RESUMO
Homocubane, a highly strained cage hydrocarbon, contains two very different positions for the introduction of a nitrogen atom into the skeleton, e. g., a position 1 exchange results in a tertiary amine whereas position 9 yields a secondary amine. Herein reported is the synthesis of 9-azahomocubane along with associated structural characterization, physical property analysis and chemical reactivity. Not only is 9-azahomocubane readily synthesized, and found to be stable as predicted, the basicity of the secondary amine was observed to be significantly lower than the structurally related azabicyclo[2.2.1]heptane, although similar to 1-azahomocubane.
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In recent years there has been a significant interest in the development of innovative lipidomics techniques capable of resolving lipid isomers. To date, methods applied to resolving sn-isomers have resolved only a limited number of species. We report a workflow based on ozone-induced dissociation for untargeted characterisation of hundreds of sn-resolved glycerophospholipid isomers from biological extracts in under 20â min, coupled with an automated data analysis pipeline. It provides an order of magnitude increase in the number of sn-isomer pairs identified as compared to previous reports and reveals that sn-isomer populations are tightly regulated and significantly different between cell lines. The sensitivity of this method and potential for de novo molecular discovery is further demonstrated by the identification of unexpected lipids containing ultra-long monounsaturated acyl chains at the sn-1 position.
Assuntos
Lipidômica , Ozônio , Isomerismo , Linhagem CelularRESUMO
Many families of lipid isomers remain unresolved by contemporary liquid chromatography-mass spectrometry approaches, leading to a significant underestimation of the structural diversity within the lipidome. While ion mobility coupled to mass spectrometry has provided an additional dimension of lipid isomer resolution, some isomers require a resolving power beyond the capabilities of conventional platforms. Here, we present the application of high-resolution traveling-wave ion mobility for the separation of lipid isomers that differ in (i) the location of a single carbon-carbon double bond, (ii) the stereochemistry of the double bond (cis or trans), or, for glycerolipids, (iii) the relative substitution of acyl chains on the glycerol backbone (sn-position). Collisional activation following mobility separation allowed identification of the carbon-carbon double-bond position and sn-position, enabling confident interpretation of variations in mobility peak abundance. To demonstrate the applicability of this method, double-bond and sn-position isomers of an abundant phosphatidylcholine composition were resolved in extracts from a prostate cancer cell line and identified by comparison to pure isomer reference standards, revealing the presence of up to six isomers. These findings suggest that ultrahigh-resolution ion mobility has broad potential for isomer-resolved lipidomics and is attractive to consider for future integration with other modes of ion activation, thereby bringing together advanced orthogonal separations and structure elucidation to provide a more complete picture of the lipidome.
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Carbono , Fosfatidilcolinas , Isomerismo , Espectrometria de Massas/métodos , Fosfatidilcolinas/análise , Cromatografia LíquidaRESUMO
Coordination cages can be used for enantio- and regioselective catalysis and for the selective sensing and separation of isomeric guest molecules. Here, stereoisomers of a family of coordination cages are resolved using ultra-high-resolution cyclic ion-mobility mass spectrometry (cIM-MS). The observed ratio of diastereomers is dependent on both the metal ion and counter ion. Moreover, the point groups can be assigned through complementary NMR experiments. This method enables the identification and interrogation of the individual isomers in complex mixtures of cages which cannot be performed in solution. Furthermore, these techniques allow the stability of individual isomers within the mixture to be probed, with the T-symmetric isomers in this case shown to be more robust than the C3 and S4 analogues.
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Gas-phase ion-molecule reactions provide structural insights across a range of analytical applications. A hindrance to the wider use of ion-molecule reactions is that they are relatively slow compared to other ion activation modalities and can thereby impose a bottleneck on the time required to analyze each sample. Here we describe a method for accelerating the rate of ion-molecule reactions involving ozone, implemented by supplementary RF-activation of mass-selected ions within a linear ion trap. Reaction rate accelerations between 15-fold (for ozonolysis of alkenes in ionised lipids) and 90-fold (for ozonation of halide anions) are observed compared to thermal conditions. These enhanced reaction rates with ozone increase sample throughput, aligning the reaction time with the overall duty cycle of the mass spectrometer. We demonstrate that the acceleration is due to the supplementary RF-activation surmounting the activation barrier energy of the entrance channel of the ion-molecule reaction. This rate acceleration is subsequently shown to aid identification of new, low abundance lipid isomers and enables an equivalent increase in the number of lipid species that can be analyzed.
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Ozônio , Ânions/química , Íons , Isomerismo , Espectrometria de Massas/métodos , Ozônio/químicaRESUMO
Coordination cages with well-defined cavities show great promise in the field of catalysis on account of their unique combination of molecular confinement effects and transition-metal redox chemistry. Here, three coordination cages are reduced from their native 16+ oxidation state to the 2+ state in the gas phase without observable structural degradation. Using this method, the reaction rate constants for each reduction step were determined, with no noticeable differences arising following either the incorporation of a C60 -fullerene guest or alteration of the cage chemical structure. The reactivity of highly reduced cage species toward molecular oxygen is "switched-on" after a threshold number of reduction steps, which is influenced by guest molecules and the structure of cage components. These new experimental approaches provide a unique window to explore the chemistry of highly-reduced cage species that can be modulated by altering their structures and encapsulated guest species.
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The nitrile imine-mediated tetrazole-ene cycloaddition is a widely used class of photoligation. Optimizing the reaction outcome requires detailed knowledge of the tetrazole photoactivation profile, which can only partially be ascertained from absorption spectroscopy, or otherwise involves laborious reaction monitoring in solution. Photodissociation action spectroscopy (PDAS) combines the advantages of optical spectroscopy and mass spectrometry in that only absorption events resulting in a mass change are recorded, thus revealing the desired wavelength dependence of product formation. Moreover, the sensitivity and selectivity afforded by the mass spectrometer enable reliable assessment of the photodissociation profile even on small amounts of crude material, thus accelerating the design and synthesis of next-generation substrates. Using this workflow, we demonstrate that the photodissociation onset for nitrile imine formation is red-shifted by ca. 50 nm with a novel N-ethylcarbazole derivative relative to a phenyl-substituted archetype. Benchmarked against solution-phase tunable laser experiments and supported by quantum chemical calculations, these discoveries demonstrate that PDAS is a powerful tool for rapidly screening the efficacy of new substrates in the quest toward efficient visible light-triggered ligation for biological applications.
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Iminas , Lasers , Reação de Cicloadição , Espectrometria de Massas , Análise EspectralRESUMO
Ultraviolet-photodissociation (UVPD) mass spectrometry is an emerging analytical tool for structural elucidation of biomolecules including lipids. Gas phase UVPD of ionised fatty acids (FAs) can promote fragmentation that is diagnostic for molecular structure including the regiochemistry of carbon-carbon double bonds and methyl branching position(s). Typically, however, lipids exhibit poor conversion to photoproducts under UVPD and thus require longer integration times to achieve the signal-to-noise required for structural assignments. Consequently, the integration of UVPD into liquid-chromatography mass spectrometry (LC-MS) workflows for FAs has been limited. To enhance photofragmentation efficiency, an alternative strategy has been devised using wet-chemical derivatization of FAs to explicitly incorporate photolabile groups. FA derivatives that include an aryl-iodide motif have photodissociation conversions of up to 28% when activated by a single 266 nm photon. The radical-directed dissociation product ions resulting from UVPD of these derivatives provide key details of molecular structure and discriminate between lipid isomers. Herein, we describe the structure-activity guided development of new FA derivatives capable of photoproduct yields of up to 97%. UVPD-action spectroscopy demonstrates that photodissociation for FAs derivatized with N-(2-aminoethyl)-4-iodobenzamide (NIBA) is maximised near 266 nm and highlights the key role of the 4-iodobenzamide motif in the efficient formation of [M - I]Ë+ radical cations (and diagnostic secondary product ions). The high photodissociation yield of NIBA-derivatized lipids is maintained across 37 commonly observed FAs with the resulting UVPD mass spectra shown to be effective in the discrimination of isomeric FAs that differ in the position(s) of carbon-carbon double bonds. Integration of this strategy with reversed-phase LC-MS workflows is confirmed with high-quality UVPD mass spectra acquired across each chromatographic peak.
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Ácidos Graxos , Raios Ultravioleta , Cromatografia Líquida , Indicadores e Reagentes , Íons , Espectrometria de MassasRESUMO
RATIONALE: Eicosanoids are short-lived bio-responsive lipids produced locally from oxidation of polyunsaturated fatty acids (FAs) via a cascade of enzymatic or free radical reactions. Alterations in the composition and concentration of eicosanoids are indicative of inflammation responses and there is strong interest in developing analytical methods for the sensitive and selective detection of these lipids in biological mixtures. Most eicosanoids are hydroxy FAs (HFAs), which present a particular analytical challenge due to the presence of regioisomers arising from differing locations of hydroxylation and unsaturation within their structures. METHODS: In this study, the recently developed derivatization reagent 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+ ) was applied to a representative set of HFAs including bioactive eicosanoids. Photodissociation (PD) mass spectra obtained at 266 nm of 4-I-AMPP+ -modified HFAs exhibit abundant product ions arising from photolysis of the aryl-iodide bond within the derivative with subsequent migration of the radical to the hydroxyl group promoting fragmentation of the FA chain and facilitating structural assignment. RESULTS: Representative polyunsaturated HFAs (from the hydroxyeicosatetraenoic acid and hydroxyeicosapentaenoic acid families) were derivatized with 4-I-AMPP+ and subjected to a reversed-phase liquid chromatography workflow that afforded chromatographic resolution of isomers in conjunction with structurally diagnostic PD mass spectra. CONCLUSIONS: PD of these complex HFAs was found to be sensitive to the locations of hydroxyl groups and carbon-carbon double bonds, which are structural properties strongly associated with the biosynthetic origins of these lipid mediators.
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Despite the impacts - both positive and negative - of atmospheric ozone for life on Earth, there remain significant gaps in our knowledge of the products, mechanisms and rates of some of its most fundamental gas phase reactions. This incomplete understanding is largely due to the experimental challenges involved in the study of gas-phase reactions of ozone and, in particular, the identification of short-lived reaction intermediates. Here we report direct observation of the stepwise reaction of the halide anions iodide (I-) and bromide (Br-) with ozone to produce XO3- (where X = I and Br, respectively). These results substantially revise the rate constant for the I- + O3 reaction to 1.1 (± 0.5) × 10-12 cm3 molecule-1 s-1 (0.13% efficiency) and the Br- + O3 reaction to 6.2 (± 0.4) × 10-15 cm3 molecule-1 s-1 (0.001% efficiency). Exploiting five-orders of temporal dynamic range on a linear ion trap mass spectrometer enabled explicit measurement of the rate constants for the highly efficient intermediate, XO- + O3 and XO2- + O3, reactions thus confirming a stepwise addition of three oxygen atoms (i.e., X- + 3O3 â XO3- + 3O2) with the first addition representing the rate determining step. Evidence is also presented for (i) slow reverse reactions of XO- and XO2-, but not XO3-, with molecular oxygen and (ii) the photodissociation of IO-, IO2- and IO3- to release I-. Collectively, these results suggest relatively short lifetimes for Br- and I- in the tropospere with direct gas-phase oxidation by ozone playing a role in both the formation of atmospheric halogen oxides and, conversely, in the ozone depletion associated with springtime polar bromine explosion events.
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Enzymes are central components of most physiological processes, and are consequently implicated in various pathologies. High-resolution maps of enzyme activity within tissues therefore represent powerful tools for elucidating enzymatic functions in health and disease. Here, we present a novel mass spectrometry imaging (MSI) method for assaying the spatial distribution of enzymatic activity directly from tissue. MSI analysis of tissue sections exposed to phospholipid substrates produced high-resolution maps of phospholipase activity and specificity, which could subsequently be compared to histological images of the same section. Functional MSI thus represents a new and generalisable method for imaging biological activity inâ situ.
Assuntos
Imagem Molecular , Fosfolipases A2/metabolismo , Animais , Espectrometria de Massas , Naja naja , Fosfolipases A2/química , Venenos de Serpentes/enzimologia , Especificidade da EspécieRESUMO
Fatty acids are a structurally diverse category of lipids with a myriad of biochemical functions, which includes their role as building blocks of more complex lipids (e.g., glycerophospholipids and triacylglycerols). Increasingly, the analysis of fatty acids is undertaken using liquid chromatography-mass spectrometry (LC-MS), due to its versatility in the detection of lipids across a wide range of concentrations and diversity of molecular structures and masses. Previous work has shown that fixed-charge pyridinium derivatives are effective in enhancing the detection of fatty acids in LC-MS workflows. Herein, we describe the development of two novel pyridinium fixed-charged derivatization reagents that incorporate a photolabile aryl iodide that is selectively activated by laser irradiation inside the mass spectrometer. Photodissociation mass spectra of fatty acids conjugated to 1-(3-(aminomethyl)-4-iodophenyl)pyridin-1-ium (4-I-AMPP+) and 1-(4-(aminomethyl)-3-iodophenyl)pyridin-1-ium (3-I-AMPP+) derivatives reveal structurally diagnostic product ions. These spectra feature radical-directed dissociation of the carbon-carbon bonds within the fatty acyl chain, enabling structural assignments of fatty acids and discrimination of isomers that differ in site(s) of unsaturation, methyl branching or cyclopropanation. These derivatives are shown to be suitable for hyphenated LC-MS methods, and their predictable photodissociation behavior allows de novo identification of unusual fatty acids within a biological context.
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Ácidos Graxos/química , Processos Fotoquímicos , Cromatografia Líquida , Iodo/química , Espectrometria de Massas , Compostos de Piridínio/químicaRESUMO
The (O-acyl)-ω-hydroxy FAs (OAHFAs) comprise an unusual lipid subclass present in the skin, vernix caseosa, and meibomian gland secretions. Although they are structurally related to the general class of FA esters of hydroxy FAs (FAHFAs), the ultra-long chain (30-34 carbons) and the putative ω-substitution of the backbone hydroxy FA suggest that OAHFAs have unique biochemistry. Complete structural elucidation of OAHFAs has been challenging because of their low abundance within complex lipid matrices. Furthermore, because these compounds occur as a mixture of closely related isomers, insufficient spectroscopic data have been obtained to guide structure confirmation by total synthesis. Here, we describe the full molecular structure of ultra-long chain OAHFAs extracted from human meibum by exploiting the gas-phase purification of lipids through multi-stage MS and novel multidimensional ion activation methods. The analysis elucidated sites of unsaturation, the stereochemical configuration of carbon-carbon double bonds, and ester linkage regiochemistry. Such isomer-resolved MS guided the first total synthesis of an ultra-long chain OAHFA, which, in turn, confirmed the structure of the most abundant OAHFA found in human meibum, OAHFA 50:2. The availability of a synthetic OAHFA opens new territory for future investigations into the unique biophysical and biochemical properties of these lipids.
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Ácidos Graxos/química , Ácidos Graxos/síntese química , Espectrometria de Massas , Técnicas de Química Sintética , Ésteres/química , Humanos , Glândulas Tarsais/química , EstereoisomerismoRESUMO
RATIONALE: Analysis of water-based personal lubricants can provide pivotal information to law enforcement regarding sexual assault allegations, particularly in the absence of biological evidence. Traditional methodology for the extraction and analysis of water-based lubricants is cumbersome, time-consuming, and is often not sufficiently selective or sensitive to fully characterise the wide range of chemical components present within complex formulations. METHODS: Liquid extraction surface analysis (LESA) was deployed in combination with high-resolution mass spectrometry (HRMS) and tandem mass spectrometry (MS/MS) to screen a range of water-based lubricants directly from contaminated cotton fabric. Rehydration of the fabric was the only sample preparation required. RESULTS: Analysis of ten different water-based lubricants by nano-electrospray ionisation mass spectrometry in negative ion mode enabled discrimination based on the presence or absence of nine compounds, which were identified by comparison of their MS/MS spectra with those of available standards. Lubricants were successfully detected by LESA from stained fabric surfaces; even following extended periods of time between deposition and sampling. CONCLUSIONS: A library encompassing the common components of water-based lubricants has been established using HRMS and tandem mass spectrometry to enable identification of personal lubricant formulations and differentiation between suppliers.
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Mass spectrometry imaging (MSI) enables the spatial distributions of molecules possessing different mass-to-charge ratios to be mapped within complex environments revealing regional changes at the molecular level. Even at high mass resolving power, however, these images often reflect the summed distribution of multiple isomeric molecules, each potentially possessing a unique distribution coinciding with distinct biological function(s) and metabolic origin. Herein, this chemical ambiguity is addressed through an innovative combination of ozone-induced dissociation reactions with MSI, enabling the differential imaging of isomeric lipid molecules directly from biological tissues. For the first time, we demonstrate both double bond- and sn-positional isomeric lipids exhibit distinct spatial locations within tissue. This MSI approach enables researchers to unravel local lipid molecular complexity based on both exact elemental composition and isomeric structure directly from tissues.
Assuntos
Ozônio/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Mama/química , Mama/metabolismo , Humanos , Isomerismo , Lipídeos/químicaRESUMO
Unambiguous identification of isomeric lipids by mass spectrometry represents a significant analytical challenge in contemporary lipidomics. Herein, the combination of collision-induced dissociation (CID) with ozone-induced dissociation (OzID) on an ion-trap mass spectrometer is applied to the identification of triacylglycerol (TG) isomers that vary only by the substitution pattern of fatty acyl (FA) chains esterified to the glycerol backbone. Isolated product ions attributed to loss of a single FA arising from CID of [TG + Na](+) ions react rapidly with ozone within the ion trap. The resulting CID/OzID spectra exhibit abundant ions that unequivocally reveal the relative position of FAs along the backbone. Isomeric TGs containing two or three different FA substituents are readily differentiated by diagnostic ions present in their CID/OzID spectra. Compatibility of this method with chromatographic separations enables the characterization of unusual TGs containing multiple short-chain FAs present in Drosophila.
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Ácidos Graxos/química , Ozônio/química , Triglicerídeos/química , Animais , Drosophila/química , Isomerismo , Espectrometria de MassasRESUMO
RATIONALE: (O-acyl)-hydroxy fatty acids (OAHFAs) are a recently discovered class of endogenous lipids, generating significant interest for their correlation with enhanced glucose tolerance. Structural variants that differ in the position of the ester linkage have been described, including the ω-OAHFA sub-class, that plays a key role in stabilizing the human tear film. Developing analytical tools for rapid and unambiguous structural elucidation of OAHFAs is essential to understanding their diverse physiological functions. METHODS: Commercially available and synthesized OAHFA standards were dissolved in chloroform and subsequently diluted into methanol with 1.5 mM ammonium acetate. Negative ion collision-induced dissociation (CID) MSn spectra were acquired using chip-based nano-electrospray ionization (Advion TriVersa NanoMate) coupled to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). RESULTS: Major product ions observed during CID of [OAHFA - H]- ions readily identify the constituent fatty acid and hydroxy fatty acid; however, isomers are not easily distinguished. Interrogation of the hydroxy fatty acid and dehydrated hydroxy fatty acid product ions by MSn and ion-molecule reactions yielded diagnostic ions that readily pinpoint hydroxylation position and, thus, the OAHFA ester location. Conversely, these ions are characteristically absent in the MS3 spectra of ω-OAHFAs. Unimolecular dissociation mechanisms are proposed, which are shown to be consistent with prior isotopic labelling experiments. CONCLUSIONS: A mechanistic rationale is provided to explain the unimolecular dissociation of [OAHFA - H]- ions in an ion trap mass spectrometer, thus enabling near-complete de novo structural elucidation of OAHFAs in shotgun lipidomics workflows, even if synthetic standards are unavailable for comparison. Copyright © 2016 John Wiley & Sons, Ltd.
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Ésteres/química , Ácidos Graxos/química , Humanos , Isomerismo , Estrutura Molecular , Espectrometria de Massas por Ionização por Electrospray , Lágrimas/químicaRESUMO
Radical-directed dissociation of gas phase ions is emerging as a powerful and complementary alternative to traditional tandem mass spectrometric techniques for biomolecular structural analysis. Previous studies have identified that coupling of 2-[(2,2,6,6-tetramethylpiperidin-1-oxyl)methyl]benzoic acid (TEMPO-Bz) to the N-terminus of a peptide introduces a labile oxygen-carbon bond that can be selectively activated upon collisional activation to produce a radical ion. Here we demonstrate that structurally-defined peptide radical ions can also be generated upon UV laser photodissociation of the same TEMPO-Bz derivatives in a linear ion-trap mass spectrometer. When subjected to further mass spectrometric analyses, the radical ions formed by a single laser pulse undergo identical dissociations as those formed by collisional activation of the same precursor ion, and can thus be used to derive molecular structure. Mapping the initial radical formation process as a function of photon energy by photodissociation action spectroscopy reveals that photoproduct formation is selective but occurs only in modest yield across the wavelength range (300-220 nm), with the photoproduct yield maximised between 235 and 225 nm. Based on the analysis of a set of model compounds, structural modifications to the TEMPO-Bz derivative are suggested to optimise radical photoproduct yield. Future development of such probes offers the advantage of increased sensitivity and selectivity for radical-directed dissociation.
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Óxidos N-Cíclicos/química , Peptídeos/química , Radicais Livres/química , Íons/química , Espectrometria de Massas , Fotólise , Raios UltravioletaRESUMO
The onset and progression of cancer is associated with changes in the composition of the lipidome. Therefore, better understanding of the molecular mechanisms of these disease states requires detailed structural characterization of the individual lipids within the complex cellular milieu. Recently, changes in the unsaturation profile of membrane lipids have been observed in cancer cells and tissues, but assigning the position(s) of carbon-carbon double bonds in fatty acyl chains carried by membrane phospholipids, including the resolution of lipid regioisomers, has proven analytically challenging. Conventional tandem mass spectrometry approaches based on collision-induced dissociation of ionized glycerophospholipids do not yield spectra that are indicative of the location(s) of carbon-carbon double bonds. Ozone-induced dissociation (OzID) and ultraviolet photodissociation (UVPD) have emerged as alternative ion activation modalities wherein diagnostic product ions can enable de novo assignment of position(s) of unsaturation based on predictable fragmentation behaviors. Here, for the first time, OzID and UVPD (193 nm) mass spectra are acquired on the same mass spectrometer to evaluate the relative performance of the two modalities for lipid identification and to interrogate the respective fragmentation pathways under comparable conditions. Based on investigations of lipid standards, fragmentation rules for each technique are expanded to increase confidence in structural assignments and exclude potential false positives. Parallel application of both methods to unsaturated phosphatidylcholines extracted from isogenic colorectal cancer cell lines provides high confidence in the assignment of multiple double bond isomers in these samples and cross-validates relative changes in isomer abundance.
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Ozônio , Humanos , Ozônio/química , Lipídeos/química , Lipídeos/análise , Raios Ultravioleta , Espectrometria de Massas/métodos , Espectrometria de Massas em Tandem/métodos , Linhagem Celular Tumoral , Íons/químicaRESUMO
Electrochemical treatment of organic matter for environmental remediation necessitates the development of cheap and robust electrodes that are chemically and structurally stable. To address this challenging requirement, we demonstrate a new electrochemical approach using a simple copper electrode under cathodic conditions to electrochemically generate reactive nitrosonium ions for the degradation of different classes of synthetic organic dyes. This could be achieved in an aqueous HNO3/KNO3 electrolyte at a relatively low cathodic potential of -0.5 V RHE at room temperature. UV-visible absorption spectroscopy, Raman spectroscopy, liquid chromatography - mass spectrometry and total organic carbon measurements revealed the rapid decolorisation and mineralisation of several dye types such as triarylmethane dyes (crystal violet, cresol red), an azo dye (methyl orange) as well as a sulfur containing thiazine dye (toluidine blue). The total organic carbon content of a 50 mg L-1 methyl orange solution was found to decrease by 83% after 1 h of electrolysis. Promisingly, locally sourced river and creek water samples spiked with 50 mg L-1 methyl orange were also successfully treated for up to 6 cycles at a simple Cu electrode, demonstrating potential for the remediation of polluted waterways.