A chemical moiety is disclosed which comprises a chemical, biochemical, or biological substance attached to one or more electrochemiluminescent organometallic compounds. In a preferred embodiment of the invention the substance is attached to one or more ruthenium-containing or osmium-containing luminescent organometallic compounds. Methods are disclosed for detecting very small amounts of the chemical moiety using chemiluminescent, electrochemiluminescent, and photoluminescent means. Compounds are disclosed which are useful for labelling substances of interest with ruthenium-containing and osmium-containing labels or other electrochemiluminescent labels. These labelled substances are useful in methods provided for detecting and quantifying analytes of interest in binding assays and competitive binding assays. The labelled substances are of particular use in homogeneous binding assays. These methods form the bases for systems designed to enable the rapid, efficient, and sensitive determination of a broad array of chemical, biochemical, and biological materials of interest.

Luminescent metal chelate labels and means for detection

Self-replication is a fundamental concept. The idea of an entity that can repeatedly create more of itself has captured the imagination of many thinkers from von Neumann to Vonnegut. Beyond the sciences and science fiction, autocatalysis has found currency in economics and language theory, and has raised ethical fears memorably summed up by the “gray goo” trope. Autocatalysis is central to the propagation of life and intrinsic to many other biological processes. This includes the modern conception of evolution, which has radically altered humanity’s image of itself. Organisms can be thought of as imperfect self-replicators which produce closely-related species, allowing for selection and evolution. Hence, any consideration of self-replication raises one of the most profound questions of all: what is life? Minimal self-replicating systems have been studied with the aim of understanding the principles underlying living systems, allowing us to refine our concepts of biological fitness and chemical stability, self-organization and emergence, and ultimately to discover how chemistry may become biology.

Mechanisms of autocatalysis.

New features in the redox coordination chemistry of metal nitrosyls {M-NO+ ; M-NO•; M-NO- (HNO)}

The ubiquitous cellular influence of nitric oxide (NO) is exerted substantially through protein S-nitrosylation. Whereas NO is highly promiscuous, physiological S-nitrosylation is typically restricted to one or very few Cys residue(s) in target proteins. The molecular basis for this specificity may derive from properties of the target protein, the S-nitrosylating species, or both. Here, we describe a protein microarray-based approach to investigate determinants of S-nitrosylation by biologically relevant low-mass S-nitrosothiols (SNOs). We identify large sets of yeast and human target proteins, among which those with active-site Cys thiols residing at N termini of α-helices or within catalytic loops were particularly prominent. However, S-nitrosylation varied substantially even within these families of proteins (e.g., papain-related Cys-dependent hydrolases and rhodanese/Cdc25 phosphatases), suggesting that neither secondary structure nor intrinsic nucleophilicity of Cys thiols was sufficient to explain specificity. Further analyses revealed a substantial influence of NO-donor stereochemistry and structure on efficiency of S-nitrosylation as well as an unanticipated and important role for allosteric effectors. Thus, high-throughput screening and unbiased proteome coverage reveal multifactorial determinants of S-nitrosylation (which may be overlooked in alternative proteomic analyses), and support the idea that target specificity can be achieved through rational design of S-nitrosothiols.

https://www.pnas.org/content/pnas/106/45/18948.full.pdf

A protein microarray-based analysis of S-nitrosylation

Synthesis, vibrational spectra and X-ray structures of copper(I) thiourea complexes

Hydroxylamine is oxidised by nitric acid to form dinitrogen monoxide and nitrous acid, the proportions varying with reaction conditions. The yield [HNO2]∞/[NH3OH+]0 is a maximum at ca. 4–5 mol dm–3 HNO3, and is also a function of the hydroxylamine concentration. In 5 mol dm–3 HNO3 the limiting yield is ca. 0.85 at very low initial hydroxylamine concentrations, but decreases towards zero at higher values of [NH3OH+]0. Reaction is only observed at sufficiently high nitric acid concentrations; at 25°C the cut-off point is ca. 2.5 mol dm–3 HNO3. The reaction is characterised by an induction period, followed by a rapid autocatalytic process. Addition of nitrite eliminates the induction period, while addition of nitrite scavengers completely prevents any reaction. Nitrous acid is an essential catalyst for the reaction, and the initial rate of reaction obeys the equation d[HNO2]/dt=V0=K[HNO2][NH3OH+]. Isotopic experiments, using 15N-enriched hydroxylamine show that virtually all of the N2O arises from reaction between HNO2 and hydroxylamine. The mechanism suggested involves oxidation of unprotonated hydroxylamine by N2O4 to form the nitroxyl diradical HNO; this is then further oxidised to HNO2, which reacts with hydroxylamine to form N2O.

Kinetics, mechanism, and stoicheiometry of the oxidation of hydroxylamine by nitric acid

Thiourea reacts rapidly with nitrous acid in aqueous solution to form an equilibrium concentration of a coloured S-nitroso-compound. The equilibrium constant has been measured, together with a preliminary value of the rate constant for the nitrosation. The kinetics of the much slower reaction to form nitrogen and thiocyanic acid have been investigated. The mechanism proposed is a rate-determining sulphur-to-nitrogen migration of the nitroso-group in the conjugate base of the S-nitroso-compound.

Electrophilic nitrosation at sulphur and nitrogen in thiourea

The kinetics of the decomposition of formamidine disulfide dihydrochloride, (NH2)2CSSC(NH2)22+ 2Cl–, in aqueous solution at 25 °C are general base catalysed, and rate constants have been measured over the pH range 1.91–9.03. Analysis of the data yields ionisation constants pK1= 5.49, pK2= 7.66 and rate constants for deprotonation of the substrate by OH– and H2O. For the doubly charged cation, reaction occurs through a small concentration of a reactive tautomer, (HN)(NH3+)CSSC+(NH2)2, which is attacked by H2O. The singly charged cation reacts by parallel pathways involving OH– and H2O as attacking bases.

Kinetic study of the stability of (NH2)2CSSC(NH2)22

The reaction between nitrous acid and hydrazine is a normal N-nitrosation reaction, and does not involve oxidation at NH2NH2 to N2H2 as previously postulated. The N-nitrosohydrazine decomposes by two parallel routes, to HN3 at high acidities and to ammonia and N2O at low acidities. An intermediate in the decomposition has been detected spectrophotometrically. Revised values are provided for the relative reactivity of some nucleophiles to nitrosating agents, and the relation between reactivity and charge in nitrosation reactions is discussed.

Kinetic and product study of the reaction between nitrous acid and hydrazine

The profile of rate constant versus[H+] for the acid catalysed pathway of the nitrous acid reaction with the hydroxyammonium ion can be qualitatively interpreted in terms of a reversible O-nitrosation, followed by an O to N migration of the nitroso group in the conjugate base species NH2ONO. There is no need to assume a non-steady-state mechanism involving NO+, as has been proposed previously.

Geoffrey Stedman

Papers

Kinetics, mechanism, and stoicheiometry of the oxidation of hydroxylamine by nitric acid

Electrophilic nitrosation at sulphur and nitrogen in thiourea

Kinetic study of the stability of (NH2)2CSSC(NH2)22

Kinetic and product study of the reaction between nitrous acid and hydrazine

Mechanism of the acid catalysed pathway for the nitrosation of hydroxylamine