clickECM: Development of a cell-derived extracellular matrix with azide functionalities.

In vitro cultured cells produce a complex extracellular matrix (ECM) that remains intact after decellularization. The biological complexity derived from the variety of distinct ECM molecules makes these matrices ideal candidates for biomaterials. Biomaterials with the ability to guide cell function are a topic of high interest in biomaterial development. However, these matrices lack specific addressable functional groups, which are often required for their use as a biomaterial. Due to the biological complexity of the cell-derived ECM, it is a challenge to incorporate such functional groups without affecting the integrity of the biomolecules within the ECM. The azide-alkyne cycloaddition (click reaction, Huisgen-reaction) is an efficient and specific ligation reaction that is known to be biocompatible when strained alkynes are used to avoid the use of copper (I) as a catalyst. In our work, the ubiquitous modification of a fibroblast cell-derived ECM with azides was achieved through metabolic oligosaccharide engineering by adding the azide-modified monosaccharide Ac4GalNAz (1,3,4,6-tetra-O-acetyl-N-azidoacetylgalactosamine) to the cell culture medium. The resulting azide-modified network remained intact after removing the cells by lysis and the molecular structure of the ECM proteins was unimpaired after a gentle homogenization process. The biological composition was characterized in order to show that the functionalization does not impair the complexity and integrity of the ECM. The azides within this "clickECM" could be accessed by small molecules (such as an alkyne-modified fluorophore) or by surface-bound cyclooctynes to achieve a covalent coating with clickECM. STATEMENT OF SIGNIFICANCE: The clickECM was produced by the incorporation of azide-functionalized sugar analogues into the extracellular glycans of fibroblast cell cultures by metabolic oligosaccharide engineering. By introducing these azide groups into the glycan structures, we enabled this cell-derived ECM for bioorthogonal click reactions. Click chemistry provides extremely specific reactions with high efficiency, high selectivity, and high reaction yields. We could show that the azide functionalities within the clickECM are chemically accessible. Based on our here described clickECM technique it will be possible to create and investigate new clickECM materials with tunable bioactive properties and additional functionalities, which offers a promising approach for basic and applied research in the field of biomaterial science, biomedical applications, and tissue engineering.

Chemoenzymatic synthesis and fluorescent visualization of cell-surface selectin-bound sialyl Lewis x derivatives.

Sialyl Lewis x (sLe(x)) derivatives conjugated to readily visualized molecular labels are useful chemical probes to study selectin-carbohydrate interactions. Localization of the selectins on the surface of leukocytes and activated endothelial cells can be detected through fluorescence of bound selectin ligands. Herein we present a short chemoenzymatic synthesis of a fluorescently labeled bivalent sLe(x) conjugate. The use of an amino-substituted monovalent sLe(x) to obtain fluorescent- and biotin-labeled sLe(x) derivatives is also described. The cell-staining utility of the fluorescent sLe(x) conjugates is demonstrated for a HUVEC cell line expressing E-selectin and for CHO-K1 cells expressing either L- or E-selectin.

Mechanism of human alpha-1,3-fucosyltransferase V: glycosidic cleavage occurs prior to nucleophilic attack.

alpha-1,3-Fucosyltransferase V (FucT V) catalyzes the transfer of 1-fucose from the donor sugar guanosine 5'-diphospho-beta-1-fucose (GDP-Fuc) to an acceptor sugar. A secondary isotope effect on the fucosyltransfer reaction with guanosine 5'-diphospho-[1-2H]-beta-1-fucose (GDP-[1-2H]-Fuc) as the substrate was observed and determined to be Dv = 1.32 +/- 0.13 and DV/K = 1.27 +/- 0.07. Competitive inhibition of FucT V by guanosine 5'-diphospho-2-deoxy-2-fluoro-beta-1-fucose (GDP-2F-Fuc) was observed with an inhibition constant of 4.2 microM which represents the most potent inhibitor of this enzyme to date. Incubation of GDP-2F-Fuc with FucT V and an acceptor molecule prior to the addition of GDP-Fuc had no effect on the potency of inhibition, indicating that GDP-2F-Fuc is neither an inactivator nor a slow substrate. Both the observed secondary isotope effect and the inhibition by GDP-2F-Fuc are consistent with a charged, sp2-hybridized, transition-state structure. A convenient and efficient synthesis of GDP-[1-2H]-Fuc and GDP-2F-Fuc and a nonradioactive, fluorescence assay for fucosyltransferase activity have been developed.

S- and C-glycopeptide derivatives of an LH-RH agonist.

The S- and C-glycosylated nonapeptides 1 and 2 were synthesized as analogs of the non-glycosylated LH-RH agonist buserelin (pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NHEt) by segment condensation in solution. 1 and 2 differ from this peptide in the amino acid in position 6. In the first case (1), D-serine (tBu) is substituted by D-cysteine carrying a rhamnosyl residue, in the second case (2) D-alanine carrying a galactosyl moiety bound as C-glycoside is incorporated. The bioactivity of both glycopeptides as fertility drugs was determined from the dose dependent LH release in male rats. Additionally, in female rats the ovulation rate was assessed. As a result the analog 1 exhibits a similar biological activity as buserelin while analog 2 shows about 25% of this potency. Compared to buserelin the solubility of the analogs 1 and 2 in aqueous buffer is improved by more than two orders of magnitude due to the carbohydrate moieties.