Polar Dibenzocyclooctynes for Selective Labeling of Extracellular Glycoconjugates of Living Cells

Abstract

Although strain-promoted alkyne-azide cycloadditions (SPAAC) have found wide utility in biological and material sciences, the low polarity and limited water solubility of commonly used cyclooctynes represents a serious shortcoming. To address this problem, an efficient synthetic route has been developed for highly polar sulfated dibenzocyclooctynylamides (S-DIBO) by a Friedel-Crafts alkylation of 1,2-bis(3-methoxyphenyl)ethylamides with trichlorocyclopropenium cation followed by a controlled hydrolysis of the resulting dichlorocyclopropenes to give bis(3-methoxyphenyl)cyclooctacyclopropenones, which were subjected to methoxy group removal of the phenols, O-sulfation, and photochemical unmasking of the cyclopropenone moiety. Accurate rate measurements of the reaction of benzyl azide with various dibenzylcyclooctyne derivatives demonstrated that aromatic substitution and the presence of the amide function had only a marginal impact on the rate constants. Biotinylated S-DIBO 8 was successfully used for labeling azido-containing glycoconjugates of living cells. Furthermore, it was found that the substitution pattern of the dibenzylcyclooctynes influences subcellular location and in particular it has been shown that DIBO derivative 4 can enter cells thereby labeling intra- and extracellular azido-modified glycoconjugates, whereas S-DIBO 8 cannot pass the cell membrane and therefore is ideally suited for selective labeling of cell surface molecules. The ability to selectively label cell surface molecules will yield unique opportunities for glycomic analysis and the study of glycoprotein trafficking.

Selective Exo-Enzymatic Labeling Detects Increased Cell Surface Sialoglycoprotein Expression upon Megakaryocytic Differentiation

“…2). S-DIBO was chosen since it does not cross the plasma membrane and therefore avoids conjugation with intracellular glycoproteins (16). In contrast to metabolically labeled cells, Western blot analysis with anti-biotin antibody showed only weak labeling following SEEL with either sialyltransferase.…”

“…CMP-Neu5Ac9N 3 , S-DIBO-Biotin, Ac 4 ManNAc, and Ac 4 ManNAz were synthesized as previously described (16,30). Vibrio cholerae neuraminidase (type II) was purchased from Sigma Aldrich (N6514).…”

Abnormal accumulation and recycling of glycoproteins visualized in Niemann–Pick type C cells using the chemical reporter strategy

“…1), followed by treatment with streptavidin-568. DIBO was chosen for this study because of its ability to detect both intracellular and extracellular glycoconjugates (21,26). Although staining of Golgi, cell surface, and extracellular matrix was detected primarily in control fibroblasts, a striking additional accumulation of sialylated glycoconjugates was observed within intracellular vesicles in NPC1-null (Fig.…”

Strain-Promoted 1,3-Dipolar Cycloaddition of Cycloalkynes and Organic Azides

“…The first probe was based on annulation of coumarin to BARAC and was developed by Jewett and Bertozzi [29]. Secondly, Friscourt et al [23] reported that the cyclopropenone derivative of Sondheimer diyne unexpectedly forms a highly fluorescent triazole upon a copper-free reaction with azide. Most recently, Lang et al [34] showed that a TAMRA-functionalized tetrazine derivative leads to fivefold to tenfold increase in fluorescence signal, similar to earlier reported for reaction with trans-cyclooctene and norbornene, upon reaction with BCN.…”