An Azido-BODIPY Probe for Glycosylation: Initiation of Strong Fluorescence upon Triazole Formation Jiun-Jie Shie, † Ying-Chih Liu, † Yu-Ming Lee, ‡ Carmay Lim, ‡ Jim-Min Fang, †,§ and Chi-Huey Wong* ,† † The Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan ‡ Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan § Department of Chemistry, National Taiwan University, Taipei 106, Taiwan * S Supporting Information ABSTRACT: We have designed a low fluorescent azido-BODIPY- based probe AzBOCEt (Az10) that undergoes copper(I)-catalyzed 1,3-dipolar cycloadditions with alkynes to yield strongly fluorescent triazole derivatives. The fluorescent quantum yield of a triazole product T10 is enhanced by 52-fold as compared to AzBOCEt upon excitation at a wavelength above 500 nm. Quantum mechanical calculations indicate that the increase in fluorescence upon triazole formation is due to the lowering of the HOMO energy level of the aryl moiety to reduce the process of acceptor photoinduced electron transfer. AzBOCEt is shown to label alkyne- functionalized proteins in vitro and glycoproteins in cells with excellent selectivity, and enables cell imaging and visualization of glycoconjugates in alkynyl-saccharide-treated cells at extremely low concentration (0.1 μM). Furthermore, the alkyne-tagged glycoproteins from cell lysates can be directly detected with AzBOCEt in gel electrophoresis. ■ INTRODUCTION Glycosylation is an important co/post-translational modifica- tion that has been shown to be crucial for the structure and function of many proteins. Aberrant glycosylation on the surface of malignant cells is often observed in pathological conditions, such as inflammation and cancer metastasis. 1,2 In particular, altered terminal sialylation and fucosylation, which presumably result from changes of expression locations and levels of sialyltransferases and fucosyltransferases, are associated with tumor malignancy. 1,3 Exploring the biological content of glycans attached to proteins or lipids as cancer biomarkers has become a major subject of research. Utilizing bioorthogonal chemical reporter is a successful strategy to study the role of glycans in numerous physiological and pathological processes as well as to visualize the process of glycosylation in vivo. In this approach, metabolic oligosaccharide engineering (MOE) is employed to modify glycans with an unnatural monosaccharide precursor containing a unique bioorthogonal chemical reporter group as substrate for the biosynthetic pathway in a living system (cell or whole organism) for incorporation into a target glycan, which then react with a complementary bioorthogonal functional group covalently linked to a set of probes, including biotin and fluorescence tags. 4 Research for specific glycan tagging in our laboratory has benefited greatly from Cu(I)-catalyzed azide-alkyne cyclo- addition (CuAAC) 5 optimized for biological conjugation. We have previously demonstrated that peracetylated alkynyl derivatives of monosaccharides, for example, Fucyne, 6a,b ManNAcyne, 6b-d and alkyne-hinged 3-fluorosialyl fluoride (DFSA), 6e are incorporated into sialylated and fucosylated glycans on the cell surface. The subsequent labeling with proper azido-containing probes via CuAAC gives the fluorescent triazole-tagged glycans that are easily visualized and profiled on a proteomic scale. We have also designed the fluorogenic 1,8- naphthalimide-based probes 6a and trifunctional coumarin-based probes 6d for in vitro and in vivo labeling of glycans. Several fluorescence-forming probes upon CuAAC reactions have also been utilized by other groups to label specific biomolecules. 7 The distinct fluorescence enhancement induced by efficient triazole formations would have broad applications in the emerging field of cell biology and functional proteomics. However, these azido- and alkynyl-functionalized probes usually require excitation in the UV region and emit blue light with poor quantum yield in aqueous solution; such optical properties are not ideal for biological applications. Therefore, there is a need to develop azido- or alkynyl-fluorogenic probes with excitation and emission wavelengths in the visible region. In addition to a fluorescein-based probe, 8 Wang and co-workers have designed an azido-containing BODIPY-based probe. 9 The azido group is located at the 3-position of the BODIPY core, so that the electron-donating effect of the α-nitrogen of the azido group can quench fluorescence via an internal charge transfer (ICT) process. Upon formation of the triazole derivative through a CuAAC reaction with alkyne, fluorescence is switched on due to the decreased electron density at the 3- position of the BODIPY core. However, the azido-BODIPY Received: January 29, 2014 Published: June 23, 2014 Article pubs.acs.org/JACS © 2014 American Chemical Society 9953 dx.doi.org/10.1021/ja5010174 | J. Am. Chem. Soc. 2014, 136, 9953-9961