Acceptor Reactivity in the Total Synthesis of Alginate Fragments Containing α‐L‐Guluronic Acid and β‐D‐Mannuronic Acid

The total synthesis of mixed‐sequence alginate oligosaccharides, featuring both β‐D ‐mannuronic acid (M) and α‐L ‐guluronic acid (G), is reported for the first time. A set of GM, GMG, GMGM, GMGMG, GMGMGM, GMGMGMG, and GMGGMG alginates was assembled using GM building blocks, having a guluronic acid acceptor part and a mannuronic acid donor side to allow the fully stereoselective construction of the cis‐glycosidic linkages. It was found that the nature of the reducing‐end anomeric center, which is ten atoms away from the reacting alcohol group in the key disaccharide acceptor, had a tremendous effect on the efficiency with which the building blocks were united. This chiral center determines the overall shape of the acceptor and it is revealed that the conformational flexibility of the acceptor is an all‐important factor in determining the outcome of a glycosylation reaction.     

Silicon and porous silicon mid-infrared photonic crystals

A 3D silicon micromachining method based on proton beam writing combined with electrochemical anodization of p-type silicon enables fabrication of mid-infrared photonic crystals made of silicon and porous silicon. Here, example structures of silicon 1D and 2D photonic crystals are demonstrated. Progress and problems of fabricating 3D photonic crystals made of silicon are discussed. The strategy of fabricating photonic crystals purely made of porous silicon, and the characterization method of all these mid-infrared structures, are discussed. Due to the flexibility of this fabrication method, photonic devices and integrated photonic circuits may be built on a single chip, for which two 2D silicon photonic crystals with one on top of the other are demonstrated.     

Synthesis and Antibody Binding Studies of Schistosome-Derived Oligo-α-(1-2)-l-Fucosides

Schistosomiasis is caused by blood-dwelling parasitic trematodes of the genus Schistosoma and is classified by the WHO as the second most socioeconomically devastating parasitic disease, second only to malaria. Schistosoma expresses a complex array of glycans as part of glycoproteins and glycolipids that can be targeted by both the adaptive and the innate part of the immune system. Some of these glycans can be used for diagnostic purposes. A subgroup of schistosome glycans is decorated with unique α-(1-2)-fucosides and it has been shown that these often multi-fucosylated fragments are prime targets for antibodies generated during infection. Since these α-(1-2)-fucosides cannot be obtained in sufficient purity from biological sources, we set out to develop an effective route of synthesis towards α-(1-2)-oligofucosides of varying length. Here we describe the exploration of two different approaches, starting from either end of the fucose chains. The oligosaccharides have been attached to gold nanoparticles and used in an enzyme-linked immunosorbent assay ELISA and a microarray format to probe antibody binding. We show that binding to the oligofucosides of antibodies in sera of infected people depends on the length of the oligofucose chains, with the largest glycans showing most binding.     

High-Resolution Bioassay Profiling with Complemented Sensitivity and Resolution for Pancreatic Lipase Inhibitor Screening

How to rapidly and accurately screen bioactive components from complex natural products remains a major challenge. In this study, a screening platform for pancreatic lipase (PL) inhibitors was established by combining magnetic beads-based ligand fishing and high-resolution bioassay profiling. This platform was well validated using a mixture of standard compounds, i.e., (-)- epigallocatechin gallate (EGCG), luteolin and schisandrin. The dose–effect relationship of high-resolution bioassay profiling was demonstrated by the standard mixture with different concentrations for each compound. The screening of PL inhibitors from green tea extract at the concentrations of 0.2, 0.5 and 1.0 mg/mL by independent high-resolution bioassay profiling was performed. After sample pre-treatment by ligand fishing, green tea extract at the concentration of 0.2 mg/mL was specifically enriched and simplified, and consequently screened through the high-resolution bioassay profiling. As a result, three PL inhibitors, i.e., EGCG, (-)-Gallocatechin gallate (GCG) and (-)-Epicatechin gallate (ECG), were rapidly identified from the complex matrix. The established platform proved to be capable of enriching affinity binders and eliminating nonbinders in sample pre-treatment by ligand fishing, which overcame the technical challenges of high-resolution bioassay profiling in the aspects of sensitivity and resolution. Meanwhile, the high-resolution bioassay profiling possesses the ability of direct bioactive assessment, parallel structural analysis and identification after separation. The established platform allowed more accurate and rapid screening of PL inhibitors, which greatly facilitated natural product-based drug screening.     

Development of a sampling and flow injection analysis technique for iron determination in the sea ice environment

A trace metal clean method for sampling and analysis of iron is set up and applied to sea ice and its associated snow, brine, and underlying seawater sampled during the Antarctic expedition “ARISE in the East” (Antarctic Remote Ice Sensing Experiment, AA03-V1, September-October 2003, 64-65°S/112-119°E, RV Aurora Australis). For clean sampling, a non-contaminating electropolished stainless steel ice corer is designed in conjunction with a polyethylene lathe equipped with Ti chisels to remove possibly contaminated outer layers of ice cores. A portable peristaltic pump with clean tubing is used on the ice to sample the underlying seawater (interface ice-water = 0, 1 and 30 m) and sea ice brine from access holes. Considering the extreme range of salinities (1-100) and Fe concentrations (0.1-100 nM) previously observed in similar environments, it is of paramount importance to set up a simple and sensitive Fe analyser adapted to such gradients. We use a flow injection analysis (FIA) technique and successfully demonstrate its capability to measure Fe concentrations directly in the sample without an on-line preconcentration/matrix separation step. We test the sensitivity, accuracy, precision and long-term stability of the analytical procedure. Also we explore and remediate interferences from a suite of other trace elements, such as Ni, Cd, Cr, Mn, Cu, Zn and Co. Analysis of reference materials NASS-5 and CASS-3 gives a good agreement with the certified values. Repeated measurements over a period of 5 months of an “in-house” Antarctic seawater standard yields a concentration of 1.02 ± 0.07 nM (n = 17, 1σ). The detection limit (3σ of the blank) is on average 0.12 nM. We report here results of the Fe distribution in sea ice that are in good agreement with previously published data. To our knowledge, this work provides the first complete profiles of total dissolvable and dissolved Fe in sea ice.     

Enzymatic Crosslinking of Polymer Conjugates is Superior over Ionic or UV Crosslinking for the On‐Chip Production of Cell‐Laden Microgels

Cell‐laden micrometer‐sized hydrogels (microgels) hold great promise for improving high throughput ex‐vivo drug screening and engineering biomimetic tissues. Microfluidics is a powerful tool to produce microgels. However, only a limited amount of biomaterials have been reported to be compatible with on‐chip microgel formation. Moreover, these biomaterials are often associated with mechanical instability, cytotoxicity, and cellular senescence. To resolve this challenge, dextran‐tyramine has been explored as a novel biomaterial for on‐chip microgel formation. In particular, dextran‐tyramine is compared with two commonly used biomaterials, namely, polyethylene‐glycol diacrylate (PEGDA) and alginate, which crosslink through enzymatic reaction, UV polymerization, and ionic interaction, respectively. Human mesenchymal stem cells (hMSCs) encapsulated in dextran‐tyramine microgels demonstrate significantly higher (95%) survival as compared to alginate (81%) and PEGDA (69%). Long‐term cell cultures demonstrate that hMSCs in PEGDA microgels become senescent after 7 d. Alginate microgels dissolve within 7 d due to Ca²⁺ loss. In contrast, dextran‐tyramine based microgels remain stable, sustain hMSCs metabolic activity, and permit for single‐cell level analysis for at least 28 d of culture. In conclusion, enzymatically crosslinking dextran‐tyramine conjugates represent a novel biomaterial class for the on‐chip production of cell‐laden microgels, which possesses unique advantages as compared to the commonly used UV and ionic crosslinking biomaterials.

     

Discovery of a Mosaic‐Like Biosynthetic Assembly Line with a Decarboxylative Off‐Loading Mechanism through a Combination of Genome Mining and Imaging

The biosynthetic gene cluster for the antiplasmodial natural product siphonazole was identified by using a combination of genome mining, imaging, and expression studies in the natural producer Herpetosiphon sp. B060. The siphonazole backbone is assembled from an unusual starter unit from the shikimate pathway that is extended by the action of polyketide synthases and non‐ribosomal peptide synthetases with unusual domain structures, including several split modules and a large number of duplicated domains and domains predicted to be inactive. Product release proceeds through decarboxylation and dehydration independent of the thioesterase SphJ and yields the diene terminus of siphonazole. High variation in terms of codon‐usage within the gene cluster, together with the dislocated domain organization, suggest a recent emergence in evolutionary terms.     

Terpene Cyclases from Social Amoebae

Genome sequences of social amoebae reveal the presence of terpene cyclases (TCs) in these organisms. Two TCs from Dictyostelium discoideum converted farnesyl diphosphate into (2S,3R,6S,9S)‐(?)‐protoillud‐7‐ene and (3S)‐(+)‐asterisca‐2(9),6‐diene. The enzyme mechanisms and EI‐MS fragmentations of the products were studied by labeling experiments.     

Spiroluchuene A Synthase: A Cyclase from Aspergillus luchuensis Forming a Spirotetracyclic Diterpene

The diterpene synthase AlTS was identified from Aspergillus luchuensis. AlTS catalyses the formation of the diterpene hydrocarbon spiroluchuene A, which exhibits a novel skeleton characterised by a spirocyclic ring system. The cyclisation mechanism towards this compound was elucidated through isotopic labelling experiments in conjunction with DFT calculations and metadynamic simulations. The biosynthetic intermediate luchudiene, besides the derivative spiroluchuene B, was captured from an enzyme variant obtained through site-directed mutagenesis. With its 10-membered ring luchudiene is structurally related to germacrenes and can undergo a Cope rearrangement to luchuelemene.