Electrochemical studies of the oxidation mechanism of polyamide on glassy carbon electrode

Polyamides containing N-methyl pyrroles and N-methyl imidazoles are a type of small molecule that can bind and recognize the bases of DNA with high affinity and specificity. Five polyamides were studied at glassy carbon electrode in acetate buffer by cyclic and differential pulse voltammetry to clarify their redox pathways. The polyamide electrochemical responses are compared by peak currents and peak potentials. The slopes of the three anodic E_p vs. pH plots of a typical polyamide are linear and show 0.059, 0.057, 0.056 V per pH in acid media, respectively, which correspond to a mechanism involving the equal number of electrons and protons. A possible mechanism for the redox pathway of various polyamides is proposed: the oxidation product of imidazole ring is acylamide and the results of in situ UV–Vis spectroscopy at Pt web electrode support the proposed mechanism. electrospray ionization mass spectroscopy (ESI-MS) indicates that one or two oxygen atoms are added into polyamide molecule after electrochemical oxidation.     

On‐Line Electrochemistry/Electrospray Ionization Mass Spectrometry (EC/ESI‐MS) for the Generation and Identification of Nucleotide Oxidation Products

The oxidation behavior of DNA and RNA nucleotides is studied by an on‐line set‐up consisting of an electrochemical thin‐layer cell (EC) directly coupled to electrospray ionization mass spectrometry (ESI‐MS). This set‐up allows the generation of nucleotide oxidation products in the electrochemical cell at increasing potentials. Moreover, the products are determined directly, without isolation or derivatization steps, by electrospray ionization time of flight mass spectrometry (ESI‐ToF/MS). The dependence of the mass spectra on the applied potential is displayed as ‘mass voltammograms’. An advanced set‐up, consisting of the electrochemical cell coupled to electrospray ionization tandem mass spectrometry (EC/ESI‐MS/MS) allows further structure elucidation based on fragmentation experiments. The electrochemical conversion is performed using a boron doped diamond (BDD) working electrode, which is known to generate hydroxyl radicals at high potentials. The capability of the EC‐MS system to generate highly relevant oxidation products which also occur upon oxidative damage in vivo is demonstrated in this study by the formation of well known biomarkers for DNA damage, including 2′‐deoxy‐8‐oxo‐guanosine 5′‐monophosphate.     

Characterization of solution-phase and gas-phase reactions in on-line electrochemistry-thermospray tandem mass spectrometry

Electrochemistry was used on-line with high-performance liquid chromatography-thermospray tandem mass spectrometry to provide insight into the solution-phase decomposition reactions of electrochemically generated oxidation products. Products formed during electrooxidation were monitored as the electrode potential was varied. The solution reactions which follow the initial electron transfer at the electrode are affected by the vaporizer tip temperature of the thermospray probe and the composition of the thermospray buffer. Either hydrolysis or ammonolysis reactions of the initial electrochemical oxidation products can occur with pH 7 ammonium acetate buffer. Both the electrochemically generated and the synthesized disulfide of 6-thiopurine decompose under thermospray conditions to produce 6-thiopurine and purine-6-sulfinate. Solution-phase studies indicate that nucleophilic and electrophilic substitution reactions with purine-6-sulfinate result in the formation of purine, adenine, and hypoxanthine. Products were identified and characterized by tandem mass spectrometry. This work shows the first example of high-performance liquid chromatography used on-line with electrochemistry to separate stable oxidation products prior to analysis by thermospray tandem mass spectrometry. In addition, solution-phase and gas-phase studies with methylamine show that the site of the nucleophilic and electrophilic reactions is probably inside the thermospray probe. Most importantly, these results also show that the on-line combination of electrochemistry with thermospray tandem mass spectrometry provides valuable information about redox and associated chemical reactions of biological molecules such as the structures of intermediates or products as well as providing insight into reaction pathways.     

Electrospray ionization mass spectrometric analysis of noncovalent complexes between DNA and polyamides containing N-methylpyrrole and N-methylimidazole

Electrospray ionization mass spectrometry was utilized to investigate the noncovalent complexes between novel polyamides and DNA containing the TCCT sequence. We analyzed the noncovalent binding of the polyamides with the DNA and assessed their relative affinities and stoichiometry. The results confirm that hairpin polyamides have higher binding affinities than three-ring polyamides. The hairpin polyamide (PyPyPyPygammaPyImImPybetaDp) has the highest affinity, and the beta-linked polyamide (PyPyPybetaImImImbetaDp) shows a dominant 1:2 binding stoichiometry. Two groups of competition experiments were undertaken to compare the binding affinities of the duplex DNA with different polyamides directly. The affinity scale thus obtained for the group-1 polyamides is PyPyPyPygammaPyImImPybetaDp > PyPyPybetaImImImbetaDp approximately PyPyPygammaImImImbetaDp > PyPyPybetaDp > PyImImbetaDp approximately ImImPybetaDp, and the order for the group-2 polyamides is PyPyPygammaImImImbetaDp > PyPyPygammaImImImbetaOEt > PyPyPygammaImImImbetaCOOH.     

Evaluation of binding selectivity of a polyamide probe to single base-pair different dna in A·T-rich region by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) was used for the evaluation of the binding selectivity of a polyamide probe to single-base pair different DNA in an A.T-rich region. In this procedure, DeltaIr(dsn) was introduced as a parameter to compare the binding affinities of the polyamides with the duplex DNA. The results show that ESI-MS is a very useful tool for analysis of binding selectivity of a polyamide probe to single-base pair different DNA.     

Effect of N-methyl substitution on the thermal decomposition processes in aliphatic–aromatic polyamides

The thermal decomposition processes of two polyamides, derived from succinic acid and two aromatic diamines, were studied by direct pyrolysis mass spectrometry. Fast atom bombardment (FAB) mass spectrometry has been also used in order to provide additional information for the elucidation of the thermal degradation mechanism of the polymers investigated. FAB mass spectra, obtained by introducing in the FAB ion source the solid residues from polymer pyrolysis performed in thermogravimetric experiments, allowed the detection of diagnostic compounds up to about 1600 amu. Our results indicate that the thermal stability of the N-methyl-substituted polyamide is higher than that of the unsubstituted polyamide. The difference in the thermal degradation mechanism accounts for the difference in the thermal stability of the two polyamides. In fact, the unsubstituted polyamide decomposes via an intramolecular exchange and a concomitant N? H hydrogen transfer process with formation of compounds with amine and/or succinimide end groups. Instead, the N-methyl-substituted polyamide decomposes via an α C? H hydrogen transfer process from the methyl group to the nitrogen atom with formation of compounds with amine and/or 2,5-piperidinedione end groups.     

Sequence‐Specific DNA Recognition by Cyclic Pyrrole–Imidazole Cysteine‐Derived Polyamide Dimers

Pyrrole–imidazole (PI) polyamides bind to the minor groove of the DNA duplex in a sequence‐specific manner and thus have the potential to regulate gene expression. To date, various types of PI polyamides have been designed as sequence‐specific DNA binding ligands. One of these, cysteine cyclic PI polyamides containing two β‐alanine molecules, were designed to recognize a 7 bp DNA sequence with high binding affinity. In this study, an efficient cyclization reaction between a cysteine and a chloroacetyl residue was used for dimerization in the synthesis of a unit that recognizes symmetrical DNA sequences. To evaluate specific DNA binding properties, dimeric PI polyamide binding was measured by using a surface plasmon resonance (SPR) method. Extending this molecular design, we synthesized a large dimeric PI polyamide that can recognize a 14 bp region in duplex DNA.     

On-line electrochemistry/liquid chromatography/mass spectrometry for the simulation of pesticide metabolism

On-line electrochemistry/liquid chromatography/mass spectrometry (EC/LC/MS) was employed to mimic the oxidative metabolism of the fungicide boscalid. High-resolution mass spectrometry and MS/MS experiments were used to identify its electrochemical oxidation products. Furthermore, the introduction of a second electrochemical cell with reductive conditions provided important additional information on the oxidation products. With this equipment, hydroxylation, dehydrogenation, formation of a covalent ammonia adduct, and dimerization were detected after initial one-electron oxidation of boscalid to a radical cation. On-line reaction with glutathione yielded different isomeric covalent glutathione adducts. The results of the electrochemical oxidation are in good accordance with previously reported in vivo experiments, showing that EC/LC/MS is a useful tool for studying biotransformation reactions of various groups of xenobiotics.     

Amino-catalyzed fragmentation characteristics of polyamides containing N-methylpyrrole and N-methylimidazole by electrospray negative ionization mass spectrometry

Different negative fragmentations of synthesized polyamides containing N-methylpyrrole and N-methylimidazole were analyzed by electrospray ionization tandem mass spectrometry with low-energy collision-induced dissociation. An interesting rearrangement derived from amino catalysis of N-methylimidazole was observed. The observation is useful for the study of the correlations between the sequences of polyamides and their mass fragmentation pathways.     

Investigation of non-covalent complexes of HIV-1 promoter DNA and polyamides containing N-methylpyrrole by electrospray ionization mass spectrometry

Eight novel polyamides containing N-methylpyrrole were designed to target the sequence (5'-CTGCATATAAGCAG-3'/5'-CTGCTTATATGCAG-3') of the TATA box element of the HIV-1 promoter DNA. The non-covalent complexes of the promoter DNA and the polyamides were investigated by electrospray ionization (ESI) mass spectrometry, which provided strong evidence for the binding of the novel polyamides to the sequence of the TATA box element. It also revealed that polyamide 2 (PyPyPyPybetaDp), a potent binder of HIV-1 promoter DNA and a lead molecule for the design of new anti-HIV-1 drugs, had the highest binding affinity with the TATA box element DNA among these polyamides by examining the stoichiometry and the selectivity.     

Sequence‐Specific DNA Alkylation by Tandem Py–Im Polyamide Conjugates

Tandem N‐methylpyrrole? N‐methylimidazole (Py? Im) polyamides with good sequence‐specific DNA‐alkylating activities have been designed and synthesized. Three alkylating tandem Py? Im polyamides with different linkers, which each contained the same moiety for the recognition of a 10 bp DNA sequence, were evaluated for their reactivity and selectivity by DNA alkylation, using high‐resolution denaturing gel electrophoresis. All three conjugates displayed high reactivities for the target sequence. In particular, polyamide 1 , which contained a β‐alanine linker, displayed the most‐selective sequence‐specific alkylation towards the target 10 bp DNA sequence. The tandem Py? Im polyamide conjugates displayed greater sequence‐specific DNA alkylation than conventional hairpin Py? Im polyamide conjugates ( 4 and 5 ). For further research, the design of tandem Py? Im polyamide conjugates could play an important role in targeting specific gene sequences.     

Mapping Polyamide–DNA Interactions in Human Cells Reveals a New Design Strategy for Effective Targeting of Genomic Sites

Targeting the genome with sequence‐specific synthetic molecules is a major goal at the interface of chemistry, biology, and personalized medicine. Pyrrole/imidazole‐based polyamides can be rationally designed to target specific DNA sequences with exquisite precision in vitro; yet, the biological outcomes are often difficult to interpret using current models of binding energetics. To directly identify the binding sites of polyamides across the genome, we designed, synthesized, and tested polyamide derivatives that enabled covalent crosslinking and localization of polyamide–DNA interaction sites in live human cells. Bioinformatic analysis of the data reveals that clustered binding sites, spanning a broad range of affinities, best predict occupancy in cells. In contrast to the prevailing paradigm of targeting single high‐affinity sites, our results point to a new design principle to deploy polyamides and perhaps other synthetic molecules to effectively target desired genomic sites in vivo.     

Electrospray mass and tandem mass spectrometry identification of ozone oxidation products of amino acids and small peptides

Aqueous ozonation of the 22 most common amino acids and some small peptides were studied by electrospray mass (ESI-MS) and tandem mass spectrometry. After 5 min of ozonation only His, Met, Trp, and Tyr form oxidation products clearly detectable by ESI-MS. For His, the main oxidation product is formed by the addition of three oxygen atoms, His + 30; for Met and Tyr by the addition of one oxygen atom, Met + O and Tyr + O, and for Trp by the addition of two oxygen atoms, Trp + 20. Ozone oxidation occurs rapidly, products are already detected after 30 s of ozonation, and the reactivity order is Met > Trp > Tyr > His. The structures of the oxygen addition products were investigated by electrospray product ion mass spectra, and by comparing these spectra to those of protonated intact amino acids, and when available, to those of model compounds. His + 30 was assigned as 2-amino-4-oxo-4-(3-formylureido)butanoic acid (1) formed by oxidation of the His imidazole ring, Met + O as methionine sulfoxide (2), Trp + 20 as N-formylkynurenine (4), and Tyr + O as a mixture of dihydroxyphenylalanines (7 and 8). Ozonation of peptides show that the same number of oxygen atoms are added as expected from the ozonation of the free amino acids. The product ion mass spectra of both the protonated intact peptides, MH+, and the main ozonation products (M + nO)H+ (n = 1-3) revealed b and y type ions as the main fragments, which allow one to assign the type and location of modified amino acid in the model peptides.     

Thermal Oxidation and Its Relation to Chemiluminescence from Polyolefins and Polyamides

Chemiluminescence (CL) from oxidation of polypropylene, polyethylene and polyamide has been compared and kinetic parameters based on the simplified kinetic scheme involving both bimolecular decomposition of hydroperoxides as an initiating event and correction for the oxidation spreading were determined. Induction times of oxidation determined from the autocatalytic shape of chemiluminescence intensity-time runs increase with an increasing initial molar mass of polypropylene within low molar masses up to 180 kDa regardless of the route of the polypropylene synthesis. The more complex chemiluminescence patterns in case of polyamides may be related with an increase of initially present defect structures including the terminal amino groups.     

聚酰胺类DNA识别分子质谱的碎裂机理

采用ESI-MS法研究了8个含有N-甲基吡咯(Py)和N-甲基咪唑(Im)杂环的聚酰胺质谱的特征和碎裂机理. MSⁿ数据表明, 聚酰胺化合物的主要碎裂路径是环与环间化学键的断裂, 即C—CO键、CO—NH键、HN—C键的断裂, 同时伴随着H原子的重排. 利用这些碎裂特征, 可以得到聚酰胺丰富的结构信息和区分它们的两种同分异构体.     

Lipid Peroxidation in Algae Oil: Antagonist Effects of Natural Antioxidants

Tandem mass spectrometry is proposed to check lipid oxidation, a free radical-mediated phenomenon which effects oxidative deterioration in polyunsaturated fatty acids. Antioxidants are used by the food industry to delay the oxidation process. This process can be controlled by antioxidants, which may occur as natural constituents of foods or may be intentionally added to products. Synthetic antioxidants such as BHT, BHA, and propyl gallate have been extensively used as antioxidants in the industry. The worldwide tendency to avoid or minimize the use of synthetic food additives has prompted the replacement of synthetic antioxidants with natural analogues. The entire process can be supported by the detection and characterization of the reacting species by suitable application of electrospray tandem mass spectrometry under collision-induced dissociation (ESI-CID-MS/MS). Natural antioxidants were tested in this study to check the oxidative stability of algae oil when adding the natural additive. Results were observed in algae oil in situ using electrospray mass spectrometry in tandem with collision-induced dissociation tandem mass spectrometry (ESI-CID-MS/MS) and the POBN spin trapper. The results indicate that alpha-tocopherol is a better antioxidant.     

Tandem trimer pyrrole–imidazole polyamide probes targeting 18 base pairs in human telomere sequences

The binding of molecules to specific DNA sequences is important for imaging genome DNA and for studying gene expression. Increasing the number of base pairs targeted by these molecules would provide greater specificity. N-Methylpyrrole–N-methylimidazole (Py–Im) polyamides are one type of such molecules and can bind to the minor groove of DNA in a sequence-specific manner without causing denaturation of DNA. Our recent work has demonstrated that tandem hairpin Py–Im polyamides conjugated with a fluorescent dye can be synthesized easily and can serve as new probes for studying human telomeres under mild conditions. Herein, to improve their selectivities to telomeres by targeting longer sequences, we designed and synthesized a fluorescent tandem trimer Py–Im polyamide probe, comprising three hairpins and two connecting regions (hinges). The new motif bound to 18 bp dsDNA in human telomeric repeats (TTAGGG)_n, the longest sequence for specific binding reported for Py–Im polyamides. We compared the binding affinities and the abilities to discriminate mismatch, the UV-visible absorption and fluorescence spectra, and telomere staining in human cells between the tandem trimer and a previously developed tandem hairpin. We found that the tandem trimer Py–Im polyamide probe has higher ability to recognize telomeric repeats and stains telomeres in chemically fixed cells with lower background signal.     

Discrimination of A/T sequences in the minor groove of DNA within a cyclic polyamide motif

Eight-ring cyclic polyamides containing pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp) aromatic amino acids recognize predetermined six base pair sites in the minor groove of DNA. Two four-ring polyamide subunits linked by (R)-2,4-diaminobutyric acid [(R)H2Ngamma] residue form hairpin polyamide structures with enhanced DNA binding properties. In hairpin polyamides, substitution of Hp/Py for Py/Py pairs enhances selectivity for T. A base pairs but compromises binding affinity for specific sequences. In an effort to enhance the binding properties of polyamides containing Hp/Py pairings, four eight ring cyclic polyamides were synthesized and analyzed on a DNA restriction fragment containing three 6-bp sites 5'-tAGNNCTt-3', where NN = AA, TA, or AT. Quantitative footprint titration experiments demonstrate that contiguous placement of Hp/Py pairs in cyclo-(gamma-ImPyPyPy-(R)H2Ngamma-ImHpHpPy-) (1) provides a 20-fold increase in affinity for the 5'-tAGAACTt-3' site (Ka = 7.5 x 10(7)M(-1)) relative to ImPyPyPy-(R)H2Ngamma-ImHpHpPy-C3-OH (2). A cyclic polyamide of sequence composition cyclo-(gamma-ImHpPyPy-(R)H2Ngamma-ImHpPyPy-) (3) binds a 5'-tAGTACTt-3' site with an equilibrium association constant KA= 3.2 x 10(9)M(-1), representing a fivefold increase relative to the hairpin analogue ImHpPyPy-(R)H2Ngamma-ImHpPyPy-C3-OH (4). Arrangement of Hp/Py pairs in a 3'-stagger regulates specificity of cyclo-(gamma-ImPyHpPy-(R)H2Ngamma-ImPyHpPy-) (5) for the 5'-tAGATCTt-3' site (Ka = 7.5 x 10(7)M(-1)) threefold increase in affinity relative to the hairpin analogue ImPyHpPy-(R)H2Ngamma-ImPyHpPy-C3-OH (6), respectively. This study identifies cyclic polyamides as a viable motif for restoring recognition properties of polyamides containing Hp/Py pairs.     

Prediction of clozapine metabolism by on-line electrochemistry/liquid chromatography/mass spectrometry

Combining electrochemical conversion, liquid chromatography and electrospray ionization mass spectrometry (EC/LC/ESI-MS) on-line allows the rapid identification of possible oxidation products of clozapine (CLZ) in the absence and in the presence of glutathione. CLZ is, depending on the applied potential, oxidized to various products in an electrochemical flow-through cell using a porous glassy carbon working electrode. Several hydroxylated and demethylated species are detected on-line using LC/MS. While hydroxy-CLZ is most abundant at a potential of 400 mV, demethylation occurs more readily at higher potentials (at around 700 mV versus Pd/H₂ reference). In the presence of glutathione (GSH), various isomeric glutathione adducts and respective products of further oxidation can be identified. The thioadducts are characterized by tandem MS. Mono-GSH and bis-GSH derivatives can be seen in the chromatograms. The results correlate well with the cyclic voltammetric profile of CLZ. The data are relevant from a pharmacological point of view, since similar metabolites (phases I and II) have been reported in the literature. The EC/LC/MS and EC/MS methods should be valuable tools that can be used to anticipate and understand the metabolization patterns of molecules of pharmacological interest and to point out reactive intermediates.     

Electrochemical oxidation and protein adduct formation of aniline: a liquid chromatography/mass spectrometry study

Historically, skin sensitization tests are typically based on in vivo animal tests. However, for substances used in cosmetic products, these tests have to be replaced according to the European Commission regulation no. 1223/2009. Modification of skin proteins by electrophilic chemicals is a key process associated with the induction of skin sensitization. The present study investigates the capabilities of a purely instrumental setup to determine the potential of commonly used non-electrophilic chemicals to cause skin sensitization by the generation of electrophilic species from the parent compound. In this work, the electrophiles were generated by the electrochemical oxidation of aniline, a basic industrial chemical which may also be released from azo dyes in cosmetics. The compound is a known sensitizer and was oxidized in an electrochemical thin-layer cell which was coupled online to electrospray ionization–mass spectrometry. The electrochemical oxidation was performed on a boron-doped diamond working electrode, which is able to generate hydroxyl radicals in aqueous solutions at high potentials. Without any pretreatment, the oxidation products were identified by electrospray ionization/time-of-flight mass spectrometry (ESI-ToF-MS) using their exact masses. A mass voltammogram was generated by plotting the obtained mass spectra against the applied potential. Oligomerization states with up to six monomeric units in different redox states of aniline were observed using this setup. This approach was extended to generate adducts between the oxidation products of aniline and the tripeptide glutathione. Two adducts were identified with this trapping experiment. Protein modification was carried out subsequently: Aniline was oxidized at a constant potential and was allowed to react with β-lactoglobulin A (β-LGA) or human serum albumin (HSA), respectively. The generated adducts were analyzed by liquid chromatography coupled to ESI-ToF-MS. For both β-LGA and HSA, aniline adducts were successfully generated and identified.