Sesquiterpene Cyclase BcBOT2 Promotes the Unprecedented Wagner-Meerwein Rearrangement of the Methoxy Group.

Presilphiperfolan-8ß-ol synthase (BcBOT2), a substrate-promiscuous sesquiterpene cyclase (STC) of fungal origin, is capable of converting two new farnesyl pyrophosphate (FPP) derivatives modified at C7 of farnesyl pyrophosphate (FPP) bearing either a hydroxymethyl group or a methoxymethyl group. These substrates were chosen based on a computationally generated model. Biotransformations yielded five new oxygenated terpenoids. Remarkably, the formation of one of these tricyclic products can only be explained by a cationically induced migration of the methoxy group, presumably via a Meerwein-salt intermediate, unprecedented in synthetic chemistry and biosynthesis. The results show the great principle and general potential of terpene cyclases for mechanistic studies of unusual cation chemistry and for the creation of new terpene skeletons.

Dextrans, Pullulan and Lentinan, New Scaffold Materials for Use as Hydrogels in Tissue Engineering.

The development of hydrogels based on dextrans, pullulan and lentinan to be used in biomedical applications including tissue engineering is reported. Despite the fact that selected polysaccharides such as hyaluronic acid are well established, little is known, how these polysaccharides can be chemically modified to create hydrogels under controlled conditions. In this study we present a small library of chemically modified polysaccharides which are used for a divergent approach to achieve biomedical relevant hydrogels. In this case the crosslinking is based on thio ether formation between thiol modified donor and vinylsulfone or maleimide modified acceptor components. Successful synthesis of the linker systems and coupling at the polysaccharides, hydrogel formation takes place under physiological conditions. We extended the study by coupling small molecules like adhesion factors for increasing cell compatibility as well as a dye for further studies. The different hydrogels were studied to their rheological properties, water uptake, their permeability, biodegrability and their cytotoxicity.

3D-Printed Microfluidic Perfusion System for Parallel Monitoring of Hydrogel-Embedded Cell Cultures.

The use of three-dimensional (3D) cell cultures has become increasingly popular in the contexts of drug discovery, disease modelling, and tissue engineering, as they aim to replicate in vivo-like conditions. To achieve this, new hydrogels are being developed to mimic the extracellular matrix. Testing the ability of these hydrogels is crucial, and the presented 3D-printed microfluidic perfusion system offers a novel solution for the parallel cultivation and evaluation of four separate 3D cell cultures. This system enables easy microscopic monitoring of the hydrogel-embedded cells and significantly reduces the required volumes of hydrogel and cell suspension. This cultivation device is comprised of two 3D-printed parts, which provide four cell-containing hydrogel chambers and the associated perfusion medium chambers. An interfacing porous membrane ensures a defined hydrogel thickness and prevents flow-induced hydrogel detachment. Integrated microfluidic channels connect the perfusion chambers to the overall perfusion system, which can be operated in a standard CO2-incubator. A 3D-printed adapter ensures the compatibility of the cultivation device with standard imaging systems. Cultivation and cell staining experiments with hydrogel-embedded murine fibroblasts confirmed that cell morphology, viability, and growth inside this cultivation device are comparable with those observed within standard 96-well plates. Due to the high degree of customization offered by additive manufacturing, this system has great potential to be used as a customizable platform for 3D cell culture applications.

Cyclopropylmethyldiphosphates are substrates for sesquiterpene synthases: experimental and theoretical results.

New homo-sesquiterpenes are accessible after conversion of presilphiperfolan-8ß-ol synthase (BcBOT2) with cyclopropylmethyl analogs of farnesyl diphosphate, and this biotransformation is dependent on subtle structural refinements. Two of the three cyclisation products are homo variants of germacrene D and germacrene D-4-ol while the third product reported contains a new bicyclic backbone for which no analogue in nature has been described so far. The findings on diphosphate activation are discussed and rationalised by relaxed force constants and dissociation energies computed at the DFT level of theory.

PvdM of fluorescent pseudomonads is required for the oxidation of ferribactin by PvdP in periplasmic pyoverdine maturation.

Fluorescent pseudomonads such as Pseudomonas aeruginosa or Pseudomonas fluorescens produce pyoverdine siderophores that ensure iron-supply in iron-limited environments. After its synthesis in the cytoplasm, the nonfluorescent pyoverdine precursor ferribactin is exported into the periplasm, where the enzymes PvdQ, PvdP, PvdO, PvdN, and PtaA are responsible for fluorophore maturation and tailoring steps. While the roles of all these enzymes are clear, little is known about the role of PvdM, a human renal dipeptidase-related protein that is predicted to be periplasmic and that is essential for pyoverdine biogenesis. Here, we reveal the subcellular localization and functional role of PvdM. Using the model organism P. fluorescens, we show that PvdM is anchored to the periplasmic side of the cytoplasmic membrane, where it is indispensable for the activity of the tyrosinase PvdP. While PvdM does not share the metallopeptidase function of renal dipeptidase, it still has the corresponding peptide-binding site. The substrate of PvdP, deacylated ferribactin, is secreted by a ΔpvdM mutant strain, indicating that PvdM prevents loss of this periplasmic biosynthesis intermediate into the medium by ensuring the efficient transfer of ferribactin to PvdP in vivo. We propose that PvdM belongs to a new dipeptidase-related protein subfamily with inactivated Zn2+ coordination sites, members of which are usually genetically linked to TonB-dependent uptake systems and often associated with periplasmic FAD-dependent oxidoreductases related to d-amino acid oxidases. We suggest that these proteins are necessary for selective binding, exposure, or transfer of specific d- and l-amino acid-containing peptides and other periplasmic biomolecules in manifold pathways.

Identification of Major Constituents of Hypericum perforatum L. Extracts in Syria by Development of a Rapid, Simple, and Reproducible HPLC-ESI-Q-TOF MS Analysis and Their Antioxidant Activities.

Hypericum perforatum Linn (St. John's wort) is a popular and widespread medicine in Syria, which is used for a wide range of conditions, including gastrointestinal diseases, heart disease, skin diseases, and psychological disorders. This widespread use prompted us to identify the main compounds of this plant from Syria that are responsible for its medicinal properties, especially since its components differ between countries according to the nature of the soil, climate, and altitude. This is, to the best of our knowledge, the first report in which St. John's wort, a plant native to Syria, is extracted using different solvents and its most important compounds are identified. In this study, the dried above-ground parts, i.e., leaves, stem, petals, and flowers, were extracted using different solvents (water, ethanol, methanol, and acetone) and extraction protocols. By increasing the polarity of the solvent, higher yields were obtained, indicating that mainly hydrophobic compounds were extracted. Therefore, we conclude that extraction using the tea method or using a mixture of water and organic solvents resulted in higher yields compared with pure organic solvents or continuous boiling with water for long periods. The obtained extracts were analyzed using high-performance liquid chromatography equipped with a diode array detector (HPLC-DAD), coupled with UV-visible spectrophotometry at a full spectrum (200-800 nm). The HPLC spectra of the extracts were almost identical at three wavelengths (260 nm for phloroglucinols (hyperforin and derivates), 590 nm for naphthodianthrones (hypericins), and 350 nm for other flavonols, flavones, and caffeoylquinic acids), with differences observed only in the intensity of the peaks. This indicates that the same compounds were obtained using different solvents, but in different amounts. Five standards (chlorogenic acid, quercetin, quercitrin hydrate, hyperoside, and hypericin) were used, and a comparison with retention times and ultraviolet (UV) spectra reported in the literature was performed to identify 10 compounds in these extracts: hyperforin, adhyperforin, hypericin, rutin, quercetin, quercitrin, quercitrin hydrate, hyperoside, biapigenin, and chlorogenic acid. Although the European Pharmacopoeia still describes ultraviolet spectroscopy as a method for determining the quantity of Hyperici herba, interference from other metabolites can occur. Combined HPLC-DAD and electrospray ionization-mass spectrometry (LC-ESI-MS) in the positive mode have therefore also been used to confirm the presence of these compounds in the extracts by correlating known masses with the identified masses or through characteristic fragmentation patterns. Total phenolic contents of the extracts were determined by the Folin-Ciocalteu assay, and antioxidant activity was evaluated as free radical scavenging capacity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The results indicate that the aqueous extracts prepared by the tea method gave the highest total phenols, while the pure organic solvents gave very low phenols. Also, the extracts that contain the largest amount of phenols gave lower IC50 values or higher antioxidant activity than that of others.

Bioaccessibility and transepithelial transportation of cranberrybush (Viburnum opulus) phenolics: Effects of non-thermal processing and food matrix.

The present study investigated the effects of non-thermal treatments and food matrix on the bioaccessibility and transepithelial transportation of phenolics from cranberrybush. High pressure processing (HPP) was applied at 600 MPa pressure for 5 min, whereas pulsed electric field (PEF) conditions were selected as 5 (PEF5) or 15 kJ/kg (PEF15). To reveal the influence of food matrix, cranberrybush juice was blended with bovine or almond milk. Results showed that PEF15 treatment enhanced the recovery of total flavonoids (TFC; increase of 3.9% ± 1.1), chlorogenic acid (increase of 29.9% ± 5.9) and antioxidant capacity after gastrointestinal digestion. The addition of bovine milk affect posivitely the bioaccessibility of total phenolics (TPC), TFC and antioxidant capacity. While untreated and treated samples exhibit comparable transportation across the epithelial cell layer, juice-bovine milk (JM) and juice-almond milk (JA) blends increased the transport efficiency of chlorogenic acid by 3.5% ± 0.8 and 3.3% ± 0.5, respectively.

RGD-Modified Titanium as an Improved Osteoinductive Biomaterial for Use in Dental and Orthopedic Implants.

This study describes the synthesis, surface analysis, and biological evaluation of bioactive titanium surfaces. The aim was to achieve an improved effect on osteoinduction in dental and orthopedic implants. For this purpose, a chemistry was developed, which allows to bind the bioactive cyclopeptide cRGDfK covalently to biomedically used titanium via polyethylene glycol linkers of different lengths. The chemical process is practicable, robust, and metal-free. The resulting chemically modified titanium plates show improved osteoinductive properties. The modification with cRGDfK targets the integrin αvß3, which is highly expressed in osteoblasts and is essential for many basic functions in the development of bone tissue. The successful immobilization of cRGDfK on titanium surfaces has been demonstrated by contact angle measurements and X-ray photoelectron spectroscopy. We show in in vitro studies that the presence of the cRGDfK peptide on titanium surfaces has a positive effect on bone formation.

Retention of polyphenols and vitamin C in cranberrybush purée (Viburnum opulus) by means of non-thermal treatments.

The effects of high pressure processing (HPP; 200-600 MPa for 5 or 15 min) and pulsed electric field (PEF; 3 kV/cm, 5-15 kJ/kg) treatment on physicochemical properties (conductivity, pH and total soluble solids content), bioactive compounds (vitamin C, total phenolic (TPC), total flavonoid (TFC), total anthocyanin (TAC) and chlorogenic acid contents), antioxidant capacities (DPPH and CUPRAC assays) and polyphenol oxidase (PPO) activity of cranberrybush purée were evaluated immediately after processing. The results were compared to an untreated purée. According to the results, conductivity increased significantly after PEF (15 kJ/kg) treatment. PEF and HPP treatments resulted in a better retention of bioactive compounds (increase in TPC in the range of ~4-11% and ~10-14% and TFC in the range of ~1-5% and ~6-8% after HPP and PEF, respectively) and antioxidant activity (as measured with CUPRAC method) compared to untreated sample. HPP reduced residual enzyme activity of PPO comparatively better than PEF.

Dextran-based scaffolds for in-situ hydrogelation: Use for next generation of bioartificial cardiac tissues.

In pursuit of a chemically-defined matrix for in vitro cardiac tissue generation, we present dextran (Dex)-derived hydrogels as matrices suitable for bioartificial cardiac tissues (BCT). The dextran hydrogels were generated in situ by using hydrazone formation as the crosslinking reaction. Material properties were flexibly adjusted, by varying the degrees of derivatization and the molecular weight of dextran used. Furthermore, to modulate dextran's bioactivity, cyclic pentapeptide RGD was coupled to its backbone. BCTs were generated by using a blend of modified dextran and human collagen (hColI) in combination with induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and fibroblasts. These hColI + Dex blends with or without RGD supported tissue formation and functional maturation of CMs. Contraction forces (hColI + Dex-RGD: 0.27 ± 0.02 mN; hColI + Dex: 0.26 ± 0.01 mN) and frequencies were comparable to published constructs. Thus, we could demonstrate that, independent of the presence of RGD, our covalently linked dextran hydrogels are a promising matrix for building cardiac grafts.

Oxidative azidations of phenols and ketones using iodine azide after release from an ion exchange resin.

The oxidative oligoazidation of phenols and ketones using iodine azide (IN3) provided by its release from an ion exchange resin is reported. Preliminary mechanistic studies indicate a previously unknown reactivity of iodine azide toward phenols and ketones.

Methyl-Shifted Farnesyldiphosphate Derivatives Are Substrates for Sesquiterpene Cyclases.

New sesquiterpene backbones are accessible after biotransformation of presilphiperfolan-8ß-ol synthase (BcBOT2), a fungal sesquiterpene synthase, with non-natural farnesyldiphosphates in which methyl groups are shifted by one position toward the diphosphate terminus. One of the macrocycles formed, a new germacrene A derivative, undergoes a Cope rearrangement to iso-ß-elemene. Three of the new terpenoids show olfactoric properties that range from an intense peppery note to a citrus, ozone-like, and fruity scent.

Photochemical Transformations with Iodine Azide after Release from an Ion-Exchange Resin.

This report discloses the photochemical homolytic cleavage of iodine azide after its formation following release from polymer-bound bisazido iodate(I) anions. A series of radical reactions are reported including the 1,2-functionlization of alkenes and the unprecedented chemoselective oxidation of secondary alcohols in the presence of primary alcohols.

Methanol-to-Olefins in a Membrane Reactor with in†situ Steam Removal - The Decisive Role of Coking.

The reaction of methanol to light olefins and water (MTO) was studied in a fixed bed tubular membrane reactor using commercial SAPO-34 catalyst. In the fixed bed reactor without membrane support, the MTO reaction collapsed after 3 h time on stream. However, if the reaction by-product steam is in situ extracted from the reactor through a hydrophilic tubular LTA membrane, the reactor produces long-term stable about 60 % ethene and 10 % propene. It is shown that the reason for the superior performance of the membrane-assisted reactor is not the prevention of catalyst damage caused by steam but the influence of the water removal on the formation of different carbonaceous residues inside the SAPO-34 cages. Catalytically beneficial methylated 1 or 2 ring aromatics have been found in a higher percentage in the MTO reaction with a water removal membrane compared to the MTO reaction without membrane support.

Novel double functional protection of cephalostatin analogues using a gas-free chlorination method.

Herewith, we report on a method that allows to simultaneously protect both the ∆14,15 bond and the carbonyl group of the symmetrical bis-steroidal diketone 2. We found that environmentally friendly and gas-free chlorination is ideally suited to achieve this goal. This method was discovered during our efforts to methoxylate 2 in a solution of dichloromethane and basic methanol in the presence of diacetoxy iodobenzene. Unexpectedly, the ∆14,15 bonds were chlorinated once as well as twice in a statistical manner. Interestingly, the singly dichlorinated desymmetrized product is an ideal precursor for conduction a series of position selective transformations. Importantly, the carbonyl group present in the nonchlorinated hemisphere can be selectively reduced, olefinated or oximated, while the other carbonyl group stays unaltered. A structurally related "monomeric" steroid derivative undergoes ∆14,15 chlorination and 11-position methoxylation under same conditions. These findings represent a powerful entry for preparing new nonsymmetrical cephalostatin derivatives.

Author Correction: Insights into a dual function amide oxidase/macrocyclase from lankacidin biosynthesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

PEGDMA Hydrogels for Cell Adhesion and Optical Waveguiding.

Hydrogels are favored materials in tissue engineering as they can be used to imitate tissues, provide scaffolds, and guide cell behavior. Recent advances in the field of optogenetics have created a need for biocompatible optical waveguides, and hydrogels have been investigated to meet these requirements. However, combining favorable waveguiding characteristics, high biocompatibility, and controllable bioactivity in a single device remains challenging. Here, we investigate the use of poly(ethylene glycol) hydrogels as carriers and illumination systems for in vitro cell culture. We present a comprehensive and reproducible protocol for selective bioactivation of the hydrogels, achieving high proliferation rates and strong cell adhesion on the treated surface. A cell model expressing the photoconvertible fluorescent protein Dendra2 confirmed that light-cell interactions occur at the hydrogel surface. Monte Carlo simulations were performed as a tool to predict the extent of these interactions. This study demonstrates a hydrogel-based waveguiding system for targeted cell stimulation in vitro and potentially in vivo environments.

Toward Chromanes by de Novo Construction of the Benzene Ring.

The work describes three principal Diels-Alder cycloaddition approaches toward chromanes that are designed for the de novo construction of the benzene ring. This study specifically focuses on the potential exploitation in the total synthesis of chromane-bearing natural products such as cebulactam A.

Author Correction: Insights into a dual function amide oxidase/macrocyclase from lankacidin biosynthesis.

In the original version of this Article, the final concentration of riboflavin in the supplemented LB medium for recombinant LkcE expression was incorrectly stated as 1 g L-1 (this was the concentration of the stock solution) and should have read 10-50 mg L-1. This error has been corrected in both the PDF and HTML versions of the Article.

A General Biomimetic Hetero-Diels-Alder Approach to the Core Skeletons of Xenovulene A and the Sterhirsutins A and B.

A biomimetic, regio- and stereoselective approach to the 5,6,11-tricyclic core skeleton of xenovulene A, as well as sterhirsutins A and B, is described. The key steps are a biomimetic inverse-electron-demand hetero-Diels-Alder cycloaddition of α-humulene and a ribose-derived vinyl ketone, followed by acid-catalyzed rearrangement of the 1,3-dioxolane that neighbors the resultant cyclic enol ether.