Mechanism-based triarylphosphine-ester probes for capture of endogenous RSNOs - PubMed (original) (raw)

. 2013 May 22;135(20):7693-704.

doi: 10.1021/ja401565w. Epub 2013 May 8.

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Mechanism-based triarylphosphine-ester probes for capture of endogenous RSNOs

Uthpala Seneviratne et al. J Am Chem Soc. 2013.

Abstract

Nitrosothiols (RSNOs) have been proposed as important intermediates in nitric oxide (NO(•)) metabolism, storage, and transport as well as mediators in numerous NO-signaling pathways. RSNO levels are finely regulated, and dysregulation is associated with the etiology of several pathologies. Current methods for RSNO quantification depend on indirect assays that limit their overall specificity and reliability. Recent developments of phosphine-based chemical probes constitute a promising approach for the direct detection of RSNOs. We report here results from a detailed mechanistic and kinetic study for trapping RSNOs by three distinct phosphine probes, including structural identification of novel intermediates and stability studies under physiological conditions. We further show that a triarylphosphine-thiophenyl ester can be used in the absolute quantification of endogenous GSNO in several cancer cell lines, while retaining the elements of the SNO functional group, using an LC-MS-based assay. Finally, we demonstrate that a common product ion (m/z = 309.0), derived from phosphine-RSNO adducts, can be used for the detection of other low-molecular weight nitrosothiols (LMW-RSNOs) in biological samples. Collectively, these findings establish a platform for the phosphine ligation-based, specific and direct detection of RSNOs in biological samples, a powerful tool for expanding the knowledge of the biology and chemistry of NO(•)-mediated phenomena.

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Figures

Figure 1

Figure 1

Structures of _S_-nitrosoglutathione (GSNO) and triarylphosphine probes used in this study.

Figure 2

Figure 2

Reaction between GSNO and phosphine probe 1 (A, B) and 2 (C, D) (in excess, in Tris-HCl, pH 7.4), analyzed by LC–TOF-MS. Upper panel (A and C): time-dependent LC of the reaction intermediates and products (at 254 nm); bottom panel (B and D): time-dependent high-resolution ESI+-MS (EIC; extracted ion chromatogram). Colors: 23 min = black, 45 min = blue, 67 min = green, 89 min = purple, 111 min = yellow, 133 min = pink. Peaks corresponding to 1, 2, 4a, and 4b were excluded on LC–MS trace for clarity. GSNO, 5 μM and 50 μM with phosphine probe (20-fold excess) were used for the LC–MS and LC–UV studies, respectively, (*) indicates the hydrolyzed 2 under our experimental conditions. Monitored/expected masses, 5; m/z = 627.1678/627.1673 [M + H]+, 6a; m/z = 750.1376/750.1373 [M + H]+, 6b; m/z = 734.1602/734.1607 [M + H]+, 7; m/z = 322.0995/322.0991 [M + H]+, 8; m/z = 444.1029/444.1029 [M + H]+. _Y_-axis represents the relative intensity.

Scheme 1

Scheme 1. Reaction Pathways Supported by 31P NMR and High-Resolution LC–MS Analysis of Intermediates and Products When GSNO Reacts with (A) Probe 1, (B) Probe 2, and (C) Probe 3 in Buffer Conditions at Physiological pH

Figure 3

Figure 3

Capturing GSNO as disulfide-iminophosphorane (9) by probe 3. Time- and concentration-dependent TOF-MS analysis of the reaction mixture comprising GSNO (5 μM) with probe 3 (50 μM to 1 mM) in phosphate buffer (pH 5.7) at 24 °C. Colors: 2 min = black, 12 min = red, 22 min = blue, 32 min = green. When the concentration of probe 3 (1 mM) is in 200-fold molar excess, the formation of 9 occurs within 15 min with <10% of the phosphoryl-disulfide 10. Peaks corresponding to 3 and 4c were excluded on EIC for clarity. Monitored/expected masses, GSNO: m/z = 337.0810/337.0812/[M + H]+; 9: m/z = 350.6044/350.6048 [M + 2H]2+; 10: m/z = 616.1334/616.1336 [M + H]+. Phosphate buffer at pH 5.7 was used to increase ionization and to aid in detection of GSNO. _Y_-axis represents the relative intensity.

Scheme 2

Scheme 2. 15N Fragments Derived from the Reaction of Probe 3 and GS15NO upon Collision Induced Dissociation (CID) in MS and Possible Resonance Stabilization of m/z = 309.0

Figure 4

Figure 4

Disulfide-iminophosphorane 9 is formed in activated macrophage lysate (106 cells) upon treatment with probe 3. (A) LC–MS (TIC; total ion chromatogram) of cell lysate. (B) LC–MS/MS (MRM) of DTT-treated cell lysate (negative control). (C) MRM of cell lysate without treatment with probe 3, analyte corresponds to GSNO. (D) m/z = 350.6 → 309.0, (G) m/z = 350.6 → 487.1, (H) m/z = 350.6 → 571.1, and (E) internal standard (m/z = 352.1 → 309.0) in cell extracts. (F) Product ion spectra of 9, m/z = 350.6 [M + 2H]2+, derived from cell lysate (top), authentic standard (middle), and 15N fragment, m/z = 351.1 [M + 2H]2+, derived from the reaction of probe 3 and GS15NO (bottom). _Y_-axis represents the relative intensity.

Scheme 3

Scheme 3. Proposed Kinetic Model for Capturing GSNO by Probe 3

Figure 5

Figure 5

(Left) Low-molecular weight nitrosothiols (LMW-RSNOs) that produce common product ion, m/z = 309.0, upon CID. (Right) Capturing LMW-RSNOs, by LC–MS/MS using the common product ion m/z 309.0 in cell lysates: (A) _S_-nitrosocysteine (9a) m/z = 309.0 → 257.6. (B) _S-_nitrosated _N_-acetyl-penicillamine (9h) m/z = 309.0 → 292.6. _Y_-axis represents the relative intensity.

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