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The Central Dogma of Biology does not allow for the study of glycans using DNA sequencing. We report a "Liquid Glycan Array" (LiGA) platform comprising a library of DNA 'barcoded' M13 virions that display 30-1500 copies of glycans per phage. A LiGA is synthesized by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido-modified glycans. Pulldown of the LiGA with lectins followed by deep sequencing of the barcodes in the bound phage decodes the optimal structure and density of the recognized glycans. The LiGA is target agnostic and can measure the glycan-binding profile of lectins such as CD22 on cells in vitro and immune cells in a live mouse. From a mixture of multivalent glycan probes, LiGAs identifies the glycoconjugates with optimal avidity necessary for binding to lectins on living cells in vitro and in vivo; measurements that cannot be performed with canonical glass slidebased glycan arrays.

A one-pot strategy for functionalizing pyranoside 1,2-cis-diols with two different ester protecting groups is reported. The approach employs regioselective acylation via orthoester hydrolysis promoted by a carboxylic acid, e.g., levulinic acid, acetic acid, benzoic acid, or chloroacetic acid. Upon removal of water and introduction of a coupling agent, the carboxylic acid is esterified to the hydroxyl group liberated during hydrolysis. Although applied to 1,2-cis-diols on pyranoside scaffolds, the method should be applicable to such motifs on any six-membered ring.

Abnormal cell surface glycosylation plays a major role in disease processes such as immune evasion. However, the underlying role of glycans is yet to be fully understood. Binding information obtained from glycan arrays can provide critical starting points for downstream applications such as the development of carbohydrate‐based inhibitors, vaccines, and other therapeutics. However, it is challenging to use powerful techniques like DNA deep sequencing to analyze glycan recognition due to the lack of 1:1 correspondence between DNA and glycan structures. Therefore, we have developed Liquid Glycan Array (LiGA), a technology that allows for genetic encoding of glycans. LiGA provides a 1:1 correspondence between the glycan displayed in multiple copies on a bacteriophage carrier and the phage genetic material. LiGA is generated by acylation of phage pVIII protein with a dibenzocyclooctyne, followed by ligation of azido‐modified glycans. The display of glycans on each phage virion can be controlled from 30‐1500 copies to probe the critical variables in glycan recognition: valency and density. A simple pulldown of the LiGA along with lectins followed by deep sequencing of the DNA in the bound phage decodes the recognized glycans. LiGA is target agnostic and measures binding profile of lectins expressed on intact cells, such as hCD22 (Siglec‐2) and DC‐SIGN (Dendritic Cell‐Specific Intercellular adhesion molecule‐3‐Grabbing Non‐integrin), and in live mice (Nat. Chem. Bio. 17, 806–816, 2021). From a mixture of 50‐100 multivalent glycan probes, LiGA identifies the glycan‐phage conjugates with optimal valency and density for binding to antibodies and lectins on cells in vitro and in vivo. Sialic acid‐binding immunoglobulin‐type lectins (Siglecs) expressed on the surface of immune cells are exploited by cancer to evade immune response. We applied LiGA to study the binding specificity of Siglec‐7, a cell surface receptor that cancer cells use to evade immune response from natural killer (NK) cells. Additionally, we explored the roles of valency and density in ganglioside interaction with Siglec‐1 using a cell‐based assay. Building on these successes, we plan to use LiGA to identify the valency and affinity required by trans‐ glycan to overcome the cis‐ masking on the surface of immune cells.

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