Letter Cite This: Org. Lett. 2018, 20, 1240−1243
pubs.acs.org/OrgLett
Carbohydrate Microarrays Containing Glycosylated Fluorescent Probes for Assessment of Glycosidase Activities Ji Young Hyun,† Na Rae Kang,† and Injae Shin* Center for Biofunctional Molecules, Department of Chemistry, Yonsei University, Seoul 03722, Republic of Korea S Supporting Information *
ABSTRACT: Carbohydrate microarrays, containing glycosylated fluorescent probes, have been constructed using N-hydroxysuccinimide (NHS) ester-conjugated BSA modified surfaces. When the carbohydrate moieties were cleaved from the fluorescent probes in the conjugates by glycosidases, the fluorescence signals of the probes were enhanced. In this study, we have applied these microarrays to profile glycosidase activities and have employed them to determine IC50 values of glycosidase inhibitors.
employed.12,13 The NIR scaffold was chosen as a fluorescent probe because it is readily synthesized and contains one hydroxyl group which can serve as a site for glycosylation.14 It also contains the functional group that is required for immobilization onto the solid surface. The five NIR probes conjugated with mono- and disaccharides were prepared according to the procedure shown in Scheme 1. Briefly, an alkyne-bearing NIR probe (NIR-AY), synthesized by using a slight modification of a known procedure,14 was subjected to glycosylation with peracetylated glycosyl halides (α-Glc(OAc)4Br, α-Gal(OAc)4-Br, α-GlcNAc(OAc)3-Cl, α-GalNAc(OAc)3Cl, or α-Lac(OAc)7-Br) in biphasic solutions to give the corresponding Sugar(OAc)-NIR-AY conjugates (9−13).15 The resulting compounds were individually coupled with N3-linkerNHNHBoc to afford Sugar(OAc)-NIR-NHNHBoc (14−18) by click chemistry.16 The hydrazide group was incorporated in the fluorogenic probes so that they could be readily attached to modified surfaces. It should be noted that attempted reactions of N3-linker-NHNH2, lacking the Boc group, with Sugar(OAc)NIR-AY conjugates by click chemistry, did not give the desired products. Sequential removal of the O-acetyl and Boc protecting groups then produced 1−5. Fluorescence responses of monosaccharide-conjugated NIR probes 1−4 to glycosidases were initially examined. Glycosylated probes exhibited very weak fluorescence,12 but an unglycosylated probe, NIR-NHNH2, showed strong fluorescence (Figure S1). When probes 1−4 were treated with the corresponding glycosidases (β-glucosidase from almonds for 1, β-galactosidase from A. oryzae for 2, and β-N-acetylhexosaminidase from S. pneumoniae for 3 and 4), fluorescence intensities were rapidly increased in a time-dependent manner (Figure S2). Enzyme kinetic studies were then conducted on
I
n the postgenomic era, functional studies of glycans in biological systems have become exceptionally important in order to provide deep insight into glycan-associated physiological and pathological processes.1 The ultimate aim of these efforts is to develop efficacious therapeutic agents and diagnostic tools. Cellular glycans are produced mainly in the form of glycoconjugates (e.g., glycoproteins and glycosphingolipids) by the action of glycosyltransferases and glycosidases (or glycoside hydrolases) in the endoplasmic reticulum (ER) and Golgi apparatus, and are degraded in lysosomes by the action of glycosidases. It is known that genetic deficiencies of lysosomal glycosidases cause lysosomal storage disorders.2 Moreover, inhibitors of glycosidases have been developed as therapeutic agents to treat viral infections, diabetes, and cancer.3 Because of the biological and pathological significance of glycosidases, it is highly important to develop new methods to assess their catalytic activities. Since their disclosure in 2002, carbohydrate microarrays, possessing a variety of glycans immobilized on solid surfaces, have been extensively applied for rapid analysis of carbohydrate associated recognition events.4−6 This microarray technology also has been successfully employed for quantitatively analyzing glycan−protein interactions,7 profiling substrate specificities of glycosyltransferases,7,8 and detecting pathogens.9 Recently, we showed that carbohydrate microarrays were useful for discovering functional glycans that elicited cellular responses.10 In spite of this high level of activity, applications of carbohydrate microarrays in profiling glycosidase activities have been rare.11 Below, we describe a new method to characterize glycosidase activities, which utilizes carbohydrate microarrays containing glycosylated fluorescent probes whose fluorescence signals increase upon glycosidase-promoted cleavage of glycosidic bonds. In the current effort, glycosylated near-infrared (NIR) based probes 1−5 as fluorogenic substrates of glycosidases were © 2018 American Chemical Society
Received: January 17, 2018 Published: February 8, 2018 1240
DOI: 10.1021/acs.orglett.8b00180 Org. Lett. 2018, 20, 1240−1243
Letter
Organic Letters Scheme 1. Synthesis of Glycosylated NIR Probes (1−5) Used for Construction of Carbohydrate Microarrays
probes 1−4 by measuring the rates of glycosidase-promoted increases in fluorescence intensities (λex = 680 nm, λem = 710 nm) as a function of the concentrations of the probes. For comparison purposes, kinetic studies were also performed using glycosylated coumarin- and p-nitrophenyl (PNP)-based probes (Figure S3). Analysis of kinetic data revealed that NIR-based probes are better substrates than coumarin- and PNP-based counterparts for glycosidases (Figure 1, Figure S3 and Table S1). This conclusion comes from the fact that NIR-based substrates have greater kcat and kcat/Km values compared to their coumarin- and PNP-based counterparts. The results also showed that GlcNAc-NIR-NHNH2 (3) is a better substrate than GalNAc-NIR-NHNH 2 (4) for β-N-acetylhexosaminidase.17 To uncover suitable inhibitors of glycosidases for use in this study, β-glucosidase, β-galactosidase, and β-N-acetylhexosaminidase were individually treated with the corresponding fluorogenic probes and various concentrations of conduritol B epoxide (CBE),18a 1-deoxygalactonojirimycin (DGJ),18b and O-(2-acetamido-2-deoxy- D -glucopyranosylidene)amino-Nphenylcarbamate (PUGNAc).18c The results showed that CBE, DGJ, and PUGNAc act as inhibitors of β-glucosidase, βgalactosidase, and β-N-acetylhexosaminidase, respectively (Figure S4). In addition, CBE was found to be a covalent irreversible inhibitor of β-glucosidase, as judged from the fact that the enzyme activity was not recovered after extensive dialysis of the incubation mixture of β-glucosidase and CBE (Figure S5). Next, carbohydrate microarrays containing glycosylated NIRbased probes were constructed and employed to determine their utility in assessing glycosidase activities. For this purpose, epoxide and N-hydroxysuccinimide (NHS) ester-conjugated BSA modified surfaces were prepared. Epoxide derivatized glass slides were fabricated from amine modified slides (Figure 2, route A),19 and NHS ester BSA coated slides were generated by treating the epoxide derivatized slides with 1% BSA followed by conversion of the carboxylic acid groups in BSA to NHS esters (Figure 2, route B). The efficiencies of immobilization of the
Figure 1. Enzyme kinetics of (A) β-glucosidase, (B) β-galactosidase, and (C) β-N-acetylhexosaminidase. Lineweaver−Burk plots were generated by varying concentrations of (A) β-Glc-NIR-NHNH2 (1), (B) β-Gal-NIR-NHNH2 (2), and (C) β-GlcNAc-NIR-NHNH2 (3, left) or β-GalNAc-NIR-NHNH2 (4, right). (D) Kinetic parameters determined from analysis of plots in A−C (mean ± s.d., n = 3).
hydrazide conjugated probes on the two types of modified slides were determined by printing a nonglycosylated, fluorescent probe using a robotic high-precision pin-type microarrayer. Analysis of microarray images indicates that NIR-NHNH2 is immobilized to a similar degree onto both epoxide and NHS ester-conjugated BSA modified surfaces 1241
DOI: 10.1021/acs.orglett.8b00180 Org. Lett. 2018, 20, 1240−1243
Letter
Organic Letters
Figure 2. Preparation of epoxide and NHS ester-conjugated BSA derivatized glass slides and the construction of carbohydrate microarrays by immobilizing hydrazide-conjugated substrates on the modified surfaces.
because similar fluorescence intensities of NIR-NHNH2 are detected onto both modified surfaces (Figure S6). Next, we compared the glycosidase-promoted cleavage of the sugar moieties attached to NHS ester BSA coated slides with that of the sugar moieties linked to epoxide slides. In this study, carbohydrate microarrays, constructed by printing 1−3 onto epoxide and NHS ester-conjugated BSA coated surfaces, were separately incubated with β-glucosidase, β-galactosidase, or βN-acetylhexosaminidase for time periods of 0−8 h. The results of microarray image analysis showed that the sugar−probe conjugates attached to the NHS ester-conjugated BSA coated surface serve as better substrates for the enzymatic reactions (Figure S7). This observation might be a consequence of the better accessibility of glycosidases to the glycosidic bonds in probes attached to BSA modified surfaces than those to epoxide derivatized surfaces. Thus, carbohydrate microarrays, created using NHS ester-conjugated BSA modified slides, were employed for further study. It was also found from this study that the rates of removal of the sugar moieties in probes 1 and 2 by β-glucosidase and β-galactosidase, respectively, are similar but are slower compared with 3 by β-N-acetylhexosaminidase. These findings are consistent with those arising from the solution-based assays (Figure 1). To determine their utility in profiling catalytic activities of glycosidases, carbohydrate microarrays were prepared by printing probes 1−5 (immobilization concentrations: 0.1, 0.25, 0.5, and 1.0 mM) on NHS ester-conjugated BSA coated surfaces. The microarrays were individually incubated with βglucosidase, β-galactosidase, and β-N-acetylhexosaminidase, or a mixture of β-glucosidase and β-galactosidase in the absence and presence of the corresponding inhibitors CBE, DGJ, and PUGNAc. Analysis of microarray images showed that Glc and Gal moieties were cleaved from 1 and 2, respectively, to a similar degree by β-glucosidase and β-galactosidase (Figure 3A and 3B). It was also found that 3 was a better substrate for β-Nacetylhexosaminidase than 4, based on the higher fluorescence intensity of 3 when compared with 4 following the enzymatic reactions (Figure 3C).17 The results arising from these studies of the enzymatic reactions conducted using carbohydrate microarrays were consistent with those obtained by solutionbased assays (as shown in Figure 1). As expected, β-lactose was cleaved from 5 only when both β-glucosidase and βgalactosidase were present but not when each individual
Figure 3. Fluorescence profiles of carbohydrate microarrays, prepared by printing four different concentrations (0.1, 0.25, 0.5, and 1.0 mM) of 1−5 on NHS ester BSA derivatized glass slides, after treatment with 500 nM of (A) β-glycosidase for 8 h, (B) β-galactosidase for 8 h, (C) β-N-acetylhexosaminidase for 2 h, and (D) a mixture of β-glycosidase and β-galactosidase for 8 h in the absence (left image) or presence (right image) of an indicated inhibitor. Bar graphs show fluorescence intensities of microarrays treated with glycosidases in the absence of inhibitors (mean ± s.d., n = 3).
enzyme is used (Figure 3D). This indicates that sequential removal of β-Gal and β-Glc from the probe 5 leads to an increase in fluorescence signals. In addition, carbohydrate microarrays were employed to detect glycosidase activity in E. coli lysates. In this study, E. coli lysates containing glycosidases were applied to the microarrays in the absence and presence of the corresponding inhibitors. As shown in Figure S8, glycosidases in cell lysates cleaved their corresponding glycans in a similar way to the purified enzymes. Carbohydrate microarrays were also applied to determine concentrations (IC50 values) of the inhibitors that lead to 50% inhibition of glycosidase activities. In this study, microarrays containing probes 1−4 were separately incubated with each glycosidase in the presence of various concentrations of CBE, DGJ, or PUGNAc. Analysis of fluorescence intensities of locations on the microarrays enabled determination of the following IC50 values: 108.6 μM of CBE using 1 toward βglucosidase, 3.2 μM of DGJ using 2 toward β-galactosidase, and 21.8 nM and 18.6 nM of PUGNAc using 3 and 4, respectively, toward β-N-acetylhexosaminidase (Figures 4 and S9). The results match those (93.3 μM of CBE using 1; 2.4 μM of DGJ using 2; 21.7 nM and 19.2 nM of PUGNAc using, respectively, 3 and 4) obtained using solution-based assays at the different 1242
DOI: 10.1021/acs.orglett.8b00180 Org. Lett. 2018, 20, 1240−1243
Organic Letters
Letter
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ACKNOWLEDGMENTS This study was financially supported by the National Creative Research Initiative Program (Grant No. 2010-0018272) in Korea.
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Figure 4. Determination of IC50 values of glycosidase inhibitors by using carbohydrate microarrays. Carbohydrate microarrays containing probes 1−4 were incubated with 500 nM of (A) β-glucosidase for 8 h, (B) β-galactosidase for 8 h, and (C) β-N-acetylhexosaminidase for 3 h in the presence of various concentrations of an indicated inhibitor. Carbohydrate microarray images for this study are shown in Figure S9.
concentrations of an inhibitor (Figure S10). The finding indicates that carbohydrate microarrays can be utilized to determine the IC50 values of glycosidase inhibitors. In the effort described above, we demonstrated that carbohydrate microarrays immobilized by glycosylated fluorescent probes are highly useful for profiling glycosidase activities. In addition, we showed for the first time that the new carbohydrate microarrays can be applied to determine IC50 values of glycosidase inhibitors although the microarrays are not able to assess the mode of inhibition. We anticipate that carbohydrate microarrays containing fluorogenic substrates will be applicable in the analysis of activities and inhibition properties of other classes of 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.8b00180. Synthetic details, microarray studies, supplementary tables and figures, and NMR spectra (PDF)
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REFERENCES
(1) (a) Park, S.; Lee, M. R.; Shin, I. Chem. Soc. Rev. 2008, 37, 1579− 1591. (b) Varki, A. Glycobiology 1993, 3, 97−130. (2) Platt, F. M. Nature 2014, 510, 68−75. (3) Asano, N. Glycobiology 2003, 13, 93R−104R. (4) (a) Park, S.; Shin, I. Angew. Chem., Int. Ed. 2002, 41, 3180−3182. (b) Wang, D. N.; Liu, S. Y.; Trummer, B. J.; Deng, C.; Wang, A. L. Nat. Biotechnol. 2002, 20, 275−281. (c) Fukui, S.; Feizi, T.; Galustian, C.; Lawson, A. M.; Chai, W. Nat. Biotechnol. 2002, 20, 1011−1017. (5) (a) Hyun, J. Y.; Pai, J.; Shin, I. Acc. Chem. Res. 2017, 50, 1069− 1078. (b) Rillahan, C. D.; Paulson, J. C. Annu. Rev. Biochem. 2011, 80, 797−823. (c) Park, S.; Gildersleeve, J. C.; Blixt, O.; Shin, I. Chem. Soc. Rev. 2013, 42, 4310−4326. (6) (a) Park, S.; Lee, M. R.; Pyo, S. J.; Shin, I. J. Am. Chem. Soc. 2004, 126, 4812−4819. (b) Lee, M. R.; Shin, I. Org. Lett. 2005, 7, 4269− 4272. (c) Park, S.; Lee, M. R.; Shin, I. Bioconjugate Chem. 2009, 20, 155−162. (d) Xia, B. Y.; Kawar, Z. S.; Ju, T. Z.; Alvarez, R. A.; Sachdev, G. P.; Cummings, R. D. Nat. Methods 2005, 2, 845−850. (e) Manimala, J. C.; Roach, T. A.; Li, Z. T.; Gildersleeve, J. C. Angew. Chem., Int. Ed. 2006, 45, 3607−3610. (f) de Paz, J. L.; Noti, C.; Seeberger, P. H. J. Am. Chem. Soc. 2006, 128, 2766−2767. (g) Tian, X.; Pai, J.; Shin, I. Chem. - Asian J. 2012, 7, 2052−2060. (h) Hyun, J. Y.; Park, C. W.; Liu, Y.; Kwon, D.; Park, S. H.; Park, S.; Pai, J.; Shin, I. ChemBioChem 2017, 18, 1077−1082. (7) Park, S.; Shin, I. Org. Lett. 2007, 9, 1675−1678. (8) Blixt, O.; Allin, K.; Bohorov, O.; Liu, X. F.; Andersson-Sand, H.; Hoffmann, J.; Razi, N. Glycoconjugate J. 2008, 25, 59−68. (9) Park, S.; Lee, M. R.; Shin, I. Nat. Protoc. 2007, 2, 2747−2758. (10) Pai, J.; Hyun, J. Y.; Jeong, J.; Loh, S.; Cho, E. H.; Kang, Y. S.; Shin, I. Chem. Sci. 2016, 7, 2084−2093. (11) McCombs, J. E.; Diaz, J. P.; Luebke, K. J.; Kohler, J. J. Carbohydr. Res. 2016, 428, 31−40. (12) Zhang, J. T.; Li, C.; Dutta, C.; Fang, M. X.; Zhang, S. W.; Tiwari, A.; Werner, T.; Luo, F. T.; Liu, H. Y. Anal. Chim. Acta 2017, 968, 97−104. (13) See other types of glycosylated NIR-based probes: (a) Han, J. Y.; Han, M. S.; Tung, C. H. Mol. BioSyst. 2013, 9, 3001−3008. (b) Oushiki, D.; Kojima, H.; Takahashi, Y.; Komatsu, T.; Terai, T.; Hanaoka, K.; Nishikawa, M.; Takakura, Y.; Nagano, T. Anal. Chem. 2012, 84, 4404−4410. (14) Yuan, L.; Lin, W. Y.; Zhao, S.; Gao, W. S.; Chen, B.; He, L. W.; Zhu, S. S. J. Am. Chem. Soc. 2012, 134, 13510−13523. (15) Park, S.; Shin, I. Org. Lett. 2007, 9, 619−622. (16) Moses, J. E.; Moorhouse, A. D. Chem. Soc. Rev. 2007, 36, 1249− 1262. (17) Imaki, H.; Tomoyasu, T.; Yamamoto, N.; Taue, C.; Masuda, S.; Takao, A.; Maeda, N.; Tabata, A.; Whiley, R. A.; Nagamune, H. J. Bacteriol. 2014, 196, 2817−2826. (18) (a) Ridley, C. M.; Thur, K. E.; Shanahan, J.; Thillaiappan, N. B.; Shen, A.; Uhl, K.; Walden, C. M.; Rahim, A. A.; Waddington, S. N.; Platt, F. M.; van der Spoel, A. C. J. Biol. Chem. 2013, 288, 26052− 26066. (b) Rigat, B. A.; Tropak, M. B.; Buttner, J.; Crushell, E.; Benedict, D.; Callahan, J. W.; Martin, D. R.; Mahuran, D. J. Mol. Genet. Metab. 2012, 107, 203−212. (c) Kim, E. J.; Perreira, M.; Thomas, C. J.; Hanover, J. A. J. Am. Chem. Soc. 2006, 128, 4234−4235. (19) (a) Lee, M. R.; Shin, I. Angew. Chem., Int. Ed. 2005, 44, 2881− 2884. (b) Pai, J.; Yoon, T.; Kim, N. D.; Lee, I. S.; Yu, J.; Shin, I. J. Am. Chem. Soc. 2012, 134, 19287−19296. (c) Pai, J.; Hyun, S.; Hyun, J. Y.; Park, S. H.; Kim, W. J.; Bae, S. H.; Kim, N. K.; Yu, J.; Shin, I. J. Am. Chem. Soc. 2016, 138, 857−867.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Injae Shin: 0000-0001-6397-0416 Author Contributions †
J.Y.H. and N.R.K. contributed equally.
Notes
The authors declare no competing financial interest. 1243
DOI: 10.1021/acs.orglett.8b00180 Org. Lett. 2018, 20, 1240−1243
