Letter pubs.acs.org/OrgLett
A Small Molecule for Controlled Generation of Peroxynitrite Vinayak S. Khodade, Apoorva Kulkarni, Ayantika Sen Gupta, Kundan Sengupta,* and Harinath Chakrapani* Indian Institute of Science Education and Research Pune, Dr. Homi Bhabha Road, Pashan, Pune 411 008, Maharashtra, India S Supporting Information *
ABSTRACT: 2-Methyl-3-[1-(N,N-dimethylamino)diazen-1-ium-1,2-diol-2-atomethyl]-naphthalene-1,4-dione 1 (HyPR-1), a small molecule containing a superoxide generator strategically linked to a diazeniumdiolate-based nitric oxide donor, is reported. Evidence for HyPR-1’s ability to generate peroxynitrite in the presence of an enzyme as well as enhance peroxynitrite within cells is provided. The utility of this tool in generating peroxynitrite for cellular studies is demonstrated. rate to generate ONOO−. In aerobic buffer, O2•− is generated during the reaction of molecular oxygen with semiquinones or hydroquinones, which in turn are produced during bioreduction of 1,4-benzoquinones.13,14 For reliable generation of NO within cells, diazeniumdiolates are frequently used, including enzyme activated generators of NO.15,16 Compound 1 (HyPR1) was considered as a potential candidate that produces both O2•− as well as NO upon entry into cells (Scheme 1).17,18 Producing proximal fluxes of O2•− as well as NO would increase the likelihood of ONOO− formation within cells.3
eroxynitrite (ONOO−), which is produced by diffusioncontrolled reaction of nitric oxide (NO) and superoxide (O2•−), is deployed by the immune system as an antimicrobial agent against infectious pathogens.1 Elevated levels of this reactive species is also associated with numerous pathophysiological conditions such as neurodegenerative, cardiovascular, and inflammatory diseases. However, generation of this reactive species is challenging, and the currently used methods have certain limitations, especially, when employed for cellular studies. The commercially available sodium salt of peroxynitrite commonly referred to as authentic ONOO− has limitations of a short shelf life and poor bioavailability. Exogenous agents such as lipopolysaccharide and cytokines are used to enhance NO and O2•−, but this methodology is suitable only for certain cell types.2 Furthermore, such agents activate a large number of signaling and metabolic cascades in parallel with NO or O2•− generation. Two component systems which can generate O2•− using a small molecule or hypoxanthine/xanthine oxidase in conjunction with an exogenous NO donor such as a diazeniumdiolate or a metal (NO) complex have been successfully used to produce ONOO−.3−7 These protocols efficiently generate ONOO− and are well suited for biochemical studies, but little evidence for their utility in cellular assays is available.8 The most commonly used ONOO− donor for cellular studies, 3-morpholino syndnonimine (SIN-1), is a mesionic heterocyclic aromatic compound and forms peroxynitrite through a complex mechanism and offers no control over ONOO− production.9,10 Recently, photochemically activated peroxynitrite generators have been reported.11,12 Although spatiotemporal control over ONOO− generation is achieved, the use of an external light source may not always be convenient for cell-based studies. Taken together, no uniform and convenient methodology for reliably enhancing ONOO− within cells is available. Here, results of our design, synthesis, and evaluation of a small molecule that generates ONOO− upon entry into cells are presented. In order to produce ONOO−, it was hypothesized that a small molecule that can be activated within cells to generate O2•− as well as release NO would be ideal. Once produced, NO and O2•− are known to react at a nearly diffusion-controlled
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© XXXX American Chemical Society
Scheme 1. Peroxynitrite Donor 1 HyPR-1 Where a Nitric Oxide Generating Diazeniumdiolate Is Linked to a 1,4Naphthoquinone Group, Which Is Known To Generate Superoxide upon Bioreduction
In order to test this hypothesis, 1 was synthesized in three steps (Scheme 2). First, 2-bromomethyl 1,4-dimethoxy-3methylnaphthalene 2 was synthesized in 43% yield by a onepot formylation and bromination of 1,4-dimethoxy-2-methylnaphthalene.19 Reaction of 2 with sodium N,N-(dimethylamino)diazen-1-ium-1,2-diolate (DMA/NO) produced 2(N,N-(dimethylamino)diazen-1-ium-1,2-diolato)methyl-1,4-dimethoxy-2-methylnaphthalene 3 in 47% yield. Oxidation of 3 by ceric ammonium nitrate gave 1 in 48% yield. Next, in order to verify the formation of ONOO−, two “turn on” fluorescence probes with distinct chemistry were chosen. First, arylboronic acids are known to react rapidly with ONOO− and produce the corresponding alcohol as the product.3,20,21 We synthesized 3-methyl-coumarin-7-(pinacol boronate ester) 4a (Figure 1), which undergoes hydrolysis in Received: January 20, 2016
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DOI: 10.1021/acs.orglett.6b00186 Org. Lett. XXXX, XXX, XXX−XXX
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to authentic ONOO− or 1 + DT-D (see Supporting Information (SI), Figure S2). As arylboronic acids are known to react with H2O2 as well, albeit at diminished rates when compared with ONOO−,22,23 we coincubated 4a and 1 + DT-D in the presence of catalase, a H2O2 quencher and found no major difference in fluorescence emission profile suggesting that the major reactive species generated was indeed ONOO− and not H2O2 (Figure 2a). We synthesized 5 (Figure 1 and Scheme S2) that generates O2•− upon exposure to DT-D. A small fluorescence increase for 5 + DT-D was observed; this was further diminished when exposed to catalase (Figure 2b). As expected, exposure of 4a to authentic H2O2 did not produce a significant signal during 15 min (Figure 2b). The commercial donor SIN-1 also produced ONOO−, but with diminished capacity in comparison with 1 + DT-D. A time course for ONOO− generation from 1 (25 μM) showed a gradual increase during 15 min (Figure 2c) with a ONOO− yield of 8.9 μM; under similar conditions, SIN-1 generated 2.4 μM. In order to independently confirm the formation of ONOO−, N-(2-aminophenyl)-5-(dimethylamino)-1-naphthalene sulfonic amide (Ds-DAB), a probe that is reported to be sensitive to ONOO−, was synthesized.24 When Ds-DAB was incubated with authentic ONOO−, a characteristic fluorescence emission at 505 nm was observed (Figure 2d). A similar shift in fluorescence was seen during incubation of 1 + DT-D, again, confirming the production of ONOO− (see Figure 2d). Next, using the probe Ds-DAB, we tested if 1 permeated cells to enhance ONOO−. Using colorectal cancer cells (DLD-1) preincubated with Ds-DAB,24 confocal microscopy revealed a significant enhancement in fluorescence signal at 505 nm when cells were exposed to 1 (see SI, Figure S4). When 1 was coincubated with uric acid (UA), a quencher of peroxynitrite,25−27 we found nearly complete abrogation of the signal attributable to ONOO− (see Figure S4). Next, cells pretreated with hydrogen sulfide, a reported antioxidant that directly interacts with ONOO−,28,29 were exposed to 1 (see SI, Figure S4). Again, we found diminished fluorescence possibly due to decomposition of ONOO− induced by hydrogen sulfide. When DLD-1 cells which were preincubated with Ds-DAB were treated with the O2•− generator 5, as expected, no signal was seen (see SI, Figure S6). Next, a similar imaging experiment was carried out in DLD-1 cells with increasing concentrations of 1 and a dose-dependent increase in the fluorescence was seen supporting the reliability of this donor in enhancing intracellular ONOO− (Figure 3a). Under similar conditions, SIN-1 had diminished capacity to enhance ONOO− in DLD-1 cells and an elevated concentration of 100 μM was necessary for a significant signal to be recorded (Figure 3a). The suitability of 1 to enhance ONOO− in a variety of cell lines including SW 480 colon cancer cells, A549 lung cancer cells, and primary corneal fibroblast cells was demonstrated by confocal microscopy (see SI, Figures S5 and S6). The ONOO− donor HyPR-1 would allow us to study the effects of enhanced ONOO− in key cellular processes such as metastasis. A key process in metastasis is an epithelial to mesenchymal transition (EMT), which is characterized by repression of key epithelial markers such as E-cadherin (that maintains cell−cell junctions in epithelial cells).30 DLD-1 cells were treated with 1 (24 μM) for 6 h. Cells were lysed, and Western blot analysis revealed a downregulation of E-cadherin (Figure 3b). When this assay was carried out with 1 in the presence of uric acid (UA), a known peroxynitrite scavenger, no significant change in levels of E-cadherin was observed. Uric
Scheme 2. Synthesis of 1
Figure 1. Structures of peroxynitrite probes and O2•− generator.
buffer to produce the boronic acid 4b. This boronic acid is not fluorescent and upon oxidation with ONOO− produces the strongly fluorescent 3-methyl-7-hydroxycoumarin 4c. When 1 was incubated in pH 7.4 buffer, fluorescence emission monitored at 460 nm showed no significant signal (Figure 2a); in the presence of DT-diaphorase (DT-D), a bioreductive enzyme, a significant increase in fluorescence signal was recorded. An HPLC-based assay was used to confirm the formation of 4c during the assay conditions when exposed
Figure 2. Peroxynitrite generation from 1: (a) Fluorescence emission spectra of 4a (100 μM) during incubation with 1 (100 μM) + DT-D in phosphate buffer pH 7.4, (λex = 315 nm); + Cat implies coincubation with catalase (100 U/mL); (b) Fluorescence intensity of 4a during incubation with 25 μM of 1 + DT-D or 5 + DT-D or SIN-1 or H2O2 for 15 min in pH 7.4 buffer. + Cat implies coincubation with catalase (100 U/mL). (c) Time course of ONOO− generation from 1 + DT-D using probe 4a. (d) Fluorescence emission spectra of Ds-DAB (10 μM) during incubation with 1 (100 μM) + DT-D in pH 7.4 buffer for 15 min (λex = 350 nm). Authentic ONOO− (100 μM) was reacted with Ds-DAB. (e) Fluorescence intensity of 4a during incubation of 1 + DT-D and in the presence of either c-PTIO or SOD or both in pH 7.4 buffer. B
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in the case of 5. In the presence of a 1 e− reducing agent,31,32 the formation of intermediate I may occur; this intermediate may react with oxygen to produce O2•− or undergo further reduction to produce II. Alternatively, II may be produced by 2 e− reductases such as DT-D and this intermediate can also generate 2 mol of O2•−. The ability of 1 to generate O2•− was tested using a reported dihydroethidine (DHE) assay.33,34 Here, DHE selectively reacts with O2•− to generate 2hydroxyethidium (2-OH-E+) and the presence of this product can be confirmed by HPLC analysis. In the presence of DT-D, we found evidence for 2-OH-E+, which is symptomatic of O2•− (see SI, Figure S11). When cotreated with superoxide dismutase (SOD), a quencher of superoxide, we find nearly complete disappearance of the peak attributable to 2-OH-E+ confirming the intermediacy of O2•− under these conditions. The ability of 1 to produce NO was assessed by a chemiluminescence assay for NO.35 When the reaction mixture of 1 + DT-D was incubated under anaerobic conditions, we find nearly instantaneous generation of NO (see SI, Figure S12a). The signal attributable to NO was nearly completely abrogated when this experiment was conducted in the presence of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide potassium salt (c-PTIO). The yield of ONOO− during 1 + DT-D is expected to decrease if either NO or O2•− is partially quenched. When 1 + DT-D is coincubated with either c-PTIO or SOD, the yield of ONOO− was expectedly diminished (Figure 2e) and further when both c-PTIO as well as SOD are present. No significant change in fluorescence was seen when the dye 4a was reacted with SOD or c-PTIO under these conditions. Similarly, in the presence of the superoxide-quencher SOD, higher NO production was observed (see SI, Figure S12b). These studies are in agreement with the proposed mechanism outlined in Scheme 3. The levels of peroxynitrite produced during incubation of cells with 1 will depend on the cellular expression of bioreductive enzymes as well as oxygen concentrations. However, our data shows that HyPR-1 uniformly generates ONOO− in a dose-dependent manner as well as in a variety of cell types (Figure S5). Although the dependence on cellular oxygen levels for O2•− generation is not peculiar to HyPR-1: to our knowledge, O2•− producing systems require the presence of molecular oxygen. The yields of ONOO− would clearly depend on oxygen concentrations within cells. The focus of much of the ONOO− donor research has been directed toward optimizing levels of NO and O2•− in order to maximize ONOO−, but little attention has been directed toward controlled cellular enhancement of this reactive species. With our design, the relative levels of O2•− and NO produced may still favor O2•−; head-to-head comparisons with SIN-1,
Figure 3. (a) ONOO− generation in DLD-1 cells treated with 1 at 25, 50, 100 μM and SIN-1 (100 μM) for 1 h using Ds-DAB (10 μM); Ctrl is untreated; scale bar: 10 μm. (b) Decrease in the expression level of E-cadherin in DLD-1 cells upon treatment with 1 (24 μM), DLD-1 cells preincubated with uric acid (100 μM) for 1 h and then 1 (24 μM); superoxide generator 5 (24 μM) for 6 h. (c) Increase in transcript levels of E-cadherin, Occulidin; decrease in Vimentin and Ncadherin normalized with GAPDH in DLD-1 cells and SW480 cells upon treatment with 1 (24 μM) for 6 h.
acid alone did not have any effect on E-cadherin levels under these conditions. When the EMT assay with 1 was conducted in SW480 colon cancer cells, we find downregulation of Ecadherin with concomitant upregulation of vimentin, a mesenchymal marker (see SI, Figure S9). Lastly, consistent with transcriptional regulation of EMT by ONOO−, RT-PCR analysis revealed transcriptional downregulation of epithelial Ecadherin and Occludin with concomitant upregulation of Ncadherin and vimentin (Figure 3c). Lastly, a mechanism for ONOO− generation from 1 involving enzyme-triggered generation of NO as well as O2•− was proposed (Scheme 3). When 1 was reacted with DT-D, HPLC analysis revealed the nearly complete disappearance of 1 within 10 min of incubation (see SI, Figure S10). A similar assay conducted with 5 and DT-D showed decomposition of 5 with the formation of 4-nitrophenol as the product. The observation of enhanced NO formation during incubation of 1 + DT-D is consistent with bioreduction of the quinone scaffold to produce II, which then triggers the rearrangement of electrons that results in the departure of the leaving group, either the diazeniumdiolate in the case of 1 or 4-nitrophenolate,
Scheme 3. Proposed Mechanism for O2•− and NO Generation during Incubation of 1 with a Bioreductive Enzyme
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Takahashi, E.; Sagara, H.; Komatsu, M.; Tanaka, K.; Akaike, T.; Nakagawa, I.; Arimoto, H. Mol. Cell 2013, 52, 794. (3) Zielonka, J.; Sikora, A.; Joseph, J.; Kalyanaraman, B. J. Biol. Chem. 2010, 285, 14210. (4) Pfeiffer, S.; Mayer, B. J. Biol. Chem. 1998, 273, 27280. (5) Hodges, G. R.; Marwaha, J.; Paul, T.; Ingold, K. U. Chem. Res. Toxicol. 2000, 13, 1287. (6) de Boer-Maggard, T. R.; Resendez, A.; Mascharak, P. K. ChemBioChem 2013, 14, 2106. (7) deBoer, T. R.; Palomino, R. I.; Idiga, S. O.; Millhauser, G. L.; Mascharak, P. K. J. Inorg. Biochem. 2014, 138, 24. (8) Wang, C.; Deen, W. M. Chem. Res. Toxicol. 2004, 17, 32. (9) Hogg, N.; Darley-Usmar, V. M.; Wilson, M. T.; Moncada, S. FEBS Lett. 1993, 326, 199. (10) Singh, R. J.; Hogg, N.; Joseph, J.; Konorev, E.; Kalyanaraman, B. Arch. Biochem. Biophys. 1999, 361, 331. (11) Ieda, N.; Nakagawa, H.; Horinouchi, T.; Peng, T.; Yang, D.; Tsumoto, H.; Suzuki, T.; Fukuhara, K.; Miyata, N. Chem. Commun. 2011, 47, 6449. (12) Ieda, N.; Nakagawa, H.; Peng, T.; Yang, D.; Suzuki, T.; Miyata, N. J. Am. Chem. Soc. 2012, 134, 2563. (13) Dharmaraja, A. T.; Jain, C.; Chakrapani, H. J. Org. Chem. 2014, 79, 9413. (14) Danson, S.; Ward, T. H.; Butler, J.; Ranson, M. Cancer Treat. Rev. 2004, 30, 437. (15) Sharma, K.; Iyer, A.; Sengupta, K.; Chakrapani, H. Org. Lett. 2013, 15, 2636. (16) Sharma, K.; Sengupta, K.; Chakrapani, H. Bioorg. Med. Chem. Lett. 2013, 23, 5964. (17) Davies, K. M.; Wink, D. A.; Saavedra, J. E.; Keefer, L. K. J. Am. Chem. Soc. 2001, 123, 5473. (18) Keefer, L. K. ACS Chem. Biol. 2011, 6, 1147. (19) Syper, L.; Młochowski, J.; Kloc, K. Tetrahedron 1983, 39, 781. (20) Sun, X.; Xu, Q.; Kim, G.; Flower, S. E.; Lowe, J. P.; Yoon, J.; Fossey, J. S.; Qian, X.; Bull, S. D.; James, T. D. Chem. Sci. 2014, 5, 3368. (21) Kim, J.; Park, J.; Lee, H.; Choi, Y.; Kim, Y. Chem. Commun. 2014, 50, 9353. (22) Sikora, A.; Zielonka, J.; Lopez, M.; Joseph, J.; Kalyanaraman, B. Free Radical Biol. Med. 2009, 47, 1401. (23) Zielonka, J.; Sikora, A.; Hardy, M.; Joseph, J.; Dranka, B. P.; Kalyanaraman, B. Chem. Res. Toxicol. 2012, 25, 1793. (24) Lin, K.-K.; Wu, S.-C.; Hsu, K.-M.; Hung, C.-H.; Liaw, W.-F.; Wang, Y.-M. Org. Lett. 2013, 15, 4242. (25) Li, X.; Tao, R.-R.; Hong, L.-J.; Cheng, J.; Jiang, Q.; Lu, Y.-M.; Liao, M.-H.; Ye, W.-F.; Lu, N.-N.; Han, F.; Hu, Y.-Z.; Hu, Y.-H. J. Am. Chem. Soc. 2015, 137, 12296. (26) Kuzkaya, N.; Weissmann, N.; Harrison, D. G.; Dikalov, S. Biochem. Pharmacol. 2005, 70, 343. (27) Sautin, Y. Y.; Johnson, R. J. Nucleosides, Nucleotides Nucleic Acids 2008, 27, 608. (28) Carballal, S.; Trujillo, M.; Cuevasanta, E.; Bartesaghi, S.; Möller, M. N.; Folkes, L. K.; García-Bereguiaín, M. A.; Gutiérrez-Merino, C.; Wardman, P.; Denicola, A.; Radi, R.; Alvarez, B. Free Radical Biol. Med. 2011, 50, 196. (29) Li, Q.; Lancaster, J. R., Jr Nitric Oxide 2013, 35, 21. (30) Lamouille, S.; Xu, J.; Derynck, R. Nat. Rev. Mol. Cell Biol. 2014, 15, 178. (31) Iyanagi, T.; Yamazaki, I. Biochim. Biophys. Acta, Bioenerg. 1969, 172, 370. (32) Iyanagi, T.; Yamazaki, I. Biochim. Biophys. Acta, Bioenerg. 1970, 216, 282. (33) Georgiou, C. D.; Papapostolou, I.; Grintzalis, K. Nat. Protoc. 2008, 3, 1679. (34) Zhao, H.; Joseph, J.; Fales, H. M.; Sokoloski, E. A.; Levine, R. L.; Vasquez-Vivar, J.; Kalyanaraman, B. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 5727. (35) Dharmaraja, A. T.; Ravikumar, G.; Chakrapani, H. Org. Lett. 2014, 16, 2610.
however, demonstrate the superiority of HyPR-1 as a donor of ONOO− cell-free (Figure 2b) as well as within cells (Figure 3a). It is likely that HyPR-1 would produce O2•− during longer incubation periods. Lastly, the likely byproduct formed after departure of the DMA/NO is III, which can either react with water to produce 2-hydroxymethyl-3-methyl-1,4-naphthoquinone or rearrange to give 2,3-dimethyl-1,4-naphthoquinone (Scheme 3). We could not characterize these byproducts formed. However, the use of 5 + DT-D as a control gave a similar HPLC profile for byproducts (see SI, Figure S10) and these byproducts may continue to produce ROS within cells and create an imbalance in favor of ROS when compared with NO. Enhanced ROS may trigger EMT: In order to address this issue, DLD-1 cells were exposed to the O2−• generator 5 and no major difference in E-cadherin levels with respect to control was seen (Figure 3b). Using an esterase activated nitric oxide donor, we find that enhanced NO did not trigger EMT (see SI, Figure S8). Taken together, these data support ONOO− as a likely mediator of EMT, which may reflect the potency and possibly the selectivity of ONOO− in mediating such key cellular processes that may have been overlooked in previous studies. Thus, our data underscore the urgent need to develop new strategies to regulate ONOO− in tumor microenvironments. It is anticipated that HyPR-1 will find use in systematically studying responses to elevated levels of this reactive species and rationalize the effects of ONOO− on specific cellular and metabolic processes.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.6b00186. Preparative procedures, characterization data and spectra and other supporting analytical data (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank IISER Pune, the Department of Biotechnology (Grant Number: BT/PR6798/MED/29/636/ 2012), and the Council for Scientific and Industrial Research (CSIR) for financial support. K.S. is supported by an intermediate fellowship from the Wellcome Trust-DBT India Alliance (Grant number: 30711044). The authors are grateful to Govindan Ravikumar (IISER Pune) for help with some initial experiments.
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REFERENCES
(1) Szabo, C.; Ischiropoulos, H.; Radi, R. Nat. Rev. Drug Discovery 2007, 6, 662. (2) Ito, C.; Saito, Y.; Nozawa, T.; Fujii, S.; Sawa, T.; Inoue, H.; Matsunaga, T.; Khan, S.; Akashi, S.; Hashimoto, R.; Aikawa, C.; D
DOI: 10.1021/acs.orglett.6b00186 Org. Lett. XXXX, XXX, XXX−XXX
