Abstract:The synthesis of cyanine dyes addressing absorption wavelengths at 550 and 648 nm is reported. Alkyne functionalized dyes were used for bioorthogonal click reactions by labeling of metabolically incorporated sugar-azides on the surface of living neuroblastoma cells, which were applied to direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of cellsurface glycans in the nm-range.
Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3‐sialyltransferase from Photobacterium phosphoreum JT‐ISH‐467 (2,3SiaTpph) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure‐guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaTpph (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (α2,3‐Neu5Ac‐Lac) from lactose in a one‐pot, two‐enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new‐to‐nature sialoconjugates for biological studies.
A series of unusual sialic acid analogs were prepared using a semi-synthetic strategy. Truncation of natural N-acetylneuraminic acid was followed by diastereoselective carbon backbone reconstruction using Barbier-type carboligations as well as different functional group interconversions, which provided access to a variety of functional motifs in the terminal carbon backbone, including examples of saturated and unsaturated, linear and branched alkyl chains, partially deoxygenated sialic acids, sialic diacids and a first truncated legionaminic acid analog. The synthetic sialic acid probes were studied for nucleotide activation by the CMPsialic acid synthetase from Neisseria meningitidis using a universal pH-shift assay for kinetic analysis. One-pot enzymatic nucleotide activation and sialyltransfer to lactose was performed using a selection of five probes together with an engineered α2,3-sialyltransferase from Photobacterium phosphoreum to furnish five new-tonature analogs of the GM3 trisaccharide, which were finally utilized to test the substrate tolerance of two bacterial sialidases. The obtained set of sialic acid analogs and neo-sialocojugates provides interesting opportunities for further glycobiology studies.
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