Detection of N‐nitrosomelatonin and other N‐nitrosotryptophan derivatives by transnitrosation of APF and DAF‐2

Abstract

S-nitrosothiols can be analyzed with some simple detection procedures and this fact strongly accelerated the understanding of the biological impact of S-nitrosothiols. Unfortunately, such simple analytic methods are presently missing for low molecular weight N-nitrosotryptophan derivatives like N-nitrosomelatonin (NOMela). Here we demonstrate that commercially available primary aromatic amines, i.e. aminophenylfluorescein (APF) and 4,5-diaminofluorescin (DAF-2), can be used for a quantitative determination of NOMela. Under optimized conditions (e.g. pH 11) of the assays, the lifetime of N-nitrosotryptophan derivatives is largely prolonged and the reactivity of S-nitrosothiols with aromatic amines can be safely ignored. The influence of reactive nitrogen oxide species like N2O3 is additionally limited at the alkaline pH and may be further decreased by working under hypoxic conditions. As a result of these optimal conditions, the APF assay has a detection limit for NOMela of about 25 nm but this assay fails to detect protein-bound N-nitrosotryptophan residues. The DAF-2 assay, however, might be used for a qualitative analysis of such residues. Due to the high efficacy of the APF assay it is safely demonstrated that in regard to peroxynitrite, N2O3 is about 50-fold more effective in nitrosating melatonin at physiological pH.

The Reaction of Peroxynitrite with Morpholine (Secondary Amines) Revisited: The Overlooked Hydroxylamine Formation

“…Significantly lower yields (ca. 1 -2%) have been observed for the peroxynitrite-dependent nitrosation of thiols [17 -19] and of indole derivatives [20] [21].…”

N‐Nitrosomelatonin: synthesis, chemical properties, potential prodrug

“…The second compound able to nitrosate N ‐terminal blocked tryptophan derivatives at physiological pH is peroxynitrite [13, 16, 18, 19]. Recently, we [20] confirmed a hypothesis of Williams [21] that the peroxynitrite‐induced nitrosation of various compounds proceeds via N 2 O 4 , which is somewhat less effective as electrophilic, amine‐nitrosating agent than N 2 O 3 [22].…”

“…35% and 65%, respectively), as proven by N‐15 nuclear magnetic resonance (NMR) spectrometry [6, 10–12]. The detailed protocol for the preparation of NOMela is given in the Journal of Pineal Research [11, 13]. As the procedure was still further improved in order to optimize the yield, we briefly describe these modifications in the following: the preparation is now completely performed in a cold (4°C) room and the reaction vessel is with the aid of an aluminium foil permanently protected against incident light.…”

“…In its absence NOMela can directly nitrosate (‘transnitrosation’, i.e. without the occurrence of a nitrosating intermediate like N 2 O 3 ) a variety of nucleophiles (Y − /YH), such as water [32], activated phenols [33, 34], catechols [35], vitamin C [6], vitamin E [36], thiols [12] and primary aromatic amines [13] (but not aliphatic secondary ones [6]) at physiological pH to yield the parent melatonin and a nitroso compound (YNO). The simplest transnitrosation, i.e.…”

“…Thus, these aromatic amines can be used as reporter molecules for NOMela [13]. Although the fluorescent dyes are produced in relatively low yields (55% and 13% in the case of DAF‐2 and APF, respectively), the detection limits drop to 100 n m (DAF‐2) and 25 n m (APF), respectively.…”