Using Bioluminescence Turn-On To Detect Cysteine in Vitro and in Vivo

3 days ago - CAS Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hef...
1 downloads 5 Views 2MB Size
Download PDF
Subscriber access provided by UNIV OF YORK

Using Bioluminescence Turn-On to Detect Cysteine in Vitro and in Vivo Miaomiao Zhang, Lin Wang, Yangyang Zhao, Fuqiang Wang, Jindao Wu, and Gaolin Liang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00682 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Using Bioluminescence Turn-On to Detect Cysteine in Vitro and in Vivo Miaomiao Zhang,† Lin Wang,§ Yangyang Zhao,§ Fuqiang Wang,‡ Jindao Wu,‡,* and Gaolin Liang†,* †CAS

Key Laboratory of Soft Matter Chemistry, Department of Chemistry, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026, China ‡Key Laboratory of Living Donor Liver Transplantation of Ministry of Public Health, Department of Liver Transplantation Center of The First Affiliated Hospital, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, China ‡School of Life Sciences, University of Science and Technology of China, Huangshan Road, Hefei, Anhui 230027, China ABSTRACT: Cysteine (Cys) is an essential amino acid and plays important roles in many biological processes. Bioluminescence (BL) is advantageous in sensitivity but BL probes that were intentionally developed for the selective detection of Cys were rarely reported. Herein, employing a fast conjugate addition between Cys and acrylic ester, we synthesized a caged BL probe acrylic ester luciferin (1) and used it to selectively detect Cys in vitro and image Cys in living cells and in tumor sites . We envision that, in the future, probe 1 might be used for evaluating the Cys roles in more biological processes.

Cysteine (Cys) is an essential amino acid to living subjects and involved in several important biological processes including protein synthesis, detoxification, and metabolism.1 Elevated levels of Cys were reported to associate with neurotoxicity2 and cardiovascular diseases,3 and Cys deficiency may lead to a lot of pathological symptoms such as edema, liver damage, skin lesions, and weakness.4 Consequently, precise assessment of Cys levels in human bodies may aid the early diagnoses of some diseases. So far, a lot of analytical methods have been reported for sensing Cys including high performance liquid chromatography (HPLC),5 capillary electrophoresis,6 voltammetry,7 mass spectrometry (MS),8 colorimetric,9 and fluorometry.10-12 Among them, fluorescence is widely used due to its high sensitivity, low limit of detection (LOD), and simple implementation. However, fluorescence always suffers from photo bleaching of the fluorophores, interference by the auto-fluorescence from the detection samples, etc. In contrast to fluorescence, bioluminescence (BL) has higher signal-to-noise ratio because it does not need light excitation.13-14 Moreover, due to their biological sources, BL probes always have relatively lower cytotoxicity than fluorescence probes. To date, various BL probes have been synthesized for sensing (or imaging) of a series of important biomakers.15-19 However, BL probes that were intentionally developed for the selective detection of Cys were rarely reported. It is well known that the condensation reaction between acrylate and Cys can be used for the preparation of substituted 1,4-thiazepines.20 Recently, many

fluorescence sensors, all of which share an acryloyl appendage, have been reported for Cys detection operating via a Michael addition and an intramolecular cyclization between Cys and the appendage.21-24 Inspired by above mentioned studies, herein we synthesized a silent BL probe acrylic ester luciferin (1), whose hydroxyl group of D-luciferin motif being caged by acrylic ester, for selective detection of Cys in vitro and sensing Cys inside cells and tumors. As outlined in Scheme 1, a conjugate addition of Cys to the acrylate of 1 occurs to yield the thioether intermediate, which instantly undergoes an intramolecular cyclization to yield a stable seven-member heterocyclic compound and D-luciferin. In the presence of adenosine triphosphate (ATP), O2, and Mg2+, D-luciferin will be converted by the enzyme firefly luciferase (fLuc) to yield decarboxylated luciferin, accompanied by a photon emission.25

Scheme 1. Proposed mechanism of probe 1 for cysteine sensing.

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 5

Figure 1. (a) Bioluminescence spectra of 25 μM probe 1 with (red) or without (black) 100 μM Cys in phosphate buffered saline (10 mM, pH 7.4) containing 0.1 mg/mL fLuc and 10 mM MgCl2. (b) The linear response of 25 μM probe 1 towards Cys in the range of 0-25 μM. (c) Selectivity of 1 (25 μM) towards Cys (25 μM), HCys (25 μM), GSH (25 μM) or other 19 natural amino acids (each at 250 μM), respectively. The error bar represents the standard deviation of three independent experiments. The relative (B-B0) (%) indicates the ratio of the bioluminescence intensity of probe 1 with the analyte to that of probe 1 with Cys.

We firstly synthesized and characterized probe 1 (Scheme S1 and Figures S1-S3). Next, we studied its chemical response to Cys. After adding 4-fold Cys to the solution of 1 and incubated the mixture for 30 minutes at 37 °C, we observed the UV absorption peak 300 nm of 1 red shifted to 330 nm which is the typical UV peak of Dluciferin (Figure S4), indicating the removal of the acryloyl appendage from 1 to yield D-luciferin. Then, BL response of 1 toward Cys was preliminarily investigated. As shown in Figure 1a, after addition of 100 μM Cys into the solution of 25 μM 1 in phosphate buffered saline (10 mM PBS, pH 7.4, with 10 mM MgCl2) and incubation at 37 °C for 30 min, addition of 1 mM ATP and 0.1 mg/mL fLuc into the mixture immediately induced the increase of the BL intensity significantly . For the control group without Cys addition, BL signal was barely observed under the same condition. This result further validated our hypothesis that probe 1 could react with Cys to yield free D-luciferin to generate BL. Under this condition, about 98.4% of probe 1 was calculated to convert to Dluciferin using HPLC analysis (Figure S5). Kinetic study of the reaction between Cys and 1 indicated that this reaction had a second-order rate constant of 60.2 M-1·S-1 (Figure S6). After confirming the rapid response of probe 1 to Cys, we performed its sensitivity studies. After incubation with different concentrations of Cys for 30 min at 37 °C, probe 1 exhibited a different increase of BL intensity in the range of 0-100 μM (Figure S7) and a good linear relationship (Y = 6.489X + 3.886, R2 = 0.997) between the BL intensity enhancement (i.e., B - B0) of 25 µM probe 1 and Cys concentration in the region of 0-25 µM (Figure 1b and Figure S8). A LOD 88.0 nM of probe 1 toward Cys was obtained based on the general 3σ method (3σ/s).26 which is comparable to the methods that have been reported (Table S1). It is known to all that the selectivity of a probe to its target analyte among potential competitive species is very important. To further evaluate its feasibility for biomedical applications, we incubated 25 μM 1 with 25 μM biothiols (Cys, homocysteine (Hcy), glutathione

(GSH)) or 250 μM of other 19 natural amino acids (Val, Tyr, Trp, Ser, Pro, Phe, Met, Lys, Leu, Arg, Ile, His, Gly, Gln, Glu, Asp, Asn, Ala, Thr), respectively. As shown in Figure 1c, compared with B - B0 value induced by other selected species in the presence of fLuc, 25 μM Cys showed significantly BL enhancement after addition to 25 µM 1 (23-182 folds of those induced by the other tested species). HPLC analysis indicated that some amino acids (e.g., Gln) could also react with probe 1 to yield tiny amount of D-luciferin to generate BL (Figure S9). This result suggested that probe 1 has excellent selectivity toward Cys over other intracellular small biological molecules. Interference tests indicated that the bioluminescence intensity of probe 1 (25 μM) with Cys (25 μM) was almost not affected in the presence of above analytes, suggesting our probe 1 could be applied for sensing Cys in real samples (Figure S10).

Figure 2. BL images of fLuc-transfected MDA-MB-231 cells in different times that pre-incubated with 2 mM MBA for 30 min followed by incubation with 25 μM Cys for 30 min and then with 25 μM 1 (top row), pre-incubated with 2 mM MBA for 30 min followed by 25 μM 1 (middle row), incubated with 25 μM 1 (bottom row) acquired at 0, 10, 20, 50, 80, 110, 140, and 170 min at 37 °C in culture medium without serum.

After verifying the good property of 1 for Cys detection in vitro, we prepared to apply probe 1 for BL imaging of Cys in living cells. Before that, we evaluated the

ACS Paragon Plus Environment

Page 3 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

cytotoxicity of 1 on MDA-MB-231 cells using 3-(4,5dimethylthiazol-2-yl) 2,5 diphenyltetrazplium bromide (MTT) assay. More than 95% of the cells survived in 100 µM 1 up to 8 h (Figure S11), suggesting that probe 1 at a concentration below 100 µM was safe for living cell imaging. We thus chose probe 1 at 25 µM for the below cell imaging experiment. As we know, cancer cells are characterized with a high concentration of intracellular GSH (~mM).27 Therefore, interference from intracellular GSH should be studied before applying 1 for sensing Cys inside cells. Through kinetic study, the second-order rate constant of the reaction between probe 1 and GSH was measured to be 14.7 M-1·S-1 (Figure S12), which was much smaller than that of 1 toward Cys. In addition, only a very small proportion of probe 1 was transformed into D-luciferin after the reaction with GSH (Figure S13). This demonstrated that probe 1 had good selectivity for imaging Cys over GSH. Indeed, as shown in figure S14, although the BL from 1 generated by 250 µM GSH was lower than that by 25 µM Cys, BL by 1 mM GSH was much higher. Therefore, we blocked the thiol activity with maleimidobutyric acid (MBA) before applying the cells for BL imaging of Cys with probe 1. MDA-MB-231 cells that transfected with fLuc were pre-incubated with 2 mM MBA for 30 min to consume all of the free thiols within the cells and washed with PBS buffer for three times. For Cys detection, after co-incubation with 25 μM Cys for 30 min, the cells were washed with PBS buffer for three times and then put in a 96-well plate at 5 × 105 cells/well. After addition of 25 μM 1, in virtue of a small animal imaging system, we gained real-time recording of BL signals that generated from the cells. As shown in Figure 2, after the addition of probe 1, BL signal from the cells in the top row was the brightest. Quantification of the BL intensity indicated that the BL signal gradually increased to its plateau (9.88 × 104 of total flux, Figure S15) at around 50 min, followed by a slow decrease. For the control cells whose thiol activity was blocked by 2 mM thiol-reactive MBA for 30 min but without Cys addition, very weak BL signal (5.01 × 104 of total flux at 50 min, Figure S15) was observed after 30 min incubation with 25 μM 1 (middle row in Figure 2), suggesting above strong BL signal was induced by the added Cys. For the cells without any treatment, weak BL signal (5.15 × 104 of total flux at 50 min, Figure S15) was also observed after 25 μM 1 addition (bottom row in Figure 2). Since Hcy is hardly detectable in healthy mammalian cell, thus the weak BL should be ascribed to the products of 1 that reacts with intracellular Cys and GSH. In addition, cellular uptake tests of 1 indicated that the probe gradually entered MDA-MB-231 cells in 2 h and rapidly reacted with Cys to yield D-luciferin (Figure S16). These results indicated that probe 1 is applicable for BL imaging of Cys in living cells.

Figure 3. BL imaging of nude mice that xenografted with fLuc-transfected MDA-MB-231 tumors at different times after i.p. injection of MBA at 0.5 mmol/kg followed by i.v. injection of Cys at 1.0 mmol/kg 30 min later and i.v. injection of 1 at 12.5 μmol/kg 30 min later (top row), i.p. injection of MBA at 0.5 mmol/kg followed by i.v. injection of 1 at 1.0 mmol/kg 30 min later (middle row), i.v. injection of 1 at 1.0 mmol/kg (bottom row) in PBS at 15, 20, 25, 30, and 40 min.

We then applied probe 1 for BL imaging of Cys in tumors in living mice. A fLuc-transfected MDA-MB-231 tumour was xenografted in the right thigh of each nude mouse and the mice were randomly divided into three groups. One group of mice were intraperitoneally (i.p.) injected with 0.5 mmol/kg MBA followed by intravenous (i.v.) injection of Cys at 1.0 mmol/kg and finally i.v. injection of probe 1 at 12.5 μmol/kg and used as the experimental group. The remaining two groups were used as control groups. In detail, one group of mice were injected with MBA followed by the injection of 1 and another group of mice were injected with 1 only. In virtue of a small animal imaging system, we gained the real-time recording of BL signal that generated from the tumours in the nude mice. As shown in the top row of Figure 3, during the observation time (i.e., 15-40 min), the BL signal from the experimental group gradually increased to reach its peak at 25 min (5.73 × 106 of total flux, Figure S17) and slowly decreased thereafter, suggesting that our probe 1 reacted with Cys to yield Dluciferin to generate BL in the tumors. For the control group whose thiol activity was blocked by injected MBA but without Cys injection, almost no bioluminescent signal (1.53 × 105 of total flux at 25 min, Figure S17) was observed after probe 1 injection (middle row in Figure 3), indicating above BL signal in the experimental group was induced by the injected Cys. Interestingly, for the control group that only injected with probe 1, weak BL

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

signal was also observed (7.20 × 105 of total flux at 25 min, Figure S17, bottom row in Figure 3). This indicated that the intracellular Cys and GSH in tumors reacted with probe 1 to yield D-luciferin to generate the BL. These results suggested that probe 1 was applicable for imaging Cys in tumours in living mice. In summary, we developed a silent bioluminescent probe 1 to selectively sense Cys in vitro and image Cys in vivo. The high selectivity of probe 1 for Cys detection lies in the fast conjugate addition of Cys to the acrylate motif of 1 and subsequent intramolecular cyclization to release D-luciferin to generate BL in the presence of fLuc. We successfully applied probe 1 to selectively detect Cys in the 0-25 μM linear concentration range with a LOD of 88 nM in vitro. Moreover, we also evaluated the capability of probe 1 for sensing Cys in living cells and tumours. We envision that, in the future, probe 1 might be applied to evaluate the Cys roles in more biological processes.

ASSOCIATED CONTENT Supporting Information General methods; Syntheses and characterizations of 1; Scheme S1; Figure S1-S17; Table S1-S3. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (J. Wu). *E-mail: [email protected] (G. Liang).

Notes

Page 4 of 5

Chem.Commun. 2008, 6173-6175. (11) Lim, S.; Escobedo, J. O.; Lowry, M.; Xu, X.; Strongin, R. Chem.Commun. 2010, 46, 5707-5709. (12) Miao, Q.; Li, Q.; Yuan, Q.; Li, L.; Hai, Z.; Liu, S.; Liang, G. Anal. Chem. 2015, 87, 3460-3466. (13) Conley, N. R.; Dragulescu-Andrasi, A.; Rao, J.; Moerner, W. E. Angew. Chem., Int. Ed. 2012, 124, 3406-3409. (14) Kojima, R.; Takakura, H.; Ozawa, T.; Tada, Y.; Nagano, T.; Urano, Y. Angew. Chem., Int. Ed. 2013, 52, 1175-1179. (15) Zheng, Z.; Li, G.; Wu, C.; Zhang, M.; Zhao, Y.; Liang, G. Chem.Commun. 2017, 53, 3567-3570. (16) Hai, Z.; Wu, J.; Wang, L.; Xu, J.; Zhang, H.; Liang, G. Anal. Chem. 2017, 89, 7017-7021. (17) Chen, P.; Zheng, Z.; Zhu, Y.; Dong, Y.; Wang, F.; Liang, G. Anal. Chem. 2017, 89, 5693-5696. (18) Zheng, Z.; Wang, L.; Tang, W.; Chen, P.; Zhu, H.; Yuan, Y.; Li, G.; Zhang, H.; Liang, G. Biosens. Bioelectron. 2016, 83, 200-204. (19) Gao, Y.; Lin, Y.; Liu, T.; Chen, H.; Yang, X.; Tian, C.; Du, L.; Li, M. Anal. Chem. 2017, 89, 12488-12493. (20) Leonard, N. J.; Ning, R. Y. J. Org. Chem. 1966, 31, 39283935. (21) Soboleva, T.; Esquer, H. J.; Benninghoff, A. D.; Berreau, L. M. J. Am. Chem. Soc. 2017, 139, 9435-9438. (22) Wang, H.; Zhou, G.; Gai, H.; Chen, X. Chem.Commun. 2012, 48, 8341-8343. (23) Yang, X.; Guo, Y.; Strongin, R. M. Angew. Chem., Int. Ed. 2011, 50, 10690-10693. (24) Yang, X.; Guo, Y.; Strongin, R. M. Org. Biomol. Chem. 2012, 10, 2739-2741. (25) Seliger, H. H.; McElroy, W. D. Arch. Biochem. Biophys. 1960, 88, 136-141. (26) Ogren, P. J.; Meetze, A.; Duer, W. C. J. Anal. Toxicol. 2009, 33, 129-142. (27) Fan, H.; Yan, G.; Zhao, Z.; Hu, X.; Zhang, W.; Liu, H.; Fu, X.; Fu, T.; Zhang, X. B.; Tan, W. Angew. Chem., Int. Ed. 2016, 128, 5567-5572.

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Ministry of Science and Technology of China (2016YFA0400904), the National Natural Science Foundation of China (Grants 21725505 and 21675145), Major program of Development Foundation of Hefei Center for Physical Science and Technology (2016FXZY006), and Jiangsu Youth Medical Talents (QNRC2016580).

REFERENCES (1) Gahl, W.; Bashan, N.; Tietze, F.; Bernardini, I.; Schulman, J. Science 1982, 217, 1263-1265. (2) Wang, X. F.; Cynader, M. S. J. Neurosci. 2001, 21, 33223331. (3) El-Khairy, L.; Ueland, P. M.; Refsum, H.; Graham, I. M.; Vollset, S. E. Circulation 2001, 103, 2544-2549. (4) Shahrokhian, S. Anal. Chem. 2001, 73, 5972-5978. (5) Amarnath, K.; Amarnath, V.; Amarnath, K.; Valentine, H. L.; Valentine, W. M. Talanta 2003, 60, 1229-1238. (6) Stamler, J. S.; Loscalzo, J. Anal. Chem. 1992, 64, 779-785. (7) Spãtaru, N.; Sarada, B. V.; Popa, E.; Tryk, D. A.; Fujishima, A. Anal. Chem. 2001, 73, 514-519. (8) Xu, K.; Zhang, Y.; Tang, B.; Laskin, J.; Roach, P. J.; Chen, H. Anal. Chem. 2010, 82, 6926-6932. (9) Huo, F.-J.; Sun, Y.-Q.; Su, J.; Chao, J.-B.; Zhi, H.-J.; Yin, C.-X. Org. Lett. 2009, 11, 4918-4921. (10) Lee, K.-S.; Kim, T.-K.; Lee, J. H.; Kim, H.-J.; Hong, J.-I.

ACS Paragon Plus Environment

Page 5 of 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

For TOC only

ACS Paragon Plus Environment