Combining CuAAC reaction enables sialylated Bi- and triantennary pseudo mannose N-glycans for investigating Siglec-7 interactions.

Naturally occurring N-glycans display much diversity in modifications, linkages, and peripheral presentation of the oligosaccharide chain. Despite continued advancements in oligosaccharide synthesis, synthetic access to these natural glycans remains challenging. Biologically relevant complex N-glycan mimetics with various natural and unnatural modifications are an alternate way for investigating glycan-protein interactions. Further supporting this pattern, we report here a new class of sialylated bi- and triantennary pseudo mannose N-glycans reproducing orientation of the underlying glycan chain and branching patterns and replacing the two inner mannopyranosyl units with 1,2,3-triazole rings. Such mimetics are straightforwardly generated by implementing multiple intermolecular Cu(I)-catalyzed azide-alkyne cycloaddition between chemoenzymatically synthesized azido sialosides and rationally designed C-3 and C-6 di-O- or C-2, C-3, and C-6 tri-O-alkynylated mannoside. Human recombinant Siglec-7-Fc fusion protein recognizes almost all sialylated pseudo mannose N-glycans in the microarray. However, a differential Sia-binding pattern was also observed. Given the library size, comparison of pairwise mannose N-glycan combinations showed that biantennary linear α(2,3)α(2,8)- and α(2,6)α(2,8)- or branched α(2,3)α(2,6)-, and triantennary branched α(2,3)α(2,6)-sialyl pseudo N-glycans possess similar binding capabilities and affinity to recombinant Siglec-7-Fc. While the full range of topological mannose arms remain elusive, the bi- and triantennary mimics are simpler structures for interrogating Siglec interactions.

Sugar nucleotide regeneration system for the synthesis of Bi- and triantennary N-glycans and exploring their activities against siglecs.

Enzymatic synthesis that is commenced by the sugar nucleotide regeneration system (SNRS) protocol can minimize 1) the consumption of exorbitant sugar nucleotides, 2) the amount of transferases required, and 3) byproduct feedback inhibition. In this study, LacNAc extensions/modifications of the N-linked mannose core were carried out efficiently with SNRS with high yields and purities on all branches in a uniform manner. In addition, we demonstrate that with SNRS, bacterial glycosyltransferases exhibit a wide acceptor tolerance for bi- and triantennary mannose core structures as substrates for target oligosaccharides. The synthesized small library of mannose core-based glycans and linear O-glycans were screened for their binding affinity against h-Siglecs 2, 4, 7, 9, 14, 15, and m-Siglec-15 to explore their structure-based binding preferences. Microarray data revealed that each Siglec showed few distinct yet overlapping specificities. An increase in branching from mono to di or tri antennary did not necessarily lead to increasing affinity. Glycans with the disialoside sequence α(2,3)α(2,8)/α(2,6)α(2,8) showed high specificity and affinity for Siglec-7, and sLex α(2,3) exhibited a strong affinity for Siglec-9. Explicit recognition of α(2,6)α(2,3)- linear and α(2,3)α(2,6)-branched glycans by Siglecs-2, 4, and 15, respectively, suggests that these structures can act as potential candidates for the further development of high-affinity ligands.

Expedient assembly of Oligo-LacNAcs by a sugar nucleotide regeneration system: Finding the role of tandem LacNAc and sialic acid position towards siglec binding.

Sialosides containing (oligo-)N-acetyllactosamine (LacNAc, Galß(1,4)GlcNAc) as core structure are known to serve as ligands for Siglecs. However, the role of tandem inner epitope for Siglec interaction has never been reported. Herein, we report the effect of internal glycan (by length and type) on the binding affinity and describe a simple and efficient chemo-enzymatic sugar nucleotide regeneration protocol for the preparative-scale synthesis of oligo-LacNAcs by the sequential use of ß1,4-galactosyltransferase (ß4GalT) and ß1,3-N-acetylglucosyl transferase (ß3GlcNAcT). Further modification of these oligo-LacNAcs was performed in one-pot enzymatic synthesis to yield sialylated and/or fucosylated analogs. A glycan library of 23 different sialosides containing various LacNAc lengths or Lac core with natural/unnatural sialylation and/or fucosylation was synthesized. These glycans were used to fabricate a glycan microarray that was utilized to screen glycan binding preferences against five different Siglecs (2, 7, 9, 14 and 15).