Hydrogen Sulfide and Nitroprusside React Directly to Give Nitroxyl (HNO)

SUPPORTING MATERIAL

Beyond H 2S and NO interplay: hydrogen sulfide and nitroprusside react directly to give nitroxyl (HNO). A new pharmacological source of HNO

Milos R. Filipovic, 1* Mirjam Eberhardt, 1,2 Vladimir Prokopovic, 1 Ana Mijuskovic, 3 Zorana Orescanin-Dusic, 3 Peter Reeh, 2 Ivana Ivanovic-Burmazovic 1*

1Department of Chemistry and Pharmacy and 2Department of Physiology and Pathophysiology, Univeristy of Erlangen-Nuremberg, Erlangen, Germany; 3Institute for Biological Research Sinisa Stankovic, University of Belgrade, Belgrade, Serbia

Keywords: nitroprusside, hydrogen sulfide, nitroxyl, CGRP, nitric oxide.

*Correspondence should be addressed to: Milos R. Filipovic, PhD. or IvanaIvanovic-Burmazovic, PhD. Department of Chemistry and Pharmacy, University of Erlangen-Nuremberg, Egerlandstrasse 1, 91058 Erlangen, Germany, Fax: 49-9131-85-27387; E-mail: [email protected] or [email protected] erlangen.de

1 Table of Contents:

Figure S1. Color changes of SNP solution (300 mM KPi, pH 7.4) upon addition of H 2S, cysteine and gluathione.

Figure S2. Ultra-high resolution cryo-spray (4 °C) ESI-TOF-mass spectrometry in negative mode of SNP (A) and reaction mixture (B).

Figure S3 . GC-MS detection of N 2O generation from SNP and H 2S reaction mixture (1:5).

Figure S4. Representative traces of H 2S electrode responses upon addition of 50 µM Na 2S into buffered solution containing increasing concentrations of SNP (0-10 mM).

3- Figure S5 . The spectrum of the [Fe(H 2O)(CN) 5] before (black) and after the addition of the excess of potassium thiocyanate.

Figure S6. GC-MS analysis of the thiocyanate coupled to pentafluorobenzyl bromide in extractive alkylation proces.

3- Figure S7 . The spectrum of [Fe(CN) 5HNO] (black) generated by total reduction of SNP with two equivalents of dithionite at pH 7.4.

Figure S1. Color changes of SNP solution (300 mM KPi, pH 7.4) upon addition of H2S, cysteine and gluathione. Upper left, before addition; upper right, immediately after addition; lower left, 5 min after additon; lower right, 15 min after addition.

Figure S2. Ultra-high resolution cryo-spray (4 °C) ESI-TOF-mass spectrometry in negative mode of SNP (A) and reaction mixture (B). Reaction mixture containing 1mM SNP and 5 mM H 2S in 50 mM KPi pH 7.4 shows only small traces of starting compound (the peak intesity is more than 250 times lower than that of 1 mM SNP). Isotopic - distribution of the observed peak (C) at 238.9 assigned to Na[Fe(CN) 5NO] and simulation of isotopic distribution for the same molecule (D).

Figure S3. GC-MS detection of N 2O generation from SNP and H 2S reaction mixture (1:5). 2 mM SNP in 50 mM KPi pH 7.4 was degassed with argon and kept in dark glass vials sealed with PTFE septa. Na 2S was added to yield a final concentration of 10 mM. GC-MS analyses were performed on a Bruker GC 450 TQ MS 300. The gas chromatograph was equipped with capillary column Varian, VF-5m. 50 µL of headspace gas samples were injected in the splitless mode. The following oven temperature program was used with helium as the carrier gas: ramp from 50 to 155 °C at a rate of 10 °C/min and then to 260 °C at a rate of 30 °C/min. Positive electron impact ionization mode was used. Detector multiplier voltage was set to 1400 V and the detection performed by selected ion monitoring of m/z 44 (N 2O) and m/z 30 (NO) using a dwell time of 50 ms and scan width for SIM of 0.7 a.u. Areas under the peaks were determined using the software provided by the manufacturer. Inset represents the characteristic MS spectrum of the observed peak.

Figure S4. Representative traces of H 2S electrode responses upon addition of 50 µM Na 2S into buffered solution containing increasing concentrations of SNP (0-10 mM).

3- Figure S5. The spectrum of the [Fe(H 2O)(CN) 5] before (black) and after the addition of the excess of potassium thiocyanate.

Figure S6. GC-MS analysis of the thiocyanate coupled to pentafluorobenzyl bromide in extractive alkylation proces. 1 mM SNP was mixed with 2 mM Na 2S in phosphate buffer pH 7.4 and after 5 min subjected to extractive alkylation following the protocol given in the Material and Methods part. A) Total GC-MS obtained for selected ion mode detection (m/z 239, 181, 161 for the pentafluorobenzyl thiocyanate product, and m/z 250, 169 for the internal standard, dibrometholuene). Inset represents the individual chromatograms of these ions, with 239, 181, 161 appearing at 7.3 min and 250 and 169 appearing at 7.7 min. B) MS spectrum of the peak at 7.3 min. C) MS spectrum of the peak at 7.7 min.

3- Figure S7. The spectrum of [Fe(CN) 5HNO] (black) generated by total reduction of SNP with two equivalents of dithionite at pH 7.4 (Montenegro, A.C.; Amorebieta, V.T.; Slep, L.D.; Martín, D.F; Roncaroli, F.; Murgida, D.H.; Bari, S.E.; Olabe, J.A. Three redox states of nitrosyl: NO+, NO., and NO-/HNO interconvert reversibly on the same pentacyanoferrate(II) platform. Angew. Chem. 2009, 48, 4213-4216). Addition of the excess of sodium sulfide (red) does not change the spectral properties, while the addition of sodium polysulfide gives immediate blue coloration with absorption maximum at ~570 nm (green).

Figure S8. Schematic representation of the experimental setup for CGRP release from isolated heart as described in Supplementary Material and Methods.