Semi‐Synthetic Sialic Acid Probes for Challenging the Substrate Promiscuity of Enzymes in the Sialoconjugation Pathway

Abstract

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.

Immobilization of CMP‐Sialic Acid Synthetase and α2,3‐Sialyltransferase for Cascade Synthesis of 3′‐Sialyl β‐D‐Galactoside with Enzyme Reuse

“…The development of multi‐enzyme catalysts for sialo‐oligosaccharide synthesis involves the fundamental decision of whether whole‐cell or cell‐free preparations of the enzymes should be used [8,10,56] . Each approach has its specific advantages, like the greater flexibility offered by cell‐free preparations in diversity‐oriented syntheses [15,57–60] . The applied scope of the two approaches is largely complementary.…”

“…[8,10,56] Each approach has its specific advantages, like the greater flexibility offered by cell-free preparations in diversity-oriented syntheses. [15,[57][58][59][60] The applied scope of the two approaches is largely complementary. Live whole cells are preferred in large-scale production.…”

“…In synthetic reactions of these different SiaTs, the CMP‐Neu5Ac substrate is generally prepared in situ from Neu5Ac and cytidine 5′‐triphosphate (CTP) [11] through the reaction of CMP‐Neu5Ac synthetase (CSS; EC 2.7.7.43) (Figure 1). [12] Diversity‐oriented chemo‐enzymatic synthesis of sialo‐oligosaccharides has often relied on the cascade reaction of CSS and SiaT [13–15] …”

“…[12] Diversity-oriented chemo-enzymatic synthesis of sialooligosaccharides has often relied on the cascade reaction of CSS and SiaT. [13][14][15] An important strategy that has been largely overlooked to increase the overall enzyme performance (i. e., activity, stability, usability) in sialo-oligosaccharide synthesis is immobilization. Solid-supported preparations of the CSS and SiaT would facilitate recycling of the enzymes and help paving a way towards a continuous (flow) synthesis of sialo-oligosaccharides.…”

Syntheses of Legionaminic Acid, Pseudaminic Acid, Acetaminic Acid, 8-epi-Acetaminic Acid, and 8-epi-Legionaminic Acid Glycosyl Donors from N-Acetylneuraminic Acid by Side Chain Exchange

“…Oxidative cleavage of the three-carbon glycerol side chain of 5 and its derivatives to the corresponding aldehyde with sodium metaperiodate is known − and has been applied, for example, in a study of siglec ligand specificity when treatment of NeuAc-α-(2→3)-Gal-β-(1→4)-GlcNAc-β-CH 2 CH 2 N 3 with aqueous sodium metaperiodate yielded aldehyde 6 (Figure ). Further transformations of such aldehydes are, however, mostly limited to reduction, ,, imine formation, and reductive amination, , with the exception of a single example of the Sakurai reaction, and recent work on the use of indium-mediated allylation and propargylation reactions. , …”