A high-throughput mass spectrometric assay for discovery of human lipoxygenase inhibitors and allosteric effectors.

Lipoxygenases (LOXs) regulate inflammation through the production of a variety of molecules whose specific downstream effects are not entirely understood due to the complexity of the inflammation pathway. The generation of these biomolecules can potentially be inhibited and/or allosterically regulated by small synthetic molecules. The current work describes the first mass spectrometric high-throughput method for identifying small molecule LOX inhibitors and LOX allosteric effectors that change the substrate preference of human lipoxygenase enzymes. Using a volatile buffer and an acid-labile detergent, enzymatic products can be directly detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS) without the need for organic extraction. The method also reduces the required enzyme concentration compared with traditional ultraviolet (UV) absorbance methods by approximately 30-fold, allowing accurate binding affinity measurements for inhibitors with nanomolar affinity. The procedure was validated using known LOX inhibitors and the allosteric effector 13(S)-hydroxy-9Z,11E-octadecadienoic acid (13-HODE)

Synthesis and alkyne-coupling chemistry of cyclomanganated 1- and 3-acetylindoles, 3-formylindole and analogues

The syntheses are reported of new cyclomanganated indole derivatives (1-acetyl-κO-indolyl-κC2)dicarbonylbis(trimethylphosphite)manganese (2), (1-methyl-3-acetyl-κO-indolyl-κC2)tetracarbonylmanganese (4), (3-formyl-κO-indolyl-κC2)tetracarbonylmanganese (5a) and (1-methyl-3-formyl-κO-indolyl-κC2)tetracarbonylmanganese (5b). The unusually complicated crystal structure of 5b has been determined, the first for a cyclomanganated aryl aldehyde. The preparations of a mitomycin-related pyrrolo-indole and related products by thermally promoted and oxidatively (Me3NO) initiated alkyne-coupling reactions of the previously known complex (1-acetyl-κO-indolyl-κC2)tetracarbonylmanganese (1) are reported for different alkynes and solvents. X-ray crystal structures are reported for the dimethyl acetylenedicarboxylate coupling product of 1 (dimethyl 1-methyl-l-hydroxypyrrolo[1,2a]-indole-2,3-dicarboxylate; 6a), and an unusually-cyclised triple insertion product 8 from the coupling of acetylene with 4, in which a cyclopentadiene moiety is η3-allyl-coordinated to Mn through only one double bond and an exocyclic carbon, but which rearranges on heating to an η5-cyclopentadienyl complex

Fluorination of Rubisco-Mimetic CO2 Capture Systems. Theoretical and Experimental Studies of Ammonium Ion Acidity Depression and Carbamylation

RuBisCO-inspired CO2 capture and release (CCR) systems featuring amines have been developed for the purpose of reversable CO2 capture from air. The enzyme active site consists of the tetrapeptide sequence Lys-Asp-Asp-Glu. The Lys sidechain amine undergoes carbamylation and an Mg2+ cation stabilizes the resulting carbamate. The Na-acyl-lysinyl-aspratyl-aspartyl-glutamide (Lys-Asp-Asp-Glu, KDDE) peptide featured maximum capture at pH ≈ 10; a pH region too high for Mg2+ ions to remain in solution. This work aims to achieve pKa depression by introducing fluorine in the proximity of the lysine’s sidechain amine. A comparative analysis was made of butylamine, (2,2,2-trifluoroethyl) butylamine, and 2,2-difluoropropylamine to examine fluorination strategies aimed at ammonium ion pKa depression and carbamylation at reduced pH (CH. 1). CF3-functionalized lysines tFKn were synthesized for the purpose of embedding the amino acids into the larger KDDE tetrapeptide (CH. 2). Two 13C NMR rotamer quartet signals were observed for the CF3 carbon of tert-butyloxycarbonyl (boc) protected intermediates. To understand the rotational dynamics, we analyzed the potential energy surface (PES) of a fluorinated tertiary carbamate model system (CH 3). The rotation-inversion barrier was measured to create a direct connection to experimentation (CH. 4). The CO2 addition pathway of the fluorinated tFLys-Asp-Asp-Glu (tFKDDE) tetrapeptide was analyzed to understand its carbamylation mechanics (CH. 5). The CF3-functionalized lysine tFK was embedded into the tetrapeptide and its carbamylation, both with and without Mg2+, was analyzed as a function of pH to determine maximum carbamylation, the pH of maximum carbamylation, and its Gibbs’ free energy (CH 6) --Abstract, p. i

Rotation-Inversion Isomerization of Tertiary Carbamates: Potential Energy Surface Analysis of Multi-Paths Isomerization using Boltzmann Statistics

The front cover artwork is provided by Prof. Rainer Glaser\u27s group at the Missouri University of Science and Technology. The image shows one of four potential energy surfaces generated from our rotation-inversion study of tertiary carbamates and highlights two of the eight possible transition state pathways between two ensembles of E- and Z-minima. In the context of synthetic studies of fluorinated carbamates R1 O-CO-N(R2 )CH2 CF3, we unexpectedly observed two sets of 13C NMR quartets for the CF3 group and we needed to understand their origin. Read the full text of the Research Article at 10.1002/cphc.2022005442

VT-NMR Analysis Of Rotation-Inversion Of N-(4-hydroxybutyl)-N-(2,2,2-trifluoroethyl) Tert-butyl Carbamate: Utilizing The −CH2CF3 Appendage As A Reporter On E/Z-Isomerization

The rotational barrier about the CN carbamate bond of N-(4-hydroxybutyl)-N-(2,2,2-trifluoroethyl) tert-butyl carbamate 1 was determined by variable temperature (VT) 13C and 19F NMR spectroscopy. The −CH2CF3 appendage reports on rotational isomerism and allows for the observation of separate signals for the E- and Z-ensembles at low temperature. The activation barrier for E/Z-isomerization was quantified using Eyring-Polanyi theory which requires the measurements of the maximum difference in Larmor frequency Δνmax and the convergence temperature Tc. Both Δνmax and Tc were interpolated by analyzing sigmoidal functions fitted to data describing signal separation and the quality of the superposition of the E- and Z-signals, respectively. Methods for generating the quality-of-fit parameters for Lorentzian line shape analysis are discussed. Our best experimental value for the rotational barrier ΔGc≠(1)=15.65±0.13 kcal/mol is compared to results of a higher level ab initio study of the model N-ethyl-N-(2,2,2-trifluoroethyl) methyl carbamate

Nuclear Magnetic Resonance Study Of CO2 Capture By Fluoroalkylamines: Ammonium Ion PKa Depression Via Fluorine Modification And Thermochemistry Of Carbamylation

We are developing energy-efficient and reversible carbon capture and release (CCR) systems that mimic the Lys201 carbamylation reaction in the active site of ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO). The multiequilibria scenario ammonium ion Xa ⇌ amine Xb ⇌ carbamic acid Xc ⇌ carbamate Xd requires the presence of both free amine and CO2 for carbamylation and is affected by the pKa(Xa). Two fluorination strategies aimed at ammonium ion pKa depression and low pH carbamylation were analyzed with (2,2,2-trifluoroethyl) butylamine 2b and 2,2-difluoropropylamine 3b and compared to butylamine 1b. The determination of K1 and ΔG1 of the carbamylation reactions requires the solution of multiequilibria systems of equations based on initial conditions, 1H NMR measurements of carbamylation yields over a wide pH range, and knowledge of K2-K5 values. K2 and K3 describe carbonic acid acidity, and ammonium ion acidities K4 were measured experimentally. We calibrated carbamic acid acidities K5 based on the measured value K6 of aminocarbamic acid using isodesmic reactions. The proton exchange reactions were evaluated with ab initio computations at the APFD/6-311+G* level in combination with continuum solvation models and explicit solvation. The utilities of 1-3 will be discussed as they pertain to the development of fluorine-modified RuBisCO-mimetic reversible CCR systems

Assessing Eolian Dust Inputs to Soils in Dry Creek Experimental Watershed, SW Idaho

A geochemical investigation of upland soils in the Dry Creek Experimental Watershed (DCEW) near Boise, Idaho was conducted to assess the potential contributions of eolian dust. Major and trace element compositions of soils within the watershed, loess deposits in the adjacent Western Snake River Plain (WSRP), and underlying granodiorite bedrock were evaluated. Multiple lines of evidence suggest a significant contribution of dust in the soils. Plots of Co/Ti, V/Ti and Cr/Ti indicate that the loess and bedrock occupy distinctly different compositional spaces and that the soils are of intermediate composition, suggesting that the soils are a mixture of the loess and bedrock. In the same compositional space, isolates of the silt + clay size fraction exhibit compositions closer to the loess field than the associated bulk soils, also consistent with a dust contribution. A two-component mixing model, using the granodiorite and loess as end-members, indicates there is an average of 28% dust in the soils. This model also indicates that the dust contribution is greater in the upper portions of soil profiles, and that the dust contribution on north facing slopes is more than double that on south facing slopes (38% and 16%, respectively). Dust appears to be the dominant contributor of fine-grained material to these soils. Theoretical removal of estimated dust contributions from DCEW soils reduced soil water storage capacity by an average of 48% in the soils, suggesting that dust deposition is an important contributor to shallow water storage capacity and subsequent summertime availability in the upland soils of this watershed

Dust and Gas in the Magellanic Clouds from the HERITAGE Herschel Key Project. II. Gas-to-Dust Ratio Variations across ISM Phases

The spatial variations of the gas-to-dust ratio (GDR) provide constraints on the chemical evolution and lifecycle of dust in galaxies. We examine the relation between dust and gas at 10-50 pc resolution in the Large and Small Magellanic Clouds (LMC and SMC) based on Herschel far-infrared (FIR), H I 21 cm, CO, and Halpha observations. In the diffuse atomic ISM, we derive the gas-to-dust ratio as the slope of the dust-gas relation and find gas-to-dust ratios of 380+250-130 in the LMC, and 1200+1600-420 in the SMC, not including helium. The atomic-to-molecular transition is located at dust surface densities of 0.05 Mo pc-2 in the LMC and 0.03 Mo pc-2 in the SMC, corresponding to AV ~ 0.4 and 0.2, respectively. We investigate the range of CO-to-H2 conversion factor to best account for all the molecular gas in the beam of the observations, and find upper limits on XCO to be 6x1020 cm-2 K-1 km-1 s in the LMC (Z=0.5Zo) at 15 pc resolution, and 4x 1021 cm-2 K-1 km-1 s in the SMC (Z=0.2Zo) at 45 pc resolution. In the LMC, the slope of the dust-gas relation in the dense ISM is lower than in the diffuse ISM by a factor ~2, even after accounting for the effects of CO-dark H2 in the translucent envelopes of molecular clouds. Coagulation of dust grains and the subsequent dust emissivity increase in molecular clouds, and/or accretion of gas-phase metals onto dust grains, and the subsequent dust abundance (dust-to-gas ratio) increase in molecular clouds could explain the observations. In the SMC, variations in the dust-gas slope caused by coagulation or accretion are degenerate with the effects of CO-dark H2. Within the expected 5--20 times Galactic XCO range, the dust-gas slope can be either constant or decrease by a factor of several across ISM phases. Further modeling and observations are required to break the degeneracy between dust grain coagulation, accretion, and CO-dark H2

Near and Mid-IR Photometry of the Pleiades, and a New List of Substellar Candidate Members

We make use of new near and mid-IR photometry of the Pleiades cluster in order to help identify proposed cluster members. We also use the new photometry with previously published photometry to define the single-star main sequence locus at the age of the Pleiades in a variety of color-magnitude planes. The new near and mid-IR photometry extend effectively two magnitudes deeper than the 2MASS All-Sky Point Source catalog, and hence allow us to select a new set of candidate very low mass and sub-stellar mass members of the Pleiades in the central square degree of the cluster. We identify 42 new candidate members fainter than Ks =14 (corresponding to 0.1 Mo). These candidate members should eventually allow a better estimate of the cluster mass function to be made down to of order 0.04 solar masses. We also use new IRAC data, in particular the images obtained at 8 um, in order to comment briefly on interstellar dust in and near the Pleiades. We confirm, as expected, that -- with one exception -- a sample of low mass stars recently identified as having 24 um excesses due to debris disks do not have significant excesses at IRAC wavelengths. However, evidence is also presented that several of the Pleiades high mass stars are found to be impacting with local condensations of the molecular cloud that is passing through the Pleiades at the current epoch.Comment: Accepted to ApJS; data tables and embedded-figure version available at http://spider.ipac.caltech.edu/staff/stauffer/pleiades07