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Synthesis of Biocompatible Glycodendrimer based on Fluorescent Perylene Bisimides and Its Bioimaging Ke-Rang Wang,* Hong-Wei An, Rui-Xue Rong, Zhi-Ran Cao, Xiao-Liu Li*
A novel water-soluble fluorescent glycodendrimer based on perylene bisimides is synthesized, which exhibits high fluorescence quantum yield of 54%. While the binding interactions of PBI-Man with Concanavalin A (Con A) are studied by fluorescence spectra and CD spectra, which show strong binding affinity for Con A with the binding constant of 3.8 × 107 M−1 for monomeric mannose, nearly four orders of magnitude higher affinity than the monovalent mannose ligand. Furthermore, the fluorescence imaging of macrophage cell with PBI-Man is investigated, and shows selectively binding interaction with the mannose receptor-medicated cell entry. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) activities of PBI-Man show that PBI-Man as a biocompatible agent is noncytotoxic to living cells.
1. Introduction Perylene bisimide derivatives are one of the most extensively studied molecules because of its prominent optical and electronic properties, such as high molar absorptivity, high fluorescence quantum yields, and prominent chemical, thermal, and photochemical stability.[1–3] Despite their excellent optical and redox properties, the biggest challenge to the use of perylene bisimides in biological systems is to develop water-soluble fluorescent analogues, because perylene bisimide derivatives existed strong π···π stacking interactions in aqueous solution, Prof. K.-R. Wang, H.-W. An, Prof. X.-L. Li Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, P. R. China Fax: (+86) 312–5971–116 E-mail: [email protected]; [email protected] Prof. K.-R. Wang, H.-W. An, Prof. X.-L. Li Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Hebei University, Baoding, P. R. China R.-X. Rong, Prof. Z.-R. Cao Department of Immunology, School of Basic Medical Science, Hebei University, Baoding, P. R. China Macromol. Rapid Commun. 2014, 35, 727−734 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
which resulted severely self-quenching effect of the fluorescence.[4,5] One of the most powerful methods to design the watersoluble fluorescent perylene bisimides is to incorporate the ionic groups such as protonated amine and quaternized aminium salts,[6–9] carboxylic[10,11] or sulfonic acids[12–14] in the imide nitrogens or in the bay positions. Another strategy is attaching of water-soluble polycerol dendrons at the imide or bay positions.[15–20] On the other hand, it is well known that carbohydrates are highly biocompatible and water-soluble, and play important roles in wide variety of biological processes, such as cell growth regulation, adhesion, cancer cell metastasis, cellular trafficking, differentiation and infection by pathogens, and the immune response.[21–24] Although, carbohydrate-modified perylene bisimides have been synthesized,[25–28] very few examples of the neutral fluorescent perylene bisimides with carbohydrate grafts have been reported.[29,30] Compared with the ionic compounds, the neutral compounds possessed the advantages of independent of pH and ionic strength (usually, the pH and ionic strengths are high in biological buffer systems), minimizing the undesired interactions of the compounds with membranes and other charged biomolecules.[25,26]
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DOI: 10.1002/marc.201300916
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Here, we wish to report a neutral and biocompatible glycodendrimer based on fluorescent perylene bisimides PBI-Man (Scheme 1) functionalized with 18 peripheral α-Dmannose functionalization at the imide position, which exhibited high fluorescence quantum yield of 54% in water. Furthermore, its selectively binding interaction with the mannose receptor-medicated cell entry was investigated.
2. Experimental Section 2.1. Synthesis 2.1.1. Synthesis of Compound 3 (Scheme 1) Compound 2 (351 mg, 0.9 mmol) was dissolved in THF (20 mL), and then compound 1 (5.3 g, 3.4 mmol), an aqueous solution of
Scheme 1. 1) CuSO4·5H2O, sodium ascorbate, THF/H2O (1/1); 2) CF3COOH, CH2Cl2; 3) Zn(AcO)2, PTCDA, Pyr.; 4) NaOH, CH3OH.
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Synthesis of Biocompatible Glycodendrimer based on Fluorescent Perylene Bisimides and Its Bioimaging
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CuSO4·5H2O (201 mg, 0.8 mmol), and sodium ascorbate (160 mg, 0.8 mmol) were added sequentially. The mixture was stirred for 12 h. The solvent was then evaporated under vacuum. The residue was purified by column chromatography with CH2Cl2 CH3OH (v/v = 15/1) as the eluent to give the product as a white solid (3.5 g) in 76% yield. M.p. 106.3–107.7 °C; 1H NMR (CDCl3, 600 MHz): δ = 1.39 (s, 9H, (CH3)3O ), 1.98 (s, 27H, COCH3), 2.03 (s, 27H, COCH3), 2.09 (s, 27H, COCH3), 2.13 (s, 27H, COCH3), 3.62–3.65 (m, 9H), 3.76–3.80 (m, 24H), 3.89–3.92 (m, 9H), 4.06 (d, J = 12 Hz, 9H), 4.10–4.14 (m, 9H), 4.20 (dd, J = 4.8 Hz, 12.6 Hz, 9H), 4.55–4.65 (m, 42H), 4.82 (s, 9H), 5.05 (s, 6H), 5.21–5.26 (s, m, 27H), 7.16 (br s, 2H, CONH ), 7.73 (s, 9H, triaz2-H), 7.79 (s, 3H, triaz1-H); 13C NMR (CDCl , 150 MHz): δ = 20.68, 20.71, 20.80, 28.32, 49.61, 3 52.72, 60.47, 62.22, 64.63, 65.72, 66.26, 68.48, 68.90, 69.02, 69.14, 97.51, 124.92, 124.70, 144.95, 165.26, 169.62, 169.95, 170.08, 170.57; MALDI (m/z): M+ calcd for C209H289N41O108, 5102.8; found, 5118.8 [M+O]+.
2.1.2. Synthesis of Compound 4 (Scheme 1) Compound 3 (1.60 g, 0.3 mmol) was dissolved in CH2Cl2 (10 mL), then TFA (10 mL) was added, the reaction mixture was stirred at room temperature for 2 h. The solvent was then evaporated under vacuum. The residue was purified by column chromatography with CH2Cl2/CH3OH (v/v = 10/1) as the eluent to give the product as a white solid (1.25 g) in 80% yield. M.p. 99.5–101.2 °C; 1H NMR (CDCl3, 600 MHz): δ = 1.98 (s, 27H, COCH3), 2.03 (s, 27H, COCH3), 2.09 (s, 27H, COCH3), 2.13 (s, 27H, COCH3), 3.63–3.65 (m, 9H), 3.75–3.80 (m, 24H), 3.89–3.92 (m, 9H), 4.06 (dd, J = 2.4 Hz, 12.6 Hz, 9H), 4.10–4.14 (m, 9H), 4.21 (dd, J = 4.8 Hz, 12.0 Hz, 9H), 4.58–4.68 (m, 42H), 4.82 (s, 9H), 5.06 (s, 6H), 5.19–5.26 (m, 27H), 7.18 (br s, 2H, CONH ), 7.43 (s, 1H, CONH ), 7.74 (s, 9H, triaz2-H), 7.82 (s, 3H, triaz1-H); 13C NMR (CDCl3, 150 MHz): δ = 20.68, 20.72, 20.80, 29.68, 49.63, 60.50, 62.26, 64.58, 65.73, 66.25, 68.50, 68.91, 69.03, 69.15, 97.51, 123.99, 144.92, 169.63, 169.97, 170.10, 170.59; MALDI (m/z): M+ calcd for C204H282N41O106, 5002.8, found, 5003.8 [M+H]+.
2.1.3. Synthesis of Compound PBI-AcMan (Scheme 1) Compound 4 (500 mg, 0.1 mmol), 3,4,9,10-perylenetetracarboxylic acid (17.8 mg, 0.045 mmol), and zinc acetate (10 mg, 0.045 mmol) were mixed in pyridine (200 mL). The reaction mixture was heated at 100 °C under N2 for 48 h. After the mixture was cooled to room temperature, the solvent was removed at reduced pressure and the residue was dissolved in chloroform, washed with water, dried over Na2SO4, and evaporated to dryness under vacuum. The residue was purified by silica-gel column chromatography with CH2Cl2 CH3OH (v/v = 12/1) as the eluent to give the product PBI-AcMan as red powder (188 mg) with a yield of 20%. M.p. 117.2–119.7 °C; 1H NMR (CDCl3, 600 MHz): δ = 1.97 (s, 54H, COCH3), 2.02 (s, 54H, COCH3), 2.08 (s, 54H, COCH3), 2.12 (s, 54H, COCH3), 3.62–3.65 (m, 18H), 3.79–3.84 (m, 18H), 3.89– 3.93 (m, 18H), 4.05 (dd, J = 2.4 Hz, 12.0 Hz, 18H), 4.10–4.14 (m, 18H), 4.20 (dd, J = 4.8 Hz, 12.6 Hz, 18H), 4.58–4.67 (m, 84H), 4.82 (s, 18H), 5.07 (s, 12H), 5.19–5.26 (m, 54H), 6.93 (br s, 2H, CONH ), 7.08 (s, 6H, CONH ), 7.72 (s, 18H, triaz2-H), 7.85 (s, 6H, triaz1-H), 8.70 (d, J = 7.8 Hz, 4H, Ar H), 8.72 (d, J = 8.4 Hz, 4H, Ar H); 13C NMR (CDCl3, 150 MHz): δ = 20.70, 20.74, 20.83, 29.68, 49.60, 52.80, 53.47, 60.25, 60.41, 62.16, 64.62, 65.63, 66.24, 68.45, 68.83,
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68.99, 69.09, 97.47, 122.95, 123.57, 123.93, 124.79, 126.57, 129.57, 131.83, 135.01, 144.94, 162.52, 163.28, 165.28, 166.72, 169.63, 169.96, 170.09, 170.60; MALDI (m/z): M+ calcd for C432H566N82O216, 10362.5; found, 10384.0 [M+Na]+.
2.1.4. Synthesis of Compound PBI-Man (Scheme 1) compound PBI-AcMan (250 mg, 2.4 μmol) was dissolved in MeOH (20 mL), NaOH (347 mg, 8.7 mmol) was added, and the reaction mixture was stirred at room temperature until the disappearance of the starting material. The deposited solid was collected by filtration and washed with MeOH. It was then dissolved in water, and the solution was placed in a cellulose dialysis tube (cutoff: 3500), dialyzed against water for 2 d, and lyophilized to give the desired solid powder (125 mg) with the yield of 71%. M.p. 143.9–146.3 °C; 1H NMR (DMSO-d6 + D2O, 600 MHz): δ = 2.99 (s, 18H), 3.39–3.56 (m, 126H), 3.70 (s, 18H), 3.87 (s, 18H), 4.39–4.57 (m, 118H), 5.06 (s, 12H), 7.87 (s, 18H, triaz2-H), 7.93 (s, 6H, triaz1-H), 8.09 (br s, 8H, Ar H); 13C NMR (D2O, 150 MHz): δ = 23.25, 50.00, 60.28, 60.63, 63.51, 65.40, 66.35, 67.33, 69.89, 70.44, 72.76, 99.52, 125.30, 125.40, 125.47, 126.22, 126.26, 143.94, 144.16, 165.93, 165.99, 166.79; MALDI (m/z): M+ calcd for C288H422N82O144, 7335.8, found, 7358.5 [M+Na]+.
3. Results and Discussion 3.1. Synthesis PBI-Man was synthesized by three steps (Scheme 1). The most important glycodendrimer 4 was synthesized using convergent approach by coupling reaction of trivalent glycoside azide 1[31] with tripropargylated compound 2 under standard Cu(I) conditions in the presence of a substoichiometric amount of CuSO4 and sodium ascorbate in homogeneous THF/water solution, then removing of the Boc protective group. The target compound PBI-Man was synthesized by reaction of glycodendrimer 4 with 3,4,9,10-perylenetetracarboxylic dianhydride in the presence of Zn(OAc)2 as catalyst, and followed by removing the acetyl group and dialyzing against water to remove the inorganic salts. The target glycodendrimer PBI-Man and its intermediate compounds were characterized by NMR and MS analyses (Figures S1–S12, Supporting Information). The purity of PBI-Man was about 92%, determined by HPLC (Figure S13, Supporting Information) using a ZORBAX SB-Aq column (3.5 μm, 2.1 × 10 mm) with a gradient elution of H2O (0.1% HCOOH) and acetonitrile at a flow rate of 0.25 mL min−1 for 30 min. The peaks were identified by setting the UV detector at 222, 497, and 534 nm in three different courses. 3.2. Optical Properties Concentration-dependent and temperature-dependent UV–vis spectra were used to investigate the π···π stacking
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behavior and optical properties of PBI-Man. At the concentration of 1.0 × 10−6 M (Figure 1a), three distinguishable absorption bands between 400 and 600 nm with the 0–0 (535 nm), 0–1 (497 nm), and 0–2 (466 nm) electronic transitions were observed, and the most intensity appeared at the first band (535 nm). It is well known that the aggregation (or lack thereof) of perylene bisimides could be determined by comparing the A0–0/A0–1 ratio.[32,33] At the lower concentration of 1 × 10−6 M, the A0–0/A0–1 ratio of PBI-Man was 1.39, indicating nonaggregated state or a very weakly aggregated state in line with a literature report.[34] However, upon gradually increasing the concentration, the apparent absorption coefficients (ε) of 0–0 (535 nm) and 0–1 (497 nm) electronic transitions decreased, while that of the 0–2 (466 nm) electronic transition increased, concurrently a new shoulder at about 560 nm appeared, and the A0–0/ A0–1 ratio reduced to 0.70 at the higher concentration of 1.5 × 10−4 M, reflecting the electronic coupling with
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Figure 1. a) Concentration-dependent (from 1 × 10−6 to 1.5 × 10−4 M) and b) temperature-dependent (4.0 × 10−5 M, from 5 to 95 °C) UV–vis spectra of PBI-Man in PBS buffer (pH 7.2, 10 × 10−3 M).
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a face-to-face stacking arrangement. Furthermore, the aggregation constant (Kagg) of PBI-Man in water was obtained as 6.9 × 104 M−1 (Figure S14, Supporting Information) using a nonlinear least-square regression analysis [Equation (S1), Supporting Information] of the concentration-dependent UV–vis spectral data by the isodesmic or equal-K model.[35,36] In addition, the π···π interactions of PBI-Man were also temperature-dependent, the spectral changes were highly comparable with those observed in concentration-dependent measurements. The apparent absorption coefficient (ε) of 0–0 (535 nm) electronic transition increased, the 0–1 electronic transition was shifted from 500 to 496 nm, and the 0–2 (466 nm) electronic transition decreased, concurrently the shoulder band at about 560 nm disappeared as temperature increasing from 5 to 95 °C (Figure 1b), which implied that the selfassembly process was enthalpy driven.[36,37] The fluorescence properties of PBI-Man are investigated by concentration-dependent fluorescence spectra. The intensity of the fluorescence emission enhanced with the concentration increasing from 1.0 × 10−6 to 6.0 × 10−6 M, and the emission band was bathochromically shifted from 560 to 565 nm (Figure S15, Supporting Information). However, the fluorescence emission was gradually quenched with the concentration further increasing from 6.0 × 10−6 to 1.5 × 10−4 M (Figure S15, Supporting Information), accompanying with the maximum fluorescence emission bathochromical shift from 565 to 592 nm because of the self-quenching effects, suggesting the transition of lower aggregates into higher aggregates.[38,39] Based on the isodesmic or equal-K model, the average dye numbers per stack, N (average aggregation number), at different concentrations can be calculated from the aggregation constant Kagg and the concentration according to the reported equation [Equation (S2), Supporting Information].[36] At the concentration of 1 × 10−7 M, the average dye numbers per stack N of PBI-Man was about 1, indicating that PBI-Man existed in the monomer form. Furthermore, benefiting from the grafting of glycodendrimer, PBI-Man possesses benign fluorescence property with the fluorescence quantum yield of 54% at the concentration of 5.0 × 10−6 M in the PBS buffer. Furthermore, the aggregated and non-aggregated morphologies of PBI-Man at the concentrations of 2.0 × 10−4 M (aggregated state) and 2.0 × 10−6 M (non-aggregated state) were intuitively distinguished from the TEM images (Figure S16, Supporting Information). The aggregation morphology of PBI-Man (2.0 × 10−4 M) was observed as the small balls with a diameter about 10 nm (Figure S16a, Supporting Information). While the non-aggregated morphology of PBI-Man showed very small blobs (Figure S16b, Supporting Information).
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3.3. Binding Interactions with Concanavalin A The behavior of the multivalent mannose-modified perylene bisimides PBI-Man binding with Concanavalin A (Con A) was studied by fluorescence spectroscopy. Con A as a well-known mannose-specific lectin has been extensively used to study carbohydrate-lectin recognition.[40] As shown in Figure 2, a progressive quenching of the fluorescence emission intensity at 546 and 589 nm was observed with increasing Con A concentrations at below 2 × 10−7 M. However, as the concentration of Con A increased beyond 2 × 10−7 M, the fluorescence increased immediately, and soon reached equilibrium. The observed fluorescence enhancement of PBI-Man might be because of hydrophobic interactions. It is known that mannose binds with Con A through hydrogen bonds, hydrophobic interactions, and coordination with metal ions.[41] Upon binding with Con A, the perylene bisimide backbones entered the hydrophobic region of Con A, which has lower polarity than that in the buffer solution. This induced fluorescence enhancement because of suppression of the possible nonradiative process.[42,43] The mechanism of the fluorescence quenching process of PBI-Man with Con A was investigated by time-resolved fluorescence measurement (Figure 3). The fluorescence decay of PBI-Man obeys the single exponential function, giving a single fluorescence lifetime (τ0 = 4.98 ns).[44] Upon addition of Con A, the fluorescence decay of PBI-Man exhibiting dual exponential function decays. Two lifetimes of 3.88 ns (80%) and 2.03 ns (20%) are observed. The results indicated that the mechanism of the the fluorescence quenching process of PBI-Man with Con A was because of a dynamic quenching process.[42,43,45] The binding inhibition assay between Con A and PBI-Man was carried out based on decreasing the binding efficiency in the presence of the α-D-mannopyranoside (α-MMP). In the beginning, with addition of α-MMP, the
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fluorescence of the complexes of PBI-Man and Con A decreased (Figure S17, Supporting Information), then the fluorescence enhanced, and finally the fluorescence kept in certain level as the α-MMP concentrations increased (Figure S17, Supporting Information). The fluorescence changes were highly comparable with those observed in the binding measurements (Figure 2). The spectral changes were because of displacement glycodendrimer PBI-Man from Con A-binding sites by α-MMP. Almost complete recovery of the fluorescence of PBI-Man could be obtained with a large excess of α-MMP. Furthermore, the specific and selective binding interactions of PBI-Man with various proteins were investigated by fluorescence spectra (Figure S18, Supporting Information). Upon addition of five equiv. Con A, the fluorescence of PBI-Man was quenched about 73%. However, titration of peanut agglutinin (PNA) and bovine serum albumin (BSA) into the solution of PBI-Man, no significant change of the fluorescence was observed. These results clearly indicated that mannose functionalized glycodendrimer PBI-Man selectively bond with Con A. Circular dichroism (CD) spectroscopy of Con A was used to elucidate its secondary structural changes caused by binding with PBI-Man. As shown in Figure 4, the spectrum of Con A showed a single negative band at about 224 nm, indicating the secondary-structure hallmark of β-sheet rich proteins.[46] Upon addition of PBI-Man, the intensity of the CD signal showed a progressive decrease, which was compatible with a loss of secondary structure because of the formation of cross-linked complexes.[47] Moreover, the binding constant of PBI-Man with Con A was obtained as 6.8 × 108 M−1 (3.8 × 107 M−1 for monomeric mannose, valency corrected) by fitting the experimental data of the maximum CD changes using the nonlinear
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least-squares curve fitting method (Equation (S3), Supporting Information; Figure 4 inset),[48,49] nearly four orders of magnitude higher affinity for Con A than the monovalent mannose ligand (8.2 × 103 M−1).[50]
diminished (Figure S19a–c, Supporting Information). On the other hand, another water-soluble fluorescent perylene bisimide PBI-CD with cyclodextrins conjugates[54] was used to investigate the fluorescence imaging with macrophage cells. As shown in Figure S20 (Supporting Information), no obvious fluorescence was observed, indicated that PBI-CD could not bind with the macrophage cells. These results suggested that PBI-Man possessed the selectively binding interactions with the surface mannose receptor of the macrophage cells. Cell viabilities of macrophage cells treated with PBI-Man were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) experiment using untreated cells as the cell viability reference. The cells were incubated with PBI-Man at 37 °C for 48 h (Table S1, Supporting Information), no statistically significant difference in MTT activity (P > 0.05) between the treatment and the reference was found. And the cell viability of PBI-Man is 110.3% at the concentration of 50 μg mL−1 (Figure S21, Supporting Information), indicating that PBI-Man was not toxic under the Confocal imaging conditions. The fluorescence imaging of PBI-Man with macrophage cells incubated for 48 h showed predominantly intracellular green fluorescence with punctate appearance (Figure S22, Supporting Information) because of endocytosis, further proving the nontoxicity of PBI-Man.
3.4. Fluorescence Imaging Having successfully examined the binding of PBI-Man with Con A, we investigate its interaction with macrophage-like cell lines, which are well known to be mannose receptor-mediated cell entry with selectively binding interaction with mannosylated derivatives.[51–53] As shown in Figure 5a–c, the green fluorescence of PBI-Man was predominantly intracellular with punctate appearance, suggesting cell surface binding and endocytosis being the cell entry mechanism that results in vesicular (endosomal) localization. As a control experiment, in the presence of α-MMP, the green fluorescence of PBI-Man was greatly
4. Conclusions A novel water-soluble fluorescent glycodendrimer based on perylene bisimides PBI-Man was synthesized. Its optical properties were investigated by UV–vis and fluorescence spectra, which indicated that compound PBI-Man existed in the monomer state under the lower concentration, exhibited the fluorescence quantum yield of 54%. While the binding interactions of PBI-Man with Con A were studied by fluorescence spectra and CD spectra, which exhibited strong binding affinity for Con A with
Figure 5. Confocal microscopic images of murine macrophage cells after incubation with PBI-Man (1 μg mL−1) (a, excited at 488 nm; b, bright field; c, merging of photos a and b) in PBS buffer at 37 °C for 1 h.
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the binding constant of 6.8 × 108 M−1 (3.8 × 107 M−1 for monomeric mannose, valency corrected), nearly four orders of magnitude higher affinity than the monovalent mannose ligand. Furthermore, the binding interaction of PBI-Man with macrophage cells was investigated by confocal laser scanning microscopy, which showed that PBI-Man had a selectively binding interaction with the surface mannose receptor of the cells. Moreover, the cytotoxicity of PBI-Man with macrophage cells was studied by MTT assay, showed noncytotoxicity to the living cells.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: The authors thank NSFC (21002020, 21372059, and 21172051), Hebei Natural Science Foundation (B2011201052, B2012201041), the Foundation of Hebei Education Department (YQ2013006), and Training Program for Innovative Research Team and Leading talent in Hebei Province University (LJRC024) for financial support. The authors also thank Prof. Bao-Xiang Gao (Hebei University) for the confocal imaging experiments. Received: December 18, 2013; Revised: January 11, 2014; Published online: February 4, 2014; DOI: 10.1002/marc.201300916 Keywords: biocompatible; fluorescence imaging; glycodendrimer; perylene bisimides [1] [2] [3] [4] [5] [6]
[7] [8]
[9] [10] [11] [12]
[13] [14]
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[15] T. Heek, J. Nikolaus, R. Schwarzer, C. Fasting, P. Welker, K. Licha, A. Herrmann, R. Haag, Bioconjugate Chem. 2013, 24, 153. [16] S. K. Yang, S. C. Zimmerman, Adv. Funct. Mater. 2012, 22, 3023. [17] S. K. Yang, X. H. Shi, S. Park, S. Doganay, T. Ha, S. C. Zimmerman, J. Am. Chem. Soc. 2011, 133, 9964. [18] B. X. Gao, H. X. Li, H. M. Liu, L. C. Zhang, Q. Q. Bai, X. W. Ba, Chem. Commun. 2011, 47, 3894. [19] T. Heek, C. Fasting, C. Rest, X. Zhang, F. Würthner, R. Haag, Chem. Commun. 2013, 46, 1884. [20] T. Heek, F. Würthner, R. Haag, Chem. Eur. J. 2013, 19, 10911. [21] D. H. Dube, C. R. Bertozzi, Nat. Rev. Drug Discovery 2005, 4, 477. [22] F. T. Liu, G. A. Rabinovich, Nat. Rev. Cancer 2005, 5, 29. [23] K. Ohtsubo, J. D. Marth, Cell 2006, 126, 855. [24] P. R. Crocker, J. C. Paulson, A. Varki, Nat. Rev. Immunol. 2007, 7, 255. [25] Y. W. Huang, J. C. Hu, W. F. Kuang, Z. X. Wei, C. F. J. Faul, Chem. Commun. 2011, 47, 5554. [26] J. Hu, W. Kuang, K. Deng, W. Zou, Y. Huang, Z. X. Wei, C. F. J. Faul, Adv. Funct. Mater. 2012, 22, 4149. [27] K. R. Wang, H. W. An, L. Wu, J. C. Zhang, L. X. Li, Chem. Commun. 2012, 48, 5644. [28] L. Xue, N. Ranjan, D. P. Arya, Biochemistry 2011, 50, 2838. [29] L. Q. Xu, L. Wang, B. Zhang, C. H. Lim, Y. Chen, K. G. Neoh, E. T. Kang, G. D. Fu, Polymer 2011, 52, 5764. [30] K. R. Wang, H. W. An, F. Qian, Y. Q. Wang, J. C. Zhang, X. L. Li, RSC Adv. 2013, 3, 23190. [31] Y. M. Chabre, P. P. Brisebois, L. Abbassi, S. C. Kerr, J. V. Fahy, I. Marcotte, R. Roy, J. Org. Chem. 2011, 76, 724. [32] W. Wang, L. S. Li, G. Helms, H. H. Zhou, A. D. Q. Li, J. Am. Chem. Soc. 2003, 125, 1120. [33] A. D. Q. Li, W. Wang, L. Q. Wang, Chem. Eur. J. 2003, 9, 4594. [34] W. Wang, A. D. Shaller, A. D. Q. Li, J. Am. Chem. Soc. 2008, 130, 8271. [35] F. Würthner, C. Thalacker, S. Diele, C. Tschierske, Chem. Eur. J. 2001, 7, 2245. [36] Z. Chen, A. Lohr, C. R. Saha-Möller, F. Würthner, Chem. Soc. Rev. 2009, 38, 564. [37] Z. J. Chen, V. Stepanenko, V. Dehm, P. Prins, L. D. A. Siebbeles, J. Seibt, P. Marquetand, V. Engel, F. Würthner, Chem. Eur. J. 2007, 13, 436. [38] J. J. Han, W. Wang, A. D. Q. Li, J. Am. Chem. Soc. 2006, 128, 672. [39] J. J. Han, A. D. Shaller, W. Wang, A. D. Q. Li, J. Am. Chem. Soc. 2008, 130, 6974. [40] G. N. Reeke, J. W. Becker, B. A. Cunningham, J. L. Wang, I. Yahara, G. M. Edelman, Adv. Exp. Med. Biol. 1975, 55, 13. [41] H. Lis, N. Sharon, Chem. Rev. 1998, 98, 637. [42] F. K. Wang, G. C. Bazan, J. Am. Chem. Soc. 2006, 128, 15786. [43] K. Y. Pu, L. P. Cai, B. Liu, Macromolecules 2009, 42, 5933. [44] R. Zhang, Z. Wang, Y. Wu, H. Fu, J. Yao, Org. Lett. 2008, 10, 3065. [45] K. Y. Pu, R. Y. Zhan, B. Liu, Chem. Commun. 2010, 46, 1470. [46] R. Loris, T. Hamelryck, J. Bouckaert, L. Wyns, Biochim. Biophys. Acta. 1998, 1383, 9. [47] C. W. Cairo, J. E. Gestwicki, M. Kanai, L. L. Kiessling, J. Am. Chem. Soc. 2002, 124, 1615. [48] K. R. Wang, Y. Q. Wang, H. W. An, J. C. Zhang, X. L. Li, Chem. Eur. J. 2013, 19, 2903.
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Communication
Synthesis of Biocompatible Glycodendrimer based on Fluorescent Perylene Bisimides and Its Bioimaging Ke-Rang Wang,* Hong-Wei An, Rui-Xue Rong, Zhi-Ran Cao, Xiao-Liu Li*
A novel water-soluble fluorescent glycodendrimer based on perylene bisimides is synthesized, which exhibits high fluorescence quantum yield of 54%. While the binding interactions of PBI-Man with Concanavalin A (Con A) are studied by fluorescence spectra and CD spectra, which show strong binding affinity for Con A with the binding constant of 3.8 × 107 M−1 for monomeric mannose, nearly four orders of magnitude higher affinity than the monovalent mannose ligand. Furthermore, the fluorescence imaging of macrophage cell with PBI-Man is investigated, and shows selectively binding interaction with the mannose receptor-medicated cell entry. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) activities of PBI-Man show that PBI-Man as a biocompatible agent is noncytotoxic to living cells.
1. Introduction Perylene bisimide derivatives are one of the most extensively studied molecules because of its prominent optical and electronic properties, such as high molar absorptivity, high fluorescence quantum yields, and prominent chemical, thermal, and photochemical stability.[1–3] Despite their excellent optical and redox properties, the biggest challenge to the use of perylene bisimides in biological systems is to develop water-soluble fluorescent analogues, because perylene bisimide derivatives existed strong π···π stacking interactions in aqueous solution, Prof. K.-R. Wang, H.-W. An, Prof. X.-L. Li Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, P. R. China Fax: (+86) 312–5971–116 E-mail: [email protected]; [email protected] Prof. K.-R. Wang, H.-W. An, Prof. X.-L. Li Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education, Hebei University, Baoding, P. R. China R.-X. Rong, Prof. Z.-R. Cao Department of Immunology, School of Basic Medical Science, Hebei University, Baoding, P. R. China Macromol. Rapid Commun. 2014, 35, 727−734 © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
which resulted severely self-quenching effect of the fluorescence.[4,5] One of the most powerful methods to design the watersoluble fluorescent perylene bisimides is to incorporate the ionic groups such as protonated amine and quaternized aminium salts,[6–9] carboxylic[10,11] or sulfonic acids[12–14] in the imide nitrogens or in the bay positions. Another strategy is attaching of water-soluble polycerol dendrons at the imide or bay positions.[15–20] On the other hand, it is well known that carbohydrates are highly biocompatible and water-soluble, and play important roles in wide variety of biological processes, such as cell growth regulation, adhesion, cancer cell metastasis, cellular trafficking, differentiation and infection by pathogens, and the immune response.[21–24] Although, carbohydrate-modified perylene bisimides have been synthesized,[25–28] very few examples of the neutral fluorescent perylene bisimides with carbohydrate grafts have been reported.[29,30] Compared with the ionic compounds, the neutral compounds possessed the advantages of independent of pH and ionic strength (usually, the pH and ionic strengths are high in biological buffer systems), minimizing the undesired interactions of the compounds with membranes and other charged biomolecules.[25,26]
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DOI: 10.1002/marc.201300916
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Here, we wish to report a neutral and biocompatible glycodendrimer based on fluorescent perylene bisimides PBI-Man (Scheme 1) functionalized with 18 peripheral α-Dmannose functionalization at the imide position, which exhibited high fluorescence quantum yield of 54% in water. Furthermore, its selectively binding interaction with the mannose receptor-medicated cell entry was investigated.
2. Experimental Section 2.1. Synthesis 2.1.1. Synthesis of Compound 3 (Scheme 1) Compound 2 (351 mg, 0.9 mmol) was dissolved in THF (20 mL), and then compound 1 (5.3 g, 3.4 mmol), an aqueous solution of
Scheme 1. 1) CuSO4·5H2O, sodium ascorbate, THF/H2O (1/1); 2) CF3COOH, CH2Cl2; 3) Zn(AcO)2, PTCDA, Pyr.; 4) NaOH, CH3OH.
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Synthesis of Biocompatible Glycodendrimer based on Fluorescent Perylene Bisimides and Its Bioimaging
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CuSO4·5H2O (201 mg, 0.8 mmol), and sodium ascorbate (160 mg, 0.8 mmol) were added sequentially. The mixture was stirred for 12 h. The solvent was then evaporated under vacuum. The residue was purified by column chromatography with CH2Cl2 CH3OH (v/v = 15/1) as the eluent to give the product as a white solid (3.5 g) in 76% yield. M.p. 106.3–107.7 °C; 1H NMR (CDCl3, 600 MHz): δ = 1.39 (s, 9H, (CH3)3O ), 1.98 (s, 27H, COCH3), 2.03 (s, 27H, COCH3), 2.09 (s, 27H, COCH3), 2.13 (s, 27H, COCH3), 3.62–3.65 (m, 9H), 3.76–3.80 (m, 24H), 3.89–3.92 (m, 9H), 4.06 (d, J = 12 Hz, 9H), 4.10–4.14 (m, 9H), 4.20 (dd, J = 4.8 Hz, 12.6 Hz, 9H), 4.55–4.65 (m, 42H), 4.82 (s, 9H), 5.05 (s, 6H), 5.21–5.26 (s, m, 27H), 7.16 (br s, 2H, CONH ), 7.73 (s, 9H, triaz2-H), 7.79 (s, 3H, triaz1-H); 13C NMR (CDCl , 150 MHz): δ = 20.68, 20.71, 20.80, 28.32, 49.61, 3 52.72, 60.47, 62.22, 64.63, 65.72, 66.26, 68.48, 68.90, 69.02, 69.14, 97.51, 124.92, 124.70, 144.95, 165.26, 169.62, 169.95, 170.08, 170.57; MALDI (m/z): M+ calcd for C209H289N41O108, 5102.8; found, 5118.8 [M+O]+.
2.1.2. Synthesis of Compound 4 (Scheme 1) Compound 3 (1.60 g, 0.3 mmol) was dissolved in CH2Cl2 (10 mL), then TFA (10 mL) was added, the reaction mixture was stirred at room temperature for 2 h. The solvent was then evaporated under vacuum. The residue was purified by column chromatography with CH2Cl2/CH3OH (v/v = 10/1) as the eluent to give the product as a white solid (1.25 g) in 80% yield. M.p. 99.5–101.2 °C; 1H NMR (CDCl3, 600 MHz): δ = 1.98 (s, 27H, COCH3), 2.03 (s, 27H, COCH3), 2.09 (s, 27H, COCH3), 2.13 (s, 27H, COCH3), 3.63–3.65 (m, 9H), 3.75–3.80 (m, 24H), 3.89–3.92 (m, 9H), 4.06 (dd, J = 2.4 Hz, 12.6 Hz, 9H), 4.10–4.14 (m, 9H), 4.21 (dd, J = 4.8 Hz, 12.0 Hz, 9H), 4.58–4.68 (m, 42H), 4.82 (s, 9H), 5.06 (s, 6H), 5.19–5.26 (m, 27H), 7.18 (br s, 2H, CONH ), 7.43 (s, 1H, CONH ), 7.74 (s, 9H, triaz2-H), 7.82 (s, 3H, triaz1-H); 13C NMR (CDCl3, 150 MHz): δ = 20.68, 20.72, 20.80, 29.68, 49.63, 60.50, 62.26, 64.58, 65.73, 66.25, 68.50, 68.91, 69.03, 69.15, 97.51, 123.99, 144.92, 169.63, 169.97, 170.10, 170.59; MALDI (m/z): M+ calcd for C204H282N41O106, 5002.8, found, 5003.8 [M+H]+.
2.1.3. Synthesis of Compound PBI-AcMan (Scheme 1) Compound 4 (500 mg, 0.1 mmol), 3,4,9,10-perylenetetracarboxylic acid (17.8 mg, 0.045 mmol), and zinc acetate (10 mg, 0.045 mmol) were mixed in pyridine (200 mL). The reaction mixture was heated at 100 °C under N2 for 48 h. After the mixture was cooled to room temperature, the solvent was removed at reduced pressure and the residue was dissolved in chloroform, washed with water, dried over Na2SO4, and evaporated to dryness under vacuum. The residue was purified by silica-gel column chromatography with CH2Cl2 CH3OH (v/v = 12/1) as the eluent to give the product PBI-AcMan as red powder (188 mg) with a yield of 20%. M.p. 117.2–119.7 °C; 1H NMR (CDCl3, 600 MHz): δ = 1.97 (s, 54H, COCH3), 2.02 (s, 54H, COCH3), 2.08 (s, 54H, COCH3), 2.12 (s, 54H, COCH3), 3.62–3.65 (m, 18H), 3.79–3.84 (m, 18H), 3.89– 3.93 (m, 18H), 4.05 (dd, J = 2.4 Hz, 12.0 Hz, 18H), 4.10–4.14 (m, 18H), 4.20 (dd, J = 4.8 Hz, 12.6 Hz, 18H), 4.58–4.67 (m, 84H), 4.82 (s, 18H), 5.07 (s, 12H), 5.19–5.26 (m, 54H), 6.93 (br s, 2H, CONH ), 7.08 (s, 6H, CONH ), 7.72 (s, 18H, triaz2-H), 7.85 (s, 6H, triaz1-H), 8.70 (d, J = 7.8 Hz, 4H, Ar H), 8.72 (d, J = 8.4 Hz, 4H, Ar H); 13C NMR (CDCl3, 150 MHz): δ = 20.70, 20.74, 20.83, 29.68, 49.60, 52.80, 53.47, 60.25, 60.41, 62.16, 64.62, 65.63, 66.24, 68.45, 68.83,
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68.99, 69.09, 97.47, 122.95, 123.57, 123.93, 124.79, 126.57, 129.57, 131.83, 135.01, 144.94, 162.52, 163.28, 165.28, 166.72, 169.63, 169.96, 170.09, 170.60; MALDI (m/z): M+ calcd for C432H566N82O216, 10362.5; found, 10384.0 [M+Na]+.
2.1.4. Synthesis of Compound PBI-Man (Scheme 1) compound PBI-AcMan (250 mg, 2.4 μmol) was dissolved in MeOH (20 mL), NaOH (347 mg, 8.7 mmol) was added, and the reaction mixture was stirred at room temperature until the disappearance of the starting material. The deposited solid was collected by filtration and washed with MeOH. It was then dissolved in water, and the solution was placed in a cellulose dialysis tube (cutoff: 3500), dialyzed against water for 2 d, and lyophilized to give the desired solid powder (125 mg) with the yield of 71%. M.p. 143.9–146.3 °C; 1H NMR (DMSO-d6 + D2O, 600 MHz): δ = 2.99 (s, 18H), 3.39–3.56 (m, 126H), 3.70 (s, 18H), 3.87 (s, 18H), 4.39–4.57 (m, 118H), 5.06 (s, 12H), 7.87 (s, 18H, triaz2-H), 7.93 (s, 6H, triaz1-H), 8.09 (br s, 8H, Ar H); 13C NMR (D2O, 150 MHz): δ = 23.25, 50.00, 60.28, 60.63, 63.51, 65.40, 66.35, 67.33, 69.89, 70.44, 72.76, 99.52, 125.30, 125.40, 125.47, 126.22, 126.26, 143.94, 144.16, 165.93, 165.99, 166.79; MALDI (m/z): M+ calcd for C288H422N82O144, 7335.8, found, 7358.5 [M+Na]+.
3. Results and Discussion 3.1. Synthesis PBI-Man was synthesized by three steps (Scheme 1). The most important glycodendrimer 4 was synthesized using convergent approach by coupling reaction of trivalent glycoside azide 1[31] with tripropargylated compound 2 under standard Cu(I) conditions in the presence of a substoichiometric amount of CuSO4 and sodium ascorbate in homogeneous THF/water solution, then removing of the Boc protective group. The target compound PBI-Man was synthesized by reaction of glycodendrimer 4 with 3,4,9,10-perylenetetracarboxylic dianhydride in the presence of Zn(OAc)2 as catalyst, and followed by removing the acetyl group and dialyzing against water to remove the inorganic salts. The target glycodendrimer PBI-Man and its intermediate compounds were characterized by NMR and MS analyses (Figures S1–S12, Supporting Information). The purity of PBI-Man was about 92%, determined by HPLC (Figure S13, Supporting Information) using a ZORBAX SB-Aq column (3.5 μm, 2.1 × 10 mm) with a gradient elution of H2O (0.1% HCOOH) and acetonitrile at a flow rate of 0.25 mL min−1 for 30 min. The peaks were identified by setting the UV detector at 222, 497, and 534 nm in three different courses. 3.2. Optical Properties Concentration-dependent and temperature-dependent UV–vis spectra were used to investigate the π···π stacking
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behavior and optical properties of PBI-Man. At the concentration of 1.0 × 10−6 M (Figure 1a), three distinguishable absorption bands between 400 and 600 nm with the 0–0 (535 nm), 0–1 (497 nm), and 0–2 (466 nm) electronic transitions were observed, and the most intensity appeared at the first band (535 nm). It is well known that the aggregation (or lack thereof) of perylene bisimides could be determined by comparing the A0–0/A0–1 ratio.[32,33] At the lower concentration of 1 × 10−6 M, the A0–0/A0–1 ratio of PBI-Man was 1.39, indicating nonaggregated state or a very weakly aggregated state in line with a literature report.[34] However, upon gradually increasing the concentration, the apparent absorption coefficients (ε) of 0–0 (535 nm) and 0–1 (497 nm) electronic transitions decreased, while that of the 0–2 (466 nm) electronic transition increased, concurrently a new shoulder at about 560 nm appeared, and the A0–0/ A0–1 ratio reduced to 0.70 at the higher concentration of 1.5 × 10−4 M, reflecting the electronic coupling with
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Figure 1. a) Concentration-dependent (from 1 × 10−6 to 1.5 × 10−4 M) and b) temperature-dependent (4.0 × 10−5 M, from 5 to 95 °C) UV–vis spectra of PBI-Man in PBS buffer (pH 7.2, 10 × 10−3 M).
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a face-to-face stacking arrangement. Furthermore, the aggregation constant (Kagg) of PBI-Man in water was obtained as 6.9 × 104 M−1 (Figure S14, Supporting Information) using a nonlinear least-square regression analysis [Equation (S1), Supporting Information] of the concentration-dependent UV–vis spectral data by the isodesmic or equal-K model.[35,36] In addition, the π···π interactions of PBI-Man were also temperature-dependent, the spectral changes were highly comparable with those observed in concentration-dependent measurements. The apparent absorption coefficient (ε) of 0–0 (535 nm) electronic transition increased, the 0–1 electronic transition was shifted from 500 to 496 nm, and the 0–2 (466 nm) electronic transition decreased, concurrently the shoulder band at about 560 nm disappeared as temperature increasing from 5 to 95 °C (Figure 1b), which implied that the selfassembly process was enthalpy driven.[36,37] The fluorescence properties of PBI-Man are investigated by concentration-dependent fluorescence spectra. The intensity of the fluorescence emission enhanced with the concentration increasing from 1.0 × 10−6 to 6.0 × 10−6 M, and the emission band was bathochromically shifted from 560 to 565 nm (Figure S15, Supporting Information). However, the fluorescence emission was gradually quenched with the concentration further increasing from 6.0 × 10−6 to 1.5 × 10−4 M (Figure S15, Supporting Information), accompanying with the maximum fluorescence emission bathochromical shift from 565 to 592 nm because of the self-quenching effects, suggesting the transition of lower aggregates into higher aggregates.[38,39] Based on the isodesmic or equal-K model, the average dye numbers per stack, N (average aggregation number), at different concentrations can be calculated from the aggregation constant Kagg and the concentration according to the reported equation [Equation (S2), Supporting Information].[36] At the concentration of 1 × 10−7 M, the average dye numbers per stack N of PBI-Man was about 1, indicating that PBI-Man existed in the monomer form. Furthermore, benefiting from the grafting of glycodendrimer, PBI-Man possesses benign fluorescence property with the fluorescence quantum yield of 54% at the concentration of 5.0 × 10−6 M in the PBS buffer. Furthermore, the aggregated and non-aggregated morphologies of PBI-Man at the concentrations of 2.0 × 10−4 M (aggregated state) and 2.0 × 10−6 M (non-aggregated state) were intuitively distinguished from the TEM images (Figure S16, Supporting Information). The aggregation morphology of PBI-Man (2.0 × 10−4 M) was observed as the small balls with a diameter about 10 nm (Figure S16a, Supporting Information). While the non-aggregated morphology of PBI-Man showed very small blobs (Figure S16b, Supporting Information).
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Times/ns Figure 3. Fluorescence decay profile of PBI-Man (λex = 498 nm) and with addition of Con A (1.0 × 10−6 M) in PBS buffer (pH 7.2, 10 × 10−3 M, 0.1 × 10−3 M CaCl2, and 0.1 × 10−3 M MnCl2).
3.3. Binding Interactions with Concanavalin A The behavior of the multivalent mannose-modified perylene bisimides PBI-Man binding with Concanavalin A (Con A) was studied by fluorescence spectroscopy. Con A as a well-known mannose-specific lectin has been extensively used to study carbohydrate-lectin recognition.[40] As shown in Figure 2, a progressive quenching of the fluorescence emission intensity at 546 and 589 nm was observed with increasing Con A concentrations at below 2 × 10−7 M. However, as the concentration of Con A increased beyond 2 × 10−7 M, the fluorescence increased immediately, and soon reached equilibrium. The observed fluorescence enhancement of PBI-Man might be because of hydrophobic interactions. It is known that mannose binds with Con A through hydrogen bonds, hydrophobic interactions, and coordination with metal ions.[41] Upon binding with Con A, the perylene bisimide backbones entered the hydrophobic region of Con A, which has lower polarity than that in the buffer solution. This induced fluorescence enhancement because of suppression of the possible nonradiative process.[42,43] The mechanism of the fluorescence quenching process of PBI-Man with Con A was investigated by time-resolved fluorescence measurement (Figure 3). The fluorescence decay of PBI-Man obeys the single exponential function, giving a single fluorescence lifetime (τ0 = 4.98 ns).[44] Upon addition of Con A, the fluorescence decay of PBI-Man exhibiting dual exponential function decays. Two lifetimes of 3.88 ns (80%) and 2.03 ns (20%) are observed. The results indicated that the mechanism of the the fluorescence quenching process of PBI-Man with Con A was because of a dynamic quenching process.[42,43,45] The binding inhibition assay between Con A and PBI-Man was carried out based on decreasing the binding efficiency in the presence of the α-D-mannopyranoside (α-MMP). In the beginning, with addition of α-MMP, the
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fluorescence of the complexes of PBI-Man and Con A decreased (Figure S17, Supporting Information), then the fluorescence enhanced, and finally the fluorescence kept in certain level as the α-MMP concentrations increased (Figure S17, Supporting Information). The fluorescence changes were highly comparable with those observed in the binding measurements (Figure 2). The spectral changes were because of displacement glycodendrimer PBI-Man from Con A-binding sites by α-MMP. Almost complete recovery of the fluorescence of PBI-Man could be obtained with a large excess of α-MMP. Furthermore, the specific and selective binding interactions of PBI-Man with various proteins were investigated by fluorescence spectra (Figure S18, Supporting Information). Upon addition of five equiv. Con A, the fluorescence of PBI-Man was quenched about 73%. However, titration of peanut agglutinin (PNA) and bovine serum albumin (BSA) into the solution of PBI-Man, no significant change of the fluorescence was observed. These results clearly indicated that mannose functionalized glycodendrimer PBI-Man selectively bond with Con A. Circular dichroism (CD) spectroscopy of Con A was used to elucidate its secondary structural changes caused by binding with PBI-Man. As shown in Figure 4, the spectrum of Con A showed a single negative band at about 224 nm, indicating the secondary-structure hallmark of β-sheet rich proteins.[46] Upon addition of PBI-Man, the intensity of the CD signal showed a progressive decrease, which was compatible with a loss of secondary structure because of the formation of cross-linked complexes.[47] Moreover, the binding constant of PBI-Man with Con A was obtained as 6.8 × 108 M−1 (3.8 × 107 M−1 for monomeric mannose, valency corrected) by fitting the experimental data of the maximum CD changes using the nonlinear
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10 5
-5
0 -5
-10
CD (mdeg)
CD (mdeg)
0
-15
-10 -15 -20
-20
0.0
-25 210
220
230
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2.0x10-7 4.0x10-7 6.0x10-7 8.0x10-7 Concentrations of PBI-18-Man
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Wavelength/nm Figure 4. Circular dichroism (CD) spectra of Con A (4.0 × 10−7 M) in PBS buffer (pH 7.2, 10 × 10−3 M, 0.1 × 10−3 M MnCl2, and 0.1 × 10−3 M CaCl2) upon addition PBI-Man (0−2 equiv.); Inset: the curve of the binding constant fitting at maximum CD signal changes.
least-squares curve fitting method (Equation (S3), Supporting Information; Figure 4 inset),[48,49] nearly four orders of magnitude higher affinity for Con A than the monovalent mannose ligand (8.2 × 103 M−1).[50]
diminished (Figure S19a–c, Supporting Information). On the other hand, another water-soluble fluorescent perylene bisimide PBI-CD with cyclodextrins conjugates[54] was used to investigate the fluorescence imaging with macrophage cells. As shown in Figure S20 (Supporting Information), no obvious fluorescence was observed, indicated that PBI-CD could not bind with the macrophage cells. These results suggested that PBI-Man possessed the selectively binding interactions with the surface mannose receptor of the macrophage cells. Cell viabilities of macrophage cells treated with PBI-Man were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) experiment using untreated cells as the cell viability reference. The cells were incubated with PBI-Man at 37 °C for 48 h (Table S1, Supporting Information), no statistically significant difference in MTT activity (P > 0.05) between the treatment and the reference was found. And the cell viability of PBI-Man is 110.3% at the concentration of 50 μg mL−1 (Figure S21, Supporting Information), indicating that PBI-Man was not toxic under the Confocal imaging conditions. The fluorescence imaging of PBI-Man with macrophage cells incubated for 48 h showed predominantly intracellular green fluorescence with punctate appearance (Figure S22, Supporting Information) because of endocytosis, further proving the nontoxicity of PBI-Man.
3.4. Fluorescence Imaging Having successfully examined the binding of PBI-Man with Con A, we investigate its interaction with macrophage-like cell lines, which are well known to be mannose receptor-mediated cell entry with selectively binding interaction with mannosylated derivatives.[51–53] As shown in Figure 5a–c, the green fluorescence of PBI-Man was predominantly intracellular with punctate appearance, suggesting cell surface binding and endocytosis being the cell entry mechanism that results in vesicular (endosomal) localization. As a control experiment, in the presence of α-MMP, the green fluorescence of PBI-Man was greatly
4. Conclusions A novel water-soluble fluorescent glycodendrimer based on perylene bisimides PBI-Man was synthesized. Its optical properties were investigated by UV–vis and fluorescence spectra, which indicated that compound PBI-Man existed in the monomer state under the lower concentration, exhibited the fluorescence quantum yield of 54%. While the binding interactions of PBI-Man with Con A were studied by fluorescence spectra and CD spectra, which exhibited strong binding affinity for Con A with
Figure 5. Confocal microscopic images of murine macrophage cells after incubation with PBI-Man (1 μg mL−1) (a, excited at 488 nm; b, bright field; c, merging of photos a and b) in PBS buffer at 37 °C for 1 h.
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Synthesis of Biocompatible Glycodendrimer based on Fluorescent Perylene Bisimides and Its Bioimaging
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the binding constant of 6.8 × 108 M−1 (3.8 × 107 M−1 for monomeric mannose, valency corrected), nearly four orders of magnitude higher affinity than the monovalent mannose ligand. Furthermore, the binding interaction of PBI-Man with macrophage cells was investigated by confocal laser scanning microscopy, which showed that PBI-Man had a selectively binding interaction with the surface mannose receptor of the cells. Moreover, the cytotoxicity of PBI-Man with macrophage cells was studied by MTT assay, showed noncytotoxicity to the living cells.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author. Acknowledgements: The authors thank NSFC (21002020, 21372059, and 21172051), Hebei Natural Science Foundation (B2011201052, B2012201041), the Foundation of Hebei Education Department (YQ2013006), and Training Program for Innovative Research Team and Leading talent in Hebei Province University (LJRC024) for financial support. The authors also thank Prof. Bao-Xiang Gao (Hebei University) for the confocal imaging experiments. Received: December 18, 2013; Revised: January 11, 2014; Published online: February 4, 2014; DOI: 10.1002/marc.201300916 Keywords: biocompatible; fluorescence imaging; glycodendrimer; perylene bisimides [1] [2] [3] [4] [5] [6]
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