Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cβ and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Semiempirical Quantum Chemical and Ab Intio Study of Co(Ii), Ni(Ii),Cu(Ii) and Zn(Ii) Complexes with the Azo Dye Derived from 4-Amino Antipyrine and 2,4-Dihydroxy Acetopheone

Semi-empirical quantum chemical calculation was made to study the nucleophilicity of the ligand and to study the mode of bonding between the ligand and the metal ions. The natural atomic charge at different atomic sites of the ligand has been calculated along with the electrostatic potential map to predict the reactive sites for electrophilic and nucleophilic attack. The theoretical spectral data such as IR, NMR and electronic have been calculated and compared with the experimentally generated data.

Plasmodium falciparum Dihydroorotate Dehydrogenase, Fragment-based Drug Design, 2D-QSAR, DFT Calculation, Lead Optimization, Induced Fit Docking

Plasmodium falciparum dihydroorotate dehydrogenase (PfDODH) is one of the enzymes currently explored in the treatment of malaria. Although there is currently no clinically approved drug targeting PfDODH, many of the compounds in clinical trials have [1, 2, 4,] triazolo [1, 5-a] pyrimidin-7-amine backbone structure. This study sought to design new compounds from the fragments of known experimental inhibitors of PfDODH. Nine experimental compounds retrieved from Drug Bank online were downloaded and broken into fragments using Schrodinger power shell; the fragments were recombined to generate new ligand structures using BREED algorithm. The new compounds were docked with PfDODH crystal structure, after which the compounds were filtered with extensive drug-likeness and toxicity parameters. A 2D-QSAR model was built using the multiple linear regression method and externally validated. The compounds electronic behaviours were studied using DFT calculations. Structural investigation of the six designed compounds, which had lower binding energies than the standard inhibitors, showed that five of them had [1, 2, 4,] triazolo [1, 5-a] pyrimidin-7-amine moieties and interacted with essential residues at the PfDODH binding site. In addition to their drug-like and pharmacokinetic properties, they also showed minimal toxicities. The externally validated 2D-QSAR model with R2 and Q2 values of 0.6852 and 0.6691, confirmed the inhibitory prowess of these compounds against PfDODH. The DFT calculations showed regions of the molecules prone to electrophilic and nucleophilic attack. The current study thus provides insight into the development of a new set of potent PfDODH inhibitors.

Nitro Fatty Acids (NO2-FAs): An Emerging Class of Bioactive Fatty Acids

Unsaturated nitro fatty acids (NO2-FAs) constitute a category of molecules that may be formed endogenously by the reaction of unsaturated fatty acids (UFAs) with secondary species of nitrogen monoxide and nitrite anions. The warhead of NO2-FAs is a nitroalkene moiety, which is a potent Michael acceptor and can undergo nucleophilic attack from thiol groups of biologically relevant proteins, showcasing the value of these molecules regarding their therapeutic potential against many diseases. In general, NO2-FAs inhibit nuclear factorκ-B (NF-κB), and simultaneously they activate nuclear factor (erythroid derived)-like 2 (Nrf2), which activates an antioxidant signaling pathway. NO2-FAs can be synthesized not only endogenously in the organism, but in a synthetic laboratory as well, either by a step-by-step synthesis or by a direct nitration of UFAs. The step-by-step synthesis requires specific precursor compounds and is in position to afford the desired NO2-FAs with a certain position of the nitro group. On the contrary, the direct nitration of UFAs is not a selective methodology; thus, it affords a mixture of all possible nitro isomers.

Chemical Decomposition of the TFSI Anion Under Aqueous Basic Conditions

Understanding the interfacial reactivity of aqueous electrolytes is crucial for their use in future batteries. We investigate the reactivity of the bis(trifluoromethane)sulfonimide (TFSI) anion when exposed to a strong alkaline medium, by means of ab initio molecular dynamics and enhanced sampling techniques. In particular, we study the nucleophilic attack by the hydroxide anion, which was proposed as a mechanism for the formation of the solid electrolyte interphase at the negative electrode with water-in-salt electrolytes. While in the gas phase we recover a stable gaseous product, namely fluoroform, we observe the formation of trifluoromethanol in strong basic conditions, which then rapidly deprotonates to form CF3O-. This anion was suggested recently as a key compound leading to the formation of a solid electrolyte interphase on an Si-C anode. Such an approach could be leveraged to discover convenient additives leading to the formation of a stable interphase.

Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome

Peptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs are properly positioned on the A site and the P site of the peptidyl transferase center (PTC) so that nucleophilic attack can occur. Here we analyse some constraints associated with the induced-fit mechanism of the PTC, that promotes this positioning through a compaction around the aminoacyl ester orchestrated by U2506. The physical basis of PTC decompaction, that allows the elongated peptidyl-tRNA to free itself from that state and move to the P site of the PTC, is still unclear. From thermodynamics considerations and an analysis of published ribosome structures, the present work highlights the rational of this mechanism, in which the free-energy released by the new peptide bond is used to kick U2506 away from the reaction center. Furthermore, we show the evidence that decompaction is impaired when the nascent peptide is not yet anchored inside the exit tunnel, which may contribute to explain why the first rounds of elongation are inefficient, an issue that has attracted much interest for about two decades. Results in this field are examined in the light of the present analysis and a physico-chemical correlation in the genetic code, which suggest that elementary constraints associated with the size of the side-chain of the amino acids penalize early elongation events.

Straightforward Regio- and Diastereoselective Synthesis, Molecular Structure, Intermolecular Interactions and Mechanistic Study of Spirooxindole-Engrafted Rhodanine Analogs

Straightforward regio- and diastereoselective synthesis of bi-spirooxindole-engrafted rhodanine analogs 5a–d were achieved by one-pot multicomponent [3 + 2] cycloaddition (32CA) reaction of stabilized azomethine ylide (AYs 3a–d) generated in situ by condensation of L-thioproline and 6-chloro-isatin with (E)-2-(5-(4-chlorobenzylidene)-2,4-dioxothiazolidin-3-yl)-N-(2-morpholinoethyl)acetamide. The bi-spirooxindole-engrafted rhodanine analogs were constructed with excellent diastereo- and regioselectivity along with high chemical yield. X-ray crystallographic investigations for hybrid 5a revealed the presence of four contiguous stereocenters related to C11, C12, C19 and C22 of the spiro structure. Hirshfeld calculations indicated the presence of many short intermolecular contacts such as Cl...C, S...S, S...H, O...H, N...H, H...C, C...C and H...H interactions. These contacts played a very important role in the crystal stability. The polar nature of the 32CA reaction was studied by analysis of the conceptual DFT reactivity indices. Theoretical study of this 32CA reaction indicated that it takes place through a non-concerted two-stage one-step mechanism associated with the nucleophilic attack of AY 3a to the electrophilic ethylene derivative.

Iron-TAML Complexes: A Computational Approach to Improving Stability

<p>Researchers at the Institute for Green Oxidation Chemistry of the Carnegie Mellon University developed a group of catalysts called tetra amido macrocyclic ligand (TAML) activators. The purpose of that research was that TAML activators would breakdown pollutants in the presence of a sacrificial oxidant. Furthermore, the catalyst was designed to decompose on a required timescale, as to not damage the environment by prolonged exposure. Since the initial designs from the 1980’s, the TAML structure has undergone significant changes to increase efficiency or selectivity. Other uses of this group of catalysts have been explored, namely, the oxidation of water to molecular oxygen. This work presents a computational study using Density Functional Theory (DFT) which addresses the issue regarding the stability of certain iron-TAML intermediates in the water oxidation mechanism. Hence, the work seeks to explore how changing certain groups on the TAML ring can affect the stability of the reactive intermediates and the activation energy of the nucleophilic attack within the mechanism. The work highlights the importance of the fluorinated tail of the TAML structure in the accessibility of the desired transition state.</p>

Iron-TAML Complexes: A Computational Approach to Improving Stability

<p>Researchers at the Institute for Green Oxidation Chemistry of the Carnegie Mellon University developed a group of catalysts called tetra amido macrocyclic ligand (TAML) activators. The purpose of that research was that TAML activators would breakdown pollutants in the presence of a sacrificial oxidant. Furthermore, the catalyst was designed to decompose on a required timescale, as to not damage the environment by prolonged exposure. Since the initial designs from the 1980’s, the TAML structure has undergone significant changes to increase efficiency or selectivity. Other uses of this group of catalysts have been explored, namely, the oxidation of water to molecular oxygen. This work presents a computational study using Density Functional Theory (DFT) which addresses the issue regarding the stability of certain iron-TAML intermediates in the water oxidation mechanism. Hence, the work seeks to explore how changing certain groups on the TAML ring can affect the stability of the reactive intermediates and the activation energy of the nucleophilic attack within the mechanism. The work highlights the importance of the fluorinated tail of the TAML structure in the accessibility of the desired transition state.</p>

Toward the Synthesis of (–)-TAN-2483B Lactam Analogues

<p>Natural products continue to be an abundant source of lead compounds for drug discovery and development. (–)-TAN-2483A and (–)-TAN-2483B, isolated from the culture of a filamentous fungus, incorporate an unusual furo[3,4-b]pyran-5-one scaffold. TAN-2483A was initially reported to inhibit the c-Src tyrosine kinase enzyme, a potential anticancer target, and parathyroid hormone-induced bone resorption. TAN-2483B, on the other hand, was not isolated in sufficient quantities for biological testing. The synthesis of TAN-2483B is therefore desirable from a drug discovery perspective. Several analogues of TAN-2483B that are functionalised at the propenyl sidechain have previously been synthesised in the Harvey group and have shown promising biological activity. For example, the (Z)-ethyl ester analogue showed micromolar inhibition of HL-60 cells and Bruton’s tyrosine kinase, a protein involved in B-cell maturation that is implicated in certain cancers. The lactone moiety of TAN-2483B and its sidechain analogues, however, appears to be unstable to nucleophilic attack. The aim of this thesis was to investigate the viability of a synthetic route toward lactam analogues of TAN-2483B. It was proposed that substituting the lactone for a lactam would increase the stability of the compound in nucleophilic media. Moreover, the lactam nitrogen may provide a site for further functionalisation of the compound for future structure-activity relationship studies. Because installation of the (Z)-ethyl ester sidechain via Wittig conditions has previously been found to be more facile than installation of the (E)-propenyl sidechain found in the natural product, investigations into forming the lactam ring system were carried out on the ethyl ester advanced intermediates. Reductive amination of a ketone intermediate was envisaged to install the amine prior to a palladium-catalysed carbonylation/lactam formation step. The promising bioactivity of the (Z)-ethyl ester analogue was anticipated to be retained in the target lactam analogues. It was found that the substrates of the proposed reductive amination, the advanced ketone intermediates, were incompatible with the tested conditions, presumably due to base sensitivity. Three by-products from the reductive amination experiments were isolated and tentatively characterised by NMR spectroscopy and HRMS. An alternative route toward lactam analogues of TAN-2483B, via intermediate amines accessed by the substitution of an activated alcohol, was briefly investigated with encouraging results. Further optimisation of the synthetic route toward analogues of TAN-2483B was also achieved. Removal of a purification step enabled the more expedient two-step synthesis of a diol intermediate. The two-step transformation to (Z)- and (E)-ethyl ester intermediates, via sodium periodate-mediated diol cleavage and Wittig olefination, proceeded in the highest yield obtained to date. Investigations into the desilylation of a trimethylsilyl-protected acetylene were also conducted. Although lactam analogues of TAN-2483B were not obtained in this study, progress was made toward their synthesis. The alternative route toward amines that was briefly explored here appears promising, and work is ongoing in the Harvey group to access lactam (and other) analogues of TAN-2483B, in addition to the natural product itself.</p>