- Email: [email protected]
The synthesis of UDP-selective fluorescent probe and its imaging application in living cells
Bioorganic & Medicinal Chemistry Letters 25 (2015) 262–265
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
The synthesis of UDP-selective fluorescent probe and its imaging application in living cells Srinivasulu Kambam a, , Xintong Ren a, , Cheng Zheng b, Fang Wang a, Yong Wang a, Haiyan Chen c,⇑, Jun Yin d, Jingjing Xie b, Xiaoqiang Chen a,⇑ a
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing Tech University, Nanjing 210009, China State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211800, China Department of Biomedical Engineering, School of Life Science and Technology, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China d Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China b c
a r t i c l e
i n f o
Article history: Received 1 August 2014 Revised 4 November 2014 Accepted 20 November 2014 Available online 27 November 2014 Keywords: Fluorescent probe UDP Perylene Cell imaging
a b s t r a c t A perylene-based probe was developed for uridine diphosphate (UDP) sensing and cell imaging. The probe presented about 4-fold fluorescence enhancement in the presence or absence of 100 equiv UDP. The selectivity toward UDP over other phosphor-containing anions was observed. The selective UDP sensing was speculated to be related to the binding affinities of Zn2+ ions in sensor with the uridine and phosphate moieties of UDP. Furthermore, this probe was also applied to image of UDP in living cells. Ó 2014 Elsevier Ltd. All rights reserved.
The recognition and sensing of anions have received increasing attentions in recent years.1 Among a variety of anions, phosphorcontaining anions including phosphates, pyrophosphates, and nucleotides are important species and play major roles in key biological processes.2 The development of detection methods for phosphor-containing anions is a favor of understanding and elucidating their functions in biological systems. Fluorogenic methods in conjunction with suitable probes can afford real information on the localization and quantity of the targets of interest. Furthermore, probes based on fluorescence ‘off–on’ for detection of phosphor-containing anions are attractive because fluorimetry is nondestructive, highly sensitive and suitable for high-throughput screening applications.2 In the recent years, great efforts have been devoted to the development of fluorescent sensors for phosphor-containing anions including phosphates (Pi),3 pyrophosphate,4 and nucleotides.5 As one of the nucleotides, uridine diphosphate (UDP) wide-spreads in living cells and play a pivotal role in various biological events. For example, UDP is involved in glycosylation process catalyzed by glycosyltransferases. Compared to other phosphor-containing ⇑ Corresponding authors. Tel./fax: +86 25 8327 1080 (H.C.), +86 25 8358 7856 (X.C.). E-mail addresses: [email protected] (H. Chen), [email protected] (X. Chen). Contributed equally to this work. http://dx.doi.org/10.1016/j.bmcl.2014.11.057 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
anions, however, few fluorescent chemosensors that specifically respond to UDP in aqueous solution have been reported so far. Perylene diimide derivatives have attracted great attention because of their high photostability and excellent optical properties. Most reported probes employing perylene diimide derivatives were based on the ‘off–on’ tuned fluorescence corresponding to aggregated and non-aggregated status.6 In 2009, we reported an ‘off–on’ type fluorescence sensor that effectively and selectively recognizes both UTP and UDP in aqueous solution.7 The probe features perylene moiety as signal unit linked two zinc-complex arms as acceptor via ethyl chain for UTP or UDP detection. The selective UTP/UDP sensing is a consequence of cooperative binding of the Zn2+ ions with the uridine and phosphate moieties of UTP/UDP. Herein, we developed a new fluorescent probe 1 for UDP-selective detection by replacing ethyl chain between perylene moiety and zinc-complex arms with propyl chain (Scheme 1). This probe based on a zinc complex exhibits high selectivity for UDP over other phosphor-containing anion derivatives under physiological conditions. The synthesis of probe 1 was performed according to the Scheme 1. N,N-Bis(2-pyridylmethyl)propane-1,3-diamine was synthesized according to the similar method reported previously.8 The mixture of 3,4,9,10-perylenetetracarboxylic dianhydride and N,N-bis(2-pyridylmethyl)propane-1,3-diamine was refluxed in propan-1-ol/H2O for 24 h. After evaporation of the solvent, the
263
S. Kambam et al. / Bioorg. Med. Chem. Lett. 25 (2015) 262–265 H2 N
O
O
O
O
O
O
N
N
O
O
N
N
O
O
N
N
N N
Propan-1-ol/H 2 O, reflux
N
N N
2
Zn(ClO4 )2 ·6H2 O CH3 CN
N Zn2+
N N
O
O
N
N
O
O
N N N
Zn2+
4ClO4
1
Scheme 1. The synthesis of probe 1.
residue was purified by Al2O3 column using CHCl3/CH3OH (99:1) as eluent. Further purification by CHCl3/CH3COCH3 precipitation afforded 0.301 g red solid (40%). Treatment of 2 with Zn(ClO4)2 6H2O in CH3CN then afforded probe 1 in 70% yield. The experimental details and characterization data for 1 and 2 are given in the Supplementary data (Figs. S1–S11). The selectivity assay of probe 1 for phosphor-containing anions was carried out in CH3CN-HEPES buffer (0.01 M, pH 7.4) (0.05:99.95, v/v). The fluorescence spectra were obtained by excitation of the perylene fluorophore at 485 nm. As shown in Figure 1, upon the addition of various phosphor-containing anions (100 equiv) including H2PO4 (Pi), pyrophosphate (PPi), ATP, CTP, GTP, ADP, AMP, UMP, UDP and UTP, only UDP induces a clear fluorescence enhancement at 550 nm. In contrast, Pi, PPi, ATP, CTP, GTP, ADP, AMP, UMP and UTP led to a little decrease in fluorescence intensity. When the amount of UDP increased to 100 equiv, about 4-fold fluorescence enhancement at 550 nm was observed (Fig. 2). The detection limit was calculated to be 0.42 lM from the fluorescent titration experiments (Fig. S12). In the absence of UDP, the initial fluorescence of compound 1 is weak (Fig. 1). In aqueous solution, one zinc ion was dissociated from compound 1 to form a mononuclear Zn2+ complex, which
Figure 1. Fluorescence changes of 1 (1 lM) with Pi, PPi, ATP, GTP, CTP, ADP, AMP, UMP, UDP and UTP (100 equiv) in CH3CN-HEPES buffer (0.01 M, pH 7.4) (0.05:99.95, v/v) (excitation at 485 nm, slit: 10 nm/5 nm).
Figure 2. The dependence of the relative fluorescence intensity of 1 (1 lM) at 550 nm on the various anions at different concentrations in CH3CN-HEPES buffer (0.01 M, pH 7.4) (0.05:99.95, v/v).
results in the electron transfer from the tertiary amine donor to the perylene singlet excited state, quenching the fluorescence (Scheme 2a). Additionally, perylene-containing compound 1 is apt to aggregation due to the p–p stacking in aqueous solution, which also causes fluorescence decreasing6 (Scheme 2b). When UDP was added to the solution containing compound 1, with the assistance of electrostatic and coordination interactions, the free Zn2+ was coordinated with DPA unit, further lead to the formation of UDP-1 complex (Scheme 2a). The result inhibits the efficiency of electron transfer from tertiary amine donor to excited perylene fluorophore, leading to fluorescence enhancement. On the other hand, the addition of UDP also results in the decomposition of p–p stacking formation, which is also helpful for the fluorescence enhancement (Scheme 2b). Other phosphor-containing analytes including ATP, GTP, ADP, and PPi did not result in the enhancement in fluorescence, which should be attributed to the specific interactions between UDP and zinc complex. Except one of the Zn2+ moieties of 1 can bind phosphate group in UDP, the selectivity of 1 toward UDP comes from the specific bind between uridine group in UDP and the other Zn2+, which have been demonstrated in previous reports.9 In our previous work, zinc-complex arms as acceptor via ethyl chain displayed the fluorescence enhancement for both UDP and UTP.7 In contrast, the new zinc-complex bearing perylene linked by propyl chain exhibited the good selectivity toward
264
S. Kambam et al. / Bioorg. Med. Chem. Lett. 25 (2015) 262–265
UDP over UTP, that means the selectivity of 1 to UDP could be tuned by controlling the length of spacer between perylene and zinc-complex moieties. Because the mixture of Pi (100 equiv) and UMP (100 equiv) did not lead to effective enhancement in the fluorescence of 1 (Fig. S13, see Supplementary data), a spacer is necessary for the cooperation between phosphate group and uridine group. The maximum emission peak of probe 1 is 550 nm, which is an ideal wavelength for biological applications. The ability of zinc complex coupling with UDP to image living cells was also evaluated (Fig. 3). After MCF-7 cells incubated with 10 lM probe for 1 h at 37 °C, weak fluorescence signal was observed by laser confocal fluorescence microscopy (Fig. 3a). However, when 20 lM of UDP was added and incubated with the cells afterwards for 30 min, an obvious fluorescence enhancement was observed from the fluorescence images (Fig. 3b). These results indicate that zinc complex 1 is cell-permeable and could be utilized to image UDP within living cells. In conclusion, a water-soluble type fluorescence sensor with high selectivity for recognizing UDP was developed. The perylene–zinc complex based sensor exhibited a fluorescence enhancement associated with formation of a complex with UDP. The selective UDP sensing may be related to the binding affinities of Zn2+ ions in sensor with the uridine and phosphate moieties of UDP. As a result, the perylene moiety in 1 plays two important roles. It is not only the key fluorophore of the sensor but also an optimum spacer template for binding. We successfully demonstrated that the sensor can be applied to image UDP in living cells. Acknowledgments
Scheme 2. The proposed binding mechanism of probe 1 with UDP.
This work was supported by the National Natural Science Foundation of China (21376117, 81371684), the Jiangsu Natural Science Funds for Distinguished Young Scholars (BK20140043), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (14KJA150005) and the Project of Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Figure 3. Laser confocal fluorescence images of the fluorescence enhancement of probe 1 by UDP in MCF-7 breast cancer cells. (a) MCF-7 cells incubated with probe 1 (10 lM) (solvent: DMSO and PBS; VDMSO:VPBS = 5:95) at 37 °C for 1 h; (b) 1 stained cells were exposed to 20 lM UDP at 37 °C for 30 min.
S. Kambam et al. / Bioorg. Med. Chem. Lett. 25 (2015) 262–265
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 11.057. References and notes 1. (a) Lee, C.-H.; Miyaji, H.; Yoon, D.-W.; Sessler, J. L. Chem. Commun. 2008, 24; (b) Gunnlaugsson, T.; Glynn, M.; Tocci, G. M.; Kruger, P. E.; Pfeffer, F. M. Coord. Chem. Rev. 2006, 250, 3094; (c) Martínez-Máñez, R.; Sancano´n, F. Chem. Rev. 2003, 103, 4419; (d) Wang, F.; Wang, L.; Chen, X.; Yoon, J. Chem. Soc. Rev. 2014, 43, 4312; (e) Yoon, J.; Kim, S. K.; Singh, N. J.; Kim, K. S. Chem. Soc. Rev. 2006, 35, 355; (f) Gale, P. A. Acc. Chem. Res. 2006, 39, 465. 2. (a) Zhou, Y.; Xu, Z.; Yoon, J. Chem. Soc. Rev. 2011, 40, 2222; (b) Kim, S. K.; Lee, D. H.; Hong, J.-I.; Yoon, J. Acc. Chem. Res. 2009, 42, 23. 3. (a) Hirsch, A. K. H.; Fischer, F. R.; Diederich, F. Angew. Chem., Int. Ed. 2007, 46, 338; (b) Xu, Z.; Kim, S.; Lee, K.-H.; Yoon, J. Tetrahedron Lett. 2007, 48, 3797; (c) Kim, S. K.; Singh, N. J.; Kim, S. J.; Kim, H. G.; Kim, J. K.; Lee, J. W.; Kim, K. S.; Yoon, J. Org. Lett. 2003, 5, 2083; (d) Yoon, J.; Kim, S. K.; Singh, N. J.; Lee, J. W.; Yang, Y. J.; Chellappan, K.; Kim, K. S. J. Org. Chem. 2004, 69, 581; (e) Xiaozhi, J.; Dawei, Z.; Jinjiang, Z.; Jianwei, Z.; Binshen, W.; Meiyun, F.; Zhiyun, D.; Guohua, G. Anal. Methods 2013, 5, 3222; (f) Kim, H. B.; Liu, Y.; Nam, D.; Li, Y.; Park, S.; Yoon, J.; Hyun, M. H. Dyes Pigments 2014, 106, 20; (g) Dawei, Z.; Xiaozhi, J.; Zhiyun, D.; Haiqiang, Y.; Alexandre, M.; Guohua, G. Tetrahedron 2013, 69, 10457; (h) Dawei, Z.; Xiaozhi, J.; Haiqiang, Y.; Zhao, S.; Enqing, G.; Alexandre, M.; Guohua, G. Chem. Commun. 2013, 6149; (i) Ze-Bao, Z.; Zhi-Ming, D.; Ying-Ying, M.; Ke-Zhi, W. Inorg. Chem. 2013, 52, 2306; (j) Nina, A. E.; Petr, K.; Tsuyoshi, M.; Lorenzo, M.; Vincent, M. L.; Grigory, V. Z.; Pavel, A. Chem. Sci. 2013, 4, 3617; (k) Jing, L.; Kai, W.; Xin, L.; Yifeng, H.; Min, X. RSC Adv. 2013, 3, 8924; (l) Kumaresh, G.; Debasis, K.; Soumen, J.; Debashis, S.; Bishwajit, G. RSC Adv. 2013, 3, 16144; (m) Kumaresh, G.; Sarfaraj Ali, S. K.; Soumen, T. Tetrahedron Lett. 2012, 53, 2054; (n) Supriti, S.; Manjira, M.; Kuheli, C.; Ipsit, H.; Subhra Kanti, M.; Pabitra, C. Org. Biomol. Chem. 2013, 11, 1537; (o) Wei-tao, G.; Shuang, B.; Fu-rui, W.; Jun-wei, Y.; Gui-ling, N.; Kazuhisa, H. Tetrahedron Lett. 2011, 52, 630; (p) Kumaresh, G.; Avik, R. S.; Amarendra, P. Tetrahedron Lett. 2009, 50, 6557; (q) Zhang, J. F.; Kim, S.; Han, J. H.; Lee, S. J.; Pradhan, T.; Cao, Q. Y.; Lee, S. J.; Kan, C.; Kim, J. S. Org. Lett. 2011, 13, 5294; (r) Zhang, J. F.; Park, M.; Ren, W. X.; Kim, Y.; Kim, S. J.; Jung, J. H.; Kim, J. S. Chem. Commun. 2011, 3568; (s) Cao, Q. Y.; Pradhan, T.; Kim, S.; Kim, J. S. Org. Lett. 2011, 4386. 4. (a) Huang, X.; Guo, Z.; Zhu, W.; Xie, Y.; Tian, H. Chem. Commun. 2008, 5143; (b) Lee, H. N.; Xu, Z.; Kim, S. K.; Swamy, K. M. K.; Kim, Y.; Kim, S.-J.; Yoon, J. J. Am. Chem. Soc. 2007, 129, 3828; (c) Singh, N. J.; Jun, E. J.; Chellappan, K.; Thangadurai, D.; Chandran, R. P.; Hwang, I.-C.; Yoon, J.; Kim, K. S. Org. Lett. 2007, 9, 485; (d) Lee, H. N.; Swamy, K. M. K.; Kim, S. K.; Kwon, J.-Y.; Kim, Y.; Kim, S.-J.; Yoon, Y. J.; Yoon, J. Org. Lett. 2007, 9, 243; (e) Kim, S. K.; Singh, N. J.; Kwon, J.; Hwang, I.-C.; Park, S. J.; Kim, K. S.; Yoon, J. Tetrahedron 2006, 62, 6065; (f) Sandip, B.;
5.
6.
7. 8. 9.
265
Biswanath, G.; Varpu, M.; Kari, R. J. Am. Chem. Soc. 2014, 136, 5543; (g) Yu, L.; Xiaohu, D.; Cheng, Z.; Zhihong, L.; Jingui, Q. Sens. Actuators, B 2013, 183, 124; (h) Yuji, M.; Anna, U.; Risa, O.; Hideo, K. Inorg. Chem. 2013, 52, 10223; (i) Barun, K. D.; Sandipan, M.; Chirantan, K.; Ramesh, A.; Gopal, D. Anal. Chem. 2013, 85, 8369; (j) Priyadip, D.; Bhanuchandar, N.; Shishir, C.; Hridesh, A.; Bishwajit, G.; Amitava, D. Inorg. Chem. 2013, 52, 11034; (k) Gandhi, S.; Thangaraj, A.; Duraisamy, C. Anal. Methods 2014, 6, 2343; (l) Sreenivasarao, A.; Subhankar, S.; Wonyoung, C.; Kyo, H. A. Org. Biomol. Chem. 2012, 10, 8410; (m) Ravikumar, I.; Pradyut, G. Inorg. Chem. 2011, 50, 4229; (n) Raul, V.-R.; Anatoly, K. Y. Chem. Commun. 2011, 2694; (o) Kim, M. J.; Swamy, K. M. K.; Kyung, M. L.; Arun, R. J.; Kim, Y.; Kim, S.-J.; Kyung, H. Y.; Yoon, J. Chem. Commun. 2009, 7215; (p) Yu, W.; Qiang, J.; Yin, J.; Srinivasulu, K.; Fang, W.; Wang, Y.; Chen, X. Org. Lett. 2014, 16, 2220. (a) Ojida, A.; Takashima, I.; Kohira, T.; Nonaka, H.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 12095; (b) Ojida, A.; Miyahara, Y.; Wongkongkatep, J.; Tamaru, S.-I.; Sada, K.; Hamachi, I. Chem. Asian J. 2006, 1, 555; (c) McCleskey, S. C.; Griffin, M. J.; Schneider, S. E.; McDevitt, J. T.; Anslyn, E. V. J. Am. Chem. Soc. 2003, 125, 1114; (d) Jose, D. A.; Mishra, S.; Ghosh, A.; Shrivastav, A.; Mishra, S. K.; Das, A. Org. Lett. 1979, 2007, 9; (e) Wang, S.; Chang, Y.-T. J. Am. Chem. Soc. 2006, 128, 10380; (f) Neelakandan, P. P.; Hariharan, M.; Ramaiah, D. J. Am. Chem. Soc. 2006, 128, 11334; (g) Kim, S. K.; Moon, B. S.; Park, J. H.; Seo, Y.; Koh, H. S.; Yoon, Y. J.; Lee, K. D.; Yoon, J. Tetrahedron Lett. 2005, 46, 6617; (h) Nisar, A.; Bahareh, S.; Il Seung, Y.; Amita, B.; Suresh, V.; Kwang, S. K. Chem. Commun. 2012, 2662; (i) Ningjie, W.; Jingbo, L.; Lipeng, Y.; Jingsong, Y. Chem. Commun. 2014, 4438; (j) Zhaochao, X.; Singh, N. J.; Jeesun, L.; Jie, P.; Kim, H. N.; Sungsu, P.; Kwang, S. K.; Yoon, J. J. Am. Chem. Soc. 2009, 131, 15528; (k) Artur, J. M.; Piotr, J. C.; Susanne, K. R.; Gerhard, J. M. Chem. Commun. 2010, 1085; (l) Jong-Ho, K.; Jin-Ho, A.; Paul, W. B.; Hong, J.; Jingqing, Z.; Daniel, A. H.; Michael, S. S. Angew. Chem., Int. Ed. 2010, 49, 1456; (m) Evgeny, K.; René, A.; Tobias, R. F.; Heinrich, L. Inorg. Chem. 2012, 51, 7948; (n) Ning, S.; Qi, G.; Jingwei, S.; Bin, Q.; Zhenyu, L.; Wong, K. Y.; Guonan, C. Anal. Methods 2014, 6, 3370; (o) Shun, N.; Masatora, F.; Tomoki, T.; Reiko, S.; Eiji, N.; Takashi, M. J. Am. Chem. Soc. 2013, 135, 3465; (p) Narinder, S.; Doo Ok, J. Tetrahedron Lett. 2011, 52, 2608; (q) Lei, S.; Ping, H.; Yanliang, R.; Guoqiang, F. Chem. Commun. 2013, 11704; (r) Sreenivasarao, A.; Dokyoung, K.; Hyoseok, N.; Hunho, J.; Ki, H. K.; Changill, B.; Kyo, H. A. Chem. Commun. 2012, 3206; (s) Muhammad, Y.; Nisar, A.; Bahareh, S.; Vijay Madhav, M.; Il Seung, Y.; Kwang, S. K. Org. Lett. 2014, 16, 2150. (a) Fang, L.; Jing, M.; Xiangyang, W.; Surajit, B.; Edwin, K. L. Y.; Bengang, X. Chem. Commun. 2014, 6200; (b) Chuibei, Z.; Wenying, L.; Jian, C.; Meiding, Y.; Yang, L.; Jintao, Z.; Cong, Y. Analyst 2014, 139, 1057; (c) Yan, W.; Jian, C.; Yang, C.; Wenying, L.; Cong, Y. Anal. Chem. 2014, 86, 4371; (d) Dongli, L.; Wenying, L.; Jian, C.; Huping, J.; Huipeng, Z.; Bin, W.; Cong, Y. Anal. Chim. Acta 2013, 797, 89. Chen, X.; Jou, M. J.; Yoon, J. Org. Lett. 2009, 11, 2181. Incarvito, C.; Lam, M.; Rhatigan, B.; Rheingold, A. L.; Qin, C. J.; Gavrilova, A. L.; Bosnich, B. J. Chem. Soc., Dalton Trans. 2001, 3478. (a) Wang, Q.; Lönnberg, H. J. Am. Chem. Soc. 2006, 128, 10716; (b) Rossiter, C. S.; Mathews, R. A.; Morrow, J. R. Inorg. Chem. 2005, 44, 9397; (c) Aoki, S.; Kimura, E. J. Am. Chem. Soc. 2000, 122, 4542.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
The synthesis of UDP-selective fluorescent probe and its imaging application in living cells Srinivasulu Kambam a, , Xintong Ren a, , Cheng Zheng b, Fang Wang a, Yong Wang a, Haiyan Chen c,⇑, Jun Yin d, Jingjing Xie b, Xiaoqiang Chen a,⇑ a
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing Tech University, Nanjing 210009, China State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing 211800, China Department of Biomedical Engineering, School of Life Science and Technology, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China d Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, China b c
a r t i c l e
i n f o
Article history: Received 1 August 2014 Revised 4 November 2014 Accepted 20 November 2014 Available online 27 November 2014 Keywords: Fluorescent probe UDP Perylene Cell imaging
a b s t r a c t A perylene-based probe was developed for uridine diphosphate (UDP) sensing and cell imaging. The probe presented about 4-fold fluorescence enhancement in the presence or absence of 100 equiv UDP. The selectivity toward UDP over other phosphor-containing anions was observed. The selective UDP sensing was speculated to be related to the binding affinities of Zn2+ ions in sensor with the uridine and phosphate moieties of UDP. Furthermore, this probe was also applied to image of UDP in living cells. Ó 2014 Elsevier Ltd. All rights reserved.
The recognition and sensing of anions have received increasing attentions in recent years.1 Among a variety of anions, phosphorcontaining anions including phosphates, pyrophosphates, and nucleotides are important species and play major roles in key biological processes.2 The development of detection methods for phosphor-containing anions is a favor of understanding and elucidating their functions in biological systems. Fluorogenic methods in conjunction with suitable probes can afford real information on the localization and quantity of the targets of interest. Furthermore, probes based on fluorescence ‘off–on’ for detection of phosphor-containing anions are attractive because fluorimetry is nondestructive, highly sensitive and suitable for high-throughput screening applications.2 In the recent years, great efforts have been devoted to the development of fluorescent sensors for phosphor-containing anions including phosphates (Pi),3 pyrophosphate,4 and nucleotides.5 As one of the nucleotides, uridine diphosphate (UDP) wide-spreads in living cells and play a pivotal role in various biological events. For example, UDP is involved in glycosylation process catalyzed by glycosyltransferases. Compared to other phosphor-containing ⇑ Corresponding authors. Tel./fax: +86 25 8327 1080 (H.C.), +86 25 8358 7856 (X.C.). E-mail addresses: [email protected] (H. Chen), [email protected] (X. Chen). Contributed equally to this work. http://dx.doi.org/10.1016/j.bmcl.2014.11.057 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
anions, however, few fluorescent chemosensors that specifically respond to UDP in aqueous solution have been reported so far. Perylene diimide derivatives have attracted great attention because of their high photostability and excellent optical properties. Most reported probes employing perylene diimide derivatives were based on the ‘off–on’ tuned fluorescence corresponding to aggregated and non-aggregated status.6 In 2009, we reported an ‘off–on’ type fluorescence sensor that effectively and selectively recognizes both UTP and UDP in aqueous solution.7 The probe features perylene moiety as signal unit linked two zinc-complex arms as acceptor via ethyl chain for UTP or UDP detection. The selective UTP/UDP sensing is a consequence of cooperative binding of the Zn2+ ions with the uridine and phosphate moieties of UTP/UDP. Herein, we developed a new fluorescent probe 1 for UDP-selective detection by replacing ethyl chain between perylene moiety and zinc-complex arms with propyl chain (Scheme 1). This probe based on a zinc complex exhibits high selectivity for UDP over other phosphor-containing anion derivatives under physiological conditions. The synthesis of probe 1 was performed according to the Scheme 1. N,N-Bis(2-pyridylmethyl)propane-1,3-diamine was synthesized according to the similar method reported previously.8 The mixture of 3,4,9,10-perylenetetracarboxylic dianhydride and N,N-bis(2-pyridylmethyl)propane-1,3-diamine was refluxed in propan-1-ol/H2O for 24 h. After evaporation of the solvent, the
263
S. Kambam et al. / Bioorg. Med. Chem. Lett. 25 (2015) 262–265 H2 N
O
O
O
O
O
O
N
N
O
O
N
N
O
O
N
N
N N
Propan-1-ol/H 2 O, reflux
N
N N
2
Zn(ClO4 )2 ·6H2 O CH3 CN
N Zn2+
N N
O
O
N
N
O
O
N N N
Zn2+
4ClO4
1
Scheme 1. The synthesis of probe 1.
residue was purified by Al2O3 column using CHCl3/CH3OH (99:1) as eluent. Further purification by CHCl3/CH3COCH3 precipitation afforded 0.301 g red solid (40%). Treatment of 2 with Zn(ClO4)2 6H2O in CH3CN then afforded probe 1 in 70% yield. The experimental details and characterization data for 1 and 2 are given in the Supplementary data (Figs. S1–S11). The selectivity assay of probe 1 for phosphor-containing anions was carried out in CH3CN-HEPES buffer (0.01 M, pH 7.4) (0.05:99.95, v/v). The fluorescence spectra were obtained by excitation of the perylene fluorophore at 485 nm. As shown in Figure 1, upon the addition of various phosphor-containing anions (100 equiv) including H2PO4 (Pi), pyrophosphate (PPi), ATP, CTP, GTP, ADP, AMP, UMP, UDP and UTP, only UDP induces a clear fluorescence enhancement at 550 nm. In contrast, Pi, PPi, ATP, CTP, GTP, ADP, AMP, UMP and UTP led to a little decrease in fluorescence intensity. When the amount of UDP increased to 100 equiv, about 4-fold fluorescence enhancement at 550 nm was observed (Fig. 2). The detection limit was calculated to be 0.42 lM from the fluorescent titration experiments (Fig. S12). In the absence of UDP, the initial fluorescence of compound 1 is weak (Fig. 1). In aqueous solution, one zinc ion was dissociated from compound 1 to form a mononuclear Zn2+ complex, which
Figure 1. Fluorescence changes of 1 (1 lM) with Pi, PPi, ATP, GTP, CTP, ADP, AMP, UMP, UDP and UTP (100 equiv) in CH3CN-HEPES buffer (0.01 M, pH 7.4) (0.05:99.95, v/v) (excitation at 485 nm, slit: 10 nm/5 nm).
Figure 2. The dependence of the relative fluorescence intensity of 1 (1 lM) at 550 nm on the various anions at different concentrations in CH3CN-HEPES buffer (0.01 M, pH 7.4) (0.05:99.95, v/v).
results in the electron transfer from the tertiary amine donor to the perylene singlet excited state, quenching the fluorescence (Scheme 2a). Additionally, perylene-containing compound 1 is apt to aggregation due to the p–p stacking in aqueous solution, which also causes fluorescence decreasing6 (Scheme 2b). When UDP was added to the solution containing compound 1, with the assistance of electrostatic and coordination interactions, the free Zn2+ was coordinated with DPA unit, further lead to the formation of UDP-1 complex (Scheme 2a). The result inhibits the efficiency of electron transfer from tertiary amine donor to excited perylene fluorophore, leading to fluorescence enhancement. On the other hand, the addition of UDP also results in the decomposition of p–p stacking formation, which is also helpful for the fluorescence enhancement (Scheme 2b). Other phosphor-containing analytes including ATP, GTP, ADP, and PPi did not result in the enhancement in fluorescence, which should be attributed to the specific interactions between UDP and zinc complex. Except one of the Zn2+ moieties of 1 can bind phosphate group in UDP, the selectivity of 1 toward UDP comes from the specific bind between uridine group in UDP and the other Zn2+, which have been demonstrated in previous reports.9 In our previous work, zinc-complex arms as acceptor via ethyl chain displayed the fluorescence enhancement for both UDP and UTP.7 In contrast, the new zinc-complex bearing perylene linked by propyl chain exhibited the good selectivity toward
264
S. Kambam et al. / Bioorg. Med. Chem. Lett. 25 (2015) 262–265
UDP over UTP, that means the selectivity of 1 to UDP could be tuned by controlling the length of spacer between perylene and zinc-complex moieties. Because the mixture of Pi (100 equiv) and UMP (100 equiv) did not lead to effective enhancement in the fluorescence of 1 (Fig. S13, see Supplementary data), a spacer is necessary for the cooperation between phosphate group and uridine group. The maximum emission peak of probe 1 is 550 nm, which is an ideal wavelength for biological applications. The ability of zinc complex coupling with UDP to image living cells was also evaluated (Fig. 3). After MCF-7 cells incubated with 10 lM probe for 1 h at 37 °C, weak fluorescence signal was observed by laser confocal fluorescence microscopy (Fig. 3a). However, when 20 lM of UDP was added and incubated with the cells afterwards for 30 min, an obvious fluorescence enhancement was observed from the fluorescence images (Fig. 3b). These results indicate that zinc complex 1 is cell-permeable and could be utilized to image UDP within living cells. In conclusion, a water-soluble type fluorescence sensor with high selectivity for recognizing UDP was developed. The perylene–zinc complex based sensor exhibited a fluorescence enhancement associated with formation of a complex with UDP. The selective UDP sensing may be related to the binding affinities of Zn2+ ions in sensor with the uridine and phosphate moieties of UDP. As a result, the perylene moiety in 1 plays two important roles. It is not only the key fluorophore of the sensor but also an optimum spacer template for binding. We successfully demonstrated that the sensor can be applied to image UDP in living cells. Acknowledgments
Scheme 2. The proposed binding mechanism of probe 1 with UDP.
This work was supported by the National Natural Science Foundation of China (21376117, 81371684), the Jiangsu Natural Science Funds for Distinguished Young Scholars (BK20140043), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (14KJA150005) and the Project of Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Figure 3. Laser confocal fluorescence images of the fluorescence enhancement of probe 1 by UDP in MCF-7 breast cancer cells. (a) MCF-7 cells incubated with probe 1 (10 lM) (solvent: DMSO and PBS; VDMSO:VPBS = 5:95) at 37 °C for 1 h; (b) 1 stained cells were exposed to 20 lM UDP at 37 °C for 30 min.
S. Kambam et al. / Bioorg. Med. Chem. Lett. 25 (2015) 262–265
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014. 11.057. References and notes 1. (a) Lee, C.-H.; Miyaji, H.; Yoon, D.-W.; Sessler, J. L. Chem. Commun. 2008, 24; (b) Gunnlaugsson, T.; Glynn, M.; Tocci, G. M.; Kruger, P. E.; Pfeffer, F. M. Coord. Chem. Rev. 2006, 250, 3094; (c) Martínez-Máñez, R.; Sancano´n, F. Chem. Rev. 2003, 103, 4419; (d) Wang, F.; Wang, L.; Chen, X.; Yoon, J. Chem. Soc. Rev. 2014, 43, 4312; (e) Yoon, J.; Kim, S. K.; Singh, N. J.; Kim, K. S. Chem. Soc. Rev. 2006, 35, 355; (f) Gale, P. A. Acc. Chem. Res. 2006, 39, 465. 2. (a) Zhou, Y.; Xu, Z.; Yoon, J. Chem. Soc. Rev. 2011, 40, 2222; (b) Kim, S. K.; Lee, D. H.; Hong, J.-I.; Yoon, J. Acc. Chem. Res. 2009, 42, 23. 3. (a) Hirsch, A. K. H.; Fischer, F. R.; Diederich, F. Angew. Chem., Int. Ed. 2007, 46, 338; (b) Xu, Z.; Kim, S.; Lee, K.-H.; Yoon, J. Tetrahedron Lett. 2007, 48, 3797; (c) Kim, S. K.; Singh, N. J.; Kim, S. J.; Kim, H. G.; Kim, J. K.; Lee, J. W.; Kim, K. S.; Yoon, J. Org. Lett. 2003, 5, 2083; (d) Yoon, J.; Kim, S. K.; Singh, N. J.; Lee, J. W.; Yang, Y. J.; Chellappan, K.; Kim, K. S. J. Org. Chem. 2004, 69, 581; (e) Xiaozhi, J.; Dawei, Z.; Jinjiang, Z.; Jianwei, Z.; Binshen, W.; Meiyun, F.; Zhiyun, D.; Guohua, G. Anal. Methods 2013, 5, 3222; (f) Kim, H. B.; Liu, Y.; Nam, D.; Li, Y.; Park, S.; Yoon, J.; Hyun, M. H. Dyes Pigments 2014, 106, 20; (g) Dawei, Z.; Xiaozhi, J.; Zhiyun, D.; Haiqiang, Y.; Alexandre, M.; Guohua, G. Tetrahedron 2013, 69, 10457; (h) Dawei, Z.; Xiaozhi, J.; Haiqiang, Y.; Zhao, S.; Enqing, G.; Alexandre, M.; Guohua, G. Chem. Commun. 2013, 6149; (i) Ze-Bao, Z.; Zhi-Ming, D.; Ying-Ying, M.; Ke-Zhi, W. Inorg. Chem. 2013, 52, 2306; (j) Nina, A. E.; Petr, K.; Tsuyoshi, M.; Lorenzo, M.; Vincent, M. L.; Grigory, V. Z.; Pavel, A. Chem. Sci. 2013, 4, 3617; (k) Jing, L.; Kai, W.; Xin, L.; Yifeng, H.; Min, X. RSC Adv. 2013, 3, 8924; (l) Kumaresh, G.; Debasis, K.; Soumen, J.; Debashis, S.; Bishwajit, G. RSC Adv. 2013, 3, 16144; (m) Kumaresh, G.; Sarfaraj Ali, S. K.; Soumen, T. Tetrahedron Lett. 2012, 53, 2054; (n) Supriti, S.; Manjira, M.; Kuheli, C.; Ipsit, H.; Subhra Kanti, M.; Pabitra, C. Org. Biomol. Chem. 2013, 11, 1537; (o) Wei-tao, G.; Shuang, B.; Fu-rui, W.; Jun-wei, Y.; Gui-ling, N.; Kazuhisa, H. Tetrahedron Lett. 2011, 52, 630; (p) Kumaresh, G.; Avik, R. S.; Amarendra, P. Tetrahedron Lett. 2009, 50, 6557; (q) Zhang, J. F.; Kim, S.; Han, J. H.; Lee, S. J.; Pradhan, T.; Cao, Q. Y.; Lee, S. J.; Kan, C.; Kim, J. S. Org. Lett. 2011, 13, 5294; (r) Zhang, J. F.; Park, M.; Ren, W. X.; Kim, Y.; Kim, S. J.; Jung, J. H.; Kim, J. S. Chem. Commun. 2011, 3568; (s) Cao, Q. Y.; Pradhan, T.; Kim, S.; Kim, J. S. Org. Lett. 2011, 4386. 4. (a) Huang, X.; Guo, Z.; Zhu, W.; Xie, Y.; Tian, H. Chem. Commun. 2008, 5143; (b) Lee, H. N.; Xu, Z.; Kim, S. K.; Swamy, K. M. K.; Kim, Y.; Kim, S.-J.; Yoon, J. J. Am. Chem. Soc. 2007, 129, 3828; (c) Singh, N. J.; Jun, E. J.; Chellappan, K.; Thangadurai, D.; Chandran, R. P.; Hwang, I.-C.; Yoon, J.; Kim, K. S. Org. Lett. 2007, 9, 485; (d) Lee, H. N.; Swamy, K. M. K.; Kim, S. K.; Kwon, J.-Y.; Kim, Y.; Kim, S.-J.; Yoon, Y. J.; Yoon, J. Org. Lett. 2007, 9, 243; (e) Kim, S. K.; Singh, N. J.; Kwon, J.; Hwang, I.-C.; Park, S. J.; Kim, K. S.; Yoon, J. Tetrahedron 2006, 62, 6065; (f) Sandip, B.;
5.
6.
7. 8. 9.
265
Biswanath, G.; Varpu, M.; Kari, R. J. Am. Chem. Soc. 2014, 136, 5543; (g) Yu, L.; Xiaohu, D.; Cheng, Z.; Zhihong, L.; Jingui, Q. Sens. Actuators, B 2013, 183, 124; (h) Yuji, M.; Anna, U.; Risa, O.; Hideo, K. Inorg. Chem. 2013, 52, 10223; (i) Barun, K. D.; Sandipan, M.; Chirantan, K.; Ramesh, A.; Gopal, D. Anal. Chem. 2013, 85, 8369; (j) Priyadip, D.; Bhanuchandar, N.; Shishir, C.; Hridesh, A.; Bishwajit, G.; Amitava, D. Inorg. Chem. 2013, 52, 11034; (k) Gandhi, S.; Thangaraj, A.; Duraisamy, C. Anal. Methods 2014, 6, 2343; (l) Sreenivasarao, A.; Subhankar, S.; Wonyoung, C.; Kyo, H. A. Org. Biomol. Chem. 2012, 10, 8410; (m) Ravikumar, I.; Pradyut, G. Inorg. Chem. 2011, 50, 4229; (n) Raul, V.-R.; Anatoly, K. Y. Chem. Commun. 2011, 2694; (o) Kim, M. J.; Swamy, K. M. K.; Kyung, M. L.; Arun, R. J.; Kim, Y.; Kim, S.-J.; Kyung, H. Y.; Yoon, J. Chem. Commun. 2009, 7215; (p) Yu, W.; Qiang, J.; Yin, J.; Srinivasulu, K.; Fang, W.; Wang, Y.; Chen, X. Org. Lett. 2014, 16, 2220. (a) Ojida, A.; Takashima, I.; Kohira, T.; Nonaka, H.; Hamachi, I. J. Am. Chem. Soc. 2008, 130, 12095; (b) Ojida, A.; Miyahara, Y.; Wongkongkatep, J.; Tamaru, S.-I.; Sada, K.; Hamachi, I. Chem. Asian J. 2006, 1, 555; (c) McCleskey, S. C.; Griffin, M. J.; Schneider, S. E.; McDevitt, J. T.; Anslyn, E. V. J. Am. Chem. Soc. 2003, 125, 1114; (d) Jose, D. A.; Mishra, S.; Ghosh, A.; Shrivastav, A.; Mishra, S. K.; Das, A. Org. Lett. 1979, 2007, 9; (e) Wang, S.; Chang, Y.-T. J. Am. Chem. Soc. 2006, 128, 10380; (f) Neelakandan, P. P.; Hariharan, M.; Ramaiah, D. J. Am. Chem. Soc. 2006, 128, 11334; (g) Kim, S. K.; Moon, B. S.; Park, J. H.; Seo, Y.; Koh, H. S.; Yoon, Y. J.; Lee, K. D.; Yoon, J. Tetrahedron Lett. 2005, 46, 6617; (h) Nisar, A.; Bahareh, S.; Il Seung, Y.; Amita, B.; Suresh, V.; Kwang, S. K. Chem. Commun. 2012, 2662; (i) Ningjie, W.; Jingbo, L.; Lipeng, Y.; Jingsong, Y. Chem. Commun. 2014, 4438; (j) Zhaochao, X.; Singh, N. J.; Jeesun, L.; Jie, P.; Kim, H. N.; Sungsu, P.; Kwang, S. K.; Yoon, J. J. Am. Chem. Soc. 2009, 131, 15528; (k) Artur, J. M.; Piotr, J. C.; Susanne, K. R.; Gerhard, J. M. Chem. Commun. 2010, 1085; (l) Jong-Ho, K.; Jin-Ho, A.; Paul, W. B.; Hong, J.; Jingqing, Z.; Daniel, A. H.; Michael, S. S. Angew. Chem., Int. Ed. 2010, 49, 1456; (m) Evgeny, K.; René, A.; Tobias, R. F.; Heinrich, L. Inorg. Chem. 2012, 51, 7948; (n) Ning, S.; Qi, G.; Jingwei, S.; Bin, Q.; Zhenyu, L.; Wong, K. Y.; Guonan, C. Anal. Methods 2014, 6, 3370; (o) Shun, N.; Masatora, F.; Tomoki, T.; Reiko, S.; Eiji, N.; Takashi, M. J. Am. Chem. Soc. 2013, 135, 3465; (p) Narinder, S.; Doo Ok, J. Tetrahedron Lett. 2011, 52, 2608; (q) Lei, S.; Ping, H.; Yanliang, R.; Guoqiang, F. Chem. Commun. 2013, 11704; (r) Sreenivasarao, A.; Dokyoung, K.; Hyoseok, N.; Hunho, J.; Ki, H. K.; Changill, B.; Kyo, H. A. Chem. Commun. 2012, 3206; (s) Muhammad, Y.; Nisar, A.; Bahareh, S.; Vijay Madhav, M.; Il Seung, Y.; Kwang, S. K. Org. Lett. 2014, 16, 2150. (a) Fang, L.; Jing, M.; Xiangyang, W.; Surajit, B.; Edwin, K. L. Y.; Bengang, X. Chem. Commun. 2014, 6200; (b) Chuibei, Z.; Wenying, L.; Jian, C.; Meiding, Y.; Yang, L.; Jintao, Z.; Cong, Y. Analyst 2014, 139, 1057; (c) Yan, W.; Jian, C.; Yang, C.; Wenying, L.; Cong, Y. Anal. Chem. 2014, 86, 4371; (d) Dongli, L.; Wenying, L.; Jian, C.; Huping, J.; Huipeng, Z.; Bin, W.; Cong, Y. Anal. Chim. Acta 2013, 797, 89. Chen, X.; Jou, M. J.; Yoon, J. Org. Lett. 2009, 11, 2181. Incarvito, C.; Lam, M.; Rhatigan, B.; Rheingold, A. L.; Qin, C. J.; Gavrilova, A. L.; Bosnich, B. J. Chem. Soc., Dalton Trans. 2001, 3478. (a) Wang, Q.; Lönnberg, H. J. Am. Chem. Soc. 2006, 128, 10716; (b) Rossiter, C. S.; Mathews, R. A.; Morrow, J. R. Inorg. Chem. 2005, 44, 9397; (c) Aoki, S.; Kimura, E. J. Am. Chem. Soc. 2000, 122, 4542.