Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry

Abstract

Vertebrate glycans constitute a large, important, and dynamic set of post-translational modifications that are notoriously difficult to manipulate and image. Although the chemical reporter strategy has been used in conjunction with bioorthogonal chemistry to image the external glycosylation state of live zebrafish and detect tumor-associated glycans in mice, the ability to image glycans systemically within a live organism has remained elusive. Here, we report a method that combines the metabolic incorporation of a cyclooctyne-functionalized sialic acid derivative with a ligation reaction of a fluorogenic tetrazine, allowing for the imaging of sialylated glycoconjugates within live zebrafish embryos.

Application of the Inverse-Electron-Demand Diels-Alder Reaction for Metabolic Glycoengineering

“…In 2015, a bicyclononyne-modified sialic acid (BCNSia) was synthesized and incorporated into the glycans of various cell lines ( Agarwal et al, 2015 ). Additionally, it was used to investigate sialylation in zebra fish embryos.…”

“…The first example is BCNSia, a sialic acid with a carbamate linkage to a BCN moiety at C-9 ( Agarwal et al, 2015 ). This position was chosen for modification, since it is known that the enzymes involved in the incorporation of sialic acids tolerate even a larger alteration ( Oetke et al, 2002 ; Han et al, 2005 ).…”

“…The incorporation efficiency by DMB-labeling was determined to be about 1% for both derivatives. Additionally, the possibility for dual labeling together with click chemistry was shown for the norbornene derivatives ( Späte et al, 2016 ) as well as BCNSia ( Agarwal et al, 2015 ).…”

Designing sugar mimetics: non-natural pyranosides as innovative chemical tools

“…Altered monosaccharides, such as a BCNSia ( 41 ), can also be injected directly in vivo in zebrafish embryos for cellular metabolism. After some development time, a fluorescent tetrazine probe designed to selectively react with the modified sialic acid was injected to ultimately create a map of the different sialylated structures present in the animal [32]. Likewise, the sugar nucleotide UDP-4-XylAz ( 40 ) was used in zebrafish to follow the incorporation of xylose at different stages of the development of the animal.…”

Concise Synthesis of Functionalized Cyclobutene Analogues for Bioorthogonal Tetrazine Ligation

“…The reaction of allyl-substituted TCO with tetrazine leads to a cascade elimination that has been widely used in the design of prodrugs [ 12 , 13 , 14 , 15 ]. Bicyclooctynes (BCN) react rapidly with tetrazine to afford pyridazine as a single product, which can facilitate downstream fluorogenic probe design [ 16 , 17 , 18 ]. Cyclopropene units have also emerged as alternative, more compact tags that can react quickly with tetrazine and have proven useful in studies of metabolic engineering [ 19 , 20 , 21 , 22 ].…”