Phase Transfer Catalysts and Role of Reaction Environment in Nucleophilc Radiofluorinations in Automated Synthesizers

Incorporation of [18F]fluorine into PET radiotracer structure has traditionally been accomplished via nucleophilic pathways. The [18F]fluoride is generated in an aqueous solution via proton irradiation of oxygen-18 enriched water and must to be introduced into water-free organic solutions in order to generate reactive species. Thus nucleophilic 18F-fluorination traditionally included steps for [18F]fluoride concentration on the anion exchange resin, followed by removal of residual water via azeotropic distillation with MeCN, a time-consuming process associated with radioactivity losses and difficult automation. To circumvent this, several adsorption/elution protocols were developed based on the minimization of water content in traditional kryptofix-based [18F]fluoride eluents. The use of pre-dried KOH/kryptofix solutions, tertiary alcohols, and strong organic bases was found to be effective. Advances in transition metal-mediated SNAr approaches for radiolabeling of non-activated aromatic substrates have prompted development of alternative techniques for reactive [18F]fluoride species generation, such as organic solutions of non-basic alkyl ammonium and pyridinium sulfonates, etc. For radiofluorinations of iodonium salts precursors, a “minimalist” approach was introduced, avoiding the majority of pitfalls common to more complex methods. These innovations allowed the development of new time-efficient and convenient work-up procedures that are easily implementable in modern automated synthesizers. They will be the subject of this review.

Highly Stable, Cold-Active Aldehyde Dehydrogenase from the Marine Antarctic Flavobacterium sp. PL002

Stable aldehyde dehydrogenases (ALDH) from extremophilic microorganisms constitute efficient catalysts in biotechnologies. In search of active ALDHs at low temperatures and of these enzymes from cold-adapted microorganisms, we cloned and characterized a novel recombinant ALDH from the psychrotrophic Flavobacterium PL002 isolated from Antarctic seawater. The recombinant enzyme (F-ALDH) from this cold-adapted strain was obtained by cloning and expressing of the PL002 aldH gene (1506 bp) in Escherichia coli BL21(DE3). Phylogeny and structural analyses showed a high amino acid sequence identity (89%) with Flavobacterium frigidimaris ALDH and conservation of all active site residues. The purified F-ALDH by affinity chromatography was homotetrameric, preserving 80% activity at 4 °C for 18 days. F-ALDH used both NAD+ and NADP+ and a broad range of aliphatic and aromatic substrates, showing cofactor-dependent compensatory KM and kcat values and the highest catalytic efficiency (0.50 µM−1 s−1) for isovaleraldehyde. The enzyme was active in the 4–60 °C-temperature interval, with an optimal pH of 9.5, and a preference for NAD+-dependent reactions. Arrhenius plots of both NAD(P)+-dependent reactions indicated conformational changes occurring at 30 °C, with four(five)-fold lower activation energy at high temperatures. The high thermal stability and substrate-specific catalytic efficiency of this novel cold-active ALDH favoring aliphatic catalysis provided a promising catalyst for biotechnological and biosensing applications.

Toward Engineering the Substrate Specificity of a PHA Synthase (PhaC)

<p>Manufacturing of high-grade plastics from petroleum-based feedstocks is a high-cost, unsustainable process resulting in expensive products. My overall goal was to engineer the pathway of bacterial bio-polyester formation, in order to produce high-grade bioplastics. More specifically, the aim was to introduce aromatic rings into the main-chain of the polyhydroxyalkanoate (PHA) polymer currently produced by specialist bacteria. This research aimed to create these bio-plastics from renewable resources, rather than relying on petroleum-based sources. A key enzyme for this process is the polyhydroxyalkanoate synthase, PhaC. This enzyme is capable of polymerizing activated hydroxybutyrate-CoA monomers. I began with the establishment of a system that allowed the use of directed evolution. I constructed a minimal plasmid for the expression of PhaC and a second plasmid with the CoA ligase genes required for substrate activation. I generated error-prone PCR libraries of the Cupriavidus necator phaCa, Chromobacterium sp. USM2 phaCb and an ancestrally reconstructed phaCb-LCA that contained differing spectra of mutations. A life-or-death selection was employed to select for PhaC variants able to polymerise aromatic substrates based upon the toxicity of the un-polymerized aromatic hydroxyacid monomers. I determined the minimum inhibitory concentrations (MICs) for six of these monomers in Escherichia coli for downstream selection. Lastly, I adapted a Nile red screening method to test wild-type PHA accumulation of PhaC enzymes. Selections for mutants capable of polymerizing aromatic monomers were implemented on the libraries generated from phaCa and phaCb. Whereas, the library generated from phaCb-LCA was screened for variants with increased wild-type activity. Selections yielded no candidates for further testing. However, the screen isolated several variants with increased wild-type activity. These variants may serve as a new scaffold for further mutagenesis experiments to achieve the overall goal; to produce a high-grade bioplastic.</p>

Toward Engineering the Substrate Specificity of a PHA Synthase (PhaC)

<p>Manufacturing of high-grade plastics from petroleum-based feedstocks is a high-cost, unsustainable process resulting in expensive products. My overall goal was to engineer the pathway of bacterial bio-polyester formation, in order to produce high-grade bioplastics. More specifically, the aim was to introduce aromatic rings into the main-chain of the polyhydroxyalkanoate (PHA) polymer currently produced by specialist bacteria. This research aimed to create these bio-plastics from renewable resources, rather than relying on petroleum-based sources. A key enzyme for this process is the polyhydroxyalkanoate synthase, PhaC. This enzyme is capable of polymerizing activated hydroxybutyrate-CoA monomers. I began with the establishment of a system that allowed the use of directed evolution. I constructed a minimal plasmid for the expression of PhaC and a second plasmid with the CoA ligase genes required for substrate activation. I generated error-prone PCR libraries of the Cupriavidus necator phaCa, Chromobacterium sp. USM2 phaCb and an ancestrally reconstructed phaCb-LCA that contained differing spectra of mutations. A life-or-death selection was employed to select for PhaC variants able to polymerise aromatic substrates based upon the toxicity of the un-polymerized aromatic hydroxyacid monomers. I determined the minimum inhibitory concentrations (MICs) for six of these monomers in Escherichia coli for downstream selection. Lastly, I adapted a Nile red screening method to test wild-type PHA accumulation of PhaC enzymes. Selections for mutants capable of polymerizing aromatic monomers were implemented on the libraries generated from phaCa and phaCb. Whereas, the library generated from phaCb-LCA was screened for variants with increased wild-type activity. Selections yielded no candidates for further testing. However, the screen isolated several variants with increased wild-type activity. These variants may serve as a new scaffold for further mutagenesis experiments to achieve the overall goal; to produce a high-grade bioplastic.</p>

An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene

Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.

A new regime of heme-dependent aromatic oxygenase superfamily

Two histidine-ligated heme-dependent monooxygenase proteins, TyrH and SfmD, have recently been found to resemble enzymes from the dioxygenase superfamily currently named after tryptophan 2,3-dioxygenase (TDO), that is, the TDO superfamily. These latest findings prompted us to revisit the structure and function of the superfamily. The enzymes in this superfamily share a similar core architecture and a histidine-ligated heme. Their primary functions are to promote O-atom transfer to an aromatic metabolite. TDO and indoleamine 2,3-dioxygenase (IDO), the founding members, promote dioxygenation through a two-step monooxygenation pathway. However, the new members of the superfamily, including PrnB, SfmD, TyrH, and MarE, expand its boundaries and mediate monooxygenation on a broader set of aromatic substrates. We found that the enlarged superfamily contains eight clades of proteins. Overall, this protein group is a more sizeable, structure-based, histidine-ligated heme-dependent, and functionally diverse superfamily for aromatics oxidation. The concept of TDO superfamily or heme-dependent dioxygenase superfamily is no longer appropriate for defining this growing superfamily. Hence, there is a pressing need to redefine it as a heme-dependent aromatic oxygenase (HDAO) superfamily. The revised concept puts HDAO in the context of thiol-ligated heme-based enzymes alongside cytochrome P450 and peroxygenase. It will update what we understand about the choice of heme axial ligand. Hemoproteins may not be as stringent about the type of axial ligand for oxygenation, although thiolate-ligated hemes (P450s and peroxygenases) more frequently catalyze oxygenation reactions. Histidine-ligated hemes found in HDAO enzymes can likewise mediate oxygenation when confronted with a proper substrate.

C-Methylation Of Organic Substrates. A Comprehensive Overview. Part Iva. Methylating Agents Other Than Methane, Methanol, And Methyl Metals

: C-Methylation of organic substrates was accomplished with a number of methylating agents other than methane, methanol, and methyl metals. They include methyl halides (MeX, X = I, Br, Cl, F), methyl-containing halogenated reagents, methyl peroxides, dimethyl carbonate (DMC), dimethylsulfoxide (DMSO), N,N-dimethyl formamide (DMF), diazomethane, formate salts, trioxane, CO/H2, CO2/H2, and dimethyl ether (DME). Under particular conditions, some methyl-containing molecules such as polymethylbenzenes, methylhydrazine, tris(diethylamino)sulfonium difluorotrimethylsilicate, methyl tosylate, long-chain alkyl alcohols, and acetic acid unexpectedly C-methylated a variety of organic substrates. A few cases of C-methylation only were reported to occur in the absence of catalysts. Otherwise, transition metal complexes as catalysts in conjunction with specific ligands and bases were ubiquitously present in most C-methylation reactions. Of the reactions, Suzuki-Miyaura-type cross-coupling remained of paramount importance in making 11CH3-bearing positron emission tomography tracers (PETs), one of the best applications of such methylation. Methylation proceeded at C(aromatic)-X, C(sp3)-X C(sp2)-X, and C(sp)-X of substrates (X = H, halogen). Ortho-methylation was regioselectively observed with aromatic substrates when they bear moieties such as pyridyl, pyrimidyl, amide, and imine functionalities, which were accordingly coined ‘ortho-directing groups’.

A multiplexed nanostructure-initiator mass spectrometry (NIMS) assay for simultaneously detecting glycosyl hydrolase and lignin modifying enzyme activities

Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.

Recent Advances in Transition Metal-Catalyzed Selective C-H Difluoromethylation Reactions

Fluorine is well-known as a very special element. Approximately 30% of agrochemicals and 20% of all drugs contain fluorine; most of those compounds have unique functions in biochemistry, pharmacy, and bioscience and those containing difluoromethyl functional groups often have irreplaceable roles. Therefore, the selective introduction of difluoromethylated functional groups into various aromatic substrates has significant practical application. This review describes recent advances in selective difluoroalkylation of aromatic substrates by using different catalytic strategies (cyclometalated ruthenium complex, transient regulating and visible-light-induced strategies).