Imaging the glycome

Abstract

Molecular imaging enables visualization of specific molecules in vivo and without substantial perturbation to the target molecule's environment. Glycans are appealing targets for molecular imaging but are inaccessible with conventional approaches. Classic methods for monitoring glycans rely on molecular recognition with probe-bearing lectins or antibodies, but these techniques are not well suited to in vivo imaging. In an emerging strategy, glycans are imaged by metabolic labeling with chemical reporters and subsequent ligation to fluorescent probes. This technique has enabled visualization of glycans in living cells and in live organisms such as zebrafish. Molecular imaging with chemical reporters offers a new avenue for probing changes in the glycome that accompany development and disease.

Fig. 1.

Examples of glycoconjugate structures found in vertebrates. The glycans can be long and linear, as in the glycosaminoglycan chondroitin sulfate, or as simple as a single monosaccharide, as in cytosolic and nuclear O-GlcNAc-modified proteins. Branched structures are typical for the N-glycans and O-glycans found on glycoproteins and in glycolipids.

Fig. 2.

The bioorthogonal chemical reporter strategy for imaging glycans. (A) Glycans can be metabolically labeled with unnatural sugars bearing a chemical reporter group. The chemical reporter group, typically an azide or terminal alkyne, can be detected in a second step by covalent reaction with a probe. (B) Bioorthogonal reactions used to visualize chemical reporters appended to unnatural sugars (R). Azides can be detected by Staudinger ligation with triaryl phosphines, resulting in the formation of an amide linkage between the reporter and the probe. Azides and terminal alkynes can be detected by reaction with each other via CuAAC, forming in a triazole linkage between the reporter and probe. The azide can also be detected by Cu-free click chemistry with strained cyclooctynes. This latter reaction avoids the use of a cytotoxic metal catalyst.

Fig. 3.

Azide- and alkyne-bearing monosaccharides used for metabolic labeling of glycans. Sialic acids may be labeled with N-azidoacetylneuraminic acid (SiaNAz), ManNAz, 9-azido N-acetylneuraminic acid, and alkynyl ManNAc. GalNAc-containing glycans and O-GlcNAc-labeled glycans may be labeled with azides by using GalNAz. O-GlcNAc-labeled glycans may also be labeled with GlcNAz. Fucose-containing glycans may be labeled with 6AzFuc or alkynyl fucose. Structures are shown in peracetylated form, which are typically used for metabolic labeling experiments. After cell entry by passive diffusion, the acetyl groups are cleaved by cytosolic esterases.

Fig. 4.

Fluorogenic phosphine dyes activated by Staudinger ligation with azides. (A) Fluorogenic phosphine in which the phosphine lone-pair electrons quench the fluorescence of the coumarin fluorophore. Staudinger ligation or nonspecific oxidation sequesters the lone-pair electrons and generates a fluorescent product. (B) A fluorogenic phosphine that utilizes a FRET quencher, which is cleaved during the Staudinger reaction but not during nonspecific oxidation.

Fig. 5.

Fluorescent dyes activated by the CuAAC reaction based on (A) 1,8-napthalimide or (B) coumarin scaffolds. After the reaction, the triazole modifies the electronics of the fluorophore to yield a fluorescent product.

Fig. 6.

Application of the bioorthogonal chemical reporter strategy for in vivo imaging of glycans. (A) Zebrafish embryos were treated with azidosugars during their development, resulting in metabolic labeling of glycans with azides. The azides were visualized by reaction with fluorescent DIFO reagents. (B) An example of a zebrafish embryo metabolically labeled with Ac₄GalNAz and reacted with Alexa Fluor 647-conjugated DIFO (DIFO-647) at 60 h postfertilization (hpf) followed by Alexa Fluor 488-conjugated DIFO (DIFO-488) at 63 hpf to detect newly synthesized glycans. (Left) Single z-plane brightfield image. (Left Center) z-projection of DIFO-647 fluorescence. (Right Center) z-projection of DIFO-488 fluorescence. (Right) z-projection of DIFO-647 and DIFO-488 fluorescence merge. (Scale bar, 100 μm.)