Trifunctional Fluorogenic Probes for Fluorescence Imaging and

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Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Trifunctional Fluorogenic Probes for Fluorescence Imaging and Isolation of Glycosidases in Cells Ji Young Hyun,† Seong-Hyun Park,† Cheol Wan Park,† Han Byeol Kim,‡ Jin Won Cho,‡ and Injae Shin*,† †

Department of Chemistry and ‡Department of Systems Biology, Yonsei University, Seoul 03722, Republic of Korea

Org. Lett. Downloaded from pubs.acs.org by BETHEL UNIV on 05/02/19. For personal use only.

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ABSTRACT: For both fluorescence imaging and isolation of glycosidases in cells, we prepared novel activity-based, trifunctional fluorogenic probes that consist of (1) a sugar moiety as a glycosidase substrate, (2) a fluoromethylated coumarin for fluorescent labeling, and (3) an alkyne tag for click reaction to enable isolation of the labeled enzyme. One probe, β-GlcNAcCM-F, was employed to fluorescently detect endogenous OGlcNAcase in cells and to isolate the labeled enzyme by affinity chromatography.

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ammalian cell-surface glycans, present in the form of glycoconjugates (e.g., glycoproteins, glycosphingolipids, and proteoglycans), play key roles in diverse biological events through their interactions with proteins.1 Glycan chains of the glycoconjugates are generated by the cooperative action of glycosyltransferases and glycosidases in the endoplasmic reticulum and the Golgi apparatus.2 Eventually, glycans in the glycoconjugates are degraded in lysosomes by lysosomal glycosidases.3 Therefore, glycosidases are crucial for biosynthesis and degradation of glycoconjugates. Consequently, methods are needed for the detection of glycosidases in cells to facilitate functional and structural studies of biologically important glycans. Mechanism-based labeling of proteins is a powerful technique for identifying and profiling enzymes in complex proteomes.4 In studies focusing on this issue, glycosidase-targeting, activitybased probes have been devised to isolate or fluorescently detect glycosidases in cells.5−7 Because most of the known activitybased glycosidase probes contain an affinity tag, they have been utilized mainly to isolate/enrich glycosidases.6 Recently, activity-based fluorogenic probes have also been developed for fluorescence imaging of glycosidases in cells and tissues.7 However, they cannot be used for isolation of the cellular enzymes owing to the lack of the affinity tag. In the investigation described below, we designed, prepared, and assessed the utility of a new type of activity-based, trifunctional fluorogenic probes, which can be employed for both fluorescence imaging and isolation of the cellular glycosidases. The newly designed probes comprise three components including (1) a sugar moiety serving as a glycosidase substrate, (2) a mono- or difluoromethylated coumarin (CM) for fluorescence labeling and imaging of the enzyme, and (3) an alkyne tag for click reaction to facilitate isolation of the labeled enzyme by affinity chromatography (Figure 1). The intact probes are nonfluorescent. However, © XXXX American Chemical Society

Figure 1. Strategy for fluorescence imaging and isolation of cellular glycosidases using trifunctional fluorogenic probes.

when the sugar portion of the probe is cleaved from the probe by the target glycosidase, fluoride will be rapidly liberated from the fluoromethylated CM group.8 The quinone methide intermediate formed in this manner then reacts with a nucleophilic site in the side chain of an amino acid(s) of the target glycosidase.8 The formed fluorescently labeled enzyme is detected using fluorescence microscopy. In addition, the alkyne Received: April 1, 2019

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DOI: 10.1021/acs.orglett.9b01147 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Scheme 1. Synthesis of Mono- (Sugar-CM-F) and Difluoromethylated (Sugar-CM-F2) Probes for Glycosidases

moiety present in the labeled glycosidase will then participate in click chemistry with an azide-conjugated biotin (N3-biotin) to enable isolation of the labeled glycosidase by affinity chromatography. To evaluate the feasibility of trifunctional fluorogenic probes to detect glycosidases, mono- (sugar-CM-F; β-Glc-CM-F, βGal-CM-F, and β-GlcNAc-CM-F) and difluoromethylated probes (sugar-CM-F2; β-Glc-CM-F2 and β-Gal-CM-F2) were synthesized using the procedure delineated in Scheme 1. Briefly, an alkyne-bearing CM fluorophore (2) was subjected to formylation to produce CM-CHO, which was also reduced to form alcohol CM-OH.9 Both CM-CHO and CM-OH were then subjected to glycosylation reactions with peracetylated glycosyl halides in biphasic solutions to generate the corresponding sugar(OAc)-CM-CHO and sugar(OAc)-CM-OH.10 The aldehyde and hydroxyl groups in these substances were converted to respective difluoromethyl and monofluoro groups in sugar(OAc)-CM-F2 and sugar(OAc)-CM-F by reactions with DAST. Finally, removal of O-acetyl protecting groups produced sugarCM-F and sugar-CM-F2. However, β-GlcNAc-CM-F2 was not prepared because the overall yield of its synthesis is very low and difluorinated probes displayed poor labeling efficiency as shown in below. With five probes in hand, we initially assessed their efficiencies of glycosidase labeling. For this purpose, glycosidases were incubated with the corresponding mono- (β-Glc-CM-F for βglucosidase, β-Gal-CM-F for β-galactosidase, and β-GlcNAcCM-F for β-N-acetylhexosaminidase) or difluoromethylated probes (β-Glc-CM-F2 for β-glucosidase and β-Gal-CM-F2 for β-galactosidase). The mixtures were subjected to click chemistry with N3-biotin. Analysis of the mixtures by SDS-PAGE showed that labeling reactions of the monofluoromethylated probes occur with efficiencies greater than those of their difluoromethylated counterparts (Figures 2a and S1a). Residual activities of glycosidases, generated in the manner described above, were then determined with near-infrared (NIR) fluorogenic substrates β-Glc-NIR, β-Gal-NIR, and βGlcNAc-NIR (Figure 2).10a,c Glycosidase activities were almost completely lost when 10 equiv of the sugar-CM-F probes was utilized (Figure 2b). However, about 100 equiv of the sugarCM-F2 probes was required to bring about the complete loss of

Figure 2. Measurements of labeling efficiency and residual activity. (a) Each glycosidase (16 pmol), treated with an indicated sugar-CM-F probe, was subjected to click reactions with N3-biotin and then separated by using SDS-PAGE (staining with (top) Coomassie blue and (bottom) Cy3-streptavidin). (b) Each glycosidase (100 nM) was treated with the corresponding sugar-CM-F probe. Remaining activities of the labeled glycosidases were measured by using β-Glc-NIR, β-GalNIR, and β-GlcNAc-NIR (λEx/λEm = 690 nm/710 nm). Because almost all of the enzyme activity is lost after reactions of each glycosidase with the corresponding sugar-CM-F probe (Figure S3), it is likely that most glycosidases are labeled by sugar-CM-F probes.

activities of the enzymes (Figure S1b). Activities were also determined after dialysis to assess stabilities of labeling of glycosidases formed by reactions with sugar-CM-F or sugarCM-F2 probes. Whereas activities of glycosidases treated with sugar-CM-F probes were not recovered after overnight dialysis, those of glycosidases treated with sugar-CM-F2 probes greatly increased (Figure S2a). In addition, fluorescence intensities of enzymes labeled by sugar-CM-F2 probes were greatly reduced after dialysis, but those of proteins labeled by sugar-CM-F probes remained nearly unchanged (Figure S2b). The findings indicate that adducts formed in reactions of glycosidases with sugar-CM-F probes are stable, but those produced using sugarCM-F2 probes are not. B

DOI: 10.1021/acs.orglett.9b01147 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

modification of proteins by the monosaccharide O-GlcNAc is highly dynamic and of great importance in various biological processes.12 Dysregulated levels of O-GlcNAc-modified proteins are also known to be associated with various human diseases.12 Owing to the pathophysiological significance of OGlcNAcase, which cleaves O-GlcNAc from O-GlcNAc-modified proteins and thus results in a decrease in their levels,12 we assessed the ability of β-GlcNAc-CM-F to fluorescently detect and isolate O-GlcNAcase in mammalian cells. In an experiment designed to test detection of cellular OGlcNAcase by β-GlcNAc-CM-F, HT-29 cells were treated for 10 h with noncytotoxic β-GlcNAc-CM-F (Figure S8) in the absence and presence of PUGNAc (Figures S9 and S10). The results of cell image analysis showed that the fluorescence intensities of cells treated with β-GlcNAc-CM-F and PUGNAc were greatly reduced compared to those of cells treated with βGlcNAc-CM-F only (Figure 4a,b). The findings suggest that

A study was conducted to evaluate the time dependence of the response of the new probes to glycosidases. The intensities of fluorescence generated by treatment of sugar-CM-F probes with glycosidases were found to increase and then reach a plateau (Figure S3a). In contrast, fluorescence signals arising from reactions of sugar-CM-F2 probes with glycosidases gradually increased and then decreased (Figure S3b). Also, the maximum increases in fluorescence intensities derived from sugar-CM-F probes by glycosidases were greater than those derived from their sugar-CM-F2 counterparts. On the basis of combined findings, mechanisms for capturing glycosidases by trifunctional fluorogenic probes were proposed, as shown in Figure S4. Reactions of the enzymes with sugar-CMF probes produce stable and fluorescent glycosidase probe adducts, along with fluorescent CM-OH (Figures S4a and S5 and Table S1). Production of CM-OH in these reactions was confirmed by HPLC analysis (Figure S6a). However, reactions of glycosidases with sugar-CM-F2 probes initially yield unstable but fluorescent “CM-OH(F) and glycosidase probe adducts”. The unstable CM-OH(F) is then converted into the very weakly fluorescent product CM-CHO (Figures S4b and S5 and Table S1), whose generation was confirmed by HPLC analysis (Figure S6b). Also, relatively unstable glycosidase probe adducts gradually decompose to form the free enzyme and CM-CHO. This explains why more sugar-CM-F2 probes are needed for labeling of glycosidases than sugar-CM-F probes, and fluorescence intensities of labeled glycosidases are markedly lower after dialysis. Overall, the findings indicate that sugar-CMF probes are superior to sugar-CM-F2 probes for capturing glycosidases.7,11 As a result, monofluorinated probes were employed for further studies. Next, we investigated the selectivity of sugar-CM-F probes for glycosidases. In this study, a mixture of three glycosidases or Escherichia coli cell lysates supplemented with each glycosidase was incubated with β-Glc-CM-F, β-Gal-CM-F, or β-GlcNAcCM-F in the absence and presence of an appropriate glycosidase inhibitor (conduritol B epocide (CBE) for β-glucosidase, 1deoxygalactonojirimycin (DGJ) for β-galactosidase, and PUGNAc for β-N-acetylhexosaminidase).10a,c The results of SDSPAGE analysis of reaction mixtures showed that glycosidases are captured by the corresponding sugar-CM-F probes and labeling is almost completely abrogated when a corresponding glycosidase inhibitor is present (Figures 3 and S7). Success of the in vitro study encouraged us to conduct an exploratory study to determine if sugar-CM-F probes can be employed for fluorescence imaging and isolation of glycosidases in mammalian cells. It is known that post-translational

Figure 4. Fluorescence imaging of O-GlcNAcase in mammalian cells using β-GlcNAc-CM-F. (a) Confocal fluorescence microscopy images of HT-29 cells treated with β-GlcNAc-CM-F in the absence and presence of PUGNAc (scale bar: 10 μm). (b) Flow cytometry of HT-29 cells treated with β-GlcNAc-CM-F in the absence and presence of PUGNAc. (c) HT-29 cells were incubated with β-GlcNAc-CM-F or βGlcNAc-CM. Before and after being washed with culture media for 2 h, cell images were obtained using confocal fluorescence microscopy.

cellular O-GlcNAcase is selectively detected by β-GlcNAc-CMF. Because a reaction with the probe causes a fluorophore to become covalently linked to the enzyme, fluorescence of the captured glycosidase should be retained in the cells even after prolonged incubation. Indeed, fluorescence arising from the labeled glycosidase was retained in the cells even after 2 h washing (Figure 4c). In marked contrast, β-GlcNAc-CM, which does not contain a fluoromethyl group, was removed from cells by washing (Figure 4c). The findings clearly indicate that βGlcNAc-CM-F can be employed for efficient and long-term imaging of the endogenous glycosidase in live cells. Finally, we evaluated if O-GlcNAcase can be isolated from cells using a sequence involving labeling with β-GlcNAc-CM-F followed by click chemistry and affinity chromatography. For this purpose, lysates of HT-29 cells, which were incubated with β-GlcNAc-CM-F in absence and presence of PUGNAc, were subjected to click reaction with N3-biotin, followed by streptavidin affinity purification. Gels, arising from SDS-PAGE analysis and streptavidin−horseradish peroxidase staining of the eluted proteins, exhibited one band whose staining intensity was greatly reduced when the labeling reaction was conducted in the presence of PUGNAc (Figure 5). Specifically, the protein labeled by β-GlcNAc-CM-F migrated at ca. 130 kDa, which corresponds to O-GlcNAcase. The identity of the labeled

Figure 3. Selective labeling of glycosidases by sugar-CM-F probes. Mixtures of three glycosidases were incubated with each sugar-CM-F probe in the absence and presence of an inhibitor (1 mM CBE; 10 μM DGJ; 100 μM PUGNAc) and were then subjected to click reactions with N3-biotin followed by separation using SDS-PAGE (staining with (top) Coomassie blue and (bottom) Cy3-streptavidin). C

DOI: 10.1021/acs.orglett.9b01147 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

Seeberger, P. Essentials of Glycobiology, 3rd ed.; Cold Spring Harbor Laboratory Press, 2015. (3) Winchester, B. Glycobiology 2005, 15, 1R−15R. (4) (a) Niphakis, M. J.; Cravatt, B. F. Annu. Rev. Biochem. 2014, 83, 341. (b) Blum, G.; Mullins, S. R.; Keren, K.; Fonovic, M.; Jedeszko, C.; Rice, M. J.; Sloane, B. F.; Bogyo, M. Nat. Chem. Biol. 2005, 1, 203. (c) Su, Y.; Ge, J.; Zhu, B.; Zheng, Y. G.; Zhu, Q.; Yao, S. Q. Curr. Opin. Chem. Biol. 2013, 17, 768. (5) Willems, L. I.; Jiang, J.; Li, K. Y.; Witte, M. D.; Kallemeijn, W. W.; Beenakker, T. J.; Schröder, S. P.; Aerts, J. M.; van der Marel, G. A.; Codée, J. D.; Overkleeft, H. S. Chem. - Eur. J. 2014, 20, 10864. (6) (a) Kwan, D. H.; Chen, H.-M.; Ratananikom, K.; Hancock, S. M.; Watanabe, Y.; Kongsaeree, P. T.; Samuels, A. L.; Withers, S. G. Angew. Chem., Int. Ed. 2011, 50, 300. (b) Witte, M. D.; Kallemeijn, W. W.; Aten, J.; Li, K.-Y.; Strijland, A.; Donker-Koopman, W. E.; van den Nieuwendijk, A. M.; Bleijlevens, B.; Kramer, G.; Florea, B. I.; Hooibrink, B.; Hollak, C. E.; Ottenhoff, R.; Boot, R. G.; van der Marel, G. A.; Overkleeft, H. S.; Aerts, J. M. Nat. Chem. Biol. 2010, 6, 907. (c) Whidbey, C.; Sadler, N. C.; Nair, R. N.; Volk, R. F.; DeLeon, A. J.; Bramer, L. M.; Fansler, S. J.; Hansen, J. R.; Shukla, A. K.; Jansson, J. K.; Thrall, B. D.; Wright, A. T. J. Am. Chem. Soc. 2019, 141, 42. (7) Doura, T.; Kamiya, M.; Obata, F.; Yamaguchi, Y.; Hiyama, T. Y.; Matsuda, T.; Fukamizu, A.; Noda, M.; Miura, M.; Urano, Y. Angew. Chem., Int. Ed. 2016, 55, 9620. (8) (a) Myers, J.; Widlanski, T. Science 1993, 262, 1451. (b) Komatsu, T.; Kikuchi, K.; Takakusa, H.; Hanaoka, K.; Ueno, T.; Kamiya, M.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2006, 128, 15946. (9) Fkyerat, A.; Dubin, G.-M.; Tabacchi, R. Helv. Chim. Acta 1999, 82, 1418. (10) (a) Hyun, J. Y.; Kang, N. R.; Shin, I. Org. Lett. 2018, 20, 1240. (b) Park, S.; Shin, I. Org. Lett. 2007, 9, 619. (c) Hyun, J. Y.; Kim, S.; Lee, H. S.; Shin, I. Cell Chem. Biol. 2018, 25, 1255. (11) (a) Tai, C. H.; Lu, C. P.; Wu, S. H.; Lo, L. C. Chem. Commun. 2014, 50, 6116. (b) Ahmed, V.; Liu, Y.; Taylor, S. D. ChemBioChem 2009, 10, 1457. (12) (a) Banerjee, P. S.; Hart, G. W.; Cho, J. W. Chem. Soc. Rev. 2013, 42, 4345. (b) Alonso, J.; Schimpl, M.; van Aalten, D. M. J. Biol. Chem. 2014, 289, 34433. (13) Brennan, J. P.; Miller, J. I.; Fuller, W.; Wait, R.; Begum, S.; Dunn, M. J.; Eaton, P. Mol. Cell. Proteomics 2006, 5, 215.

Figure 5. Isolation of O-GlcNAcase in mammalian cells using βGlcNAc-CM-F. HT-29 cells were incubated with β-GlcNAc-CM-F in the absence and presence of PUGNAc. Lysates of treated cells were subjected to click reaction with N3-biotin. The labeled proteins were isolated by affinity chromatography. The red triangle indicates OGlcNAcase. The protein band at ca. 75 kDa represents an endogenous biotinylated protein (ref 13).

protein was confirmed by using Western blotting with the OGlcNAcase antibody (Figure 5). To further confirm its identity, the protein gel band was excised and digested with trypsin. LCMS analysis showed that the protein labeled by GlcNAc-CM-F is O-GlcNAcase (Table S2). Because β-GlcNAc-CM-F is mainly located to the cytosol, it is likely that the cytosolic O-GlcNAcase is mostly captured by the probe. In conclusion, we designed, prepared, and characterized a novel type of mechanism-based, trifunctional fluorogenic probes for glycosidases. One of the probes was employed for fluorescence imaging of endogenous O-GlcNAcase and isolation of the captured enzyme from mammalian cells by affinity chromatography. It is anticipated that the strategy employed to design the glycosidase probes explored in this effort will be exploited in developing activity-based fluorogenic probes for other biologically important enzymes.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b01147. Experimental procedures, chemical characterization, and additional data (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Injae Shin: 0000-0001-6397-0416 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported financially by the National Creative Research Initiative program (Grant No. 2010-0018272 to I.S.) and the National Research Foundation (Grant No. NRF2016R1A5A1010764 to J.W.C).

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REFERENCES

(1) (a) Varki, A. Glycobiology 2017, 27, 3. (b) Park, S.; Lee, M.-R.; Shin, I. Chem. Soc. Rev. 2008, 37, 1579. (2) Varki, A.; Cummings, R.; Esko, J.; Stanley, P.; Hart, G.; Aebi, M.; Darvill, A.; Kinoshita, T.; Packer, N.; Prestegard, J.; Schnaar, R.; D

DOI: 10.1021/acs.orglett.9b01147 Org. Lett. XXXX, XXX, XXX−XXX