Novel Dextran-Supported Biological Probes Decorated with Disaccharide Entities for Investigating the Carbohydrate–Protein Interactions of Gal-3

The quest for novel natural-like biomolecular probes that can be used to gain information on biological recognition events is of topical interest to several scientific areas. In particular, the recognition of carbohydrates by proteins modulates a number of important biological processes. These molecular recognition events are, however, difficult to study by the use of naturally occurring oligosaccharides and polysaccharides owing to their intrinsic structural heterogeneity and to the many technical difficulties encountered during the isolation of sufficient quantities of pure material for detailed structural and biological studies. Therefore, the construction of homogenous biomolecular probes that can mimic both the biophysical properties of polysaccharide backbones and the properties of bioactive oligosaccharide fragments are highly sought after. Herein, synthetic methodology for the construction of well-defined bioconjugates consisting of biologically relevant disaccharide fragments grafted onto a dextran backbone is presented, and a preliminary NMR spectroscopy study of their interactions with galectin-3 as a model lectin is conducted.Peer reviewe

Single cell mutant selection for metabolic engineering of actinomycetes

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying highperforming individual cells from a mutant population directly from liquid cultures. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g-1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l- 1 in a second round of mutant selection. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.Peer reviewe

Synthesis of an Azide- and Tetrazine-Functionalized [60]Fullerene and Its Controlled Decoration with Biomolecules

Bingel cyclopropanation between Buckminster fullerene and a heteroarmed malonate was utilized to produce a hexakis-functionalized C-60 core, with azide and tetrazine units. This orthogonally bifunctional C-60 scaffold can be selectively one-pot functionalized by two pericyclic click reactions, that is, inverse electron-demand Diels-Alder and azide-alkyne cycloaddition, which with appropriate ligands (monosaccharides, a peptide and oligonucleotides tested) allows one to control the assembly of heteroantennary bioconjugates.Peer reviewe

Acetyl group migration across the saccharide units in oligomannoside model compound

Acetylated oligosaccharides are common in nature. While they are involved in several biochemical and biological processes, the role of the acetyl groups and the complexity of their migration has largely gone unnoticed. In this work, by combination of organic synthesis, NMR spectroscopy and quantum chemical modeling, we show that acetyl group migration is a much more complex phenomenon than previously known. By use of synthetic oligomannoside model compounds, we demonstrate, for the first time, that the migration of acetyl groups in oligosaccharides and polysaccharides may not be limited to transfer within a single monosaccharide moiety, but may also involve migration over a glycosidic bond between two different saccharide units. The observed phenomenon is not only interesting from the chemical point of view, but it also raises new questions about the potential biological role of acylated carbohydrates in nature.Peer reviewe

Novel Dextran-Supported Biological Probes Decorated with Disaccharide Entities for Investigating the Carbohydrate–Protein Interactions of Gal-3

The quest for novel natural‐like biomolecular probes that can be used to gain information on biological recognition events is of topical interest to several scientific areas. In particular, the recognition of carbohydrates by proteins modulates a number of important biological processes. These molecular recognition events are, however, difficult to study by the use of naturally occurring oligosaccharides and polysaccharides owing to their intrinsic structural heterogeneity and to the many technical difficulties encountered during the isolation of sufficient quantities of pure material for detailed structural and biological studies. Therefore, the construction of homogenous biomolecular probes that can mimic both the biophysical properties of polysaccharide backbones and the properties of bioactive oligosaccharide fragments are highly sought after. Herein, synthetic methodology for the construction of well‐defined bioconjugates consisting of biologically relevant disaccharide fragments grafted onto a dextran backbone is presented, and a preliminary NMR spectroscopy study of their interactions with galectin‐3 as a model lectin is conducted.</p

Single cell mutant selection for metabolic engineering of actinomycetes

Actinomycetes are important producers of pharmaceuticals and industrial enzymes. However, wild type strains require laborious development prior to industrial usage. Here we present a generally applicable reporter-guided metabolic engineering tool based on random mutagenesis, selective pressure, and single-cell sorting. We developed fluorescence-activated cell sorting (FACS) methodology capable of reproducibly identifying high-performing individual cells from a mutant population directly from liquid cultures. Actinomycetes are an important source of catabolic enzymes, where product yields determine industrial viability. We demonstrate 5-fold yield improvement with an industrial cholesterol oxidase ChoD producer Streptomyces lavendulae to 20.4 U g−1 in three rounds. Strain development is traditionally followed by production medium optimization, which is a time-consuming multi-parameter problem that may require hard to source ingredients. Ultra-high throughput screening allowed us to circumvent medium optimization and we identified high ChoD yield production strains directly from mutant libraries grown under preset culture conditions. Genome-mining based drug discovery is a promising source of bioactive compounds, which is complicated by the observation that target metabolic pathways may be silent under laboratory conditions. We demonstrate our technology for drug discovery by activating a silent mutaxanthene metabolic pathway in Amycolatopsis. We apply the method for industrial strain development and increase mutaxanthene yields 9-fold to 99 mg l−1 in a second round of mutant selection. In summary, the ability to screen tens of millions of mutants in a single cell format offers broad applicability for metabolic engineering of actinomycetes for activation of silent metabolic pathways and to increase yields of proteins and natural products.</p

Synthesis of an Azide- and Tetrazine-Functionalized [60]Fullerene and Its Controlled Decoration with Biomolecules

Bingel cyclopropanation between Buckminster fullerene and a heteroarmed malonate was utilized to produce a hexakis-functionalized C60 core, with azide and tetrazine units. This orthogonally bifunctional C60 scaffold can be selectively one-pot functionalized by two pericyclic click reactions, that is, inverse electron-demand Diels-Alder and azide-alkyne cycloaddition, which with appropriate ligands (monosaccharides, a peptide and oligonucleotides tested) allows one to control the assembly of heteroantennary bioconjugates.</p

Controlled Monofunctionalization of Molecular Spherical Nucleic Acids on a Buckminster Fullerene Core

An azide-functionalized 12-armed Buckminster fullerene has been monosubstituted in organic media with a substoichiometric amount of cyclooctyne-modified oligonucleo-tides. Exposing the intermediate products then to the same reaction (i. e., strain-promoted alkyne-azide cycloaddition, SPAAC) with an excess of slightly different oligonucleotide constituents in an aqueous medium yields molecularly defined monofunctionalized spherical nucleic acids (SNAs). This procedure offers a controlled synthesis scheme in which one oligonucleotide arm can be functionalized with labels or other conjugate groups (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, and Alexa-488 demonstrated), whereas the rest of the 11 arms can be left unmodified or modified by other conjugate groups in order to decorate the SNAs' outer sphere. Extra attention has been paid to the homogeneity and authenticity of the C60-azide scaffold used for the assembly of full-armed SNAs

Multivalency in Carbohydrate Chemistry : From Oligosaccharides to Oligovalency and Beyond

Carbohydrates are among the most abundant biomolecules on earth and can be found in all living things. Their role in nature ranges from structural components such as cellulose in plants and chitin in the exoskeletons of arthropods, to fundamental building blocks of DNA and RNA, to the most important energy storage molecules in organisms in the form of starch and glycogen. The practically unlimited ways in which individual monosaccharides can be connected make the information storage potential of this class of compounds exponentially greater than that of DNA or proteins. This property does, however, also result in some complications as, while the potential for storing information in carbohydrate structures is great, the process of doing so and reading the information back is complicated by this exact same phenomenon. A majority of the early carbohydrate investigations focused on the elucidation of the structures of carbohydrates and the pioneering work in this field was done by Emil Fischer at the end of the 19th century. Later the focus shifted towards creating glycosidic linkages, and over the years, countless methods have been developed allowing for the preparation of complex structures. The advances in the field have enabled the investigation of the biological roles of this class of compounds. This thesis explores the world of carbohydrates starting from small oligosaccharides, expanding towards multivalent structures and finally into modification of polysaccharides for preparing well-defined mimics of natural polysaccharides. Potential biological applicability is always a consideration when designing the compound and a recurring theme throughout the thesis are β-(1→2) linked mannosides which are an interesting class of compounds due to their biological impact, their unique structure and problems associated with their synthesis. Particular attention has been paid to the analysis of products by NMR spectroscopy and complete assignment of the spectra. Where feasible, the spectra were simulated by NMR simulation software PERCH to allow a more complete interpretation