compounds 1–4 were evaluated for their cytotoxicity against three cell lines, A549, MCF-7, and HepG2.

A rhodanine derivative as a potential antibacterial and anticancer agent: Crystal structure, spectral characterization, DFT calculations, Hirshfeld surface analysis, in silico molecular docking and ADMET studies

A novel rhodanine derivative, namely 5-(4-dimethylaminobenzylidene) rhodanine “C12H12N2OS2”, was successfully crystallized from condensation route and characterized by NMR, FT-IR, UV–Vis spectral methods as well as single-crystal X-ray diffraction. This rhodanine derivate crystallizes in the monoclinic P21/c space group, with the cell parameters: a = 3.93590(10), b = 11.2480(3), c = 26.6703(7) Å and β = 93.8530(10)°. The molecular structure displays intra- (C − S···H) and intermolecular (N − H···O) hydrogen-bonding interactions. Micro-spectroscopy performed on single-crystals of the studied compound revealed the first absorption transition at 2.25 eV, and a well-structured luminescence peaked at 2.01 eV (0.15 eV broad). Density functional theory (DFT) calculations allowed the structure optimization, the electronic properties, the IR-vibrational modes and frequencies as well as the 1H and 13C NMR chemical shifts’ calculation. Furthermore, time-dependent DFT (TD-DFT) calculations were performed for the vertical transition energies. Hirshfeld surface analysis (HSA) showed the presence of non-conventional C–H···H–C, C–H···π and π···lp interactions and π−π stacking. The anti-cancer and anti-bacterial activities of the studied compound towards the Polo-like kinase PLK1 and the Escherichia coli MurB enzymes were in silico evaluated by performing molecular docking simulations. The results suggest that the molecule can significantly inhibit the enzymes’ active sites. Additionally, the physicochemical and pharmacokinetic characteristics of the molecule were evaluated through absorption, distribution, metabolism, excretion and toxicity (ADMET) analysis, and the results ensure its good drug-likeness properties.

Characterizing inhibitors of human AP endonuclease 1.

AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds (MLS000552981, MLS000419194) inhibit APE1 in vitro with low micromolar IC50 and do not appear to aggregate in this concentration range. However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme.

Benchmarking computational chemistry approaches on iminodiacetic acid

In the present study, theoretical harmonic vibrational frequencies (IR and Raman), carbon and proton NMR chemical shifts, geometric parameters, atomic charges (only for heteroatoms), reactivity indices (eLUMO, eHOMO, electronegativity, and hardness), and thermodynamical data (inner energy, enthalpy, Gibbs free energy, and entropy) of iminodiacetic acid (IDA) molecule have been investigated. We utilized ORCA software for B3LYP and HF (combined with Pople and Karlsruhe basis sets) calculations and MOPAC2016 software for semi-empirical calculations (AM1, PM3, and PM6). Theoretical vibrational frequencies and carbon and proton NMR chemical shifts have been compared with the corresponding experimental data. Although there was a strong correlation between the experimental and computational vibrational frequencies at low frequencies (<2200 cm−1), the computational predictions of vibrational frequencies were unsuccessful at high frequencies (>2200 cm−1). Distinctly, the studied computational approaches appeared to perform better in the prediction of carbon and proton NMR chemical shifts. Theoretical vibrational frequencies were also compared to each other to understand the impact of method choice (HF vs B3LYP D3 vs semi-empirical methods), dispersion correction (B3LYP D3 vs B3LYP), water solvation (SMD supplemented vs non-supplemented calculations), the family of basis set (Pople vs Karlsruhe basis sets), numbers of zeta (double vs triple zeta), polarization function (polarized vs nonpolarized basis sets), and diffusion function (diffusion supplemented vs non-supplemented basis sets). Moreover, geometric parameters, heteroatom charges, reactivity indices, and thermodynamical data produced by distinct computational approaches, as well, were compared to each other. Based on these comparisons, we detected critical factors (such as water solvation) acting on the computation of geometries, energies, and charges.

New organic ionic plastic crystals based on pyrrolidinium dication for a solid‐phase electrolyte

AbstractA series of 1,4‐bis(N‐alkylpyrrolidinium)butane salts combined with Br−, I−, PF6−, and Tf2N− anions were newly designed and synthesized. In nonpolar CDCl3 solution, the axial and equatorial proton peaks of the bis‐pyrrolidinium cation ring are distinguished in 1H NMR. Also, the strengths of ion paring could be estimated from the 1H NMR chemical shift changes of the bridge and pyrrolidinium proton peaks. 1,4‐Bis(N‐hepthylpyrrolidinium)butane I− is an organic ionic plastic crystal showing the lowest fusion entropy value (ΔSf = 3.4 J K·mol−1). Softer crystalline morphologies were found by polarized optical microscope images at plastic crystal phase of each sample and the results were corresponding to differential scanning calorimetry results. 1,4‐Bis(N‐ethylpyrrolidinium)butane Tf2N− showed the softest crystal phase. Ionic conductivity changes and electrochemical stabilities of the bis‐pyrrolidinium PF6− and Tf2N− salts were also measured, and they were stable up to 4.3 V (vs. Li/Li+) and expected to be applied as an electrolyte component.

Accurate and Cost-Effective NMR Chemical Shift Predictions for Nucleic Acids Using a Molecules-in-Molecules Fragmentation-Based Method.

We have developed, implemented, and assessed an efficient protocol for the prediction of NMR chemical shifts of large nucleic acids using our molecules-in-molecules (MIM) fragment-based quantum chemical approach. To assess the performance of our approach, MIM-NMR calculations are calibrated on a test set of three nucleic acids, where the structure is derived from solution-phase NMR studies. For DNA systems with multiple conformers, the one-layer MIM method with trimer fragments (MIM1trimer) is benchmarked to get the lowest energy structure, with an average error of only 0.80 kcal/mol with respect to unfragmented full molecule calculations. The MIMI-NMRdimer calibration with respect to unfragmented full molecule calculations shows a mean absolute deviation (MAD) of 0.06 and 0.11 ppm, respectively, for 1H and 13C nuclei, but the performance with respect to experimental NMR chemical shifts is comparable to the more expensive MIM1-NMR and MIM2-NMR methods with trimer subsystems. To compare with the experimental chemical shifts, a standard protocol is derived using DNA systems with Protein Data Bank (PDB) IDs 1SY8, 1K2K, and 1KR8. The effect of structural minimizations is employed using a hybrid mechanics/semiempirical approach and used for computations in solution with implicit and explicit-implicit solvation models in our MIM1-NMRdimer methodology. To demonstrate the applicability of our protocol, we tested it on seven nucleic acids, including structures with nonstandard residues, heteroatom substitutions (F and B atoms), and side chain mutations with a size ranging from ∼300 to 1100 atoms. The major improvement for predicted MIM1-NMRdimer calculations is obtained from structural minimizations and implicit solvation effects. A significant improvement with the explicit-implicit solvation model is observed only for two smaller nucleic acid systems (1KR8 and 7NBK), where the expensive first solvation shell is replaced by the microsolvation model, in which a single water molecule is added for each solvent-exposed amino and imino protons, along with the implicit solvation. Overall, our target accuracy of ∼0.2-0.3 ppm for 1H and ∼2-3 ppm for 13C has been achieved for large nucleic acids. The proposed MIM-NMR approach is accurate and cost-effective (linear scaling with system size), and it can aid in the structural assignments of a wide range of complex biomolecules.

Improving the accuracy of 31P NMR chemical shift calculations by use of scaling methods.

Calculation of 31P NMR chemical shifts for a series of tri- and tetracoordinate phosphorus compounds using several basis sets and density functional theory (DFT) functionals gave a modest fit to experimental chemical shifts, but an excellent linear fit when plotted against the experimental values. The resultant scaling methods were then applied to a variety of "large" compounds previously selected by Latypov et al. and a set of stereoisomeric and unusual compounds selected here. No one method was best for all structural types. For compounds that contain P-P bonds and P-C multiple bonds, the Latypov et al. method using the PBE0 functional was best (mean absolute deviation/root mean square deviation (MAD/RMSD) = 6.9/8.5 ppm and 6.6/8.2 ppm, respectively), but for the full set of compounds gave higher deviations (MAD/RMSD = 8.2/12.3 ppm), and failed by over 60 ppm for a three-membered phosphorus heterocycle. Use of the M06-2X functional for both the structural optimization and NMR chemical shift calculation was best overall for the compounds without P-C multiple bonds (MAD/RMSD = 5.4/7.1 ppm), but failed by 30-49 ppm for compounds having any P-C multiple-bond character. Failures of these magnitudes have not been reported previously for these widely used functionals. These failures were then used to screen a variety of recommended functionals, leading to better overall methods for calculation of these chemical shifts: optimization with the M06-2X functional and NMR calculation with the PBE0 or ωB97x-D functionals gave values for MAD/RMSD = 6.9/8.5 ppm and 6.8/9.1 ppm, respectively, over an experimental chemical shift range of -181 to 356 ppm. Due to the unexplained failures observed, we recommend use of more than one method when looking at novel structures.

Rosetta's Predictive Ability for Low-Affinity Ligand Binding in Fragment-Based Drug Discovery.

Fragment-based drug discovery begins with the identification of small molecules with a molecular weight of usually less than 250 Da which weakly bind to the protein of interest. This technique is challenging for computational docking methods as binding is determined by only a few specific interactions. Inaccuracies in the energy function or slight deviations in the docking pose can lead to the prediction of incorrect binding or difficulties in ranking fragments in in silico screening. Here, we test RosettaLigand by docking a series of fragments to a cysteine-depleted variant of the TIM-barrel protein, HisF (UniProtKB Q9X0C6). We compare the computational results with experimental NMR spectroscopy screens. NMR spectroscopy gives details on binding affinities of individual ligands, which allows assessment of the ligand-ranking ability using RosettaLigand and also provides feedback on the location of the binding pocket, which serves as a reliable test of RosettaLigand's ability to identify plausible binding poses. From a library screen of 3456 fragments, we identified a set of 31 ligands with intrinsic affinities to HisF with dissociation constants as low as 400 μM. The same library of fragments was blindly screened in silico. RosettaLigand was able to rank binders before non-binders with an area under the curve of the receiver operating characteristics of 0.74. The docking poses observed for binders agreed with the binding pocket identified by NMR chemical shift perturbations for all fragments. Taken together, these results provide a baseline performance of RosettaLigand in a fragment-based drug discovery setting.