RESUMO
The incidence of head and neck squamous cell carcinoma (HNSCC) is increasing and the conventional treatments for this form of cancer can be tough. Despite the success of existing immunotherapies in some HNSCC patients, many do not respond to this type of treatment. Thus, the development of novel anti-cancer therapies should be prioritized. In the current study, the anticancer activity of a panel of novel compounds, herein termed marine product mimics (MPMs), against HNSCC cell lines is explored. The previously reported compound MPM-1, which is structurally related to the novel MPMs, was shown to have promising effects on the HNSCC cell line HSC-3. The results from the current study indicate that the novel MPMs are more potent than MPM-1 but cause a similar type of cell death. The results indicated that the MPMs must cross through the cell membrane to exert their action and that they are lysosomotropic. Further experiments showed that some of the MPMs could induce phosphorylation of eukaryotic initiation factor 2α (eIF2α) in HSC-3 and UT-SCC-24A cells, which indicates that they can activate the integrated stress response that is strongly associated with immunogenic cell death. Cell surface expression of calreticulin and release of HMGB1 and ATP, which are all hallmarks of immunogenic cell death, was also demonstrated in HSC-3 and UT-SCC-24A cells treated with MPMs. This suggests that the MPMs are interesting candidates for future HNSCC cancer therapies.
RESUMO
Mimics of antimicrobial peptides (AMPs) have been proposed as a promising class of antimicrobial agents. We report the analysis of five tetrasubstituted, cationic, amphipathic heterocycles as potential AMP mimics. The analysis showed that the heterocyclic scaffold had a strong influence on the haemolytic activity of the compounds, and the hydantoin scaffold was identified as a promising template for drug lead development. Subsequently, a total of 20 hydantoin derivatives were studied for their antimicrobial potency and haemolytic activity. We found 19 of these derivatives to have very low haemolytic toxicity and identified three lead structures, 2dA, 6cG, and 6dG with very promising broad-spectrum antimicrobial activity. Lead structure 6dG displayed minimum inhibitory concentration (MIC) values as low as 1 µg/mL against Gram-positive bacteria and 4-16 µg/mL against Gram-negative bacteria. Initial mode of action (MoA) studies performed on the amine derivative 6cG, utilizing a luciferase-based biosensor assay, suggested a strong membrane disrupting effect on the outer and inner membrane of Escherichia coli. Our findings show that the physical properties and structural arrangement induced by the heterocyclic scaffolds are important factors in the design of AMP mimics.
Assuntos
Anti-Infecciosos , Hidantoínas , Hidantoínas/farmacologia , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Bactérias Gram-Negativas , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Antibacterianos/químicaRESUMO
We have synthesised short analogues of the marine antimicrobial peptide Turgencin A from the colonial Arctic ascidian Synoicum turgens. In this study, we focused on a central, cationic 12-residue Cys-Cys loop region within the sequence. Modified (tryptophan- and arginine-enriched) linear peptides were compared with Cys-Cys cyclic derivatives, and both linear and Cys-cyclic peptides were N-terminally acylated with octanoic acid (C8), decanoic acid (C10) or dodecanoic acid (C12). The highest antimicrobial potency was achieved by introducing dodecanoic acid to a cyclic Turgencin A analogue with low intrinsic hydrophobicity, and by introducing octanoic acid to a cyclic analogue displaying a higher intrinsic hydrophobicity. Among all tested synthetic Turgencin A lipopeptide analogues, the most promising candidates regarding both antimicrobial and haemolytic activity were C12-cTurg-1 and C8-cTurg-2. These optimized cyclic lipopeptides displayed minimum inhibitory concentrations of 4 µg/mL against Staphylococcus aureus, Escherichia coli and the fungus Rhodothorula sp. Mode of action studies on bacteria showed a rapid membrane disruption and bactericidal effect of the cyclic lipopeptides. Haemolytic activity against human erythrocytes was low, indicating favorable selective targeting of bacterial cells.
Assuntos
Anti-Infecciosos , Lipopeptídeos , Humanos , Lipopeptídeos/farmacologia , Lipopeptídeos/química , Ciclização , Peptídeos Antimicrobianos , Anti-Infecciosos/farmacologia , Anti-Infecciosos/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/química , Antibacterianos/farmacologia , Antibacterianos/química , Escherichia coli , HemóliseRESUMO
An amphipathic barbiturate mimic of the marine eusynstyelamides is reported as a promising class of antimicrobial agents. We hereby report a detailed analysis of the structure-activity relationship for cationic amphipathic N,N'-dialkylated-5,5-disubstituted barbiturates. The influence of various cationic groups, hydrocarbon linkers and lipophilic side chains on the compounds' antimicrobial potency and haemolytic activity was studied. A comprehensive library of 58 compounds was prepared using a concise synthetic strategy. We found cationic amine and guanidyl groups to yield the highest broad-spectrum activity and cationic trimethylated quaternary amine groups to exert narrow-spectrum activity against Gram-positive bacteria. n-Propyl hydrocarbon linkers proved to be the best compromise between potency and haemolytic activity. The combination of two different lipophilic side chains allowed for further fine-tuning of the biological properties. Using these insights, we were able to prepare both, the potent narrow-spectrum barbiturate 8a and the broad-spectrum barbiturates 11lG, 13jA and 13jG, all having low or no haemolytic activity. The guanidine derivative 11lG demonstrated a strong membrane disrupting effect in luciferase-based assays. We believe that these results may be valuable in further development of antimicrobial lead structures.
Assuntos
Anti-Infecciosos , Bactérias Gram-Negativas , Aminas , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Barbitúricos/farmacologia , Cátions/química , Cátions/farmacologia , Hemólise , Humanos , Testes de Sensibilidade Microbiana , Relação Estrutura-AtividadeRESUMO
We report a series of synthetic cationic amphipathic barbiturates inspired by the pharmacophore model of small antimicrobial peptides (AMPs) and the marine antimicrobials eusynstyelamides. These N,N'-dialkylated-5,5-disubstituted barbiturates consist of an achiral barbiturate scaffold with two cationic groups and two lipophilic side chains. Minimum inhibitory concentrations of 2-8 µg/mL were achieved against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum ß-lactamase-carbapenemase production. The guanidine barbiturate 7e (3,5-di-Br) demonstrated promising in vivo antibiotic efficacy in mice infected with clinical isolates of Escherichia coli and Klebsiella pneumoniae using a neutropenic peritonitis model. Mode of action studies showed a strong membrane disrupting effect and was supported by nuclear magnetic resonance and molecular dynamics simulations. The results express how the pharmacophore model of small AMPs and the structure of the marine eusynstyelamides can be used to design highly potent lead peptidomimetics against multi-resistant bacteria.
Assuntos
Antibacterianos/farmacologia , Barbitúricos/farmacologia , Produtos Biológicos/farmacologia , Guanidinas/farmacologia , Indóis/farmacologia , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Tensoativos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Barbitúricos/síntese química , Barbitúricos/química , Produtos Biológicos/síntese química , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Guanidinas/síntese química , Guanidinas/química , Indóis/síntese química , Indóis/química , Testes de Sensibilidade Microbiana , Estrutura Molecular , Proteínas Citotóxicas Formadoras de Poros/síntese química , Proteínas Citotóxicas Formadoras de Poros/química , Relação Estrutura-Atividade , Tensoativos/síntese química , Tensoativos/químicaRESUMO
The rapid emergence and spread of multi-resistant bacteria have created an urgent need for new antimicrobial agents. We report here a series of amphipathic α,α-disubstituted ß-amino amide derivatives with activity against 30 multi-resistant clinical isolates of Gram-positive and Gram-negative bacteria, including isolates with extended spectrum ß-lactamase - carbapenemase (ESBL-CARBA) production. A variety of halogenated aromatic side-chains were investigated to improve antimicrobial potency and minimize formation of Phase I metabolites. Net positive charge and cationic character of the derivatives had an important effect on toxicity against human cell lines. The most potent and selective derivative was the diguanidine derivative 4e with 3,5-di-brominated benzylic side-chains. Derivative 4e displayed minimum inhibitory concentrations (MIC) of 0.25-8⯵g/mL against Gram-positive and Gram-negative reference strains, and 2-32⯵g/mL against multi-resistant clinical isolates. Derivative 4e showed also low toxicity against human red blood cells (EC50â¯>â¯200⯵g/mL), human hepatocyte carcinoma cells (HepG2: EC50â¯>â¯64⯵g/mL), and human lung fibroblast cells (MRC-5: EC50â¯>â¯64⯵g/mL). The broad-spectrum antimicrobial activity and low toxicity of diguanylated derivatives such as 4e make them attractive as lead compounds for development of novel antimicrobial drugs.
Assuntos
Amidas/química , Anti-Infecciosos/química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Amidas/síntese química , Amidas/farmacologia , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Halogenação , Humanos , Camundongos , Testes de Sensibilidade MicrobianaRESUMO
The present study describes the synthesis and biological studies of a small series of head-to-tail cyclic tetrapeptides of the general structure c(Lys-ß2,2 -Xaa-Lys) containing one lipophilic ß2,2 -amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram-positive and gram-negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum ß-lactamase-carbapenemase (ESBL-CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram-positive clinical isolates with minimum inhibitory concentrations of 4-8 µg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic ß2,2 -amino acids form a valuable scaffold for designing novel antimicrobial agents.
Assuntos
Antibacterianos/síntese química , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Peptídeos Cíclicos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Desenho de Fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Halogenação , Estrutura Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Relação Estrutura-AtividadeRESUMO
We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a ß2,2-amino acid with either two 2-naphthyl-methylene or two para-CF3-benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC50) 1170 µM] compared to the linear tetrapeptide counterparts (IC50 18.7 to >413 µM). The most potent cyclic tetrapeptide c3 had a 16.5-fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9-fold) preference for Ramos cells (IC50 23 µM) compared to healthy human lung fibroblast cells (MRC-5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 2228 µM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide-based cancer drugs.
Assuntos
Antineoplásicos/farmacologia , Oligopeptídeos/farmacologia , Peptídeos Cíclicos/farmacologia , Albumina Sérica/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Linhagem Celular , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Ressonância Magnética Nuclear Biomolecular , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacocinética , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacocinética , Ligação ProteicaRESUMO
We report the anticancer activity from screening of a series of synthetic ß(2,2)-amino acid derivatives that were prepared to confirm the pharmacophore model of short cationic antimicrobial peptides with high anti-Staphylococcal activity. The most potent derivatives against human Burkitt's lymphoma (Ramos) cells displayed IC(50) values below 8 µM, and low toxicity against human red blood cells (EC(50) > 200 µM). A more than 5-fold preference for Ramos cancer cells compared to human lung fibroblasts (MRC-5 cells) was also obtained for the most promising ß(2,2)-amino acid derivative 3-amino-N-(2-aminoethyl)-2,2-bis(naphthalen-2-ylmethyl)propanamide (5c). Screening of 5c at the National Cancer Institute (NCI, USA) confirmed its anticancer potency and revealed a very broad range of anticancer activity with IC(50) values of 0.32-3.89 µM against 59 different cancer cell lines. Highest potency was obtained against the colon cancer cell lines, a non-small cell lung cancer, a melanoma, and three leukemia cell lines included in the NCI screening panel. The reported ß(2,2)-amino acid derivatives constitute a promising new class of anticancer agents based on their high anticancer potency, ease of synthesis, mode-of-action, and optimized pharmacokinetic properties compared to much larger antimicrobial peptides.
Assuntos
Aminoácidos/farmacologia , Antineoplásicos/farmacologia , Aminoácidos/síntese química , Aminoácidos/química , Antineoplásicos/síntese química , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Eritrócitos/efeitos dos fármacos , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
We have recently reported a series of synthetic anticancer heptapeptides (H-KKWß(2,2) WKK-NH(2) ) containing a central achiral and lipophilic ß(2,2) -amino acid that display low toxicity against non-malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising ß(2,2) -amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC-5). The results demonstrated a considerable increase in anticancer potency following head-to-tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High-resolution NMR studies and molecular dynamics simulations together with an annexin-V-FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents.
Assuntos
Aminoácidos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linfoma de Burkitt/tratamento farmacológico , Peptídeos Cíclicos/farmacologia , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Ciclização , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Eritrócitos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Relação Estrutura-AtividadeRESUMO
We have investigated the in vitro metabolism of three small antimicrobial ß(2,2)-amino acid derivatives (M (w) < 500) that are highly potent against methicillin resistant Staphylococcus aureus, and are among the first compounds designed from small cationic antimicrobial peptides with potential for oral administration. The ß(2,2)-amino acid derivatives are virtually completely resistant against degradation by proteases, and to further explore their drug potential, we have investigated the hepatic Phase I metabolism of this class of antimicrobial compounds. The ß(2,2)-amino acid derivatives were incubated with murine liver microsomes and the metabolites analyzed semi-quantitatively by HPLC-MS and qualitatively by ultra performance liquid chromatography coupled to a tandem mass spectrometer which enabled identification of the metabolites by careful interpretation of the collision activated dissociation spectra. The study shows that sterically hindered ß(2,2)-amino acid derivatives that otherwise are stable against proteolytic degradation underwent Phase I metabolism and were oxidized to a number of different metabolites depending on the structure of the ß(2,2)-amino acid side-chains.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Microssomos Hepáticos/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/análise , Peptídeos Catiônicos Antimicrobianos/química , Cromatografia Líquida de Alta Pressão , Masculino , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas em TandemRESUMO
Sewage and sewage sludge is known to contain pharmaceuticals, and since sewage sludge is often used as fertilizer within agriculture, the reduction of the selective serotonin reuptake inhibitors (SSRIs) Citalopram, Sertraline, Paroxetine, Fluvoxamine and Fluoxetine during composting has been investigated. Sewage sludge was spiked with the SSRIs before the composting experiment started, and the concentration of the SSRIs in the sludge during a 21 day composting period was measured by liquid phase microextraction (LPME) and high-performance liquid chromatography-mass spectrometry. All the SSRIs had a significant decrease in concentration during the composting process. The highest reduction rates were measured for Fluoxetine and Paroxetine and the lowest for Citalopram. In addition three out of four known SSRI metabolites were found in all the samples, and two of them showed a significant increase in concentration during the composting period.
Assuntos
Inibidores Seletivos de Recaptação de Serotonina/análise , Esgotos/química , Poluentes do Solo/análise , Eliminação de Resíduos Líquidos/métodos , Bactérias Aeróbias/metabolismo , Biodegradação Ambiental , Reatores Biológicos/microbiologia , Cromatografia Líquida de Alta Pressão , Citalopram/análise , Citalopram/metabolismo , Fluoxetina/análise , Fluoxetina/metabolismo , Paroxetina/análise , Paroxetina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/metabolismoRESUMO
A method for the determination of five selective serotonin reuptake inhibitors (citalopram, sertraline, fluoxetine, fluvoxamine and paroxetine) and four of their metabolites (desmethylcitalopram, didesmethylcitalopram, norfluoxetine and desmethylsertraline) in seawater and sewage influents and effluents, has been developed and validated. The method is based on a three-phase hollow-fibre supported liquid phase microextraction of 1.1L samples, followed by high performance liquid chromatography with electrospray ionization and mass spectrometric detection. The detection limits varied between 17 pg/L (citalopram) and 618 ng/L (desmethylsertraline), and the quantification limits between 57 pg/L (citalopram) and 4.1 ng/L (desmethylsertraline). Sampling was done from February to August in 2007 on three different locations with dissimilarities concerning waste water treatment procedures. No significant difference in SSRI cleansing efficiency between merely sieving (Langnes STP, Tromsø) and a more advanced sewage treatment (VEAS STP, Oslo) was seen. All the investigated compounds are present in all waste water samples from these STPs, and a total concentration of SSRIs and metabolites up to 840 ng/L has been found. Untreated sewage samples have been collected in the small town Longyearbyen at Spitsbergen. Despite few inhabitants (2000), it was still possible to find traces of SSRIs in the waste water. In Tromsø and Longyearbyen the waste water is discharged into the sea, therefore seawater samples have been collected close to the outlets. The results show higher concentrations of SSRIs outside Longyearbyen than Tromsø, possibly due to the stronger tidal currents around Tromsø. However, the concentrations are quite low, not exceeding total concentrations of 3 ng/L.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Inibidores Seletivos de Recaptação de Serotonina/análise , Esgotos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Água/análise , Noruega , Sensibilidade e Especificidade , Inibidores Seletivos de Recaptação de Serotonina/química , Inibidores Seletivos de Recaptação de Serotonina/isolamento & purificação , SvalbardRESUMO
Hollow fiber protected liquid-phase microextraction (LPME) was developed for large sample volume extractions in a single step, with special emphasis on extraction of basic drugs from environmental waters. Five antidepressant drugs were extracted from 1100 or 100 mL water samples, through approximately 50 microL of dihexyl ether immobilized in the pores in the wall of a porous hollow fiber (liquid membrane), and into 20 microL of 10 mM HCl or HCOOH as the acceptor solution. Extractions were performed for 60 or 120 min supported by magnetic stirring at 800 rpm, and hereafter the acceptor solution was directly injected in HPLC-UV or HPLC-MS. Compared with earlier work on LPME from small sample volumes, both closing the hollow fiber and the type of liquid membrane was found to be critical for large volume extractions. The hollow fibers were carefully closed with a small piece of metal wire, dihexyl ether was used as the liquid membrane, and pH in the sample was adjusted to 11.8 with NaOH. Recoveries from 1100 mL samples were in the range 33-49%, and enrichments were in the range 18,000-27,000 after 120 min of extraction. With HPLC-MS, the drugs were detected down to the 5-30 pg L(-1) level. Within-day precision was within 12.4-20.6% R.S.D. (n=6), whereas between-day precision was within 17.6 and 37.2% R.S.D. Linearity was obtained in the range 1-500 ng L(-1) with r2-values between 0.982 and 0.994. The proposed LPME system was utilized to detect the five antidepressants in wastewater from the city of Tromsø in Northern Norway.
Assuntos
Cromatografia Líquida/métodos , Concentração de Íons de Hidrogênio , Espectrometria de Massas , Espectrofotometria UltravioletaRESUMO
In the present work, the unsaturated fatty acid substituents of some phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol species were converted to their 1,2-di-hydroxy derivatives by OsO(4). The subsequent electrospray ionization tandem low-energy mass spectrometry analysis of the deprotonated species allowed positional determination of the double bonds by the production of specific product-ions. The product-ions are formed by charge-remote and charge-proximate homolytic cleavages as well as charge-directed heterolytic cleavages and rearrangements. The commercial availability of pure species of the phospholipids in question was limited, and a number of species were therefore synthesized. The developed method was used to fully characterize the two isobaric phosphatidylglycerol species 16:0/16:1Delta(9) and 16:0/16:1Delta(10) extracted from the bacteria Methylococcus capsulatus. The presence of these fatty acids was supported by a gas-chromatography mass spectrometry investigation of the dimethyloxazoline derivatives of the species of the lipid extract. The present work demonstrates that a total structure characterization of acidic unsaturated phospholipids in isolate or in mixtures is accomplished by vicinal di-hydroxylation of olefinic sites and subsequent electrospray ionization mass spectrometry of the derivatized phospholipids.
Assuntos
Ácidos Graxos Insaturados/química , Fosfolipídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Gasosa-Espectrometria de Massas , Hidroxilação , Methylococcus capsulatus/química , Estrutura Molecular , Ácidos Fosfatídicos/química , Fosfatidilgliceróis/química , Fosfatidilinositóis/química , Fosfatidilserinas/químicaRESUMO
We report a novel method allowing the complete structural characterization of intact species of the phospholipid classes phosphatidylcholine and phosphatidylethanolamine by utilizing negative electrospray ionization quadrupole tandem mass spectrometry (MS/MS). Information on the molecular weight of the intact phospholipid species, the class to which it belongs, the molecular mass of the fatty acid substituents and their regioisomerism, is easily revealed by MS/MS. Throughout our investigations the R2COO- ions were more abundant than the R1COO- ions, and this observation is used for regioisomeric assignment of the two fatty acids. However, for phospholipid species containing an unsaturated fatty acid, information on the position of the double bond is not achieved in this way. By converting the olefinic sites to their 1,2-dihydroxylated derivatives, information on the position of the hydroxyl groups (and hence of the double bond) is obtained by performing a second MS/MS experiment. Thus, a complete structural characterization of intact phosphatidylcholine and phosphatidylethanolamine species is obtained by performing these two MS/MS experiments. In order to ensure structural distinction of isobaric species, a number of phosphatidylethanolamine and phosphatidylcholine species were synthesized from lyso-phosphatidylcholine and analyzed by the present method. The applicability of the method to real samples is also demonstrated by the complete structural elucidation of the two phosphatidylcholine species 16:0/18:1Delta9 and 16:0/18:1Delta11 from egg yolk.