One site fits both: A model for the ternary complex of folate + NADPH in R67 dihydrofolate reductase, a D2 symmetric enzyme

R67 dihydrofolate reductase (DHFR) is a novel enzyme that confers resistance to the antibiotic trimethoprim. The crystal structure of R67 DHFR displays a toroidal structure with a central active-site pore. This homotetrameric protein exhibits 222 symmetry, with only a few residues from each chain contributing to the active site, so related sites must be used to bind both substrate (dihydrofolate) and cofactor (NADPH) in the productive R67 DHFR?NADPH?dihydrofolate complex. Whereas the site of folate binding has been partially resolved crystallographically, an interesting question remains: how can the highly symmetrical active site also bind and orient NADPH for catalysis? To model this ternary complex, we employed DOCK and SLIDE, two methods for docking flexible ligands into proteins using quite different algorithms. The bound pteridine ring of folate (Fol I) from the crystal structure of R67 DHFR was used as the basis for docking the nicotinamide-ribose-P_i (NMN) moiety of NADPH. NMN was positioned by both DOCK and SLIDE on the opposite side of the pore from Fol I, where it interacts with Fol I at the pore's center. Numerous residues serve dual roles in binding. For example, Gln 67 from both the B and D subunits has several contacts with the pteridine ring, while the same residue from the A and C subunits has several contacts with the nicotinamide ring. The residues involved in dual roles are generally amphipathic, allowing them to make both hydrophobic and hydrophilic contacts with the ligands. The result is a `hot spot' binding surface allowing the same residues to co-optimize the binding of two ligands, and orient them for catalysis.     

Investigation of chiral recognition by molecular micelles with molecular dynamics simulations

Molecular dynamics simulations were used to characterize the binding of the chiral drugs chlorthalidone and lorazepam to the molecular micelle poly-(sodium undecyl-(L)-leucine-valine). The project’s goal was to characterize the nature of chiral recognition in capillary electrophoresis separations that use molecular micelles as the chiral selector. The shapes and charge distributions of the chiral molecules investigated, their orientations within the molecular micelle chiral binding pockets, and the formation of stereoselective intermolecular hydrogen bonds with the molecular micelle were all found to play key roles in determining where and how lorazepam and chlorthalidone enantiomers interacted with the molecular micelle.     

Photocontrolled Release of Chemicals from Nano‐ and Microparticle Containers

A benzoin‐derived diol linker was synthesized and used to generate biocompatible polyesters that can be fully decomposed on demand upon UV irradiation. Extensive structural optimization of the linker unit was performed to enable the defined encapsulation of diverse organic compounds in the polymeric structures and allow for a well‐controllable polymer cleavage process. Selective tracking of the release kinetics of encapsulated model compounds from the polymeric nano‐ and microparticle containers was performed by confocal laser scanning microscopy in a proof‐of‐principle study. The physicochemical properties of the incorporated and released model compounds ranged from fully hydrophilic to fully hydrophobic. The demonstrated biocompatibility of the utilized polyesters and degradation products enables their use in advanced applications, for example, for the smart packaging of UV‐sensitive pharmaceuticals, nutritional components, or even in the area of spatially selective self‐healing processes.     

The N‐Methylpyrrolidone (NMP) Effect in Iron‐Catalyzed Cross‐Coupling with Simple Ferric Salts and MeMgBr

The use of N‐methylpyrrolidone (NMP) as a co‐solvent in ferric salt catalyzed cross‐coupling reactions is crucial for achieving the highly selective, preparative scale formation of cross‐coupled product in reactions utilizing alkyl Grignard reagents. Despite the critical importance of NMP, the molecular level effect of NMP on in situ formed and reactive iron species that enables effective catalysis remains undefined. Herein, we report the isolation and characterization of a novel trimethyliron(II) ferrate species, [Mg(NMP)₆][FeMe₃]₂ ( 1 ), which forms as the major iron species in situ in reactions of Fe(acac)₃ and MeMgBr under catalytically relevant conditions where NMP is employed as a co‐solvent. Importantly, combined GC analysis and ⁵⁷Fe Mössbauer spectroscopic studies identified 1 as a highly reactive iron species for the selective formation generating cross‐coupled product. These studies demonstrate that NMP does not directly interact with iron as a ligand in catalysis but, alternatively, interacts with the magnesium cations to preferentially stabilize the formation of 1 over [Fe₈Me₁₂]^? cluster generation, which occurs in the absence of NMP.     

Hexakis(2,4,6‐triisopropylphenyl)cyclotristannoxane – a Molecular Diorganotin Oxide with Kinetically Inert Sn–O Bonds

The single‐crystal X‐ray structure analysis of hexakis(2,4,6‐triisopropylphenyl)cyclotristannoxane, cyclo‐[(2,4,6‐i‐Pr₃‐C₆H₂)₂SnO]₃ ( 1 ), is reported and reveals this compound to contain an almost planar six‐membered ring. Redistribution reactions of 1 with cyclo‐(t‐Bu₂SnO)₃ and t‐Bu₂SiCl₂, respectively, failed and indicate an unusual kinetic inertness of the Sn–O bonds in 1 as compared to related molecular diorganotin oxides containing less bulkier substituents. The redistribution reaction of cyclo‐(t‐Bu₂SnO)₃ with cyclo‐(t‐Bu₂SnS)₂ leads to an equilibrium involving the trimeric diorganotin oxysulphides cyclo‐t‐Bu₂Sn(OSnt‐Bu₂)₂S ( 2 a ) and cyclo‐t‐Bu₂Sn(SSnt‐Bu₂)₂O ( 2 b ).     

Spectral assignments and anisotropy data of cellulose I(alpha): 13C-NMR chemical shift data of cellulose I(alpha) determined by INADEQUATE and RAI techniques applied to uniformly 13C-labeled bacterial celluloses of different Gluconacetobacter xylinus strains

Solid-state (13)C-NMR spectroscopy was used to characterize native cellulose pellicles from two strains of Gluconacetobacter xylinus (ATCC 53582, ATCC 23769), which had been statically cultivated in Hestrin-Schramm (HS) medium containing fully (13)C-labeled beta-D-glucose-U-(13)C(6) as the sole source of carbon. For both samples, the (13)C-NMR chemical shifts were completely assigned for each (13)C-labeled site of cellulose I(alpha) with the aid of 2D refocused INADEQUATE NMR. To determine the principal chemical shift tensor components, a pulse sequence based on the recoupling of anisotropy information (RAI) was applied at 10 kHz MAS. The detailed (13)C tensors of cellulose I(alpha) from different bacterial celluloses are thus available now for the first time, and these results have been compared with previously published data of nonenriched material and with theoretical predictions.     

Gas-phase carbene radical anions: new mechanistic insights

The gas-phase reactivity of the CHCl*- anion has been investigated with a series of halomethanes (CCl4, CHCl3, CH2Cl2, and CH3Cl) using a FA-SIFT instrument. Results show that this anion primarily reacts via substitution and by proton transfer. In addition, the reactions of CHCl*- with CHCl3 and CH2Cl2 form minor amounts of Cl2*- and Cl-. The isotopic distribution of these two products is consistent with an insertion-elimination mechanism, where the anion inserts into a C-Cl bond to form an unstable intermediate, which eliminates either Cl2*- or Cl- and Cl*. Neutral and cationic carbenes are known to insert into single bonds; however, this is the first observation of such reactivity for carbene anions.     

A simple DNase model system comprising a dinuclear Zn(II) complex in methanol accelerates the cleavage of a series of methyl aryl phosphate diesters by 10(11)-10(13)

The di-Zn(II) complex of 1,3-bis[ N1, N1'-(1,5,9-triazacyclododecyl)]propane with an associated methoxide ( 3:Zn(II) 2: (-)OCH 3) was prepared and its catalysis of the methanolysis of a series of fourteen methyl aryl phosphate diesters ( 6) was studied at s (s)pH 9.8 in methanol at 25.0 +/- 0.1 degrees C. Plots of k obs vs [ 3:Zn(II) 2: (-)OCH 3] free for all members of 6 show saturation behavior from which K(M) and kcat (max) were determined. The second order rate constants for the catalyzed reactions (kcat (max)/K(M)) for each substrate are larger than the corresponding methoxide catalyzed reaction (k 2 (-OMe)) by 1.4 x 10(8) to 3 x 10 (9)-fold. The values of k cat (max) for all members of 6 are between 4 x 10(11) and 3 x 10(13) times larger than the solution reaction at s (s)pH 9.8, with the largest accelerations being given for substrates where the departing aryloxy unit contains ortho-NO 2 or C(O)OCH 3 groups. Based on the linear Br?nsted plots of k cat (max) vs s (s)pKa of the phenol, beta lg values of -0.57 and -0.34 are determined respectively for the catalyzed methanolysis of "regular" substrates that do not contain the ortho-NO 2 or C(O)OCH 3 groups, and those substrates that do. The data are consistent with a two step mechanism for the catalyzed reaction with rate limiting formation of a catalyst-coordinated phosphorane intermediate, followed by fast loss of the aryloxy leaving group. A detailed energetics calculation indicates that the catalyst binds the transition state comprising [CH 3O (-): 6], giving a hypothetical [ 3:Zn(II) 2:CH 3O (-): 6] complex, by -21.4 to -24.5 kcal/mol, with the strongest binding being for those substrates having the ortho-NO 2 or C(O)OCH 3 groups.     

Protein analysis of laser capture micro-dissected tissues revealed cell-type specific biological functions in developing barley grains

Both the nucellar projection (NP) and endosperm transfer cells (ETC) of the developing barley grain (harvested 8 days after flowering) were isolated by laser capture micro-dissection combined with pressure catapulting. Protein extracts were analyzed by nanoUPLC separation combined with ESI-Q-TOF mass spectrometry. The majority of the ～160 proteins identified were involved in translation, protein synthesis, or protein destination. The NP proteome was enriched for stress defense molecules, while proteins involved in assimilate transport and the mobilization of nutrients were common to both the NP and the ETC. The combined qualitative and quantitative protein profiling allowed for the identification of several proteins showing tissue specificity in their expression, which underlines the distinct biological functions of these two tissues within the developing barley grain.