Elsevier

Journal of Inorganic Biochemistry

Volume 118, January 2013, Pages 140-147
Journal of Inorganic Biochemistry

Water soluble acyloxy nitroso compounds: HNO release and reactions with heme and thiol containing proteins

https://doi.org/10.1016/j.jinorgbio.2012.07.023Get rights and content

Abstract

Nitroxyl (HNO) has gained interest as a potential treatment of congestive heart failure through the ability of the HNO donor, Angeli's salt (AS), to evoke positive inotropic effects in canine cardiac muscle. The release of nitrite during decomposition limits the use of AS requiring other HNO sources. Acyloxy nitroso compounds liberate HNO and small amounts of nitrite upon hydrolysis and the synthesis of the water-soluble 4-nitrosotetrahydro-2H-pyran-4-yl acetate and pivalate allows for pig liver esterase (PLE)-catalysis increasing the rate of decomposition and HNO release. The pivalate derivative does not release HNO, but the addition of PLE catalyzes hydrolysis (t1/2 = 39 min) and HNO formation (65% after 30 min). In the presence of PLE, this compound converts metmyoglobin (MetMb) to iron nitrosyl Mb and oxyMb to metMb indicating that these compounds only react with heme proteins as HNO donors. The pivalate in the presence and the absence of PLE inhibits aldehyde dehydrogenase (ALDH) with IC50 values of 3.5 and 3.3 μM, respectively, in a time-dependent manner. Reversibility assays reveal reversible inhibition of ALDH in the absence of PLE and partially irreversible inhibition with PLE. Liquid chromatography–mass spectrometry (LC–MS) reveals formation of a disulfide upon incubation of an ALDH peptide without PLE and a mixture of disulfide and sulfinamide in the presence of PLE. A dehydroalanine residue forms upon incubation of this peptide with excess AS. These results identify acyloxy nitroso compounds as unique HNO donors capable of thiol modification through direct electrophilic reaction or HNO release.

Graphical abstract

Water-soluble 4-nitrosotetrahydro-2H-pyran-4-yl acetate and pivalate were prepared and pig liver esterase catalysis increases the rate of decomposition and HNO release. In the presence of esterase, these compounds react with heme proteins as HNO donors and inhibit a thiol-containing enzyme through direct reaction or HNO release.

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Highlights

► Synthesis of water-soluble 4-nitrosotetrahydro-2H-pyran-4-yl acetate and pivalate ► Pig liver esterase catalyzes hydrolysis and HNO release of these compounds. ► Reaction converts metmyoglobin (Mb) to iron nitrosyl Mb and oxyMb to metMb. ► Reaction inhibits aldehyde dehydrogenase through thiol modification. ► Reaction of HNO with cysteine residue yields dehydro-alanine.

Introduction

Nitroxyl (HNO/NO), is the one electron reduced relative of nitric oxide (NO), an important biological signaling agent [1], [2], [3], [4], [5]. Nitroxyl has distinct biological properties compared to NO and is a potential treatment of myocardial reperfusion injury and congestive heart failure [6], [7], [8]. Nitroxyl avidly reacts with heme and thiol containing proteins and these reactions likely mediate its biological actions [8], [9], [10], [11]. Nitroxyl must be generated from donors due to its fast dimerization to nitrous oxide (N2O, k = 8 × 106 M 1 s 1) and a limited number of donors exist [12], [13], [14]. Angeli's salt (NaN2O3, AS) is the most widely used HNO donor, but at physiological pH AS forms an equivalent of nitrite during decomposition [15], Nitrite reacts with all of the redox forms of hemoglobin and possesses its own biological profile, which must be accounted for in biological evaluations of HNO activity [15], [16], [17], [18], [19]. Other limitations of AS include the fast rate of decomposition (k = 10 4 s 1) and difficulty in modifying the structure [14], [15]. The ability of AS to elicit positive inotropic effects in canine cardiac muscle highlights the need for new HNO donors as potential new therapies [7], [20].

Acyloxy nitroso compounds (13, Fig. 1) act as HNO donors through ester hydrolysis to give an unstable intermediate that decomposes to HNO without generation of nitrite in buffered conditions [21]. Varying the pH and the R group of the ester of these structures varies their stability, HNO donor activity and ability to relax pre-constricted rat aorta [22]. Compounds 1 and 2 only slowly hydrolyze and competitively react with other nucleophiles (thiolates) without HNO formation, but 3 hydrolyzes to HNO under all conditions [22]. While compounds 13 provide information concerning the reactivity and HNO release of acyloxy nitroso compounds, their lack of water solubility limits their use in biological systems. Incorporating oxygen into the ring of 1 and 2 gives 4 and 5, and should greatly increase water solubility allowing for esterase mediated hydrolysis to increase the rate of decomposition (Fig. 1) [23].1

The increased water solubility of 45 and their structural similarity to C-nitroso compounds, which also react with thiols [24], allow evaluation with myoglobin (Mb), a heme protein, and aldehyde dehydrogenase (ALDH), a thiol containing protein, known HNO targets in the presence of pig liver esterase (PLE) to enhance HNO release [11], [25], [26]. Our results show that acyloxy nitroso compounds can act as efficient HNO donors in the presence of PLE, which may be useful in further probing HNO biology.

Section snippets

General

All chemicals were purchased from Sigma Aldrich Chemical Company and used as received. AS and 15N-AS were prepared as described, and stored dry at − 20 °C [27]. Analytical TLC was performed on silica gel plates with C-4 Spectroline 254 indicator. Visualization was accomplished with UV light. Solvents for extraction and purification were of technical grade and used as received. Liquid chromatography–mass spectrometry (LC–MS) solvents were HPLC grade. 1H NMR and 13C NMR spectra were recorded using

Synthesis

Substitution of the cyclohexane ring of 12 with an oxygen-containing tetrahydropyran ring gives new acyloxy nitroso compounds (45) that can be prepared using previously reported methodology (Fig. 2) [21], [22]. Condensation of tetrahydropyran-4-one with hydroxylamine hydrochloride results in dihydro-2H-pyran-4(3H)-one oxime and lead (IV) tetraacetate oxidation in the absence and presence of 5 equivalents of 2, 2-dimethylpropanoic acid gives 4-nitrosotetrahydro-2H-pyran-4-yl acetate and

Conclusions

In summary, replacement of a methylene group with an oxygen atom (compare compounds 2 and 5) gives new acyloxy nitroso compounds (4 and 5) that demonstrate improved water solubility. The acetate derivative (4) slowly hydrolyzes to HNO but the pivalate (5) does not hydrolyze or release HNO under buffered conditions. Addition of pig liver esterase (PLE) catalyzes hydrolysis of 5 and increases HNO release, making this compound a unique esterase-mediated HNO donor that produces minimal amounts of

Acknowledgments

This work was supported by the NIH (HL 062198, SBK) as well as Cardioxyl Pharmaceuticals (SBK). The Bruker NMR spectrometers used in this work were purchased with partial support from the NSF (CHE-9708077) and North Carolina Biotechnology Center (9703-IDG-1007).

JFD and SBK kindly thank Dr. Susan Mitroka (Wake Forest University) for helpful discussion.

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