Sulfated phenolic acids in plants.

MAIN CONCLUSION: Sulfated phenolic acids are widely occurring metabolites in plants, including fruits, vegetables and crops. The untargeted UHPLC-QTOF-MS metabolomics of more than 50 samples from plant, fungi and algae lead to the discovery of a small group of sulfated metabolites derived from phenolic acids. These compounds were detected in land plants for the first time. In this study, zosteric acid, 4-(sulfooxy)benzoic acid, 4-(sulfoooxy)phenylacetic acid, ferulic acid 4-sulfate and/or vanillic acid 4-sulfate were detected in a number of edible species/products, including oat (Avena sativa L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), tomato (Solanum lycopersicum L.), carrot (Daucus carota subsp. Sativus Hoffm.), broccoli (Brassica oleracea var. Italica Plenck), celery (Apium graveolens L.), cabbage (Brassica oleracea convar. sabauda L.), banana tree (Musa tropicana L.), pineapple fruit (Ananas comosus L.), radish bulb (Raphanus sativus L.) and olive oil (Olea europaea L.). The structural identification of sulfated compounds was performed by comparing retention times and mass spectral data to those of synthesized standards. In addition to above-mentioned compounds, isoferulic acid 3-sulfate and caffeic acid 4-sulfate were putatively identified in celery bulb (Apium graveolens L.) and broccoli floret (Brassica oleracea var. Italica Plenck), respectively. While sulfated phenolic acids were quantified in concentrations ranging from 0.34 to 22.18 µg·g-1 DW, the corresponding non-sulfated acids were mostly undetected or present at lower concentrations. The subsequent analysis of oat symplast and apoplast showed that they are predominantly accumulated in the symplast (> 70%) where they are supposed to be biosynthesized by sulfotransferases.

Chemoenzymatic Hunsdiecker-Type Decarboxylative Bromination of Cinnamic Acids.

In this contribution, we report chemoenzymatic bromodecarboxylation (Hunsdiecker-type) of α,ß-unsaturated carboxylic acids. The extraordinarily robust chloroperoxidase from Curvularia inaequalis (CiVCPO) generated hypobromite from H2O2 and bromide, which then spontaneously reacted with a broad range of unsaturated carboxylic acids and yielded the corresponding vinyl bromide products. Selectivity issues arising from the (here undesired) addition of water to the intermediate bromonium ion could be solved by reaction medium engineering. The vinyl bromides so obtained could be used as starting materials for a range of cross-coupling and pericyclic reactions.

Water-Soluble Anthraquinone Photocatalysts Enable Methanol-Driven Enzymatic Halogenation and Hydroxylation Reactions.

Peroxyzymes simply use H2O2 as a cosubstrate to oxidize a broad range of inert C-H bonds. The lability of many peroxyzymes against H2O2 can be addressed by a controlled supply of H2O2, ideally in situ. Here, we report a simple, robust, and water-soluble anthraquinone sulfonate (SAS) as a promising organophotocatalyst to drive both haloperoxidase-catalyzed halogenation and peroxygenase-catalyzed oxyfunctionalization reactions. Simple alcohols, methanol in particular, can be used both as a cosolvent and an electron donor for H2O2 generation. Very promising turnover numbers for the biocatalysts of up to 318 000 have been achieved.

Chemoenzymatic Halocyclization of 4-Pentenoic Acid at Preparative Scale.

The scale-up of chemoenzymatic bromolactonization to 100 g scale is presented, together with an identification of current limitations. The preparative-scale reaction also allowed for meaningful mass balances identifying current bottlenecks of the chemoenzymatic reaction.

Chemoenzymatic Halocyclization of γ,δ-Unsaturated Carboxylic Acids and Alcohols.

Invited for this month's cover is the group of Prof. Dr. Frank Hollmann at Delft University of Technology in the Netherlands. The Front Cover shows the vanadium-dependent haloperoxidase from the marine organism Curcuvaria inaequalis, which efficiently activates halides as hypohalites that can then initiate spontaneous halo-lactonization and halo-etherification reactions. The Communication itself is available at 10.1002/cssc.201902240.

Chemoenzymatic Halocyclization of γ,δ-Unsaturated Carboxylic Acids and Alcohols.

A chemoenzymatic method for the halocyclization of unsaturated alcohols and acids by using the robust V-dependent chloroperoxidase from Curvularia inaequalis (CiVCPO) as catalyst has been developed for the in situ generation of hypohalites. A broad range of halolactones and cyclic haloethers are formed with excellent performance of the biocatalyst.

Marine Vanadium-Dependent Haloperoxidases, Their Isolation, Characterization, and Application.

Vanadium-dependent haloperoxidases in seaweeds, cyanobacteria, fungi, and possibly phytoplankton play an important role in the release of halogenated volatile compounds in the environment. These halocarbons have effects on atmospheric chemistry since they cause ozone depletion. In this chapter, a survey is given of the different sources of these enzymes, some of their properties, the various methods to isolate them, and the bottlenecks in purification. The assays to detect and quantify haloperoxidase activity are described as well as their kinetic properties. Several practical tips and pitfalls are given which have not yet been published explicitly. Recent developments in research on structure and function of these enzymes are reviewed. Finally, the application of vanadium-dependent haloperoxidases in the biosynthesis of brominated and other compounds is discussed.

Selective aerobic oxidation reactions using a combination of photocatalytic water oxidation and enzymatic oxyfunctionalisations.

Peroxygenases offer attractive means to address challenges in selective oxyfunctionalisation chemistry. Despite their attractiveness, the application of peroxygenases in synthetic chemistry remains challenging due to their facile inactivation by the stoichiometric oxidant (H2O2). Often atom inefficient peroxide generation systems are required, which show little potential for large scale implementation. Here we show that visible light-driven, catalytic water oxidation can be used for in situ generation of H2O2 from water, rendering the peroxygenase catalytically active. In this way the stereoselective oxyfunctionalisation of hydrocarbons can be achieved by simply using the catalytic system, water and visible light.

Vanadium Chloroperoxidases: The Missing Link in the Formation of Chlorinated Compounds and Chloroform in the Terrestrial Environment?

It is well established that the majority of chlorinated organic substances found in the terrestrial environment are produced naturally. The presence of these compounds in soils is not limited to a single ecosystem. Natural chlorination is also a widespread phenomenon in grasslands and agricultural soils typical for unforested areas. These chlorinated compounds are formed from chlorination of natural organic matter consisting of very complex chemical structures, such as lignin. Chlorination of several lignin model compounds results in the intermediate formation of trichloroacetyl-containing compounds, which are also found in soils. These decay, in general, through a haloform-type reaction mechanism to CHCl3 . Upon release into the atmosphere, CHCl3 will produce chlorine radicals through photolysis, which will, in turn, lead to natural depletion of ozone. There is evidence that fungal chloroperoxidases able to produce HOCl are involved in the chlorination of natural organic matter. The objective of this review is to clarify the role and source of the various chloroperoxidases involved in the natural formation of CHCl3 .

Halofunctionalization of alkenes by vanadium chloroperoxidase from Curvularia inaequalis.

The vanadium-dependent chloroperoxidase from Curvularia inaequalis is a stable and efficient biocatalyst for the hydroxyhalogenation of a broad range of alkenes into halohydrins. Up to 1 200 000 TON with 69 s-1 TOF were observed for the biocatalyst. A bienzymatic cascade to yield epoxides as reaction products is presented.

Dissolving Lignin in Water through Enzymatic Sulfation with Aryl Sulfotransferase.

We introduce the concept of using site-specific sulfation of various lignins for increasing their aqueous solubility and thereby their processability. Using p-nitrophenylsulfate as a sulfate source and an aryl sulfotransferase enzyme as catalyst, lignins are easily sulfated at ambient conditions. We demonstrate the specific sulfation of phenolic hydroxyl groups on five different lignins: Indulin AT (Kraft softwood), Protobind 1000 (mixed wheat straw/Sarkanda grass soda) and three organosolv lignins. The reaction proceeds smoothly and the increase in solubility is visible to the naked eye. We then examine the reaction kinetics, and show that these are easily monitored qualitatively and quantitatively using UV/Vis spectroscopy. The UV/Vis results are validated with 31 P NMR spectroscopy of the lignin phenol groups after derivatization with phosphorylation reagent II. In general, the results are more significant with organosolv lignins, as Kraft and soda lignins are produced from aqueous lignocellulose extraction processes.

5¹V NMR Crystallography of Vanadium Chloroperoxidase and Its Directed Evolution P395D/L241V/T343A Mutant: Protonation Environments of the Active Site.

Vanadium-dependent haloperoxidases (VHPOs) perform two-electron oxidation of halides using hydrogen peroxide. Their mechanism, including the factors determining the substrate specificity and the pH-dependence of the catalytic rates, is poorly understood. The vanadate cofactor in the active site of VHPOs contains "spectroscopically silent" V(V), which does not change oxidation state during the reaction. We employed an NMR crystallography approach based on (51)V magic angle spinning NMR spectroscopy and Density Functional Theory, to gain insights into the structure and coordination environment of the cofactor in the resting state of vanadium-dependent chloroperoxidases (VCPO). The cofactor environments in the wild-type VCPO and its P395D/L241V/T343A mutant exhibiting 5-100-fold improved catalytic activity are examined at various pH values. Optimal sensitivity attained due to the fast MAS probe technologies enabled the assignment of the location and number of protons on the vanadate as a function of pH. The vanadate cofactor changes its protonation from quadruply protonated at pH 6.3 to triply protonated at pH 7.3 to doubly protonated at pH 8.3. In contrast, in the mutant, the vanadate protonation is the same at pH 5.0 and 8.3, and the cofactor is doubly protonated. This methodology to identify the distinct protonation environments of the cofactor, which are also pH-dependent, could help explain the different reactivities of the wild-type and mutant VCPO and their pH-dependence. This study demonstrates that (51)V-based NMR crystallography can be used to derive the detailed coordination environments of vanadium centers in large biological molecules.

The role of vanadium haloperoxidases in the formation of volatile brominated compounds and their impact on the environment.

Vanadium haloperoxidases differ strongly from heme peroxidases in substrate specificity and stability and in contrast to a heme group they contain the bare metal oxide vanadate as a prosthetic group. These enzymes specifically oxidize halides in the presence of hydrogen peroxide into hypohalous acids. These reactive halogen intermediates will react rapidly and aspecifically with many organic molecules. Marine algae and diatoms containing these iodo- and bromoperoxidases produce short-lived brominated methanes (bromoform, CHBr3 and dibromomethane CH2Br2) or iodinated compounds. Some seas and oceans are supersaturated with these compounds and they form an important source of bromine to the troposphere and lower stratosphere and contribute significantly to the global budget of halogenated hydrocarbons. This perspective focuses, in particular, on the biosynthesis of these volatile compounds and the direct or indirect involvement of vanadium haloperoxidases in the production of huge amounts of bromoform and dibromomethane. Some of the global sources are discussed and from the literature a picture emerges in which oxidized brominated species generated by phytoplankton, seaweeds and cyanobacteria react with dissolved organic matter in seawater, resulting in the formation of intermediate brominated compounds. These compounds are unstable and decay via a haloform reaction to form an array of volatile brominated compounds of which bromoform is the major component followed by dibromomethane.

Synthesis of carbohydrates in a continuous flow reactor by immobilized phosphatase and aldolase.

Herein, we report a new flow process with immobilized enzymes to synthesize complex chiral carbohydrate analogues from achiral inexpensive building blocks in a three-step cascade reaction. The first reactor contained immobilized acid phosphatase, which phosphorylated dihydroxyacetone to dihydroxyacetone phosphate using pyrophosphate as the phosphate donor. The second flow reactor contained fructose-1,6-diphosphate aldolase (RAMA, rabbit muscle aldolase) or rhamnulose-1-phosphate aldolase (RhuA from Thermotoga maritima) and acid phosphatase. The immobilized aldolases coupled the formed dihydroxyacetone phosphate to aldehydes, resulting in phosphorylated carbohydrates. A final reactor containing acid phosphatase that dephosphorylated the phosphorylated product yielded the final product. Different aldehydes were used to synthesize carbohydrates on a gram scale. To demonstrate the feasibility of the flow systems, we synthesized 0.6 g of the D-fagomine precursor. By using immobilized aldolase RhuA we were also able to obtain other stereoisomers of the D-fagomine precursor.

Vanadium pentoxide nanoparticles mimic vanadium haloperoxidases and thwart biofilm formation.

Marine biofouling--the colonization of small marine microorganisms on surfaces that are directly exposed to seawater, such as ships' hulls--is an expensive problem that is currently without an environmentally compatible solution. Biofouling leads to increased hydrodynamic drag, which, in turn, causes increased fuel consumption and greenhouse gas emissions. Tributyltin-free antifouling coatings and paints based on metal complexes or biocides have been shown to efficiently prevent marine biofouling. However, these materials can damage the environment through metal leaching (for example, of copper and zinc) and bacteria resistance. Here, we show that vanadium pentoxide nanowires act like naturally occurring vanadium haloperoxidases to prevent marine biofouling. In the presence of bromide ions and hydrogen peroxide, the nanowires catalyse the oxidation of bromide ions to hypobromous acid (HOBr). Singlet molecular oxygen ((1)O(2)) is formed and this exerts strong antibacterial activity, which prevents marine biofouling without being toxic to marine biota. Vanadium pentoxide nanowires have the potential to be an alternative approach to conventional anti-biofouling agents.

Selective oxidative decarboxylation of amino acids to produce industrially relevant nitriles by vanadium chloroperoxidase.

Industrial nitriles from biomass: Vanadium-chloroperoxidase is successfully used to transform selectively glutamic acid into 3-cyanopropanoic acid, a key intermediate for the synthesis of bio-succinonitrile and bio-acrylonitrile, by using a catalytic amount of a halide salt. This clean oxidative decarboxylation can be applied to mixtures of amino acids obtained from plant waste streams, leading to easily separable nitriles.

Continuous-flow reactor-based enzymatic synthesis of phosphorylated compounds on a large scale.

Acid phosphatase, an enzyme that is able to catalyze the transfer of a phosphate group from cheap pyrophosphate to alcoholic substrates, was covalently immobilized on polymethacrylate beads with an epoxy linker (Immobeads-150 or Sepabeads EC-EP). After immobilization 70% of the activity was retained and the immobilized enzyme was stable for many months. With the immobilized enzyme we were able to produce and prepare D-glucose-6-phosphate, N-acetyl-D-glucosamine-6-phosphate, allyl phosphate, dihydroxyacetone phosphate, glycerol-1-phosphate, and inosine-5'-monophosphate from the corresponding primary alcohol on gram scale using either a fed-batch reactor or a continuous-flow packed-bed reactor.

Vanadium K-edge XAS studies on the native and peroxo-forms of vanadium chloroperoxidase from Curvularia inaequalis.

Vanadium K-edge X-ray Absorption Spectra have been recorded for the native and peroxo-forms of vanadium chloroperoxidase from Curvularia inaequalis at pH 6.0. The Extended X-ray Absorption Fine Structure (EXAFS) regions provide a refinement of previously reported crystallographic data; one short V=O bond (1.54A) is present in both forms. For the native enzyme, the vanadium is coordinated to two other oxygen atoms at 1.69A, another oxygen atom at 1.93A and the nitrogen of an imidazole group at 2.02A. In the peroxo-form, the vanadium is coordinated to two other oxygen atoms at 1.67A, another oxygen atom at 1.88A and the nitrogen of an imidazole group at 1.93A. When combined with the available crystallographic and kinetic data, a likely interpretation of the EXAFS distances is a side-on bound peroxide involving V-O bonds of 1.67 and 1.88A; thus, the latter oxygen would be 'activated' for transfer. The shorter V-N bond observed in the peroxo-form is in line with the previously reported stronger binding of the cofactor in this form of the enzyme. Reduction of the enzyme with dithionite has a clear influence on the spectrum, showing a change from vanadium(V) to vanadium(IV).

Improvement of an acid phosphatase/DHAP-dependent aldolase cascade reaction by using directed evolution.

To enhance the phosphorylating activity of the bacterial nonspecific acid phosphatase from Salmonella enterica ser. typhimurium LT2 towards dihydroxyacetone (DHA), a mutant library was generated from the native enzyme. Three different variants that showed enhanced activity were identified after one round of epPCR. The single mutant V78L was the most active and showed an increase in the maximal DHAP concentration to 25 % higher than that of the wild-type enzyme at pH 6.0. This variant is 17 times more active than the wild-type acid phosphatase from Salmonella enterica ser. typhimurium LT2 in the acid phosphatase/aldolase cascade reaction at pH 6.0 and is also six times more active than the phosphatase from Shigella flexneri that we previously used.

Synergy between chemo- and bio-catalysts in multi-step transformations.

Cascade synthetic pathways, which allow multi-step conversions to take place in one reaction vessel, are crucial for the development of biomimetic, highly efficient new methods of chemical synthesis. Theoretically, the complexity introduced by combining processes could lead to an improvement of the overall process; however, it is the current general belief that it is more efficient to run processes separately. Inspired by natural cascade procedures we successfully combined a lipase catalyzed amidation with palladium catalyzed coupling reactions, simultaneously carried out on the same molecule. Unexpectedly, the bio- and chemo-catalyzed processes show synergistic behaviour, highlighting the complexity of multi-catalyst systems.