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Synthesis and labeling of a-(2,9)-trisialic acid with cyanine dyes for imaging of glycan-binding receptors on living cells†
Received 6th March 2015, Accepted 10th April 2015
Xiao-tai Zhang,‡a Zhen-yuan Gu,‡a Libing Liu,b Shu Wangb and Guo-wen Xing*a
DOI: 10.1039/c5cc01907a www.rsc.org/chemcomm
A sugar epitope, a-(2,9)-trisialic acid, was synthesized and labeled by cyanine dyes through Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC). The cyanine tagged oligosialic acid can be utilized as an efficient fluorescent probe to image the glycan-binding receptors on PC-12 cells. The distribution of Sia-binding immunoglobulin-like lectins (Siglecs) for a-(2,9)-trisialic acid was visualized by Cy3/Cy5 or FRET channel fluorescence imaging.
Sialic acids (Sia) are found to be the typical monosaccharides located at the terminal position of various glycoconjugates on cell surfaces or in intracellular membranes. These unique carboxylated nine-carbon sugars exhibit complex structural diversity, and the Sia family comprises over 60 members with variations at C-5 or modifications of the hydroxyl groups at C-4, C-7, C-8 and C-9.1 Due to the significance of Sia in human health and disease, tremendous effort has been exerted in the research of Sia chemistry, biochemistry and cell biology.2 More importantly, the Sia-binding immunoglobulin-like lectins (Siglecs), which belong to the immunoglobulin superfamily, have drawn great research interest in recent years. The sialoglycoconjugate–Siglecs interactions have crucial influences on various biological processes including signal transduction, cell adhesion, pathogen recognition, etc.3 One of the most challengeable issues in exploring the multibiological functions of Sia is how to in vivo image sialylated glycans or Siglecs in cells, especially in cancer cells, since many tumor-associated carbohydrate antigens are found to be sialoglycoconjugates, oligosialic or polysialic acids, which were overexpressed in various tumors.4 In recent years, two strategies have been developed for in vivo imaging of Sia containing glycans or the sialylation process.5 The first one is metabolic labeling, a
Department of Chemistry, Beijing Normal University, Beijing 100875, China. E-mail: [email protected] b Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China † Electronic supplementary information (ESI) available: Detailed experimental procedures. See DOI: 10.1039/c5cc01907a ‡ X.-T. Zhang and Z.-Y. Gu contributed equally to this work.
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in which Sia precursors, particularly N-acetylmannosamine (ManNAc) analogues, were employed for the living cell’s Sia biosynthesis to produce unnatural Sia modified with a special functional group, such as azide,6 alkyne7 or diazirine,6g on the sugar ring for further fluorescence labeling via bioorthogonal chemistry. The second one is direct recognition of Sia or the sialoglycoconjugate by fluorescent probes equipped with boronic and functional groups as Sia receptors.8 More recently, the crystal structures of immunoglobulin-like type II receptors PILRa and PILRb bearing Siglecs fold were solved by X-ray crystallography to image the molecular mechanism of Sia recognition.9 However, methods for glycan-specific imaging of Siglecs, especially unknown Siglecs, are still largely lacking. One of the bottlenecks to address this issue is the limited supply and efficient fluorescent labeling of a chemically pure sialoglycoconjugate. In this paper, we report the synthesis and labeling of sialylated glycan, and its use as a probe for the imaging of glycan-binding acceptors on living cells. Since a-(2,9)-di/oligosialic acids have been found as sugar epitopes in human teratocarcinoma10a and mouse neuroblastoma cells,10b a-(2,9)-trisialic acid (triSia), a complex oligosialic acid, was chosen for chemical synthesis, fluorescent labeling and cell imaging. As shown in Fig. 1, a a-(2,9)-trisialic acid derivative pendant with an azide group was prepared by multi-step chemical reactions. Then, the oligosialic acid was labeled by cyanine dyes (Cy3 or Cy5) via CuAAC addition assisted by the ligand tris(benzyltriazolylmethyl)amine (TBTA). Finally, the dye-tagged trisialic acid probes were used for the fluorescence imaging of Siglecs on PC-12 cells. In the past decade, although several research groups have reported the synthesis of a-(2,9)-oligosialic acid using different N-5 protected sialic acid donors including sialyl phosphite,11a,b phosphate,11c and thiosialoside,11d,e the development of a new efficient and stereoselective strategy for oligosialic acid synthesis is still continuously required.11 f On the basis of our previous studies on Sia chemistry,12 we prepared the azide derivative of a-(2,9)trisialic acid (triSia-N3) 9 from the known thiosialoside 1 reported by us12a in 12 steps with 10% overall yield (Scheme S1, ESI†).
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Fig. 1
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Schematic of the trisialic acid synthesis, labeling and imaging.
The crucial glycosylation in the multi-step synthesis employed the preactivation protocol with (p-Tol)2SO/Tf2O as a promoter, which was developed by our group in the O- and C-sialylations.12d–f With 5-N,4-O-carbonyl-protected p-toluenethiosialoside 2 as a sialyl donor, the corresponding a-(2,9)-mono/di/trisialosides 3, 5 and 7 were successfully constructed in 93%, 88% and 69% yield with exclusive a-selectivity, respectively. The structures of the sialylation products were determined by NMR spectroscopy, which are consistent with the observations by the Lin group11e (see ESI† for details). Global deprotection of all the protective groups in trisaccharide 8 provided triSia-N3 9 in 75% yield. Cyanine, one of the most important biological dyes, is widely used as fluorescence labels and sensors for in vivo and in vitro biological and medical applications.13 For this purpose, we tried to label a cyanine dye to trisialic acid 9 through Cu(I)-catalyzed azide–alkyne cycloaddition.14 Notably, although a few azide functionalized glycan epitopes bearing Sia residues were synthesized,11c,e,15 to the best of our knowledge, there is no report on the addition of cyanine dyes and Sia containing sugars via CuAAC reaction. To address this issue, initially, 3-azidopropyl a-(2,9)-monosialoside (monoSia-N3) 10 was chosen as a reference compound for the CuAAC reaction to attach a Cy5 dye molecule. Surprisingly, as shown in Table 1 (entry 1), no desired product 12 was obtained under the classical CuAAC coupling conditions with 2.5 mol% copper(II) sulfate and 25 mol% sodium ascorbate (VcNa). PMDETA16a and DIEPA16b have been used as additives for the cyanine dye involved CuAAC reaction to ensure its high coupling efficiency. However, the same negative results were produced with the combination of PMDETA/DIEPA, CuI, and DMF as the CuAAC system (Table 1, entries 2 and 3). Polytriazolylamines,17 including TBTA17a and its water soluble analogues, were other kinds of useful
Table 1
Entry 1 2 3 4
CuAAC reaction of monoSia-N3 (10) and Cy5 alkyne (11)
Catalyst VcNa (0.25 eq.), CuSO4 (0.025 eq.) CuI (0.75 eq.) CuI (0.1 eq.) VcNa (0.4 eq.), CuSO4 (0.2 eq.)
Additive
Solvent
None
t
PMDETAc (1.5 eq.) DIEPAd (0.2 eq.) TBTAe (0.4 eq.)
DMF DMF t BuOH–H2Oa
BuOH–H2O
Yield a
—b —b —b 86%
a The ratio of the solvent mixture is 1 : 1. b No reaction. c PMDETA = N,N,N 0 ,N 0 ,N00 -pentamethyldiethylenetriamine. d DIEPA = diisopropylethylamine. e TBTA = tris-(benzyltriazolylmethyl)amine.
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additives for CuAAC due to the powerful ability to stabilize Cu(I) and accelerate the cycloadditions. To our delight, when TBTA served as the ligand for the traditional CuAAC irradiated by ultrasound (Table 1, entry 4), the Cy5 tagged monoSia (monoSia-Cy5, 12) was finally obtained in high yield (86%). Encouraged by these results, Cy5 and Cy3-tagged a-(2,9)trisialic acids, triSia-Cy5 (14) and triSia-Cy3 (15), were prepared under the similar CuAAC conditions assisted by the ligand TBTA from Cy5/Cy3 alkyne (11/13) and triSia-N3 (9) in 87% and 76% yield, respectively (Fig. 2). The dye labeled oligosialic acids were purified by P-2 biogel size exclusion column chromatography eluted with H2O to afford the corresponding glycoconjugates. TriSia-Cy5 was validated by NMR spectroscopy and high resolution mass spectrometry, which recorded a signal at m/z 1522.7097 corresponding to [triSia-Cy5 + 3H]+ with an expected m/z of 1522.7093. Similar results were obtained for triSia-Cy3 (see ESI† for details). The absorption and fluorescence properties of cyanine alkynes and cyanine labeled trisialic acids used in this study are shown in Table S1 and Fig. S1, ESI†. There is no remarkable difference in the optical characteristics of cyanine dyes before/after labeling trisialic acid. Next, we introduced triSia-Cy3 or triSia-Cy5 into cultured PC-12 cells to observe the distribution of target molecules by fluorescence microscopy. Interestingly, the fluorescently-labeled triSia was principally gathered on the membrane of PC-12 cells, and the imaging region was discrete (Fig. 3). The amount of triSia-Cy3/triSia-Cy5 in the cytoplasm was negligible, because the negative charges of triSia remarkably restrict its cell permeability. It is worth noting that the natural Sia was abundantly expressed on the surface of PC-12 cells. Meanwhile, some glycan-binding receptors could be co-accreted with these sialylated glycoproteins or glycolipids. According to the results of imaging experiments, we considered that the labeled triSia should specifically bind to the Siglecs located on the PC-12 cell membranes. Additionally,
Fig. 2 Structures of Cy3 alkyne 13 and cyanine dye tagged a-(2,9)-trisialic acid 14–15.
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Fig. 3 Confocal laser scanning microscopy images of PC-12 cells incubated with triSia-Cy3 or triSia-Cy5 (5 mM) for 1 h. The middle column: Cy3 fluorescence (excitation: 559 nm/emission: 570–635 nm) or Cy5 fluorescence (excitation: 635 nm/emission: 650–720 nm). Scale bars, 10 mm.
the fluorescence of exogenous triSia-Cy3/triSia-Cy5 reflected the uneven distribution of Siglecs to some extent. As a comparison, monoSia-Cy5 (12) was also used for the imaging of PC-12 cells. To our surprise, monoSia-Cy5 entered into the cells rapidly. Further colocalization experiments involving 12 were performed using a commercially mitochondrionspecific fluorescent probe, MitoTracker Green (MTG) (Fig. S2, ESI†). A high Pearson’s colocalization coefficient (0.91) was obtained, indicating that the staining of monoSia-Cy5 fits well with that of mitochondrial dye MTG. The results show that monoSia-Cy5 is preferentially distributed in the mitochondria of PC-12 cells, which is totally different from the cases of triSiaCy3 and triSia-Cy5. As discussed above, triSia-Cy3/triSia-Cy5 was hardly brought into the PC-12 cells. To further verify the properties of triSia-Cy3
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and triSia-Cy5, we found that poly(ethylene imine) (PEI),18 a transfection agent, can assist the oligosialic acid to break through the membrane of PC-12 cells via endocytosis, and then the fluorescent imaging of triSia in the cytoplasm was achieved (Fig. S3, ESI†). In contrast to the images in Fig. 3b and e, clusters of fluorescent signals were observed obviously in Fig. S3b and e (ESI†), indicating that PEI can condense triSia-Cy3 or triSiaCy5 into positively-charged or electrically neutral particles. ¨rster resonance energy The Cy3 and Cy5 dyes are the typical Fo transfer (FRET) pair in fluorometry.19 The data in Fig. S1, ESI† show that the emission of triSia-Cy3 overlaps with the absorption of triSia-Cy5 to ensure the overlap integral requirement for resonance energy transfer, which allows real-time monitoring of various important cellular events. On this basis, to further obtain the information of spatial presentation of Siglecs on cells, we incubated PC-12 with equal amounts of triSia-Cy3 and triSia-Cy5 simultaneously for 1 h, and both the dual-channel (Cy3 and Cy5 fluorescence) and FRET-induced fluorescence images were collected using a confocal microscope (Fig. 4). As expected, the FRET effect of triSia-Cy3 to triSia-Cy5 was observed (Fig. 4c, g and k). Compared with Fig. 4j, the fluorescence intensity of energy acceptor triSia-Cy5 in Fig. 4k was much stronger under the same excitation conditions (lex = 559 nm). Although different kinds of Siglecs may coexist on the surface of the living cells, it is reasonable to consider that both the triSia-Cy3 and triSia-Cy5 targeted the same type of glycan-binding receptors due to their similar molecular structures. A pair of adjacent Siglec moieties on the membrane of the PC-12 cell with the spatial distance less than 10 nm had a 50% probability of binding with different cyaninelabeled triSias, which gave rise to an efficient FRET process from Cy3 to Cy5 consequently. The energy transfer efficiency E19 is B0.37, which was determined by the fluorescence intensity of the
Fig. 4 Fluorescence confocal images of PC-12 cells incubated with triSia-Cy3 (5 mM) and/or triSia-Cy5 (5 mM) for 1 h. The first row: Cy3 channel (excitation: 559 nm/emission: 570–635 nm); the second row: Cy5 channel (excitation: 635 nm/emission: 650–720 nm); the third row: FRET channel (excitation: 559 nm/emission: 650–720 nm); the fourth row: treatment of the images in (c), (g) and (k) by Origin 8.0, respectively. Scale bars, 5 mm.
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Cy3 channel in the absence/presence of triSia-Cy5. The simulated images (Fig. 4d, h and l) corresponding to Fig. 4c, g and k illustrated the distribution of Siglecs more clearly. There are some distinctions between the point cloud of Fig. 4d and h, indicating that triSia-Cy3 and triSia-Cy5 targeted different Siglec moieties on the cell surface. Additionally, the fluorescence region in Fig. 4l is a subaggregate of that in Fig. 4h, suggesting the special region of the targeted Siglecs in higher spatial density with a certain probability. In summary, cyanine dye labeled oligosialic acids for fluorescence imaging of glycan-binding receptors were deeply investigated in this study. A glycan epitope, a-(2,9)-trisialic acid, was successfully synthesized with (p-Tol)2SO/Tf2O as a promoter in the key a-stereoselective sialylations. It is the first time to efficiently label a negatively charged Sia bearing glycan by cyanine dyes through CuAAC reaction assisted by TBTA as the ligand. By collecting fluorescence in the dual-channel as well as the FRET channel, we showed that the triSia-Cy3/triSia-Cy5 could be enriched on the membrane of PC-12 cells to illustrate the spatial distribution of glycan-binding receptors like Siglecs. Taken together, it is deduced that triSia-Cy3/triSia-Cy5 is a specific fluorescent probe to interact with the glycan-binding receptors on PC-12 cells. To the best of our knowledge, there is no report about the Siglecs existence on PC-12 cells. Further studies on the isolation, identification and bioactivity of Siglecs on PC-12 cells are currently underway. Remarkably, the labeling strategy developed in this study provides a powerful tool to assess and visualize new glycan-binding receptors on the membrane of living cells. The project was financially supported by the National Natural Science Foundation of China (21272027), Beijing National Natural Science Foundation (2122031), and Beijing Municipal Commission of Education.
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Cite this: Chem. Commun., 2015, 51, 8606
Synthesis and labeling of a-(2,9)-trisialic acid with cyanine dyes for imaging of glycan-binding receptors on living cells†
Received 6th March 2015, Accepted 10th April 2015
Xiao-tai Zhang,‡a Zhen-yuan Gu,‡a Libing Liu,b Shu Wangb and Guo-wen Xing*a
DOI: 10.1039/c5cc01907a www.rsc.org/chemcomm
A sugar epitope, a-(2,9)-trisialic acid, was synthesized and labeled by cyanine dyes through Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC). The cyanine tagged oligosialic acid can be utilized as an efficient fluorescent probe to image the glycan-binding receptors on PC-12 cells. The distribution of Sia-binding immunoglobulin-like lectins (Siglecs) for a-(2,9)-trisialic acid was visualized by Cy3/Cy5 or FRET channel fluorescence imaging.
Sialic acids (Sia) are found to be the typical monosaccharides located at the terminal position of various glycoconjugates on cell surfaces or in intracellular membranes. These unique carboxylated nine-carbon sugars exhibit complex structural diversity, and the Sia family comprises over 60 members with variations at C-5 or modifications of the hydroxyl groups at C-4, C-7, C-8 and C-9.1 Due to the significance of Sia in human health and disease, tremendous effort has been exerted in the research of Sia chemistry, biochemistry and cell biology.2 More importantly, the Sia-binding immunoglobulin-like lectins (Siglecs), which belong to the immunoglobulin superfamily, have drawn great research interest in recent years. The sialoglycoconjugate–Siglecs interactions have crucial influences on various biological processes including signal transduction, cell adhesion, pathogen recognition, etc.3 One of the most challengeable issues in exploring the multibiological functions of Sia is how to in vivo image sialylated glycans or Siglecs in cells, especially in cancer cells, since many tumor-associated carbohydrate antigens are found to be sialoglycoconjugates, oligosialic or polysialic acids, which were overexpressed in various tumors.4 In recent years, two strategies have been developed for in vivo imaging of Sia containing glycans or the sialylation process.5 The first one is metabolic labeling, a
Department of Chemistry, Beijing Normal University, Beijing 100875, China. E-mail: [email protected] b Key Laboratory of Organic Solids, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China † Electronic supplementary information (ESI) available: Detailed experimental procedures. See DOI: 10.1039/c5cc01907a ‡ X.-T. Zhang and Z.-Y. Gu contributed equally to this work.
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in which Sia precursors, particularly N-acetylmannosamine (ManNAc) analogues, were employed for the living cell’s Sia biosynthesis to produce unnatural Sia modified with a special functional group, such as azide,6 alkyne7 or diazirine,6g on the sugar ring for further fluorescence labeling via bioorthogonal chemistry. The second one is direct recognition of Sia or the sialoglycoconjugate by fluorescent probes equipped with boronic and functional groups as Sia receptors.8 More recently, the crystal structures of immunoglobulin-like type II receptors PILRa and PILRb bearing Siglecs fold were solved by X-ray crystallography to image the molecular mechanism of Sia recognition.9 However, methods for glycan-specific imaging of Siglecs, especially unknown Siglecs, are still largely lacking. One of the bottlenecks to address this issue is the limited supply and efficient fluorescent labeling of a chemically pure sialoglycoconjugate. In this paper, we report the synthesis and labeling of sialylated glycan, and its use as a probe for the imaging of glycan-binding acceptors on living cells. Since a-(2,9)-di/oligosialic acids have been found as sugar epitopes in human teratocarcinoma10a and mouse neuroblastoma cells,10b a-(2,9)-trisialic acid (triSia), a complex oligosialic acid, was chosen for chemical synthesis, fluorescent labeling and cell imaging. As shown in Fig. 1, a a-(2,9)-trisialic acid derivative pendant with an azide group was prepared by multi-step chemical reactions. Then, the oligosialic acid was labeled by cyanine dyes (Cy3 or Cy5) via CuAAC addition assisted by the ligand tris(benzyltriazolylmethyl)amine (TBTA). Finally, the dye-tagged trisialic acid probes were used for the fluorescence imaging of Siglecs on PC-12 cells. In the past decade, although several research groups have reported the synthesis of a-(2,9)-oligosialic acid using different N-5 protected sialic acid donors including sialyl phosphite,11a,b phosphate,11c and thiosialoside,11d,e the development of a new efficient and stereoselective strategy for oligosialic acid synthesis is still continuously required.11 f On the basis of our previous studies on Sia chemistry,12 we prepared the azide derivative of a-(2,9)trisialic acid (triSia-N3) 9 from the known thiosialoside 1 reported by us12a in 12 steps with 10% overall yield (Scheme S1, ESI†).
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Fig. 1
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Schematic of the trisialic acid synthesis, labeling and imaging.
The crucial glycosylation in the multi-step synthesis employed the preactivation protocol with (p-Tol)2SO/Tf2O as a promoter, which was developed by our group in the O- and C-sialylations.12d–f With 5-N,4-O-carbonyl-protected p-toluenethiosialoside 2 as a sialyl donor, the corresponding a-(2,9)-mono/di/trisialosides 3, 5 and 7 were successfully constructed in 93%, 88% and 69% yield with exclusive a-selectivity, respectively. The structures of the sialylation products were determined by NMR spectroscopy, which are consistent with the observations by the Lin group11e (see ESI† for details). Global deprotection of all the protective groups in trisaccharide 8 provided triSia-N3 9 in 75% yield. Cyanine, one of the most important biological dyes, is widely used as fluorescence labels and sensors for in vivo and in vitro biological and medical applications.13 For this purpose, we tried to label a cyanine dye to trisialic acid 9 through Cu(I)-catalyzed azide–alkyne cycloaddition.14 Notably, although a few azide functionalized glycan epitopes bearing Sia residues were synthesized,11c,e,15 to the best of our knowledge, there is no report on the addition of cyanine dyes and Sia containing sugars via CuAAC reaction. To address this issue, initially, 3-azidopropyl a-(2,9)-monosialoside (monoSia-N3) 10 was chosen as a reference compound for the CuAAC reaction to attach a Cy5 dye molecule. Surprisingly, as shown in Table 1 (entry 1), no desired product 12 was obtained under the classical CuAAC coupling conditions with 2.5 mol% copper(II) sulfate and 25 mol% sodium ascorbate (VcNa). PMDETA16a and DIEPA16b have been used as additives for the cyanine dye involved CuAAC reaction to ensure its high coupling efficiency. However, the same negative results were produced with the combination of PMDETA/DIEPA, CuI, and DMF as the CuAAC system (Table 1, entries 2 and 3). Polytriazolylamines,17 including TBTA17a and its water soluble analogues, were other kinds of useful
Table 1
Entry 1 2 3 4
CuAAC reaction of monoSia-N3 (10) and Cy5 alkyne (11)
Catalyst VcNa (0.25 eq.), CuSO4 (0.025 eq.) CuI (0.75 eq.) CuI (0.1 eq.) VcNa (0.4 eq.), CuSO4 (0.2 eq.)
Additive
Solvent
None
t
PMDETAc (1.5 eq.) DIEPAd (0.2 eq.) TBTAe (0.4 eq.)
DMF DMF t BuOH–H2Oa
BuOH–H2O
Yield a
—b —b —b 86%
a The ratio of the solvent mixture is 1 : 1. b No reaction. c PMDETA = N,N,N 0 ,N 0 ,N00 -pentamethyldiethylenetriamine. d DIEPA = diisopropylethylamine. e TBTA = tris-(benzyltriazolylmethyl)amine.
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additives for CuAAC due to the powerful ability to stabilize Cu(I) and accelerate the cycloadditions. To our delight, when TBTA served as the ligand for the traditional CuAAC irradiated by ultrasound (Table 1, entry 4), the Cy5 tagged monoSia (monoSia-Cy5, 12) was finally obtained in high yield (86%). Encouraged by these results, Cy5 and Cy3-tagged a-(2,9)trisialic acids, triSia-Cy5 (14) and triSia-Cy3 (15), were prepared under the similar CuAAC conditions assisted by the ligand TBTA from Cy5/Cy3 alkyne (11/13) and triSia-N3 (9) in 87% and 76% yield, respectively (Fig. 2). The dye labeled oligosialic acids were purified by P-2 biogel size exclusion column chromatography eluted with H2O to afford the corresponding glycoconjugates. TriSia-Cy5 was validated by NMR spectroscopy and high resolution mass spectrometry, which recorded a signal at m/z 1522.7097 corresponding to [triSia-Cy5 + 3H]+ with an expected m/z of 1522.7093. Similar results were obtained for triSia-Cy3 (see ESI† for details). The absorption and fluorescence properties of cyanine alkynes and cyanine labeled trisialic acids used in this study are shown in Table S1 and Fig. S1, ESI†. There is no remarkable difference in the optical characteristics of cyanine dyes before/after labeling trisialic acid. Next, we introduced triSia-Cy3 or triSia-Cy5 into cultured PC-12 cells to observe the distribution of target molecules by fluorescence microscopy. Interestingly, the fluorescently-labeled triSia was principally gathered on the membrane of PC-12 cells, and the imaging region was discrete (Fig. 3). The amount of triSia-Cy3/triSia-Cy5 in the cytoplasm was negligible, because the negative charges of triSia remarkably restrict its cell permeability. It is worth noting that the natural Sia was abundantly expressed on the surface of PC-12 cells. Meanwhile, some glycan-binding receptors could be co-accreted with these sialylated glycoproteins or glycolipids. According to the results of imaging experiments, we considered that the labeled triSia should specifically bind to the Siglecs located on the PC-12 cell membranes. Additionally,
Fig. 2 Structures of Cy3 alkyne 13 and cyanine dye tagged a-(2,9)-trisialic acid 14–15.
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Fig. 3 Confocal laser scanning microscopy images of PC-12 cells incubated with triSia-Cy3 or triSia-Cy5 (5 mM) for 1 h. The middle column: Cy3 fluorescence (excitation: 559 nm/emission: 570–635 nm) or Cy5 fluorescence (excitation: 635 nm/emission: 650–720 nm). Scale bars, 10 mm.
the fluorescence of exogenous triSia-Cy3/triSia-Cy5 reflected the uneven distribution of Siglecs to some extent. As a comparison, monoSia-Cy5 (12) was also used for the imaging of PC-12 cells. To our surprise, monoSia-Cy5 entered into the cells rapidly. Further colocalization experiments involving 12 were performed using a commercially mitochondrionspecific fluorescent probe, MitoTracker Green (MTG) (Fig. S2, ESI†). A high Pearson’s colocalization coefficient (0.91) was obtained, indicating that the staining of monoSia-Cy5 fits well with that of mitochondrial dye MTG. The results show that monoSia-Cy5 is preferentially distributed in the mitochondria of PC-12 cells, which is totally different from the cases of triSiaCy3 and triSia-Cy5. As discussed above, triSia-Cy3/triSia-Cy5 was hardly brought into the PC-12 cells. To further verify the properties of triSia-Cy3
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and triSia-Cy5, we found that poly(ethylene imine) (PEI),18 a transfection agent, can assist the oligosialic acid to break through the membrane of PC-12 cells via endocytosis, and then the fluorescent imaging of triSia in the cytoplasm was achieved (Fig. S3, ESI†). In contrast to the images in Fig. 3b and e, clusters of fluorescent signals were observed obviously in Fig. S3b and e (ESI†), indicating that PEI can condense triSia-Cy3 or triSiaCy5 into positively-charged or electrically neutral particles. ¨rster resonance energy The Cy3 and Cy5 dyes are the typical Fo transfer (FRET) pair in fluorometry.19 The data in Fig. S1, ESI† show that the emission of triSia-Cy3 overlaps with the absorption of triSia-Cy5 to ensure the overlap integral requirement for resonance energy transfer, which allows real-time monitoring of various important cellular events. On this basis, to further obtain the information of spatial presentation of Siglecs on cells, we incubated PC-12 with equal amounts of triSia-Cy3 and triSia-Cy5 simultaneously for 1 h, and both the dual-channel (Cy3 and Cy5 fluorescence) and FRET-induced fluorescence images were collected using a confocal microscope (Fig. 4). As expected, the FRET effect of triSia-Cy3 to triSia-Cy5 was observed (Fig. 4c, g and k). Compared with Fig. 4j, the fluorescence intensity of energy acceptor triSia-Cy5 in Fig. 4k was much stronger under the same excitation conditions (lex = 559 nm). Although different kinds of Siglecs may coexist on the surface of the living cells, it is reasonable to consider that both the triSia-Cy3 and triSia-Cy5 targeted the same type of glycan-binding receptors due to their similar molecular structures. A pair of adjacent Siglec moieties on the membrane of the PC-12 cell with the spatial distance less than 10 nm had a 50% probability of binding with different cyaninelabeled triSias, which gave rise to an efficient FRET process from Cy3 to Cy5 consequently. The energy transfer efficiency E19 is B0.37, which was determined by the fluorescence intensity of the
Fig. 4 Fluorescence confocal images of PC-12 cells incubated with triSia-Cy3 (5 mM) and/or triSia-Cy5 (5 mM) for 1 h. The first row: Cy3 channel (excitation: 559 nm/emission: 570–635 nm); the second row: Cy5 channel (excitation: 635 nm/emission: 650–720 nm); the third row: FRET channel (excitation: 559 nm/emission: 650–720 nm); the fourth row: treatment of the images in (c), (g) and (k) by Origin 8.0, respectively. Scale bars, 5 mm.
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Cy3 channel in the absence/presence of triSia-Cy5. The simulated images (Fig. 4d, h and l) corresponding to Fig. 4c, g and k illustrated the distribution of Siglecs more clearly. There are some distinctions between the point cloud of Fig. 4d and h, indicating that triSia-Cy3 and triSia-Cy5 targeted different Siglec moieties on the cell surface. Additionally, the fluorescence region in Fig. 4l is a subaggregate of that in Fig. 4h, suggesting the special region of the targeted Siglecs in higher spatial density with a certain probability. In summary, cyanine dye labeled oligosialic acids for fluorescence imaging of glycan-binding receptors were deeply investigated in this study. A glycan epitope, a-(2,9)-trisialic acid, was successfully synthesized with (p-Tol)2SO/Tf2O as a promoter in the key a-stereoselective sialylations. It is the first time to efficiently label a negatively charged Sia bearing glycan by cyanine dyes through CuAAC reaction assisted by TBTA as the ligand. By collecting fluorescence in the dual-channel as well as the FRET channel, we showed that the triSia-Cy3/triSia-Cy5 could be enriched on the membrane of PC-12 cells to illustrate the spatial distribution of glycan-binding receptors like Siglecs. Taken together, it is deduced that triSia-Cy3/triSia-Cy5 is a specific fluorescent probe to interact with the glycan-binding receptors on PC-12 cells. To the best of our knowledge, there is no report about the Siglecs existence on PC-12 cells. Further studies on the isolation, identification and bioactivity of Siglecs on PC-12 cells are currently underway. Remarkably, the labeling strategy developed in this study provides a powerful tool to assess and visualize new glycan-binding receptors on the membrane of living cells. The project was financially supported by the National Natural Science Foundation of China (21272027), Beijing National Natural Science Foundation (2122031), and Beijing Municipal Commission of Education.
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