Asymmetric azidohydroxylation of styrene derivatives mediated by a biomimetic styrene monooxygenase enzymatic cascade.

Enantioenriched azido alcohols are precursors for valuable chiral aziridines and 1,2-amino alcohols, however their chiral substituted analogues are difficult to access. We established a cascade for the asymmetric azidohydroxylation of styrene derivatives leading to chiral substituted 1,2-azido alcohols via enzymatic asymmetric epoxidation, followed by regioselective azidolysis, affording the azido alcohols with up to two contiguous stereogenic centers. A newly isolated two-component flavoprotein styrene monooxygenase StyA proved to be highly selective for epoxidation with a nicotinamide coenzyme biomimetic as a practical reductant. Coupled with azide as a nucleophile for regioselective ring opening, this chemo-enzymatic cascade produced highly enantioenriched aromatic α-azido alcohols with up to >99% conversion. A bi-enzymatic counterpart with halohydrin dehalogenase-catalyzed azidolysis afforded the alternative ß-azido alcohol isomers with up to 94% diastereomeric excess. We anticipate our biocatalytic cascade to be a starting point for more practical production of these chiral compounds with two-component flavoprotein monooxygenases.

Controlled Production of Zearalenone-Glucopyranoside Standards with Cunninghamella Strains Using Sulphate-Depleted Media.

In recent years, conjugated mycotoxins have gained increasing interest in food safety, as their hydrolysis in human and animal intestines leads to an increase in toxicity. For the production of zearalenone (ZEN) glycosides reference standards, we applied Cunninghamellaelegans and Cunninghamella echinulata fungal strains. A sulphate-depleted medium was designed for the preferred production of ZEN glycosides. Both Cunninghamella strains were able to produce zearalenone-14-ß-D-glucopyranoside (Z14G), zearalenone-16-ß-D-glucopyranoside (Z16G) and zearalenone-14-sulphate (Z14S). In a rich medium, Cunninghamellaelegans preferably produced Z14S, while Cunninghamellaechinulata preferably produced Z14G. In the sulphate-depleted medium a dramatic change was observed for Cunninghamellaelegans, showing preferred production of Z14G and Z16G. From 2 mg of ZEN in sulphate-depleted medium, 1.94 mg of Z14G and 0.45 mg of Z16G were produced. Following preparative Liquid Chromatography-Mass Spectrometry (LC-MS) purification, both fractions were submitted to 1H and 13C NMR and High-Resolution Mass Spectrometry (HRMS). These analyses confirmed that the purified fractions were indeed Z14G and Z16G. In conclusion, the presented research shows that a single Cunninghamella strain can be an effective and efficient tool for the controlled biotransformation of ZEN glycosides and other ZEN metabolites. Additionally, the biotransformation method was extended to zearalanone, ß-zearalenol and other mycotoxins.

Toward a Systematic Nomenclature for (Neo)Lignanamides.

Phenylalkenoic acid amides, often referred to as phenol amides or hydroxycinnamic acid amides, are bioactive phytochemicals, whose bioactivity can be enhanced by coupling to form dimers or oligomers. Phenylalkenoic acid amides consist of a (hydroxy)cinnamic acid derivative (i.e., the phenylalkenoic acid subunit) linked to an amine-containing compound (i.e., the amine subunit) via an amide bond. The phenylalkenoic acid moiety can undergo oxidative coupling, either catalyzed by oxidative enzymes or due to autoxidation, which leads to the formation of (neo)lignanamides. Dimers described in the literature are often named after the species in which the compound was first discovered; however, the naming of these compounds lacks a systematic approach. We propose a new nomenclature, inspired by the existing system used for hydroxycinnamic acid dimers and lignin. In the proposed systematic nomenclature for (neo)lignanamides, compound names will be composed of three-letter codes and prefixes denoting the subunits, and numbers that indicate the carbon atoms involved in the linkage between the monomeric precursors. The proposed nomenclature is consistent, future-proof, and systematic.

Eat1-Like Alcohol Acyl Transferases From Yeasts Have High Alcoholysis and Thiolysis Activity.

Esters are important flavor and fragrance compounds that are present in many food and beverage products. Many of these esters are produced by yeasts and bacteria during fermentation. While ester production in yeasts through the alcohol acyl transferase reaction has been thoroughly investigated, ester production through alcoholysis has been completely neglected. Here, we further analyze the catalytic capacity of the yeast Eat1 enzyme and demonstrate that it also has alcoholysis and thiolysis activities. Eat1 can perform alcoholysis in an aqueous environment in vitro, accepting a wide range of alcohols (C2-C10) but only a small range of acyl donors (C2-C4). We show that alcoholysis occurs in vivo in several Crabtree negative yeast species but also in engineered Saccharomyces cerevisiae strains that overexpress Eat1 homologs. The alcoholysis activity of Eat1 was also used to upgrade ethyl esters to butyl esters in vivo by overexpressing Eat1 in Clostridium beijerinckii. Approximately 17 mM of butyl acetate and 0.3 mM of butyl butyrate could be produced following our approach. Remarkably, the in vitro alcoholysis activity is 445 times higher than the previously described alcohol acyl transferase activity. Thus, alcoholysis is likely to affect the ester generation, both quantitatively and qualitatively, in food and beverage production processes. Moreover, mastering the alcoholysis activity of Eat1 may give rise to the production of novel food and beverage products.

Laser Ablation Electrospray Ionization Hydrogen/Deuterium Exchange Ambient Mass Spectrometry Imaging.

Identification and confirmation of known as well as unknown (bio)chemical entities in ambient mass spectrometry (MS) and MS imaging (MSI) mostly involve accurate mass determination, often in combination with MS/MS or MSn work flows. To further improve structural assignment, additional molecular information is required. Here we present an ambient hydrogen/deuterium exchange (HDX) laser ablation electrospray ionization (LAESI) MS method in which, apart from the accurate mass and MS/MS data, the number of exchangeable protons in (un)known molecules is obtained. While eventually presenting ambient HDX-LAESI-MSI, samples were not preincubated with deuterated solvents, but instead HDX occurred following fusion of ablated sample material with microdroplets generated by ESI of deuterated solvents. Therefore, the degree of HDX was first studied following ablation of nondeuterated sample solutions of melamine and monosaccharides. From these experiments, it was concluded that the set-up used could provide meaningful HDX data in support of molecular structure elucidation by significantly reducing the number of structure options from a measured elemental composition. This reduction was demonstrated with an unknown accurate m/z value obtained in the analysis of an orange slice, reducing the possible number of molecular structures having the same elemental composition by 87% due to the number of H/D exchanges observed. Next, deuterated and nondeuterated MS/MS experiments showed the number of exchangeable protons in the substructures from deuterated neutral losses in the product ion spectra, confirming the compound to be arginine. Finally, the potential of ambient HDX-LAESI-MSI was demonstrated by the imaging of (secondary) plant metabolites in a Phalaenopsis petal.

TiO2 Photocatalyzed Oxidation of Drugs Studied by Laser Ablation Electrospray Ionization Mass Spectrometry.

In drug discovery, it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism is oxidation reactions, oxidation of drugs with TiO2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient ionization mass spectrometry (MS). Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. Here, we introduce a novel (time-resolved) TiO2-photocatalysis laser ablation electrospray ionization (LAESI) MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of verapamil, buspirone, testosterone, andarine, and ostarine. Finally, a time-resolved LAESI MS setup was developed and initial results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least 1 h. Graphical Abstract.

Kauniolide synthase is a P450 with unusual hydroxylation and cyclization-elimination activity.

Guaianolides are an important class of sesquiterpene lactones with unique biological and pharmaceutical properties. They have been postulated to be derived from germacranolides, but for years no progress has been made in the elucidation of their biosynthesis that requires an unknown cyclization mechanism. Here we demonstrate the isolation and characterization of a cytochrome P450 from feverfew (Tanacetum parthenium), kauniolide synthase. Kauniolide synthase catalyses the formation of the guaianolide kauniolide from the germacranolide substrate costunolide. Unlike most cytochrome P450s, kauniolide synthase combines stereoselective hydroxylation of costunolide at the C3 position, with water elimination, cyclization and regioselective deprotonation. This unique mechanism of action is supported by in silico modelling and docking experiments. The full kauniolide biosynthesis pathway is reconstructed in the heterologous hosts Nicotiana benthamiana and yeast, paving the way for biotechnological production of guaianolide-type sesquiterpene lactones.

One-Step Generation of Reactive Superhydrophobic Surfaces via SiHCl3-Based Silicone Nanofilaments.

Superhydrophobic surfaces gain ever-growing attention because of their applicability in many (consumer) products/materials as they often display, among others, antifouling, anti-icing, and/or self-cleaning properties. A simple way to achieve superhydrophobicity is through the growth of silicone nanofilaments. These nanofilaments, however, are very often nonreactive and thus difficult to utilize in subsequent chemistries. In response, we have developed a single-step procedure to grow (SiHCl3-based) silicone nanofilaments with selective reactivity that are intrinsically superhydrophobic. The silicone nanofilaments could be further functionalized via Pt-catalyzed hydrosilylation of exposed Si-H moieties. These surfaces are easily obtained using mild conditions and are stable under hydrolytic conditions (neutral water, 24 h at 80 °C) while remaining highly transparent, which makes them well suited for optical and photochemical experiments.

Reactive Laser Ablation Electrospray Ionization Time-Resolved Mass Spectrometry of Click Reactions.

Reactions in confined compartments like charged microdroplets are of increasing interest, notably because of their substantially increased reaction rates. When combined with ambient ionization mass spectrometry (MS), reactions in charged microdroplets can be used to improve the detection of analytes or to study the molecular details of the reactions in real time. Here, we introduce a reactive laser ablation electrospray ionization (reactive LAESI) time-resolved mass spectrometry (TRMS) method to perform and study reactions in charged microdroplets. We demonstrate this approach with a class of reactions new to reactive ambient ionization MS: so-called click chemistry reactions. Click reactions are high-yielding reactions with a high atom efficiency, and are currently drawing significant attention from fields ranging from bioconjugation to polymer modification. Although click reactions are typically at least moderately fast (time scale of minutes to a few hours), in a reactive LAESI approach a substantial increase of reaction time is required for these reactions to occur. This increase was achieved using microdroplet chemistry and followed by MS using the insertion of a reaction tube-up to 1 m in length-between the LAESI source and the MS inlet, leading to near complete conversions due to significantly extended microdroplet lifetime. This novel approach allowed for the collection of kinetic data for a model (strain-promoted) click reaction between a substituted tetrazine and a strained alkyne and showed in addition excellent instrument stability, improved sensitivity, and applicability to other click reactions. Finally, the methodology was also demonstrated in a mass spectrometry imaging setting to show its feasibility in future imaging experiments.

Laccase-Mediated Grafting on Biopolymers and Synthetic Polymers: A Critical Review.

Laccase-mediated grafting on lignocelluloses has gained considerable attention as an environmentally benign method to covalently modify wood, paper and cork. In recent decades this technique has also been employed to modify fibres with a polysaccharide backbone, such as cellulose or chitosan, to infer colouration, antimicrobial activity or antioxidant activity to the material. The scope of this approach has been further widened by researchers, who apply mediators or high redox potential laccases and those that modify synthetic polymers and proteins. In all cases, the methodology relies on one- or two-electron oxidation of the surface functional groups or of the graftable molecule in solution. However, similar results can very often be achieved through simple deposition, even after extensive washing. This unintended adsorption of the active substance could have an adverse effect on the durability of the applied coating. Differentiating between actual covalent binding and adsorption is therefore essential, but proves to be challenging. This review not only covers excellent research on the topic of laccase-mediated grafting over the last five to ten years, but also provides a critical comparison to highlight either the lack or presence of compelling evidence for covalent grafting.

Elucidating the mechanism behind the laccase-mediated modification of poly(ethersulfone).

Laccase-mediated oligomerisation of 4-hydroxybenzoic acid (4-HBA) derivatives and simultaneous in situ surface modification has proven to be a cost-effective, easily applicable and eco-friendly strategy for preventing biofouling of poly(ethersulfone) (PES) water filtration membranes. Modification of the membrane surface has previously been hypothesised to occur through covalent bonding of enzymatically generated phenolic radicals to the polymeric membrane. The current study shows, however, that in situ formation of soluble phenolic oligomers does not result in covalent membrane modification. We studied in situ laccase-mediated oligomerisation of custom-synthesised positively charged and commercially available negatively charged monomeric phenols, and demonstrated that their mode of binding to PES is not covalent. In addition, soluble, non-soluble and on-resin PES model compounds were synthesised and used in the laccase-mediated oligomerisation of 4-HBA. Covalent bond formation between these model compounds and (oligomeric) 4-HBA could not be observed either. Furthermore, extensive washing of PES membranes modified through laccase-mediated oligomerisation of 4-HBA resulted in substantial discolouration of the membrane surface, showing that the layer of oligomerised phenolics could easily be removed. Altogether, it was concluded that laccase-assisted modification of PES membranes resulted from strong physical adsorption of phenolic oligomers and polymers rather than from covalent bonding of those.

Identification of a drimenol synthase and drimenol oxidase from Persicaria hydropiper, involved in the biosynthesis of insect deterrent drimanes.

The sesquiterpenoid polygodial, which belongs to the drimane family, has been shown to be an antifeedant for a number of herbivorous insects. It is presumed to be synthesized from farnesyl diphosphate via drimenol, subsequent C-12 hydroxylation and further oxidations at both C-11 and C-12 to form a dialdehyde. Here, we have identified a drimenol synthase (PhDS) and a cytochrome P450 drimenol oxidase (PhDOX1) from Persicaria hydropiper. Expression of PhDS in yeast and plants resulted in production of drimenol alone. Co-expression of PhDS with PhDOX1 in yeast yielded drimendiol, the 12-hydroxylation product of drimenol, as a major product, and cinnamolide. When PhDS and PhDOX1 were transiently expressed by agro-infiltration in Nicotiana benthamiana leaves, drimenol was almost completely converted into cinnamolide and several additional drimenol derivatives were observed. In vitro assays showed that PhDOX1 only catalyses the conversion from drimenol to drimendiol, and not the further oxidation into an aldehyde. In yeast and heterologous plant hosts, the C-12 position of drimendiol is therefore likely to be further oxidized by endogenous enzymes into an aldehyde and subsequently converted to cinnamolide, presumably by spontaneous hemiacetal formation with the C-11 hydroxyl group followed by oxidation. Purified cinnamolide was confirmed by NMR and shown to be deterrent with an effective deterrent dose (ED50 ) of about 200-400 µg g-1 fresh weight against both whiteflies and aphids. The putative additional physiological and biochemical requirements for polygodial biosynthesis and stable storage in plant tissues are discussed.

Ambient Characterization of Synthetic Fibers by Laser Ablation Electrospray Ionization Mass Spectrometry.

Direct analysis of synthetic fibers under ambient conditions is highly desired to identify the polymer, the finishes applied and irregularities that may compromise its performance and value. In this paper, laser ablation electrospray ionization ion mobility time-of-flight mass spectrometry (LAESI-IMS-TOF-MS) was used for the analysis of synthetic polymers and fibers. The key to this analysis was the absorption of laser light by aliphatic and aromatic nitrogen functionalities in the polymers. Analysis of polyamide (PA) 6, 46, 66, and 12 pellets and PA 6, 66, polyaramid and M5 fibers yielded characteristic fragment ions without any sample pretreatment, enabling their unambiguous identification. Synthetic fibers are, in addition, commonly covered with a surface layer for improved adhesion and processing. The same setup, but operated in a transient infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mode, allowed the detailed characterization of the fiber finish layer and the underlying polymer. Differences in finish layer distribution may cause variations in local properties of synthetic fibers. Here we also show the feasibility of mass spectrometry imaging (MSI) of the distribution of a finish layer on the synthetic fiber and the successful detection of local surface defects.

Covalent attachment of 1-alkenes to oxidized platinum surfaces.

We report the formation of covalently bound alkyl layers onto oxidized Pt (PtOx) substrates by reaction with 1-alkenes as a novel way to bind organic molecules to metal surfaces. The organic layers were characterized by static contact angle, infrared reflection absorption spectroscopy (IRRAS), X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). The grafted alkyl layers display a hydrolytic stability that is comparable to that of alkyl thiols on Au. PtOx-alkene attachment is compatible with terminal ester moieties enabling further anchoring of functional groups, such as redox-active ferrocene, and thus has great potential to extend monolayer chemistry on noble metals.

Microwave-assisted formation of organic monolayers from 1-alkenes on silicon carbide.

The rate of formation of covalently linked organic monolayers on HF-etched silicon carbide (SiC) is greatly increased by microwave irradiation. Upon microwave treatment for 60 min at 100 °C (60 W), 1-alkenes yield densely packed, covalently attached monolayers on flat SiC surfaces, a process that typically takes 16 h at 130 °C under thermal conditions. This approach was extended to SiC microparticles. The monolayers were characterized by X-ray photoelectron spectroscopy and static water contact angle measurements. The microwave-assisted reaction is compatible with terminal functionalities such as alkenes that enable subsequent versatile "click" chemistry reactions, further broadening the range and applicability of chemically modified SiC surfaces.

DHAP-dependent aldolases from (hyper)thermophiles: biochemistry and applications.

Generating new carbon-carbon (C-C) bonds in an enantioselective way is one of the big challenges in organic synthesis. Aldolases are a natural tool for stereoselective C-C bond formation in a green and sustainable way. This review will focus on thermophilic aldolases in general and on dihydroxyacetone phosphate-dependent aldolases in particular. Biochemical properties and applications for synthesis of rare sugars and carbohydrates will be discussed.

Biosynthesis of sesquiterpene lactones in pyrethrum (Tanacetum cinerariifolium).

The daisy-like flowers of pyrethrum (Tanacetum cinerariifolium) are used to extract pyrethrins, a botanical insecticide with a long history of safe and effective use. Pyrethrum flowers also contain other potential defense compounds, particularly sesquiterpene lactones (STLs), which represent problematic allergenic residues in the extracts that are removed by the pyrethrum industry. The STLs are stored in glandular trichomes present on the pyrethrum achenes, and have been shown to be active against herbivores, micro-organisms and in the below-ground competition with other plants. Despite these reported bioactivities and industrial significance, the biosynthetic origin of pyrethrum sesquiterpene lactones remains unknown. In the present study, we show that germacratrien-12-oic acid is most likely the central precursor for all sesquiterpene lactones present in pyrethrum. The formation of the lactone ring depends on the regio- (C6 or C8) and stereo-selective (α or ß) hydroxylation of germacratrien-12-oic acid. Candidate genes implicated in three committed steps leading from farnesyl diphosphate to STL and other oxygenated derivatives of germacratrien-12-oic acid were retrieved from a pyrethrum trichome EST library, cloned, and characterized in yeast and in planta. The diversity and distribution of sesquiterpene lactones in different tissues and the correlation with the expression of these genes are shown and discussed.

Immobilised enzymes in biorenewables production.

Oils, fats, carbohydrates, lignin, and amino acids are all important raw materials for the production of biorenewables. These compounds already play an important role in everyday life in the form of wood, fabrics, starch, paper and rubber. Enzymatic reactions do, in principle, allow the transformation of these raw materials into biorenewables under mild and sustainable conditions. There are a few examples of processes using immobilised enzymes that are already applied on an industrial scale, such as the production of High-Fructose Corn Syrup, but these are still rather rare. Fortunately, there is a rapid expansion in the research efforts that try to improve this, driven by a combination of economic and ecological reasons. This review focusses on those efforts, by looking at attempts to use fatty acids, carbohydrates, proteins and lignin (and their building blocks), as substrates in the synthesis of biorenewables using immobilised enzymes. Therefore, many examples (390 references) from the recent literature are discussed, in which we look both at the specific reactions as well as to the methods of immobilisation of the enzymes, as the latter are shown to be a crucial factor with respect to stability and reuse. The applications of the renewables produced in this way range from building blocks for the pharmaceutical and polymer industry, transport fuels, to additives for the food industry. A critical evaluation of the relevant factors that need to be improved for large-scale use of these examples is presented in the outlook of this review.

Lipase-catalyzed aza-Michael reaction on acrylate derivatives.

A methodology has been developed for an efficient and selective lipase-catalyzed aza-Michael reaction of various amines (primary and secondary) with a series of acrylates and alkylacrylates. Reaction parameters were tuned, and under the optimal conditions it was found that Pseudomonas stutzeri lipase and Chromobacterium viscosum lipase showed the highest selectivity for the aza-Michael addition to substituted alkyl acrylates. For the first time also, some CLEAs were examined that showed a comparable or higher selectivity and yield than the free enzymes and other formulations.

Enzyme-catalyzed modification of PES surfaces: reduction in adsorption of BSA, dextrin and tannin.

Poly(ethersulfone) (PES) can be modified in a flexible manner using mild, environmentally benign components such as 4-hydroxybenzoic acid and gallic acid, which can be attached to the surface via catalysis by the enzyme laccase. This leads to grafting of mostly linear polymeric chains (for 4-hydroxybenzoic acid, and for gallic acid at low concentration and short modification time) and of networks (for gallic acid at high concentration and long exposure time). The reaction is stopped at a specific time, and the modified surfaces are tested for adsorption of BSA, dextrin and tannin using in-situ reflectometry and AFM imaging. At short modification times, the adsorption of BSA, dextrin and tannin is significantly reduced. However, at longer modification times, the adsorption increases again for both substrates. As the contact angle on modified surfaces at short modification times is reduced (indicative of more hydrophilic surfaces), and keeps the same low values at longer modification times, hydrophilicity is not the only determining factor for the measured differences. At longer modification times, intra-layer reactivity will increase the amount of cross-linking (especially for gallic acid), branching (for 4-hydroxybenzoic acid) and/or collapse of the polymer chains. This leads to more compact layers, which leads to increased protein adsorption. The modifications were shown to have clear potential for reduction of fouling by proteins, polysaccharides, and polyphenols, which could be related to the surface morphology.