Two‐Color Glycan Labeling of Live Cells by a Combination of Diels–Alder and Click Chemistry

TLDR: This work shows that ManNAc derivatives containing a terminal alkene in the acyl side chain are metabolically incorporated into cell-surface sialic acids and can subsequently be labeled by the DARinv, and identifies monosubstituted (terminal) alkenes as a new class of chemical reporters.

Abstract: Protein glycosylation is a complex form of posttranslational modification and has been shown to be crucial for the function of many proteins. Sialic acid is prominently positioned at the outer end of membrane glycoproteins. It plays a critical role for the regulation of a myriad of cellular functions and it forms a shield around the cell. Furthermore, it constantly interacts with the environment of cells and contributes to histocompatibility. This makes studying sialylation an interesting field of research, but monitoring sialic acid in vivo is challenging. While proteins are routinely labeled by genetic methods, such as expression as GFP fusion proteins, comparable methods are not available for secondary gene products, such as glycans of glycoconjugates. Metabolic oligosaccharide engineering (MOE) is a successful new strategy to visualize the localization of glycans in vitro and in vivo. In this approach, cells are cultivated in the presence of non-natural monosaccharide derivatives that carry a chemical reporter group and are nonetheless accepted by the biosynthetic machinery of a cell. For instance, peracetylated N-azidoacetylmannosamine (Ac4ManNAz) is taken up by the cell, deacetylated by cellular esterases, and owing to the promiscuity of the enzymes of sialic acid biosynthesis, is converted into N-azidoacetyl neuraminic acid and incorporated into sialoglycoconjugates. Once presented on the cell surface, the azide-containing sialylated glycan can be visualized through a bioorthogonal ligation reaction. Besides Ac4ManNAz, several monosaccharide derivatives of N-acetylgalactosamine, N-acetylglucosamine, and l-fucose are suitable for MOE providing further insights into the role of cellular structures and functions of glycans in the cell. Currently, mainly Staudinger ligation and azide–alkyne [3+2] cycloaddition (copper-catalyzed or strain-promoted, also known as the click reaction) are applied as ligation reactions in MOE. However, both of them rely on the reaction of azides and thus cannot be used for the concurrent detection of two different metabolically incorporated carbohydrates. A labeling strategy that can be carried out in the presence of azides and alkynes would significantly expand the scope of chemical labeling reactions in living cells and is thus highly desirable. Recently, it was shown that the Diels–Alder reaction with inverse electron demand (DARinv) of 1,2,4,5-tetrazines with strained dienophiles, such as trans-cyclooctenes, cyclobutenes, norbornenes, 13] cyclooctynes, and substituted cyclopropenes, fulfills the requirements of a bioorthogonal ligation reaction and furthermore is orthogonal to the azide–alkyne cycloaddition. However, these cyclic alkenes or kinetically stable tetrazines are expected to be too large for being efficiently metabolized by the sialic acid biosynthetic pathway, starting from the corresponding Nacylmannosamine derivative. In search for smaller dienophiles suitable for MOE, we identified monosubstituted (terminal) alkenes as a new class of chemical reporters. We recently reported the successful application of the DARinv between terminal alkenes and 1,2,4,5-tetrazines in the preparation of carbohydrate microarrays. The fact that terminal alkenes are hardly found in biological systems and are completely absent in proteins makes them a promising reporter group. Herein, we show that ManNAc derivatives containing a terminal alkene in the acyl side chain are metabolically incorporated into cell-surface sialic acids and can subsequently be labeled by the DARinv (Figure 1). Moreover, we demonstrate that double labeling of two differently modified, metabolically incorporated monosaccharides is possible by combining the DARinv with strainpromoted azide–alkyne cycloaddition (SPAAC). As the reaction rate of the DARinv of acyclic olefins with tetrazines is very sensitive to steric hindrance, double bonds with more than one substituent react very slowly. Terminal alkenes, on the other hand, can react rapidly without any further activation. This prompted us to design mannosamine derivatives 2 and 4 (Figure 2) that were synthesized in three steps from mannosamine hydrochloride (see the Supporting Information). Based on previous work by Keppler et al., we expected both derivatives to be accepted by cells with Npentenoylmannosamine 2 (owing to the shorter acyl side chain) being incorporated with higher efficiency. On the other [*] Dipl.-Chem. A. Niederwieser, M. Sc. A.-K. Sp te, M. Sc. C. J ngst, Prof. Dr. V. Wittmann University of Konstanz, Department of Chemistry and Konstanz Research School Chemical Biology (KoRS-CB) 78457 Konstanz (Germany) E-mail: mail@valentin-wittmann.de