Inhibition of the key enzyme of sialic acid biosynthesis by C6-Se modified N-acetylmannosamine analogs.

Synthetically accessible C6-analogs of N-acetylmannosamine (ManNAc) were tested as potential inhibitors of the bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE/MNK), the key enzyme of sialic acid biosynthesis. Enzymatic experiments revealed that the modification introduced at the C6 saccharide position strongly influences the inhibitory potency. A C6-ManNAc diselenide dimer showed the strongest kinase inhibition in the low µM range among all the substrates tested and successfully reduced cell surface sialylation in Jurkat cells.

Applying Acylated Fucose Analogues to Metabolic Glycoengineering.

Manipulations of cell surface glycosylation or glycan decoration of selected proteins hold immense potential for exploring structure-activity relations or increasing glycoprotein quality. Metabolic glycoengineering describes the strategy where exogenously supplied sugar analogues intercept biosynthetic pathways and are incorporated into glycoconjugates. Low membrane permeability, which so far limited the large-scale adaption of this technology, can be addressed by the introduction of acylated monosaccharides. In this work, we investigated tetra-O-acetylated, -propanoylated and -polyethylene glycol (PEG)ylated fucoses. Concentrations of up to 500 µM had no substantial effects on viability and recombinant glycoprotein production of human embryonic kidney (HEK)-293T cells. Analogues applied to an engineered Chinese hamster ovary (CHO) cell line with blocked fucose de novo synthesis revealed an increase in cell surface and recombinant antibody fucosylation as proved by lectin blotting, mass spectrometry and monosaccharide analysis. Significant fucose incorporation was achieved for tetra-O-acetylated and -propanoylated fucoses already at 20 µM. Sequential fucosylation of the recombinant glycoprotein, achieved by the application of increasing concentrations of PEGylated fucose up to 70 µM, correlated with a reduced antibody's binding activity in a Fcγ receptor IIIa (FcγRIIIa) binding assay. Our results provide further insights to modulate fucosylation by exploiting the salvage pathway via metabolic glycoengineering.

Chemoselective Staudinger-phosphite reaction of symmetrical glycosyl-phosphites with azido-peptides and polygycerols.

In this paper we present the synthesis of glyco-phosphoramidate conjugates as easily accessible analogs of glyco-phosphorous esters via the Staudinger-phosphite reaction. This protocol takes advantage of synthetically accessible symmetrical carbohydrate phosphites in good overall yields, in which ethylene or propylene linkers can be introduced between phosphorous and galactose or lactose moieties. The phosphites were finally applied for the chemoselective reaction with azido-peptides and polyazido(poly)glycerols.

Site-specific modification of proteins by the staudinger-phosphite reaction.

Chemoselective reactions are important tools for the modification of peptides and proteins. Thereby the modification is desired to be site specific and bioorthogonal. Here we describe the site-specific modification of azido-proteins via a Staudinger-type phosphite ligation. The reaction was carried out in aqueous system on proteins containing p-azido-phenylalanine in a single position introduced by the amber codon technique. A selective introduction of branched polyethylene scaffolds can be achieved with the application of the methodology reported herein.

Efficient metabolic oligosaccharide engineering of glycoproteins by UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) knock-down.

Improving the accessibility and functions of therapeutic and diagnostic glycoproteins is one of the major goals of glycobiotechnology. Here we present that stable knock-down of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE), the key enzyme in the sialic acid biosynthetic pathway, dramatically increases incorporation of N-acetylmannosamine analogues into glycoproteins of HEK293 cells. By means of these GNE-deficient cells highly sialylated glycoproteins can efficiently be decorated with reactive functional groups, which can be employed in bioorthogonal functionalization strategies for fluorescence labelling or biotinylation.

Site-specific functionalisation of proteins by a Staudinger-type reaction using unsymmetrical phosphites.

Unsymmetrical phosphites react efficiently in a Staudinger reaction with p-azido-phenylalanine, which can be applied for the biotinylation of proteins, thereby expanding the scope of the chemoselective Staudinger-phosphite reaction of aryl azides with symmetrical phosphites to the corresponding phosphoramidates.

The gas-phase chemistry of cis-diammineplatinum(II) complexes: a joint experimental and theoretical study.

Prior to reactions with DNA, the anticancer drug cisplatin [Pt(II)(NH(3))(2)Cl(2)] forms a series of solvolysis intermediates by successive replacement of the chloro ligands by water or hydroxyl groups. The bonding of water to Pt(II) is weak, and it is easily substituted by donor ligands present in the solution, for example, amines or alcohols. We studied such compounds using high-resolution electrospray mass spectrometry with a linear ion trap and DFT computations. This combination allows for the first time a detailed description of the reactions initiated by the central atom of the complexes. Positively charged cisplatin adducts with primary and secondary alcohols ([Pt(II)(NH(3))(2)(ROH)Cl](+)) show unexpected reactions when fragmented in a linear ion trap. Either water loss is accompanied by formation of the corresponding carbene complex, or loss of the corresponding aldehyde/ketone leads to the formation of the complex [Pt(NH(3))(2)(H(2))Cl](+). Complete loss of the alcohol ligand is not observed for kinetic reasons. A detailed investigation by DFT and molecular dynamics for the cisplatin/methanol complex [Pt(II)(NH(3))(2)(CH(3)OH)Cl](+) allowed identification of the reaction mechanisms leading to the observed fragmentation patterns. The initial step for both fragmentation pathways is activation of the alpha-CH bond and subsequent H transfer within the complex. Direct activation of the OH or CO bond is less favorable. Ligands bound to the Pt(II) center such as the chloro ligand can directly catalyze the reaction by intermediate binding of H atoms. Upon collision activation, adducts without an alpha-H atom such as [Pt(NH(3))(2){(CH(3))(3)COH}Cl](+) show loss of water or the corresponding alkene.

A concise synthesis of (-)-centrolobine via a diastereoselective ring rearrangement metathesis-isomerisation sequence.

A total synthesis of (-)-centrolobine (1) based on a diastereoselective ring rearrangement metathesis-double bond isomerisation sequence and a one-pot cross metathesis-hydrogenation procedure is described.