Azide-sialic acid

The methods used for in vivo incorporation of azido-monomers and performing a labeling reaction with live cells are relatively simple. The following protocol is based on the methods of Saxon and Bertozzi (2000), which uses acetylated azidoacetylmannosamine as the azido-monomer source and a biotin-PEG-phosphine compound to biotinylate cell surface glycoproteins at the specific azide-sialic acid incorporation sites (Figure 17.19). 

To facilitate accesses to suitably functionalized sialic acid derivatives and complex sialyloligosaccharides for other usehil neoglycoconjugates, phase transfer catalysis (PTC) has been exploited extensively [for reviews see 42]. This process provided a wide range of carbohydrate derivatives under essentially clean Sn2 transformations. In the case of acetochloroneuraminic acid 1, the PTC reactions always provided inverted a-sialic acid derivatives [43]. para-Formylphenyl sialoside 7 [44], together with many other sialoside derivatives such as 8-10 [43], including thioacetate 12 [45] and azide 14 [46], were thus obtained (Scheme 1). Aldehyde 7 and similar glycosides are of particular interest since they could be directly conjugated to protein by reductive amina-tion after suitable deprotection [44]. 

C) Strategy for surface labeling of sialic acid-displayed azide groups... 

Several non-natural a2,6-linked sialosides 36-40 with azide or alkyne-modified sialic acid residues were also prepared in excellent yields (86%-93%) from their C2- or C6- modified ManNAc or mannose bearing corresponding azide or alkyne functional groups 30-34 using the one-pot three-enzyme approach and GaipOMe (35) as an acceptor for Pd2,6ST (Scheme 5). 

Figure 42 Structures of aryl-azide mannosamine and sialic acid analogs (a) SiaNAAz, (b) 9-AAz-NeuAc, (c) ManNAAz.

The incorporation of new functional groups can also be accomplished using the metabolic machinery for posttranslational protein modifications. These methods rely on the ability of some modification enzymes to process and install analogs of their natural substrates containing reactive handles of interest. In an early demonstration of this technique, it was shown that derivatives of N-acetylmannosamine 40a bearing ketones 40b) [62] or azides 40c [63] in the acyl moiety are tolerated by enzymatic pathways that produce sialic acid. By feeding these unnatural building blocks to cell cultures,... 

Fig. 10.3-16 Introduction of unnatural functional groups through posttranslational modification, (a) Ketones and azides can be introduced onto cell surfaces by feeding cells with unnatural sialic acid precursors, such as mannosamine derivatives 40b and c. These are incorporated into cell-surface glycans, which can be further elaborated using additional bioconjugation reactions, (b) Specific amino acid sequences can be modified using biotin ligase. Interestingly, ketobiotin" is also recognized as a substrate for the enzyme, allowing a...

The use of this reaction in the biological context was first demonstrated for the chemospecific labeling of Jurkat cell surfaces [63]. Metabolic engineering with N-acetylmannosamine derivative 40c was used to incorporate azides into sialic acid groups on cell surfaces. The cells were then incubated with biotinylated phosphine 49, and the extent of the reaction was quantified by flow cytometry after treatment with fluorescent avidin. Importantly, neither the azide nor the phosphine displayed any reactivity with the cell-surface groups in the absence of its reactive partner. In addition, the cells showed unchanged growth rates after modification. 

---