Chien-Fu Liang

Synthesis of sialic acid-containing saccharides.

Sialic acids are a diverse family of negatively charged monosaccharides with a shared nine-carbon carboxylated backbone, and they often serve as the terminal positions of cell surface glycoproteins and glycolipids. Sialic acids play essential roles in mediating or modulating numerous pathological, biological, and immunological recognition events. Advances in synthesis have provided chemically well-defined and structurally homogeneous sialic acid-containing carbohydrates that are crucial for studying glycobiology. This review highlights recent innovations in the chemical and chemoenzymatic synthesis of difficult α-sialosides, with a particular focus on methods developed for α-selective sialylation in the synthesis of O-linked and S-linked oligosialic acids.

Synthesis of S-Linked α(2→9) Octasialic Acid via Exclusive α S-Glycosidic Bond Formation

A new approach for the synthesis of S-linked alpha(2-->9) oligosialic acids was developed by using an asymmetric tert-butyl disulfide linkage as an anomeric thiol protecting group. Compared with conventional thiosialosides, the asymmetric disulfide sialosides can tolerate the conditions under which functional groups are modified without producing the undesired elimination and racemization products. In addition, the asymmetric tert-butyl disulfide protecting group can be efficiently removed to afford a thiol nucleophile at the alpha anomeric position without flipping the anomeric stereochemistry. By this strategy, the syntheses of alpha(2-->9) tetra-, hexa-, and octasialic acids were achieved.

Site-selective protein immobilization through 2-cyanobenzothiazole-cysteine condensation.

We described a rapid site‐selective protein immobilization strategy on glass slides and magnetic nanoparticles, at either the N or C terminus, by a 2‐cyanobenzothiazole (CBT)‐cysteine (Cys) condensation reaction. A terminal cysteine was generated at either terminus of a target protein by a combination of expressed protein ligation (EPL) and tobacco etch virus protease (TEVp) digestion, and was reacted with the CBT‐solid support to immobilize the protein. According to microarray analysis, we found that glutathione S‐transferase immobilized at the N terminus allowed higher substrate binding than for immobilization at the C terminus, whereas there were no differences in the activities of N‐ and C‐terminally immobilized maltose‐binding proteins. Moreover, immobilization of TEVp at the N terminus preserved higher activity than immobilization at the C terminus. The success of utilizing CBT‐Cys condensation and the ease of constructing a terminal cysteine using EPL and TEVp digestion demonstrate that this method is feasible for site‐selective protein immobilization on glass slides and nanoparticles. The orientation of a protein is crucial for its activity after immobilization, and this strategy provides a simple means to evaluate the preferred protein immobilization orientation on solid supports in the absence of clear structural information.

Sequential one-pot enzymatic synthesis of oligo-N-acetyllactosamine and its multi-sialylated extensions

A simple and efficient protocol for the preparative-scale synthesis of various lengths of oligo-N-acetyllactosamine (oligo-LacNAc) and its multi-sialylated extensions is described. The strategy utilizes one thermophilic bacterial thymidylyltransferase (RmlA) coupled with corresponding sugar-1-phosphate kinases to generate two uridine diphosphate sugars, UDP-galactose and UDP-N-acetylglucosamine. By incorporating glycosyltransferases, oligo-LacNAcs and their sialylated analogs were synthesized.

Synthesis and evaluation of a photoactive probe with a multivalent carbohydrate for capturing carbohydrate-lectin interactions.

Lectins are ubiquitous carbohydrate-binding proteins of nonimmune origin that are characterized by their specific recognition of defined monosaccharide or oligosaccharide structures. However, the use of carbohydrates to study lectin has been restricted by the weak binding affinity and noncovalent character of the interaction between carbohydrates and lectin. In this report, we designed and synthesized a multifunctional photoaffinity reagent composed of a trialkyne chain, a masked latent amine group, and a photoreactive 3-trifluoromethyl-3-phenyl-diazirine group in high overall yield. Two well-defined chemistries, Huisgen-Sharpless click chemistry and amide bond coupling, were the key steps for installing the multivalent character and tag in our designed photoaffinity probe. The photolabeling results demonstrated that the designed probe selectively labeled the target lectin, RCA120 ( Ricinus communis Agglutinin), in an E. coli lysate and an asialoglycoprotein receptor (ASGP-R) on intact HepG2 cell membranes. Moreover, the probe also enabled the detection of weak protein-protein interactions between RCA120 and ovalbumin (OVA).

A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein

The use of photo-crosslinking glycoprobes represents a powerful strategy for the covalent capture of labile protein complexes and allows detailed characterization of carbohydrate-mediated interactions. The selective release of target proteins from solid support is a key step in functional proteomics. We envisaged that light activation can be exploited for releasing labeled protein in a dual photo-affinity probe-based strategy. To investigate this possibility, we designed a trifunctional, galactose-based, multivalent glycoprobe for affinity labeling of carbohydrate-binding proteins. The resulting covalent protein-probe adduct is attached to a photo-cleavable biotin affinity tag; the biotin moiety enables specific presentation of the conjugate on streptavidin-coated beads, and the photolabile linker allows the release of the labeled proteins. This dual probe promotes both the labeling and the facile cleavage of the target protein complexes from the solid surfaces and the remainder of the cell lysate in a completely unaltered form, thus eliminating many of the common pitfalls associated with traditional affinity-based purification methods.