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WO 2012/094603 Al O (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2012/094603 Al 12 July 2012 (12.07.2012) P O P C T (51) International Patent Classification: (72) Inventors; and G 1/22 (2006.01) G01N 33/00 (2006.01) (75) Inventors/Applicants (for US only): PENG, Hanjing G0 21/64 (2006.01) [CN/US]; 3236 Mercer University Drive, Apartment 205, Chamblee, GA 30341 (US). WANG, Binghe [US/US]; (21) International Application Number: 1222 Rosette Way, Marietta, GA 30062 (US). PCT/US20 12/020497 (74) Agents: SHYNTUM, Yvonne, Y. et al; Pabst Patent (22) International Filing Date: Group LLP, 1545 Peachtree Street, N.e., Suite 320, At 6 January 2012 (06.01 .2012) lanta, GA 30309 (US). English (25) Filing Language: (81) Designated States (unless otherwise indicated, for every (26) Publication Language: English kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (30) Priority Data: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, 61/430,390 6 January 201 1 (06.01 .201 1) US DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, (71) Applicant (for all designated States except US): GEOR¬ HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, GIA STATE UNIVERSITY RESEARCH FOUNDA¬ KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, TION, INC. [US/US]; 30 Courtland Street Ne, Suite 217, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, Atlanta, GA 30303 (US). OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. [Continued on nextpage] (54) Title: CHEMOSENSORS FOR HYDROGEN SULFIDE (57) Abstract: Selective chemosensing agents for hydrogen sulfide are provided. 300 The chemosensing agents can act fast un der mild conditions, are chemically stable for long-term storage, are sensitive for 250 detection under near physiological condi r tions, show a linear concentration-signal relationship within physiologically relev 5 ant hydrogen sulfide concentration ranges 200 for easy quantitation, show minimal or no interference by other anions in the blood, and are functional in aqueous solutions 150 and blood plasma. 100 ϋ 50 0 0 20 40 60 SO 100 120 140 160 180200 o Time (s) FIGURE 4B w o 2012/094603 Al I 1 11 1 I 1 1 I 11 1 I II I 1 I (84) Designated States (unless otherwise indicated, for every Declarations under Rule 4.17: Mnd of regional protection available): ARIPO (BW, GH, — of inventorship (Rule 4. 17(iv)) GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, Published: RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, — with international search report (Art. 21(3)) DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, — before the expiration of the time limit for amending the SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, claims and to be republished in the event of receipt of GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). amendments (Rule 48.2(h)) CHEMOSENSORS FOR HYDROGEN SULFIDE FIELD OF THE INVENTION The invention is generally related to the field of chemosensors, more particularly to methods and compositions for detecting hydrogen sulfide. BACKGROUND OF THE INVENTION Traditionally, hydrogen sulfide (H2S), well known for its unpleasant odor, was considered a toxic gas. However, recent studies have demonstrated that H2S is an endogenously produced gaseous signaling compound (gasotransmitter) with importance on par with that of the other two known endogenous gasotransmitters, nitric oxide (NO) (E. Culotta, D. E. Koshland, Science 1992, 258, 1862-1865) and carbon monoxide (CO) (T. Morita, M. A. Perrella, M. E. Lee, S. Kourembanas, P. Natl. Acad. Sci. U. S. A . 1995, 92, 1475-1479). Studies have indicated that H2S plays a regulatory role in the cardiovascular system (D. J. Lefer, P. Natl. Acad. Sci. U. S. A. 2007, 104, 17907-17908) by acting as a K-ATP channel opener (W. M. Zhao, J. Zhang, Y. J. Lu, R. Wang, EMBOJ. 2001, 20, 6008-6016). H2S also functions as a modulator in the central nervous system (K. Abe, H. Kimura , J. Neurosci. 1996, 16, 1066-1071; D. Boehning, S. H. Snyder, Annu. Rev. Neurosci. 2003, 26, 105-131; H. Kimura, Mol. Neurobiol. 2002, 26, 13-19), respiratory system, gastrointestinal system, and endocrine system (A. Martelli, et al, Med. Res. Rev. 201 1). These findings suggest that H2S exhibits many of the beneficial effects of NO without generating toxic reactive oxygen species (ROS). In fact, H2S acts as an anti-oxidant or scavenger of ROS. As a result, there has been a steady increase in the interest in understanding hydrogen sulfide's physiological and pathological functions (A. Martelli, et al, Med. Res. Rev. 201 1; P. K. Moore, M. Bhatia, S. Moochhala, Trends Pharmacol. Sci. 2003, 24, 609-61 1; R. Wang, FASEB J. 2002, 16, 1792-1798). Progress in studying the role of H2S in biological systems has been significantly limited by the lack of sensors and agents that allow for the rapid and accurate detection of H2S. The standard method for sulfide analysis is a colorimetric assay based on the reaction between sulfide and N,N-dimethyl- /?-phenylenediamine in the presence of Fe + and hydrochloric acid (W. Lei, P. K. Dasgupta, Anal. Chim. Acta 1989, 226, 165-170; M. N. Hughes, M. N. Centelles, K. P. Moore, Free Radical Bio.Med. 2009, 47, 1346-1353). As shown below, the reaction produces methylene blue, a common dye which possesses a characteristic maximum absor tion at 670 nm. In practice, the colorometric assay involves the addition of zinc acetate, N,N- dimethyl-/?-phenylenediamine dihydrochloride in 7.2 M HCI, and FeCl3 in 1.2 M HCI to an aqueous sample containing an unknown quantity of sulfide. After incubation for 10 to 30 minutes, trichloroacetic acid is added to the reaction mixture. The sample is then centrifuged, and the absorption of methylene blue in the supernatant is measured using a spectrophotometer. The quantity of sulfide in the sample is determined by comparing the absorbance of the supernatant to a standard calibration curve. While useful for some applications, this method of H2S detection possesses many intrinsic disadvantages. First, the assay requires a long reaction time (10-30 min), toxic reagents, and relatively harsh reaction conditions. In addition, the relationship between the absorption at 670 nm and the concentration of H2S is not linear due to the tendency for methylene blue to form dimers and trimers in solution (M. N. Hughes, M. N. Centelles, K. P. Moore, Free Radical Bio.Med. 2009, 47, 1346-1353). An alternative colorimetric assay was developed by Martinez-Manez and co-workers (D. Jimenez, R. Martinez-Manez, F. Sancenon, J. V. Ros- Lis, A. Benito, J. Soto, J. Am. Chem. Soc. 2003, 125, 9000-9001). This assay is based on a pyrylium-thiopyrylium transformation. Upon addition of sulfide in a water/acetonitrile (1:1) solution and further treatment with H2S0 4, a dye containing an aniline-pyrylium backbone is transformed into a dye containing an aniline-thiopyrylium backbone. This transformation is accompanied by a large color change from magenta to blue. While simple and easy to perform, the detection limit for this assay is only about 200 µΜ . For many applications, lower detection limits are required. For example, physiological concentrations of H2S are typically in the range of -10-100 µΜ (J.C. Savage, D.H. Gould, J. Chromatogr. Biomed. 1990, 526, 540-545; L.R. Goodwin, t al, J Anal. Toxicol. 1989, 13, 105- 109). As a result, the utility of this probe is limited. Sensitive electrochemical methods for the detection of H2S have also been reported. However, these assays require sophisticated instrumentation and long equilibration times, greatly limiting their potential applications (M. N. Hughes, M. N. Centelles, K. P. Moore, Free Radical Bio.Med. 2009, 47, 1346-1353). For example, H2S catabolism is known to be rapid, resulting in H2S concentrations that continuously fluctuate in vivo. As a result, assays which require substantial equilibration times cannot be used as biosensors to measure H2S levels. Therefore, there is a need for the development of new methods for the fast and selective detection of sulfide in biological systems. It is therefore an object of the invention to provide improved chemosensing agents for the detection and quantification of H2S in aqueous solution. It is also an object of the invention to provide chemosensing agents for the detection and quantification of H2S with lower detection limits and/or decreased equilibration time. It is also an object of the invention to provide chemosensing agents for the detection and quantification of H2S that do not require additional reagents, other than the chemosensing agent, to react with and/or stabilize H2S. It is a further object of the invention to provide methods of detecting sulfide in biological systems. SUMMARY OF THE INVENTION Chemosensing agents for the detection and/or quantification of H2S are provided. The chemosensing agents contain one or more reactive functional groups covalently connected to a fluorophore. The reactive functional groups can be any moieties which undergo a chemical reaction in the presence of H2S. The reactive functional groups and the fluorophore are covalently connected such that the reaction of the reactive function group with H2S elicits one or more spectroscopically observable changes in the fluorophore's photophysical properties.
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